Research ArticleSplenectomy Promotes Macrophage Polarization in a MouseModel of Concanavalin A- (ConA-) Induced Liver Fibrosis

YongjuanWang 12 Xiaopei Guo1 Guohui Jiao 1 Lili Luo3 Lu Zhou 1

Jie Zhang 1 and BangmaoWang1

1Department of Gastroenterology and Hepatology Tianjin Medical University General Hospital Tianjin China2Department of Gastroenterology and Hepatology e Second Affiliated Hospital of Hebei Medical University Hebei China3Department of Geriatric Medicine Tianjin Medical University General Hospital Tianjin China

Correspondence should be addressed to Lu Zhou lzhou01tmueducn and Jie Zhang zhangjie xhktmueducn

Received 2 November 2018 Accepted 24 December 2018 Published 6 January 2019

Guest Editor Hengjia Ni

Copyright copy 2019 YongjuanWang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Splenectomy can improve liver function and survival in patients with autoimmune hepatitis (AIH) and liver cirrhosisWe investigated the underlying mechanism in a mouse model of concanavalin A- (ConA-) induced liver fibrosis Methods Weused ConA to induce immune liver fibrosis in BALBc mice Splenectomy was performed alone or with the administration ofdexamethasone (DEX) Changes in blood and liver tissues were evaluated ResultsMice treated with ConA for 7 weeks developedadvanced liver fibrosis while splenectomy suppressed liver fibrosis Although the populations of macrophagesmonocytesand M1 macrophages decreased after splenectomy the inflammatory factors associated with M2 macrophages increased aftersplenectomy Furthermore the population of circulating CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) increasedafter splenectomy After ConA treatment elevated levels of activated and total NF-kBp65p50 combined with DNA were observedin hepatic tissues In contrast the levels of NF-120581Bp65p50 decreased after splenectomyConclusions Splenectomymay promote thepolarization of CD11b+Ly6Chigh MDSCs and the differentiation of M2 macrophages while restricting the level of NF-120581B p65-p50heterodimersThese factors may suppress the progression of liver fibrosis

1 Introduction

Autoimmune hepatitis (AIH) is characterized by the infiltra-tion of mononuclear cells into the liver which together withelevated levels of gamma globulins and autoantibodies caninduce fibrosis and cirrhosis [1] Liver cirrhosis frequentlycauses portal hypertension and splenomegaly and eventuallyliver failure Recent studies in various clinical settings haveindicated that splenectomy can lead to improvements inboth liver function parameters and thrombocytopenia [23] Moreover a study by Maruoka et al demonstrated thatsplenectomy could treat corticosteroid insufficiency in miceand increased the duration of positive effects from dexam-ethasone (DEX) [4] Nonetheless the mechanism behind thisphenomenon remained unknown

Kupffer cells (KCs) are a subset of highly heteroge-neous macrophages that can be stratified into types M1 andM2 [5 6] Bacterial lipopolysaccharide (LPS) can stimulate

the differentiation of macrophages to the M1 type whichproduce the inflammatory factors tumor necrosis factor-(TNF-) 120572 interleukin- (IL-) 12 IL-23 and inducible nitricoxide synthase (iNOS) In contrast IL-4 IL-13 IL-10 andglucocorticoids can induce macrophage polarization to theM2 type which produce cytokines such as IL-4 IL-5 andIL-10 which can inhibit inflammation while promotingblood and lymphatic vessel formation digestion and extra-cellular matrix repair [7 8] Therefore macrophages andmacrophage-related factorsmight play an essential role in thedevelopment and pathogenesis of hepatic lesions and fibrosis[9ndash11] However the polarization of M1M2 macrophages inAIH remains unclear

Myeloid-derived suppressor cells (MDSCs) comprise aheterogeneous population of cells that inhibit the pro-liferation and regular functions of T cells suppress thecytotoxicity of NK cells and accelerate the polarization ofregulatory T cells (Tregs) in tumor-bearing hosts all of

HindawiBioMed Research InternationalVolume 2019 Article ID 5756189 12 pageshttpsdoiorg10115520195756189

2 BioMed Research International

which play key regulatory roles in tumor-related diseases[12 13] In mice MDSCs are CD11b+Gr1+ myeloid cellswhich can be subdivided into CD11b+Gr1+Ly6Ghigh Ly6Clow

and CD11b+Gr1+Ly6GlowLy6Chigh MDSCs Although manyreports have discussed the immunosuppressive functionof MDSCs in various cancer-related diseases the spe-cific regulatory mechanisms of these cells in immunolog-ical liver disease remained unclear [13 14] Zhang et alestablished a model of acute immunological liver injuryby injecting BALBc mice with concanavalin A (ConA)and found that rapamycin could promote the prolifera-tion of CD11b+Gr1Ly6ChighMDSCs which protected againstimmunological hepatic injury in this model [15] This led ustowonder whether splenectomy could inhibit liver fibrosis bypromoting the polarization of MDSCs

NF-120581B a key transcription factor is found in cell typesthroughout the body This factor is a homo- or heterodimercomprising the p50 and p65 (RelA) subunits [16] Theregulation of NF-120581B signaling may lead to macrophage-driven inflammation in both autoimmune and hematologicaldiseases [17 18] Furthermore NF-120581B p50 can regulate M2polarization in the context of LPS-induced inflammation [19]However the correlation between macrophage infiltrationand underlying NF-120581B function in immune-related liverfibrosis remained unclear

ConA-induced hepatitis is a widely accepted and success-ful mouse model of AIH that resembles advanced immune-related liver fibrosis in humans [20 21] In this study weinvestigated the effects of splenectomy in a mouse modelof ConA-induced liver fibrosis and determined whetherMDSCs and NF-120581B were necessary to the protective effectsof splenectomy against liver cirrhosis in these animals

2 Methods

21 Animal Models The study was approved by the MedicalEthics Committee of the General Hospital of Tianjin MedicalUniversity All animals used experimentally were treatedhumanely and all procedures were conducted according tothe guidelines set forth by the Animal Care Committee ofthe General Hospital of Tianjin Medical University Specificpathogen-free female BALBc mice (7 weeks of age) wereacquired from Beijing China A total of 24 mice were usedfor the experiments The mice were randomly assigned tofour groups the control group ConA model group splenec-tomy group and splenectomy combinedwith dexamethasone(DEX) group Except mice in the control group all micereceived a weekly dose of 125 mgkg body weight of ConAvia tail vein injection Splenectomy was conducted 1 dayafter the sixth injection Mice in the splenectomy combinedwith DEX group also received an intraperitoneal injection ofDEX at 1 mgkg body weight every second day To mimicthe presence of immune damage in vivo ConA treatmentwas maintained until death All mice were sacrificed after7 weeks and blood and liver tissue samples were collectedThe liver tissues were embedded in paraffin and subjectedto hematoxylin and eosin (HampE) staining and Massonrsquostrichrome staining

22 Splenectomy For splenectomy the abdominal wall ofthe mouse was opened by making a left subcostal minimalincision under chloral hydrate The splenic arteries and veinswere ligated at the splenic hilum with a 3ndash0 silk sutureand divided All surgical procedures were conducted undercompletely sterile conditions

23 Histological Analysis The histological analysis proce-dures have already been outlined according to previousreports [22 23] Liver tissues were fixed in 10 buffered for-malin and embedded in paraffin Subsequently the sampleswere stained with HampE and Massonrsquos trichrome (to assessfibrosis) and evaluated under a light microscope

24 Immunohistochemical Staining Samples were also sub-jected to immunohistochemistry A goat monoclonal anti-body (mAb) specific for human CD68 (Santa Cruz Biotech-nology Dallas TX USA) and mouse mAb specific forhumanCD206 (Santa Cruz Biotechnology) were used to labelmacrophages and M2 macrophages A goat mAb specific formouse 120572-smooth muscle actin (SMA Santa Cruz Biotech-nology) was used to label 120572-SMA The labeled sections werefurther incubated with a biotin-free secondary antibodyHorseradish peroxidase (Santa Cruz Biotechnology) wasthen applied and the labeled tissues were developed usingdiaminobenzidine (Sigma St Louis MO USA) followed byhematoxylin counterstaining

25 Immunofluorescence Staining A goat mAb specific forhuman CD68 and mouse mAb specific for human CD206(both Santa Cruz Biotechnology) were used to immunoflu-orescently label macrophages and M2 macrophages GoatmAbs specific for mouse F480 and mouse iNOS (bothAbcam CambridgeMA USA) were used to immunofluores-cently labelmacrophages andM1macrophagesThe followingreagents were used in all immunofluorescence experimentsAlexa 488-labeled donkey anti-rat Alexa 647-labeled donkeyanti-rabbit antibodies (Molecular Probes Eugene OR USA)Alexa Fluor 568-labeled donkey anti-mouse and Alexa Fluor488-labeled rabbit anti-goat DAPI was used for nuclearcounterstaining

26 Quantitative RT-PCR (Real-Time PCR) The RT-PCRanalysis was conducted as previously described [24 25]Table 1 lists the primers used in this experiment The datawere analyzed using SDS 21 software For all target genesthe expression is represented as a ldquofold changerdquo relative to thecontrol sample (2-comparative threshold)

27 MAbs and Flow Cytometry MAbs specific for CD11b(M170) F480 (BM8) and Ly6C were obtained from BioLe-gend (San Diego CA USA) The cells were suspended inbuffer and incubated with the above antibody for 30 minutesSubsequently the cells were analyzed on a FACSCaliburflow cytometer (Becton Dickinson San Jose CA USA)or Beckman Coulter Epics XL bench-top flow cytometer(BeckmanCoulter Brea CAUSA)Datawere analyzed usingFlowJo software (TreeStar Ashland OR USA) The number

BioMed Research International 3

Table 1 Oligo sequences for RT-PCR used in this study

genes Forward Reverse120573-actin 51015840-TGTGTCCGTCGTGGATCTGA-31015840 51015840-CCTGCTTCACCACCTTCTTGA-31015840

IL-10 51015840-TGGACAACATACTGCTAACCG-31015840 51015840-GGATCATTTCCGATAAGGCT-31015840

ARG-1 51015840-TGGCTTGCGAGACGTAGAC-31015840 51015840-GCTCAGGTGAATCGGCCTTTT-31015840

IL-4 51015840-CACCAGCTATGCATTGGAGA-31015840 51015840-TTTGGCGGTCAATGTATTTCT-31015840

of cells in several different populations was calculated bymultiplying the percentages of the counted targeted cells bythe number of total cells

28Western Blotting Polyvinylidene fluoride (PVDF) mem-branes were incubated overnight at 4∘C with antibod-ies specific for NF-120581B p50 and NF-120581B p65 (Santa CruzBiotechnology) Samples were then washed in Tris-bufferedsaline with Tween-20 and labeled further with a goat anti-mouse secondary antibody (LI-CORBiotechnology LincolnNE USA) Labeled protein bands were detected using anOdyssey infrared imaging system (LI-COR) Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an inter-nal reference control

29 Electrophoretic Mobility Shi Assay (EMSA) PCRprimers were labeled with [-33P]-ATP (Perkin ElmerWaltham MA USA) using T4 DNA polynucleotide kinase(New England Biolabs Ipswich MA USA) Subsequent-ly DNA probes were created by labeling the forwardprimers and unlabeling the reverse primers via PCR Twomicrograms of nuclear protein were incubated with abiotin-labeled NF-120581B p65 binding-site DNA probe (51015840-AGTTGAGGGGACTTTCCCAGGC-31015840 Sigma Genosys) inbuffer for 30 minutes on ice Gels were placed on Whatmanpaper vacuum-dried and exposed to a phosphoscreen for 12hours Subsequently the screen was scanned using a storm860 PhosphorImager and the data were analyzed usingImageQuant software (Molecular DynamicsGE HealthcareAmersham UK)

210 Statistical Analysis All numeric values are expressedas means plusmn standard deviations (SD) Studentsrsquo t-test orthe MannndashWhitney U test was used to detect the statisticalsignificance of differences between groups Multiple groupcomparisons were assessed using a one-way analysis ofvariance (ANOVA) combinedwith Bonferronirsquos post hoc testA P valuelt005 was considered the statistical significancethreshold

3 Results

31 Effects of Splenectomy in a Mouse Liver Fibrosis ModelClear increases in splenic volume were observed after a 5-week course of intravenous ConA injection and the resultingimmune hepatic injury was characterized by hepatocellularnecrosis portal inflammation mononuclear cell infiltrationinto the parenchyma and sinusoidal hyperemia (Figures1(a) 1(b) and 1(c)) These tissue changes were concomitant

with significant increases in the serum levels of aspar-tate aminotransferase (AST) and alanine aminotransferase(ALT (Plt005) (Figure 1(d)) Most importantly immuno-histochemical staining of 120572-SMA revealed a marked andsignificant increase in the number of activated hepatic stellatecells (Plt005) (Figure 1(e))

32 Splenectomy Attenuated ConA-Induced Liver FibrosisIn the ConA model group the hepatic surfaces were darkred and granular whereas mice were subjected to splenec-tomy had smooth hepatic surfaces (Figure 2(a)) Notablysplenectomy was associated with significant decreases inserum ALT and AST levels (Plt005) (Figure 2(b)) An anal-ysis of liver tissue from the splenectomy group revealed adecrease in interface hepatitis (Figure 2(c)) and significantattenuation of liver fibrosis as indicated by a semiquan-titative analysis of 120572-SMA-positive areas (Plt005) (Figures2(d) and 2(e)) Masson-stained areas (Figure 2(f)) andIshak scores (Plt005) (Figure 2(g)) The Ishak score waslower in the DEX group relative to the splenectomy group(Plt005)

33 Percentages of MacrophagesMonocytes and M2Macrophages Weused flow cytometry immunofluorescencestaining and RT-PCR to detect changes in the numbersof monocytes in peripheral blood and liver macrophagepopulations Notably the ratio of F480-positive monocytesin the peripheral blood increased significantly in theConA model group but decreased in the splenectomygroup (Plt005) (Figure 3) Immunofluorescence staining(Figure 4(a)) revealed increases in the percentages ofF480-positive macrophages and M1 (F480+iNOS+) mac-rophages after ConA treatment as well as decreases inboth populations after splenectomy (Plt005) (Figure 4(b))Furthermore RT-PCR revealed the increased expressionof mRNAs encoding M2 macrophage-associated factorssuch as IL-10 ARG-1 and IL-4 after splenectomy (Plt005)(Figure 4(c))

34 Splenectomy Promoted the Differentiation of11986211986311119887+1198711199106119862ℎ119894119892ℎ MDSCs in the Peripheral Blood Toevaluate the potential involvement of MDSCs in ConA-induced immune liver fibrosis we used flow cytometryto analyze peripheral MDSCs Notably the proportion ofCD11b+Ly6C+ MDSCs decreased after ConA treatment butincreased significantly after splenectomy (Plt005) (Figures5(a) and 5(b)) We also used flow cytometry to analyzechanges in the percentage of CD11b+Ly6Chigh MDSCs in theperipheral blood Here the percentage of CD11b+Ly6Chigh

4 BioMed Research International

Control ConA

Control ConA

(a)

Control ConA

(b)

Control ConA

200X

(c)lowastlowastlowast

lowastlowastlowast

ALT

(UL

)

AST

(UL

)

0

100

200

300

400

500

0

100

200

300

400

Control

ConA

Control

ConA

(d)Contro

l

ConA

lowastlowastlowast

0

20

40

60

80Th

e num

ber o

f act

ivat

ed H

SC

(e)

Figure 1 Morphological and immunohistochemical analysis of liver tissues in control and concanavalin A- (ConA-) treated mice (a) Miceand liver in the control andConA-treated groups (b)Hematoxylin and eosin staining showing inflammatory cell infiltration in the portal area(c) Immunohistochemical analysis of 120572-smooth muscle actin (SMA) expression in liver tissues (immunohistochemical stain magnificationtimes200) (d) Alanine (ALT) and aspartate aminotransferase (AST) levels detected using an enzyme-linked immunosorbent assay (e) Thenumbers of activated hepatic stellate cells (HSC) lowast lowast lowast indicates P lt 0001

MDSCs decreased after ConA treatment but increasedsignificantly after splenectomy (Plt005) (Figures 5(a) and5(b))

35 Splenectomy Inhibited NF-120581B Signaling in Immune Hep-atic Fibrosis We next used Western blotting to analyze thelevels of NF-120581B p65 and NF-120581B p50 proteins in the liverThe level of p65 decreased dramatically after splenectomy(Plt005) whereas the expression of p50 was increased aftersplenectomy (Plt005) (Figure 5(c))

Finally to analyze the DNA-binding capacity of nuclearp65 we used EMSA to measure the amounts of the

NF-120581B p50p65 heterodimer and p50p50 homodimer thathad bound to a specific site using a 32P-labeled probecontaining the minus178 site A band larger than the p50homodimer band was extracted from cells cotransfectedwith p50 and p65 and indicated that p50p65 could bindto the specific site suggesting that the levels of bothp50p65 and p50p50 increased significantly after ConAtreatment (Plt005) In contrast the level of the p50p65 het-erodimer decreased significantly after splenectomy (Plt005)Furthermore the levels of both p50p65 and p50p50were significantly decreased in the DEX group (Plt005)(Figure 5(d))

BioMed Research International 5

ControlConA

SplenectomySplenectomy + DEX

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

100

200

300

400

500

AST

(UL

)

0

100

200

300

400

500

ALT

(UL

)

(b)Control ConA Splenectomy Splenectomy + DEX

(c)

Control ConA Splenectomy Splenectomy + DEX

(d)

Figure 2 Continued

6 BioMed Research International

Control

ConA

Splenect

omy

Splenect

omy+DEX

The n

umbe

r of

lowastlowastlowast

lowastlowastlowast

0

10

20

30

40

HSC

(e)

Control ConA Splenectomy Splenectomy + DEX

(f)

Control

ConA

Splenect

omy

Splenect

omy+EDX

lowastlowastlowast

lowastlowastlowast

lowast

minus2

0

2

4

6

8

Isha

k sc

ore

(g)

Figure 2 Morphologic features histological and immunohistochemical staining (magnification times200) and alanine (ALT) and aspartateaminotransferase (AST) levels in liver tissues from mice in four treatment groups (a) Gross liver tissues (b) The levels of ALT and ASTdetected by enzyme-linked immunosorbent assay (c) Hematoxylin and eosin (HE) revealing inflammatory cell infiltration in the portal area(d) Immunohistochemistry used to detect 120572-smooth muscle actin (SMA) expression in liver tissues (e) Numbers of activated hepatic stellatecells (HSC) evaluated (immunohistochemical stain) (f) Masson trichrome stain Blue areas indicate collagen fiber deposition in the livertissues (g) Ishak scores lowast indicates P lt 005 lowast lowast lowast indicates P lt 0001

BioMed Research International 7

F480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset141

101 102 103 104 105

(a) ControlF480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset176

1020 103 104 105

(b) ConA

F480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset144

102 103 104 105

(c) SplenectomyF480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset109

102 103 104 105

(d) Splenectomy + DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

25

The r

atio

of m

acro

phag

es

(e)

Figure 3 Flow cytometric analysis of monocytes from mice in (a) control (b) ConA-treated (c) splenectomy and (d) splenectomy+DEXgroups and (e) the ratios of F480 monocytes lowast lowast lowast indicates P lt 0001

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

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Submit your manuscripts atwwwhindawicom

BioMed Research International 3

Table 1 Oligo sequences for RT-PCR used in this study

genes Forward Reverse120573-actin 51015840-TGTGTCCGTCGTGGATCTGA-31015840 51015840-CCTGCTTCACCACCTTCTTGA-31015840

IL-10 51015840-TGGACAACATACTGCTAACCG-31015840 51015840-GGATCATTTCCGATAAGGCT-31015840

ARG-1 51015840-TGGCTTGCGAGACGTAGAC-31015840 51015840-GCTCAGGTGAATCGGCCTTTT-31015840

IL-4 51015840-CACCAGCTATGCATTGGAGA-31015840 51015840-TTTGGCGGTCAATGTATTTCT-31015840

of cells in several different populations was calculated bymultiplying the percentages of the counted targeted cells bythe number of total cells

28Western Blotting Polyvinylidene fluoride (PVDF) mem-branes were incubated overnight at 4∘C with antibod-ies specific for NF-120581B p50 and NF-120581B p65 (Santa CruzBiotechnology) Samples were then washed in Tris-bufferedsaline with Tween-20 and labeled further with a goat anti-mouse secondary antibody (LI-CORBiotechnology LincolnNE USA) Labeled protein bands were detected using anOdyssey infrared imaging system (LI-COR) Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an inter-nal reference control

29 Electrophoretic Mobility Shi Assay (EMSA) PCRprimers were labeled with [-33P]-ATP (Perkin ElmerWaltham MA USA) using T4 DNA polynucleotide kinase(New England Biolabs Ipswich MA USA) Subsequent-ly DNA probes were created by labeling the forwardprimers and unlabeling the reverse primers via PCR Twomicrograms of nuclear protein were incubated with abiotin-labeled NF-120581B p65 binding-site DNA probe (51015840-AGTTGAGGGGACTTTCCCAGGC-31015840 Sigma Genosys) inbuffer for 30 minutes on ice Gels were placed on Whatmanpaper vacuum-dried and exposed to a phosphoscreen for 12hours Subsequently the screen was scanned using a storm860 PhosphorImager and the data were analyzed usingImageQuant software (Molecular DynamicsGE HealthcareAmersham UK)

210 Statistical Analysis All numeric values are expressedas means plusmn standard deviations (SD) Studentsrsquo t-test orthe MannndashWhitney U test was used to detect the statisticalsignificance of differences between groups Multiple groupcomparisons were assessed using a one-way analysis ofvariance (ANOVA) combinedwith Bonferronirsquos post hoc testA P valuelt005 was considered the statistical significancethreshold

3 Results

31 Effects of Splenectomy in a Mouse Liver Fibrosis ModelClear increases in splenic volume were observed after a 5-week course of intravenous ConA injection and the resultingimmune hepatic injury was characterized by hepatocellularnecrosis portal inflammation mononuclear cell infiltrationinto the parenchyma and sinusoidal hyperemia (Figures1(a) 1(b) and 1(c)) These tissue changes were concomitant

with significant increases in the serum levels of aspar-tate aminotransferase (AST) and alanine aminotransferase(ALT (Plt005) (Figure 1(d)) Most importantly immuno-histochemical staining of 120572-SMA revealed a marked andsignificant increase in the number of activated hepatic stellatecells (Plt005) (Figure 1(e))

32 Splenectomy Attenuated ConA-Induced Liver FibrosisIn the ConA model group the hepatic surfaces were darkred and granular whereas mice were subjected to splenec-tomy had smooth hepatic surfaces (Figure 2(a)) Notablysplenectomy was associated with significant decreases inserum ALT and AST levels (Plt005) (Figure 2(b)) An anal-ysis of liver tissue from the splenectomy group revealed adecrease in interface hepatitis (Figure 2(c)) and significantattenuation of liver fibrosis as indicated by a semiquan-titative analysis of 120572-SMA-positive areas (Plt005) (Figures2(d) and 2(e)) Masson-stained areas (Figure 2(f)) andIshak scores (Plt005) (Figure 2(g)) The Ishak score waslower in the DEX group relative to the splenectomy group(Plt005)

33 Percentages of MacrophagesMonocytes and M2Macrophages Weused flow cytometry immunofluorescencestaining and RT-PCR to detect changes in the numbersof monocytes in peripheral blood and liver macrophagepopulations Notably the ratio of F480-positive monocytesin the peripheral blood increased significantly in theConA model group but decreased in the splenectomygroup (Plt005) (Figure 3) Immunofluorescence staining(Figure 4(a)) revealed increases in the percentages ofF480-positive macrophages and M1 (F480+iNOS+) mac-rophages after ConA treatment as well as decreases inboth populations after splenectomy (Plt005) (Figure 4(b))Furthermore RT-PCR revealed the increased expressionof mRNAs encoding M2 macrophage-associated factorssuch as IL-10 ARG-1 and IL-4 after splenectomy (Plt005)(Figure 4(c))

34 Splenectomy Promoted the Differentiation of11986211986311119887+1198711199106119862ℎ119894119892ℎ MDSCs in the Peripheral Blood Toevaluate the potential involvement of MDSCs in ConA-induced immune liver fibrosis we used flow cytometryto analyze peripheral MDSCs Notably the proportion ofCD11b+Ly6C+ MDSCs decreased after ConA treatment butincreased significantly after splenectomy (Plt005) (Figures5(a) and 5(b)) We also used flow cytometry to analyzechanges in the percentage of CD11b+Ly6Chigh MDSCs in theperipheral blood Here the percentage of CD11b+Ly6Chigh

4 BioMed Research International

Control ConA

Control ConA

(a)

Control ConA

(b)

Control ConA

200X

(c)lowastlowastlowast

lowastlowastlowast

ALT

(UL

)

AST

(UL

)

0

100

200

300

400

500

0

100

200

300

400

Control

ConA

Control

ConA

(d)Contro

l

ConA

lowastlowastlowast

0

20

40

60

80Th

e num

ber o

f act

ivat

ed H

SC

(e)

Figure 1 Morphological and immunohistochemical analysis of liver tissues in control and concanavalin A- (ConA-) treated mice (a) Miceand liver in the control andConA-treated groups (b)Hematoxylin and eosin staining showing inflammatory cell infiltration in the portal area(c) Immunohistochemical analysis of 120572-smooth muscle actin (SMA) expression in liver tissues (immunohistochemical stain magnificationtimes200) (d) Alanine (ALT) and aspartate aminotransferase (AST) levels detected using an enzyme-linked immunosorbent assay (e) Thenumbers of activated hepatic stellate cells (HSC) lowast lowast lowast indicates P lt 0001

MDSCs decreased after ConA treatment but increasedsignificantly after splenectomy (Plt005) (Figures 5(a) and5(b))

35 Splenectomy Inhibited NF-120581B Signaling in Immune Hep-atic Fibrosis We next used Western blotting to analyze thelevels of NF-120581B p65 and NF-120581B p50 proteins in the liverThe level of p65 decreased dramatically after splenectomy(Plt005) whereas the expression of p50 was increased aftersplenectomy (Plt005) (Figure 5(c))

Finally to analyze the DNA-binding capacity of nuclearp65 we used EMSA to measure the amounts of the

NF-120581B p50p65 heterodimer and p50p50 homodimer thathad bound to a specific site using a 32P-labeled probecontaining the minus178 site A band larger than the p50homodimer band was extracted from cells cotransfectedwith p50 and p65 and indicated that p50p65 could bindto the specific site suggesting that the levels of bothp50p65 and p50p50 increased significantly after ConAtreatment (Plt005) In contrast the level of the p50p65 het-erodimer decreased significantly after splenectomy (Plt005)Furthermore the levels of both p50p65 and p50p50were significantly decreased in the DEX group (Plt005)(Figure 5(d))

BioMed Research International 5

ControlConA

SplenectomySplenectomy + DEX

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

100

200

300

400

500

AST

(UL

)

0

100

200

300

400

500

ALT

(UL

)

(b)Control ConA Splenectomy Splenectomy + DEX

(c)

Control ConA Splenectomy Splenectomy + DEX

(d)

Figure 2 Continued

6 BioMed Research International

Control

ConA

Splenect

omy

Splenect

omy+DEX

The n

umbe

r of

lowastlowastlowast

lowastlowastlowast

0

10

20

30

40

HSC

(e)

Control ConA Splenectomy Splenectomy + DEX

(f)

Control

ConA

Splenect

omy

Splenect

omy+EDX

lowastlowastlowast

lowastlowastlowast

lowast

minus2

0

2

4

6

8

Isha

k sc

ore

(g)

Figure 2 Morphologic features histological and immunohistochemical staining (magnification times200) and alanine (ALT) and aspartateaminotransferase (AST) levels in liver tissues from mice in four treatment groups (a) Gross liver tissues (b) The levels of ALT and ASTdetected by enzyme-linked immunosorbent assay (c) Hematoxylin and eosin (HE) revealing inflammatory cell infiltration in the portal area(d) Immunohistochemistry used to detect 120572-smooth muscle actin (SMA) expression in liver tissues (e) Numbers of activated hepatic stellatecells (HSC) evaluated (immunohistochemical stain) (f) Masson trichrome stain Blue areas indicate collagen fiber deposition in the livertissues (g) Ishak scores lowast indicates P lt 005 lowast lowast lowast indicates P lt 0001

BioMed Research International 7

F480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset141

101 102 103 104 105

(a) ControlF480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset176

1020 103 104 105

(b) ConA

F480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset144

102 103 104 105

(c) SplenectomyF480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset109

102 103 104 105

(d) Splenectomy + DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

25

The r

atio

of m

acro

phag

es

(e)

Figure 3 Flow cytometric analysis of monocytes from mice in (a) control (b) ConA-treated (c) splenectomy and (d) splenectomy+DEXgroups and (e) the ratios of F480 monocytes lowast lowast lowast indicates P lt 0001

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

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Hindawiwwwhindawicom Volume 2018

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Nucleic AcidsJournal of

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Submit your manuscripts atwwwhindawicom

4 BioMed Research International

Control ConA

Control ConA

(a)

Control ConA

(b)

Control ConA

200X

(c)lowastlowastlowast

lowastlowastlowast

ALT

(UL

)

AST

(UL

)

0

100

200

300

400

500

0

100

200

300

400

Control

ConA

Control

ConA

(d)Contro

l

ConA

lowastlowastlowast

0

20

40

60

80Th

e num

ber o

f act

ivat

ed H

SC

(e)

Figure 1 Morphological and immunohistochemical analysis of liver tissues in control and concanavalin A- (ConA-) treated mice (a) Miceand liver in the control andConA-treated groups (b)Hematoxylin and eosin staining showing inflammatory cell infiltration in the portal area(c) Immunohistochemical analysis of 120572-smooth muscle actin (SMA) expression in liver tissues (immunohistochemical stain magnificationtimes200) (d) Alanine (ALT) and aspartate aminotransferase (AST) levels detected using an enzyme-linked immunosorbent assay (e) Thenumbers of activated hepatic stellate cells (HSC) lowast lowast lowast indicates P lt 0001

MDSCs decreased after ConA treatment but increasedsignificantly after splenectomy (Plt005) (Figures 5(a) and5(b))

35 Splenectomy Inhibited NF-120581B Signaling in Immune Hep-atic Fibrosis We next used Western blotting to analyze thelevels of NF-120581B p65 and NF-120581B p50 proteins in the liverThe level of p65 decreased dramatically after splenectomy(Plt005) whereas the expression of p50 was increased aftersplenectomy (Plt005) (Figure 5(c))

Finally to analyze the DNA-binding capacity of nuclearp65 we used EMSA to measure the amounts of the

NF-120581B p50p65 heterodimer and p50p50 homodimer thathad bound to a specific site using a 32P-labeled probecontaining the minus178 site A band larger than the p50homodimer band was extracted from cells cotransfectedwith p50 and p65 and indicated that p50p65 could bindto the specific site suggesting that the levels of bothp50p65 and p50p50 increased significantly after ConAtreatment (Plt005) In contrast the level of the p50p65 het-erodimer decreased significantly after splenectomy (Plt005)Furthermore the levels of both p50p65 and p50p50were significantly decreased in the DEX group (Plt005)(Figure 5(d))

BioMed Research International 5

ControlConA

SplenectomySplenectomy + DEX

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

100

200

300

400

500

AST

(UL

)

0

100

200

300

400

500

ALT

(UL

)

(b)Control ConA Splenectomy Splenectomy + DEX

(c)

Control ConA Splenectomy Splenectomy + DEX

(d)

Figure 2 Continued

6 BioMed Research International

Control

ConA

Splenect

omy

Splenect

omy+DEX

The n

umbe

r of

lowastlowastlowast

lowastlowastlowast

0

10

20

30

40

HSC

(e)

Control ConA Splenectomy Splenectomy + DEX

(f)

Control

ConA

Splenect

omy

Splenect

omy+EDX

lowastlowastlowast

lowastlowastlowast

lowast

minus2

0

2

4

6

8

Isha

k sc

ore

(g)

Figure 2 Morphologic features histological and immunohistochemical staining (magnification times200) and alanine (ALT) and aspartateaminotransferase (AST) levels in liver tissues from mice in four treatment groups (a) Gross liver tissues (b) The levels of ALT and ASTdetected by enzyme-linked immunosorbent assay (c) Hematoxylin and eosin (HE) revealing inflammatory cell infiltration in the portal area(d) Immunohistochemistry used to detect 120572-smooth muscle actin (SMA) expression in liver tissues (e) Numbers of activated hepatic stellatecells (HSC) evaluated (immunohistochemical stain) (f) Masson trichrome stain Blue areas indicate collagen fiber deposition in the livertissues (g) Ishak scores lowast indicates P lt 005 lowast lowast lowast indicates P lt 0001

BioMed Research International 7

F480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset141

101 102 103 104 105

(a) ControlF480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset176

1020 103 104 105

(b) ConA

F480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset144

102 103 104 105

(c) SplenectomyF480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset109

102 103 104 105

(d) Splenectomy + DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

25

The r

atio

of m

acro

phag

es

(e)

Figure 3 Flow cytometric analysis of monocytes from mice in (a) control (b) ConA-treated (c) splenectomy and (d) splenectomy+DEXgroups and (e) the ratios of F480 monocytes lowast lowast lowast indicates P lt 0001

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

ControlConA

SplenectomySplenectomy + DEX

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

100

200

300

400

500

AST

(UL

)

0

100

200

300

400

500

ALT

(UL

)

(b)Control ConA Splenectomy Splenectomy + DEX

(c)

Control ConA Splenectomy Splenectomy + DEX

(d)

Figure 2 Continued

6 BioMed Research International

Control

ConA

Splenect

omy

Splenect

omy+DEX

The n

umbe

r of

lowastlowastlowast

lowastlowastlowast

0

10

20

30

40

HSC

(e)

Control ConA Splenectomy Splenectomy + DEX

(f)

Control

ConA

Splenect

omy

Splenect

omy+EDX

lowastlowastlowast

lowastlowastlowast

lowast

minus2

0

2

4

6

8

Isha

k sc

ore

(g)

Figure 2 Morphologic features histological and immunohistochemical staining (magnification times200) and alanine (ALT) and aspartateaminotransferase (AST) levels in liver tissues from mice in four treatment groups (a) Gross liver tissues (b) The levels of ALT and ASTdetected by enzyme-linked immunosorbent assay (c) Hematoxylin and eosin (HE) revealing inflammatory cell infiltration in the portal area(d) Immunohistochemistry used to detect 120572-smooth muscle actin (SMA) expression in liver tissues (e) Numbers of activated hepatic stellatecells (HSC) evaluated (immunohistochemical stain) (f) Masson trichrome stain Blue areas indicate collagen fiber deposition in the livertissues (g) Ishak scores lowast indicates P lt 005 lowast lowast lowast indicates P lt 0001

BioMed Research International 7

F480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset141

101 102 103 104 105

(a) ControlF480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset176

1020 103 104 105

(b) ConA

F480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset144

102 103 104 105

(c) SplenectomyF480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset109

102 103 104 105

(d) Splenectomy + DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

25

The r

atio

of m

acro

phag

es

(e)

Figure 3 Flow cytometric analysis of monocytes from mice in (a) control (b) ConA-treated (c) splenectomy and (d) splenectomy+DEXgroups and (e) the ratios of F480 monocytes lowast lowast lowast indicates P lt 0001

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

Control

ConA

Splenect

omy

Splenect

omy+DEX

The n

umbe

r of

lowastlowastlowast

lowastlowastlowast

0

10

20

30

40

HSC

(e)

Control ConA Splenectomy Splenectomy + DEX

(f)

Control

ConA

Splenect

omy

Splenect

omy+EDX

lowastlowastlowast

lowastlowastlowast

lowast

minus2

0

2

4

6

8

Isha

k sc

ore

(g)

Figure 2 Morphologic features histological and immunohistochemical staining (magnification times200) and alanine (ALT) and aspartateaminotransferase (AST) levels in liver tissues from mice in four treatment groups (a) Gross liver tissues (b) The levels of ALT and ASTdetected by enzyme-linked immunosorbent assay (c) Hematoxylin and eosin (HE) revealing inflammatory cell infiltration in the portal area(d) Immunohistochemistry used to detect 120572-smooth muscle actin (SMA) expression in liver tissues (e) Numbers of activated hepatic stellatecells (HSC) evaluated (immunohistochemical stain) (f) Masson trichrome stain Blue areas indicate collagen fiber deposition in the livertissues (g) Ishak scores lowast indicates P lt 005 lowast lowast lowast indicates P lt 0001

BioMed Research International 7

F480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset141

101 102 103 104 105

(a) ControlF480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset176

1020 103 104 105

(b) ConA

F480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset144

102 103 104 105

(c) SplenectomyF480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset109

102 103 104 105

(d) Splenectomy + DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

25

The r

atio

of m

acro

phag

es

(e)

Figure 3 Flow cytometric analysis of monocytes from mice in (a) control (b) ConA-treated (c) splenectomy and (d) splenectomy+DEXgroups and (e) the ratios of F480 monocytes lowast lowast lowast indicates P lt 0001

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

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Hindawiwwwhindawicom Volume 2018

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GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

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Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

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Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Cell BiologyInternational Journal of

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MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

F480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset141

101 102 103 104 105

(a) ControlF480

FSC

50K

100K

150K

200K

250K

0

Comp-PE-A FSC-H subset176

1020 103 104 105

(b) ConA

F480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset144

102 103 104 105

(c) SplenectomyF480

FSC

50K

100K

150K

200K

250K

0

0

Comp-PE-A FSC-H subset109

102 103 104 105

(d) Splenectomy + DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

25

The r

atio

of m

acro

phag

es

(e)

Figure 3 Flow cytometric analysis of monocytes from mice in (a) control (b) ConA-treated (c) splenectomy and (d) splenectomy+DEXgroups and (e) the ratios of F480 monocytes lowast lowast lowast indicates P lt 0001

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

DAPI F480 iNOS Merge

Control

ConA

Splenectomy

Splenectomy

+ DEX

400 x

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

The n

umbe

r ofF

480

+ce

lls

0

5

10

15

20

25

The n

umbe

r of F

480

+iN

OS+

cells

(b)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

1

2

3

Rela

tive I

L-4

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

Rela

tive A

RG-1

leve

l

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

00

05

10

15

20

Rela

tive I

L-10

leve

l

(c)

Figure 4 F480+ macrophages and F480+ inducible nitric oxide synthase (NOS)+ macrophages were detected by (a) immunofluorescentstaining and (b) the ratios were calculatedTheM2macrophage-related cytokines (c) interleukin- (IL-) 10 arginase- (ARG-) 1 and IL-4 weredetected by RT-PCR ConA concanavalin A DEX dexamethasone lowast lowast lowast indicates P lt 0001

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

Control ConA Splenectomy Splenectomy + DEX

CD11b

Ly6C

FSC

Ly6C

(a)

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowast

lowastlowastlowast

0

5

10

15

20

The r

atio

of L

y6C

high

MD

SCs

The r

atio

of

Control

ConA

Splenect

omy

Splenect

omy+DEX0

5

10

15

20

25

CD11

b+Ly

6C+

MD

SCs

(b)

GAPDH

P50

P65

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowastlowast

0

50

100

150

200

P65

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowast

60708090

100110120

P50

expr

essi

on

(c)

Figure 5 Continued

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

10 BioMed Research International

P50P65

P50P50

Control

ConA

Splenectomy

Splenectomy

+DEX

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowast

020406080

100

p50-

p50

expr

essi

on

Control

ConA

Splenect

omy

Splenect

omy+DEX

lowastlowastlowastlowastlowast

0

50

100

150

P50-

P65

expr

essi

on

(d)

Figure 5 The ratios of CD11b+Ly6C and CD11b+Ly6Chigh myeloid-derived suppressor cells (MDSCs) were detected by flow cytometry (a)Flow cytometry analysis (b) the ratios of CD11b+Ly6C and CD11b+Ly6Chigh MDSCs (c) the levels of p65 and p50 and (d) ratios of p65p50and p50p50 in treatment different groups of mice ConA concanavalin A DEX dexamethasonelowast indicates P lt 0005lowastlowast indicates P lt 001and lowast lowast lowast indicates P lt 0001

4 Discussion

Liver cirrhosis greatly limits the use of immunosuppressantsin patients with AIH [1] Several studies have reported thepotential protective effects of splenectomy liver functionparameters in this patient population [2 4] For example ina series of 12 patients with end-stage AIH who underwentliver transplantation Xu et al found that splenectomy couldprevent the posttransplantation recurrence of AIH [3] Inprevious studies ConA has always been used at a dose of15ndash20mgkg body weight to induce acute liver injury in mice[26] However we successfully established a mouse modelof immune liver fibrosis using a ConA dose of 125 mgkgbody weight for up to 5 weeks In our ConA model weobserved significant increases in serum ALT and AST levelsthe spleenbody weight ratio and the tissue level of 120572-SMAWe also found that splenectomy could significantly reducethese signs of ConA-induced liver fibrosis

Macrophages and macrophage-related factors have beenreported to play an essential role in the pathogenesis ofhepatic lesions and fibrosis [9ndash11] Jindal et al observed asignificant increase in F480-positive macrophages in NASHmice and significant decreases in the expression of M1macrophage-related markers such as IL-10 and CXCL10 [27]However the polarization of M1M2 macrophages in thecontext of AIH remained unclear Interestingly we foundthat the total andM1macrophage populations were increasedin AIH patients whereas the M2 macrophage populationhad decreased In our ConA-induced mouse model of liverfibrosis we similarly observed increases in macrophagesand M1 macrophages and a decrease in M2 macrophagesconsistent findings in AIH patients After splenectomy thenumbers of macrophages and M1 macrophages in our modelmice decreased while the expression of M2 macrophage-related cytokines increased These findings suggest that M2macrophages play an important role in suppressing liverfibrosis in AIH

Umemura et al reported that MDSCs exhibit pleiotropiccharacteristics of both M1 and M2 monocytesmacrophages[28] In recent years MDSCs have been identified in the

context of various inflammatory and immune responsesincluding parasitic infections and autoimmune reactions [2930] However few studies have evaluated MDSCs in patientswith AIH In a BALBc mouse model of ConA-induced acuteimmunological liver injury Zhang et al found that rapamycinpromoted the proliferation of CD11b+Gr1Ly6ChighMDSCswhich protected against immunological hepatic injury [15]In this study we observed a significant decrease in thefrequency of CD11b+Ly6ChighMDSCs after ConA treatmentbut a significant increase in this population after splenectomyTherefore we suggest that CD11b+Ly6ChighMDSCs may bekey inhibitors of liver inflammation in AIH We also suggestthat CD11b+Ly6ChighMDSCs could potentially transforminto M2 macrophages

MDSCs can differentiate into M1 and M2 macrophagesin a process that is mainly regulated by STAT1 and NF-120581B [31] The NF-120581B signaling pathway is known to regulatemacrophage-driven inflammation in autoimmune diseasesand the NF-120581Bp5050 homodimer is a regulator of M2polarization19 Using Western blotting we demonstratedsignificant increases in the protein levels of NF-120581Bp50 andP65 after treatment with ConA as well as a significantdecrease in the level of NF-120581Bp65 after splenectomy Asintracellular NF-120581B can only exert transcriptional activityafter binding toDNAwe also performed an EMSA and foundthat the expression of NF-120581Bp65p50 and NF-120581Bp50p50both increased significantly after ConA treatment whereasthe expression of NF-120581Bp65p50 decreased after splenec-tomy Most interestingly the expression of NF-120581Bp5050decreased significantly after splenectomy combined withDEX whereas no significant change in this expression levelwas observed after splenectomy alone We speculate thathormone therapy may inhibit NF-120581B signaling pathwayactivity whereas splenectomy only inhibits the formation ofthe NF-120581Bp65p50 heterodimer

Ryutaro Maruoka et al reported that splenectomy couldprolong the effects of corticosteroids in a mouse model ofAIH [4] Specifically these authors suggested that althoughcorticosteroid treatment protects against AIH it allows

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 11

Splenectomy

MDSCs Monocytes

NF-BP65p50 NF-BP65p50

M1 M2

inflammation

Figure 6 Proposed mechanisms by which splenectomy suppressesliver fibrosis in a mouse model

residual splenic failed to regulate TFH cells to remain after thetreatment Few previous reports have compared splenectomywith DEX therapy In our study we compared splenectomyalone or combined with DEX therapy Our results indicatethat splenectomy combined with DEX led to significantlyreduced liver fibrosis scores compared with the splenectomyalone Moreover the NF-120581B signaling pathway was signif-icantly inhibited after splenectomy combined with DEXThese results suggest that DEX and splenectomy have asynergistic effect in the treatment of ConA-induced liverfibrosis in mice However additional studies of the specificmechanism and long-term safety are needed

This study provides basic information regarding thepotential mechanism underlying the ability of splenectomy toinhibit liver inflammation However the exact mechanismsinvolving MDSCs macrophages and the NF-120581B signalingpathway will require further study Future research regardingtherapeutic applications should aim to understand the mech-anism underlying macrophage phenotype switches in vivo

In summary we have evaluated the role of splenectomy inthe observed accumulation of CD11b+Ly6ChighMDSCs in amouse model of liver fibrosis Notably splenectomy inducedthe proliferation of M2 macrophages possibly by inhibitingactivation of the NF-120581B signaling pathway (Figure 6) Thisfinding suggests that MDSCs could be amplified in vitro andused therapeutically and indicates a new concept for thetreatment of autoimmune diseases

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Additional Points

Key Summary Our research findings enabled us tonewly clarify the molecular mechanism underlying themacrophagemonocyte activation and the inflammatoryresponse in autoimmune hepatitis (AIH) We also newly

detected the role of splenectomy in CD11b+Ly6ChighMDSCsaccumulation in a mouse model of liver fibrosis anddemonstrated that splenectomy could induce the prolif-eration of M2 macrophages possibly by inhibiting NF-120581Bsignaling pathway activation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

Yongjuan Wang and Xiaopei Guo contributed equally to thismanuscript

Acknowledgments

This work was supported by Science and Technology Devel-opment Fund Project of Tianjin no 17ZXMFSY00210 and theNational Natural Science Foundation of China (81600509)

References

[1] European Association for the Study of the Liver ldquoEASL clinicalpractice guidelines autoimmune hepatitisrdquo Journal of Hepatol-ogy vol 63 no 4 pp 971ndash1004 2015

[2] K Murata K Ito K Yoneda K Shiraki H Sakurai and MIto ldquoSplenectomy improves liver function in patients with livercirrhosisrdquoHepatogastroenterology vol 55 pp 1407ndash1411 2008

[3] S Yamada Y Morine S Imura et al ldquoLiver regenerationafter splenectomy in patients with liver cirrhosisrdquo HepatologyResearch vol 46 no 5 pp 443ndash449 2016

[4] R Maruoka N Aoki M Kido et al ldquoSplenectomy prolongsthe effects of corticosteroids in mouse models of autoimmunehepatitisrdquo Gastroenterology vol 145 no 1 pp 209ndash220 2013

[5] R D Stout C Jiang B Matta I Tietzel S K Watkins andJ Suttles ldquoMacrophages sequentially change their functionalphenotype in response to changes in microenvironmentalinfluencesrdquo e Journal of Immunology vol 175 pp 342ndash3492005

[6] IM JWolfsMM P CDonners andM P J deWinther ldquoDif-ferentiation factors and cytokines in the atherosclerotic plaquemicro-environment as a trigger for macrophage polarisationrdquorombosis and Haemostasis vol 106 no 5 pp 763ndash771 2011

[7] AMantovaniA Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

[8] S Gordon and F O Martinez ldquoAlternative activation ofmacrophagesmechanism and functionsrdquo Immunity vol 32 no5 pp 593ndash604 2010

[9] K K Wijesundera T Izawa A H Tennakoon et al ldquoM1-and M2-macrophage polarization in rat liver cirrhosis inducedby thioacetamide (TAA) focusing on Iba1 and galectin-3rdquoExperimental and Molecular Pathology vol 96 no 3 pp 382ndash392 2014

[10] Y Sun X Li X Meng C Huang L Zhang and J LildquoMacrophage Phenotype in Liver Injury and Repairrdquo Scandina-vian Journal of Immunology vol 85 no 3 pp 166ndash174 2017

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

12 BioMed Research International

[11] H Fukushima S Yamashina A Arakawa et al ldquoFormationof p62-positive inclusion body is associated with macrophagepolarization in non-alcoholic fatty liver diseaserdquo HepatologyResearch vol 48 no 9 pp 757ndash767 2018

[12] S Ostrand-Rosenberg and P Sinha ldquoMyeloid-derived suppres-sor cells linking inflammation and cancerrdquo e Journal ofImmunology vol 182 no 8 pp 4499ndash4506 2009

[13] A A Al-Khami P C Rodriguez and A C Ochoa ldquoMetabolicreprogramming of myeloid-derived suppressor cells (MDSC)in cancerrdquo Oncoimmunology vol 5 no 8 Article ID e12007712016

[14] M Shen J Wang W Yu et al ldquoA novel MDSC-induced PD-1(-)PD-L1(+) B-cell subset in breast tumor microenvironmentpossesses immuno-suppressive propertiesrdquo Oncoimmunologyvol 7 no 4 Article ID e1413520 2018

[15] Y Zhang Y Bi H Yang et al ldquomTOR limits the recruitment ofCD11b+Gr1+Ly6Chighmyeloid-derived suppressor cells in pro-tecting against murine immunological hepatic injuryrdquo Journalof Leukocyte Biology vol 95 no 6 pp 961ndash970 2014

[16] M S Hayden and S Ghosh ldquoSignaling toNF-kappaBrdquoGenes ampDevelopment vol 18 no 18 pp 2195ndash2224 2004

[17] D W Kang M Park H Oh et al ldquoPhospholipase D1 has apivotal role in interleukin-1beta-driven chronic autoimmunearthritis through regulation of NF-kappaB hypoxia-induciblefactor 1alpha and FoxO3ardquoMolecular and Cellular Biology vol33 no 14 pp 2760ndash2772 2013

[18] A Valaperti ldquoDrugs targeting the canonical NF-kappaB path-way to treat viral and autoimmune myocarditisrdquo Current Phar-maceutical Design vol 22 pp 440ndash449 2016

[19] C Porta M Rimoldi G Raes et al ldquoTolerance and M2(alternative) macrophage polarization are related processesorchestrated by p50 nuclear factor kappaBrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 106 no 35 pp 14978ndash14983 2009

[20] J Liang B Zhang R-W Shen et al ldquoPreventive effect ofhalofuginone on concanavalin A-induced liver fibrosisrdquo PLoSONE vol 8 no 12 Article ID e82232 2013

[21] J Liang B Zhang R W Shen et al ldquoThe effect of antifibroticdrug halofugine on Th17 cells in concanavalin A-induced liverfibrosisrdquo Scandinavian Journal of Immunology vol 79 no 3 pp163ndash172 2014

[22] L Chen F Lu D Chen et al ldquoBMSCs-derived miR-223-containing exosomes contribute to liver protection in experi-mental autoimmune hepatitisrdquoMolecular Immunology vol 93pp 38ndash46 2018

[23] B C de Sousa C B Miguel W F Rodrigues et al ldquoEffects ofshort-term consumption ofMorinda citrifolia (Noni) fruit juiceon mice intestine liver and kidney immune modulationrdquo Foodand Agricultural Immunology vol 28 no 3 pp 528ndash542 2017

[24] W Ren P Wang J Yan et al ldquoMelatonin alleviates weanlingstress in mice Involvement of intestinal microbiotardquo Journal ofPineal Research vol 64 no 2 Article ID e12448 2018

[25] J Yin J Duan Z Cui W Ren T Li and Y Yin ldquoHydrogenperoxide-induced oxidative stress activates NF-kappa B andNrf2Keap1 signals and triggers autophagy in pigletsrdquo RSCAdvances vol 5 no 20 pp 15479ndash15486 2015

[26] M I Arshad M Rauch A LrsquoHelgoualcrsquoh et al ldquoNKT cellsare required to induce high IL-33 expression in hepatocytesduring ConA-induced acute hepatitisrdquo European Journal ofImmunology vol 41 no 8 pp 2341ndash2348 2011

[27] A Jindal S Bruzzı S Sutti et al ldquoFat-laden macrophagesmodulate lobular inflammation in nonalcoholic steatohepatitis(NASH)rdquo Experimental and Molecular Pathology vol 99 no 1pp 155ndash162 2015

[28] N Umemura M Saio T Suwa et al ldquoTumor-infiltratingmyeloid-derived suppressor cells are pleiotropic-inflamedmonocytesmacrophages that bear M1- and M2-type charac-teristicsrdquo Journal of Leukocyte Biology vol 83 no 5 pp1136ndash1144 2008

[29] K R Crook M Jin M F Weeks et al ldquoMyeloid-derivedsuppressor cells regulate T cell and B cell responses duringautoimmune diseaserdquo Journal of Leukocyte Biology vol 97 no3 pp 573ndash582 2015

[30] C Guo F Hu H Yi et al ldquoMyeloid-derived suppressor cellshave a proinflammatory role in the pathogenesis of autoim-mune arthritisrdquo Annals of the Rheumatic Diseases vol 75 no1 pp 278ndash285 2016

[31] A Lin F Liang E A Thompson et al ldquoRhesus MacaqueMyeloid-Derived Suppressor Cells Demonstrate T CellInhibitory Functions and Are Transiently Increased afterVaccinationrdquo e Journal of Immunology vol 200 no 1 pp286ndash294 2018

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom