aeromonas hydrophila infection and alters cd4 /cd8 t...

Research ArticleHesperidin Inhibits Inflammatory ResponseInduced by Aeromonas hydrophila Infection andAlters CD4+CD8+ T Cell Ratio

Abdelaziz S A Abuelsaad12 Gamal Allam12 and Adnan A A Al-Solumani3

1 Department of Microbiology (Immunology Section) College of Medicine Taif University Taif 21974 Saudi Arabia2Department of Zoology Faculty of Science Beni-Suef University Beni-Suef Egypt3 Department of Pediatric College of Medicine Taif University Taif 21974 Saudi Arabia

Correspondence should be addressed to Abdelaziz S A Abuelsaad elsaad1yahoocom

Received 21 December 2013 Accepted 21 March 2014 Published 6 May 2014

Academic Editor Muzamil Ahmad

Copyright copy 2014 Abdelaziz S A Abuelsaad et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Background Aeromonas hydrophila is an opportunistic bacterial pathogen that is associated with a number of human diseasesHesperidin (HES) has been reported to exert antioxidant and anti-inflammatory activities Objectives The aim of this study wasto investigate the potential effect of HES treatment on inflammatory response induced by A hydrophila infection in murineMethodsA hydrophila-infectedmicewere treatedwithHES at 250mgkg bwtweek for 4 consecutiveweeks Phagocytosis reactiveoxygen species production CD4+CD8+ T cell ratio and CD14 expression on intestinal infiltrating monocytes were evaluatedTheexpression of E-selectin and intercellular adhesionmolecule 1 on stimulatedHUVECs andRAWmacrophagewas evaluatedResultsPercentage of CD4+ T cells in the intestinal tissues of infected treated mice was highly significantly increased however phagocyticindex ROSproduction CD8+ T cells percentage andCD14 expression onmonocyteswere significantly reducedOn the other handHES significantly inhibited A-LPS- and A-ECP-induced E-selectin and ICAM-1 expression on HUVECs and ICAM-1 expressionon RAWmacrophageConclusion Present data indicated that HES has a potential role in the suppression of inflammatory responseinduced by A hydrophila toxins through downmodulation of ROS production and CD14 and adhesion molecules expression aswell as increase of CD4+CD8+ cell ratio

1 Introduction

Aeromonas species are facultative aerobes and motile andgram negative bacteria They are widely distributed innature and involved in sepsis wound infections and food-borne gastroenteritis [1] The virulence of Aeromonas (A)hydrophila is based upon its extracellular proteins (ECP)such as aerolysins hemolysins enterotoxins and proteolyticenzymes as well as its extracellular polysaccharides (EPS)and lipopolysaccharides (LPS endotoxin) Nam and Kiseong[2] showed that Aeromonas aerolysin can form channelsby heptamerization of the host cell membrane The porechannels impair epithelial integrity by promoting intestinaltight junction protein redistribution and thus affect woundclosure [3 4] Meanwhile the EPS of Aeromonas medi-ate the interaction between pathogenic bacteria and their

environment through adhesion to the host cells [5 6] Inparticular A hydrophila infection rapidly alters a numberof potentially critical lectins chemokines interleukins andother mucosal factors in a manner predicted to enhanceits ability to adhere to and invade host tissues [7] Anequally important nonfimbrial adhesion factor that has beenimplicated in the pathogenesis of Aeromonas spp is LPS Asan adhesin S-type LPS is indispensable for initial attachmentof bacteria to host tissue and is necessary during infectionevents where it protects bacteria from antimicrobial peptidesand complement-mediated killing [8 9]

CD14 is expressed on the surface of monocytes macro-phages and neutrophils and occurs as a membrane-boundform and a soluble form [10 11] It has been implicatedin the development and maturation of the innate immunesystem [12ndash15] Several studies have reported the relationship

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 393217 11 pageshttpdxdoiorg1011552014393217

2 Mediators of Inflammation

between CD14 and its role in the polarisation of T lympho-cytes intoTh1 andTh2 subsets [16ndash20]

Classical immunoregulatory tissues control and deter-mine the success of critical early steps in pathogenesis includ-ing microbe adhesion entry and replication [7] Even whenmucosal tissues are healthy they are bathed in low levels ofE-selectin intercellular adhesion molecule 1 (ICAM-1) andinterleukin 8 (IL-8) Of these IL-8 forms a gradient of expres-sion that is greatest near the bacteriaepithelial cell interface[21 22] E-selectin meanwhile is a membrane glycoproteinand is expressed by endothelial cells in order to mediatethe adhesion of leukocytes It is upregulated rapidly duringinflammation resulting in increased leukocyte-endothelialcell adhesion [23] Adhesion molecules play importantroles in cellular interactions during inflammatory responsesExpression of ICAM-1 for example plays an important rolein the adhesion of monocytes to endothelial cells [24]

Regarding flavonoids these have metal chelating freeradical scavenging properties such as neutralization of thesinglet oxygen and superoxide and inhibition of the hydro-gen peroxide-induced lipid peroxidation (LPO) [25 26]Flavonoids inhibit the expression of isoforms of cyclooxy-genase inducible nitric oxide synthase and lipooxygenasewhich are responsible for the production of NO prostanoidsand leukotrienes as well as inflammatory mediators such ascytokines chemokines or adhesion molecules [27]

Hesperidin (HES) is a flavanone glycoside commonlyfound in the diet in citrus fruits or citrus fruit derivedproducts [26 28] The anti-inflammatory effects of HES havebeen characterized in vitro in both rodent and human celllines [29 30]The scavenging effect of free radicals associatedwithHES has been evidenced by different neurochemical andneurobehavioral parameters with HES treatment appearingto reduce expression of proinflammatory mediators likeinducible nitric oxide synthase (iNOS) TNF-120572 and IL-1120573[31 32] Recently HES has been shown to exhibit pronouncedimmunological activities serving to inhibit inflammatory cellinfiltration and mucus hypersecretion in a murine model ofasthma [33] In addition HES counteracted the upregulationof proinflammatory cytokines such as the expression of TNF-120572 and IL-1120573 in cerebral ischemia [31 34 35] as well as IL-8IL-6 IL-12 and vascular cell adhesion molecule 1 (VCAM-1)in the case of acute lung inflammation induced by LPS in vivo[36]

The aim of the present study was to investigate theantiadhesion and anti-inflammatory role of HES in the caseof gastrointestinal Aeromonas infection in a murine model

2 Materials and Methods21 Bacteria and Growth Conditions A standard A hydroph-ila strain (ATCC catalogue number 7966) was kindlyprovided by the Fish Department Faculty of VeterinaryCairo University Giza EgyptThe bacteriumwasmaintainedand subcultured three times before the experiments Briefly100 120583L ofA hydrophilawas inoculated into 150mL of a liquidpeptone broth (Oxoid) and incubated for 30∘C for 24 h withcontinuous shaking at 250 rpm The harvested bacteria werecentrifuged at 6000 g for 10min and the dried pellet was

suspended twice in phosphate-buffered saline (PBS) to thefinal dose of 2 times 108 CFUmL

211 Preparation of A hydrophila Lipopolysaccharides (A-LPS) LPS was prepared as described by Westphal and Jann[37] Briefly the bacteriawere inoculated in 250mL of a LuriaBertani (LB) broth and incubated for 24 h at 30∘C on a shakerat 250 rpm The culture was then centrifuged at 10000 rpmfor 10min at 4∘C resuspended in 166mL of TAE buffer(40mM Tris-acetate pH85 2mM EDTA) and then mixedwith 332mL alkaline solution (containing 3 g of SDS 06 gof Trizma (Sigma) and 160mL of 2N NaOH in 1000mL ofwater) The suspension was heated at 55 to 60∘C for 70minand then mixed with phenol and chloroform in the ratio of1 1 (VV) The mixture was spun at 10 000 rpm for 10min at4∘C and the supernatant obtained was mixed with 332mLof water and 83mL of 3M sodium acetate buffer (pH 52)LPS was precipitated by adding twice the volume of ethanolThe precipitate was dissolved in 332mL of 50mM Tris-HClpH 80 (Sigma) and 100mM sodium acetate mixed well andwas then reprecipitated with twice the volume of ethanolThecombined water extract was dialyzed for 2ndash4 days againstdistilled water and then freeze-dried

212 Preparation of A hydrophila Extracellular Proteins (A-ECP) The bacterial isolate was grown overnight in 5mLLBbroth for preculturing 100120583L of this culture suspension(inoculum) was added to 50mLLB broth and incubatedovernight at 37∘C at a shaker speed of 200 rpm The culturesuspension was harvested at 5000 rpm at 4∘C for 15min Thesupernatant was precipitated by the addition of 10 (wv)trichloroacetic acidwith overnight incubation at 4∘C Furthercentrifugation at 11000 rpm for 20minutes resulted in a pelletcontaining extracellular proteins which was suspended in50 120583L of 1M Tris-HCl buffer (pH 8) and dialyzed overnightagainst the same buffer The freeze-dried protein content wasdetermined as described by Lowry et al [38] The purifiedprotein was ascertained as endotoxin-free with the limulusamebocyte lysate (LAL) test

22 Animals MaleMF1 albinomice (7-8weeks old weighing20ndash25 g King Fahd Specialist Medical Centre Jeddah KSA)were used in the experiments and housed in a barrier roomunder standard conditions The animals were kept in wire-mesh polycarbonate cages with autoclaved bedding wereacclimatized to laboratory conditions (12 h dark 12 h lightcycles 240 plusmn 10∘C) and had free access to food and waterad libitum The food containers were refilled daily with freshstandard diet and were fitted with bars to reduce losses Rou-tine clinical observations and body weight were measuredregularly throughout the experiments Animal use and thecare protocol were approved by the Research Ethics Commit-tee College of Medicine Taif University Saudi Arabia

23 Natural Products Hesperidin (HES) used in this studywas of analytical grade and purchased from Sigma ChemicalCo (St Louis Missouri USA) and dissolved in 1 dimethylsulphoxide (DMSO) immediately before use

Mediators of Inflammation 3

24 Experiment Design For in vivo studies mice were ran-domly assigned to four groups (119899 = 10group) as follows (1)Control group (C) received only the standard diet had freeaccess to sterile water and was orally fed with PBS (pH 7402mLmice) using intragastric intubation at intervals par-allel to the treated groups (2) Bacteria group (B) was orallyfed once per week with bacterial suspension of A hydrophila(02mL containing 2 times 108 CFUmouse) for four consecutiveweeks This dose was selected according to Abuelsaad et al[39] (3) In infected-treated group (B-HES) bacteria-infectedmice were orally fed with 250mg HESkgweek for fourconsecutive weeks according to Abuelsaad et al [39] At theend of week four following infection and treatment bloodwas collected from the retroorbital sinus into sodium citrate(038)

25 Quantification of Phagocytic Index in Blood Phagocyticability of neutrophils was performed according to a modifiedversion of a previously described assay for the intracellularconversion of nitroblue tetrazolium (NBT) to formazan bysuperoxide anion [40 41] Briefly 01mL of blood was mixedwith 01mL of 02 NBT solution (Sigma) in sterile plastictest tubes for 30min at room temperature The formazancontent of the cells was then solubilized with 960 120583L 2MKOH and 1120 120583L DMSO and the extinction was measuredspectrophotometrically in 1 cm cuvettes at optical density(OD) of the cells was 630 nm Values of the extinction weretransposed according to a standard curve into mg NBTformate per 1mL of blood A standard curve was preparedby adding KOH and DMSO to known amounts of NBT Asa positive control 100mM hydrogen peroxide was added tocells and the amount of formazan formed was measuredAt the same time the total number of the leukocytes wasexamined in order to calculate the absolute number of bloodneutrophils Individual mouse blood samples were applied intriplicate and the mean was calculated The NBT index wasdetermined by using the following equation

Phagocytic index in blood (NBT conversion)

=

mg of NBT formate1mL bloodNeutrophil count in thousands

(1)

26 Quantification of Reactive Oxygen Species in IntestinalTissues Intracellular conversion of NBT to formazan bysuperoxide anion (O

2

∙minus) was used to measure the generationof reactive oxygen species [40ndash42] About 01mL of intestinaltissue homogenate was incubated with 01mL of 10 120583MNBT (Sigma) for 30min to allow O

2

∙ generated from thecollected intestinal tissues to reduce NBT to formazan Theformazan content of was then solubilized with 960 120583L 2MKOHand 1120120583LDMSOdetermined spectrophotometricallyat 630 nm against a mixture of KOH and DMSO as a blankAs a positive control 100 120583MH

2O2was added to cells and the

amount of formazan formed was measured Standard curvesof NBT (0ndash10 120583M)were constructed by using themixture as avehicle The SOD-inhibitable NBT reduction was calculatedby subtracting the average of the negative controls from allother samples Final O

2

∙ production was expressed as nmoles

of NBT per milligram protein per 30min incubation timeIndividual mouse samples were applied in triplicate and themean was calculated

27 Flow Cytometry (FACS) Analysis for CD Markers271 Total Lymphocytes and Monocytes Isolation Smallslices from intestine tissues were homogenized using40 120583m cell strainers (BD Falcon Bedford MA) Red bloodcells were osmotically lysed using lysis buffer containing0165MNH

4Cl2 Lymphocytes are resolved from other

white blood cells (granulocytes monocytes) based ondensity gradient centrifugation using lymphocyte separationmedium (LSM 1077 PAA Laboratories Germany) asdescribed by Badr et al [43] Monocytes were isolatedfrom lymphocytes to evaluate CD14 expression by positiveselection using magnetic CD14 microbeads (human Catnumber 130-050-201Miltenyi Biotec Germany) as describedby Neu et al [44]

Lymphocytes and monocytes were washed with phos-phate-buffered saline (PBS pH 74) counted using trypanblue exclusion test and cultured in complete R-10 medium(RPMI 1640 medium supplemented with 10 FCS 2mML-glutamine 100 IUmL penicillin 100 120583gmL streptomycin1mM sodium pyruvate and 50 120583M 2-mercaptoethanol) Thepurity of cells was assessed using flow cytometry and wasgreater than 90 Cells were cultured in R-10 medium

272 Antibodies and Flow Cytometry Cells were stainedwith mAbs and analyzed using a FACSCalibur (BD FranklinLakes NJ) according to Neu et al [44] Briefly purifiedlymphocytes and monocytes from intestinal tissues (1 times 106cells50 120583L PBS) were washed once with washing buffer (3(vv) FBS and 01 (wv) NaN

3in PBS) resuspended in

blocking buffer (3 (vv) FBS 5 (vv) normal human ABserum Cat number C11-020 PAA Laboratories Germanyand 01 NaN

3(wv) in PBS) with purified CD16CD32

FccIIIIImAb (AbDSerotec Co USA) to prevent nonspecificbinding Subsequently cells were incubated with mAb for20min at room temperature in dark area with the followingFluor-conjugated FITC rat anti-mouse antibodies purchasedfrom AbD Serotec Co USA as follows CD3-FITC CD4-FITC (Cat numbers MCA500FT and MCA1767FT resp)and PE-conjugated anti-CD8 (CAT MCA1768PE) and anti-CD14 (CATMCA2745PE) Subsequently cells were washedfixed in paraformaldehyde (PFA 4 (vv) in PBS Sigma-Aldrich Germany) and stored at 4∘C in washing buffer untilfurther use

A FACS Calibur flow cytometer was used for dataacquisition with Diva software (BD Biosciences) for dataanalysis After gating on viable cells 10000 events per samplewere analyzed For each marker the threshold of positivitywas defined beyond the nonspecific binding observed in thepresence of a relevant control mAb

28 Expression of Adhesion Molecules on HUVECs andRAW Macrophage Human umbilical vein endothelial cells(HUVECs) and RAW macrophage cell lines were obtainedand cultured as described by Takami et al [45] and Leitinger


et al [46] Monolayer of HUVECs and RAW cells (passages4ndash6) was incubated with 100 150 or 200120583MmL HES fortwo hours in the presence or absence of Aeromonas LPS(100 ngmL) or Aeromonas ECP (100 ngmL) in medium 199(M199) containing 20 supplemented fetal bovine serum(FBS) 1 unitmL heparin 50120583gmL bovine endothelial cellgrowth supplement (Technoclone Vienna Austria) 2mMglutamine 100 unitsmL penicillin and 100120583gmL strepto-mycin Antibodies for whole-cell ELISAs using cell-surface-expressed method for E-selectin or intercellular adhesionmolecule 1 (ICAM-1) were obtained from RampD Systems(MinneapolisMinnesota)Detection is performedusing goatanti-mouse antibody conjugated to peroxidase O-Phenylenediamine (OPD Sigma) was used for colour development thereaction was stopped using 3M H

2SO4 and optical density

(OD) was read at 492 nm using a microtiter plate reader(ANTHOS Salzburg Austria)

29 Statistical Analysis Analysis of variance on SPSS soft-ware package (version 16) was used to test the present dataOne-way analysis of variance (ANOVA)was used to study thesignificant differences In the case of significant difference themultiple range comparisons (Duncanrsquos test) was selected fromthe post hoc window on the same statistical package to detectthe distinct variance between means For further analysis allvalues are given as the means plusmn SD Differences with 119875 lt 005were considered statistically significant

3 Results

31 Changes in Body and Organ Weights Concerning chan-ges in body and organ weights Figure 1(a) shows that bodyweight did not significantly (119875 gt 005) change betweenthe groups (2486 plusmn 2847 g in the infected group versus23682 plusmn 1728 and 23211 plusmn 3244 g in the control and HES-treated groups resp) Liver weight recorded a nonsignificantincrease (119875 gt 005 Figure 1(b)) in the infected group (1461 plusmn0271 g in the infected group versus 1346 plusmn 0028 and1331 plusmn 0133 g in the control andHES-treated groups resp)Similarly spleenweight (Figure 1(c)) showed a nonsignificantincrease (119875 gt 005) in the infected group (0141 plusmn 00280215 plusmn 0121 and 0204 plusmn 0099 g for control infected andHES-treated groups resp) Meanwhile the intestine weight(Figure 1(d)) was not significantly (119875 gt 005) altered in thedifferent groups (3572 plusmn 0373 3291 plusmn 0861 and 3010 plusmn0609 g for control infected and HES-treated groups resp)

32 Quantification of Phagocytic Activity and ROS ProductionRegarding the quantification of the phagocytic ability ofneutrophils in blood Figure 2(a) shows that there was ahighly significant (119875 lt 0001) increase in the A hydrophila-infected group (1073 plusmn 0117) in comparison to the control(080 plusmn 0048) and HES-treated (0881 plusmn 0208) groupsThis data should be discussed in parallel with the quan-tification of reactive oxygen species in intestinal tissues asmeasured by the intracellular conversion of NBT to formazanby the superoxide anion (O

2

∙minus) Intestinal ROS production(nM NBTmg protein tissues30min Figure 2(b)) showed

a highly significant (119875 lt 0001) increase in the infectedgroup (11545 plusmn 1052 nM NBTmg protein tissues30min)in comparison to the control (7099 plusmn 1161) andHES-treated(8736 plusmn 086) groups

33 Flow Cytometry (FACS) Analysis for CDMarkers Quan-tification of the CD markers of the intestinal infiltratinglymphocytes and monocytes obtained from a selection ofmice is illustrated in Figure 3 The results showed thatHES treatment significantly increased CD4+ T cells in theintestinal infiltrating lymphocytes (9173 plusmn 655 with 119875 lt0001) versus 5555 plusmn 1110 and 5845 plusmn 821 for the controland infected groups respectively (Figure 3(b)) On the otherhand A hydrophila infection induced a highly significantelevation in CD8+ T cells (119875 lt 0001) while HES treat-ment significantly suppressed this increase in the numberof CD8+ T cells (7600 plusmn 050 12858 plusmn 23 4290 plusmn 094for control bacteria-infected and HES-treated groups resp)(Figure 3(c)) Taken together the present data shows that theratio of CD4+CD8+ T lymphocytes inA hydrophila-infectedmice was significantly increased by HES treatment

Moreover A hydrophila infection induced a highly sig-nificant expression of CD14+ on the surface on intestinalinfiltrating monocytes (69322 plusmn 591 with 119875 lt 0001)while HES treatment significantly suppressed this elevation(51168 plusmn 225 and 51734 plusmn 567 for control and HES-treatedgroups resp) (Figure 3(d))

34 Expression of Adhesion Molecules Using Modified CellELISA Expression of E-selectin was explored in vitro bymodified cell enzyme linked immunosorbent assay (ELISA)Pretreatment of human umbilical vein endothelial cells(HUVECs) with A-LPS (100 ngmL) significantly increasedthe expression of both E-selectin and ICAM-1 (0525 plusmn 0082and 1519 plusmn 0092 resp) as shown in Table 1 Treatment ofHUVECs with different concentrations of HES meanwhilesignificantly suppressed the A-LPS-induced expression of E-selectin (0214 plusmn 0007 0122 plusmn 0002 and 0225 0031 for100 150 and 200 120583M HESmL resp) and the expression ofICAM-1 (0209 plusmn 0011 0181 plusmn 0016 and 0145 plusmn 001 for100 150 and 200 120583MHESmL resp) as shown in Table 1

Moreover A-ECP induced a highly significant expression(119875 lt 0001) of E-selectin and ICAM-1 (0833 plusmn 0068 and1491 plusmn 0099 resp) as shown in Table 1 HES treatment onthe other hand significantly suppressed this A-ECP-inducedexpression of E-selectin (0195 plusmn 0052 0114 plusmn 0002 and0136 plusmn 0018 for 100 150 and 200120583M HESmL resp) andthat of ICAM-1 (0143 plusmn 00050126 plusmn 001 and 0096 plusmn 0005for 100 150 and 200120583MHESmL resp) (Table 1)

The expression of ICAM-1 on RAW macrophage wasexplored in vitro with the results set out in Table 1 Pre-treatment of RAW cells with A-LPS and A-ECP (100 ngmL)significantly increased the expression of ICAM-1 (1452 plusmn0074 and 1401 plusmn 0063 resp) The data showed that HES


A

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Control group HES-treated groupB-infected group

(a)

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Figure 1 In vivo effect of hesperidin inoculation on body weight (a) liver weightg (b) spleen weightg (c) and intestine weightg (d)Mice were infected each with 2 times 108 CFU of Aeromonas hydrophila per week for four consecutive weeks (B-infected group) and treatedsimultaneously with hesperidin at a dose of 250mgkgweek for four consecutive weeks (HES-treated group) At the end of week 4 followingexposure and treatment mice were sacrificed and weighted the liver spleen and intestine were weighted phagocytic activity was estimatedin fresh blood and intestinal ROS production was evaluated in intestinal homogenate Values not sharing common superscripts denotesignificant differences

suppressed A-LPS-induced expression of ICAM-1 on RAWcells (1224 plusmn 012 1096 plusmn 0087 and 104 plusmn 0212 for 100150 and 200120583M HESmL resp) Similarly HES suppressedA-ECP-induced expression of ICAM-1 on RAWcells (1148 plusmn0159 1061 plusmn 0045 and 1215 plusmn 0029 for 100 150 and200120583MHESmL resp) (Table 1)

4 Discussion

Previously it was reported that all mice injected ip withAeromonas hydrophila had died within twenty days of infec-tion [39] Pretreatment with HES (250mgkg bwt) how-ever was effective and significantly (119875 lt 005) prolonged


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Phag

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(a)

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Alowastlowastlowast

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Control group

HES-treated group

B-infected group

(b)

Figure 2 In vivo effect of hesperidin inoculation on phagocytic activities in blood (NBT index) (a) and intestinal reactive oxygen species(ROS) production (b) Mice were infected each with 2 times 108 CFU of Aeromonas hydrophila per week for four consecutive weeks (B-infectedgroup) and treated simultaneously with hesperidin at a dose of 250mgkgweek for four consecutive weeks (HES-treated group) At the endof week 4 following exposure and treatment mice were sacrificed and weighted the liver spleen and intestine were weighted phagocyticactivity was estimated in fresh blood and intestinal ROS production was evaluated in intestinal homogenate Values not sharing commonsuperscripts denote significant differences

Table 1 In vitro effect of different concentrations of hesperidin on the expression of E-selectin and ICAM-1 on HUVECs and RAWcells in response to Aeromonas hydrophila antigen stimulation Human umbilical vein endothelial cells (HUVECs) and RAW macrophagewere incubated for 2 h with 100 150 and 200120583MmL hesperidin in the presence or absence of Aeromonas hydrophila antigen (Ag)lipopolysaccharides (A-LPS 100 ngmL) and extracellular proteins (A-ECP 100 ngmL) Expression of E-selectin on HUVECs andintercellular adhesion molecule 1 (ICAM-1) on HUVECs and RAWmacrophage were estimated by using modified cell ELISA Data reportedas mean optical density (OD) plusmn standard deviation (SD) Values of the same parameter not sharing common superscripts denote significantdifferences

A-LPS A-ECPE-selectin HUVECs ICAM-1 HUVECs ICAM-1 RAW E-selectin HUVECs ICAM-1 HUVECs ICAM-1 RAW

Control 0090 plusmn 0007d 0092 plusmn 0003d 0084 plusmn 0002e 0090 plusmn 0007d 0092 plusmn 0003d 0084 plusmn 0002e

Ag 0525 plusmn 0082a 1519 plusmn 0092a 1452 plusmn 0074a 0833 plusmn 0068a 1491 plusmn 0099a 1401 plusmn 0063a

HES-100 0114 plusmn 0002cd 0170 plusmn 0053c 0079 plusmn 0009e 0114 plusmn 0002d 0170 plusmn 0053ac 0079 plusmn 0009e

HES-150 0114 plusmn 0001cd 0795 plusmn 0082b 0273 plusmn 0063d 0114 plusmn 0001d 0795 plusmn 0082b 0273 plusmn 0063d

HES-200 0170 plusmn 0028bc 0145 plusmn 0009cd 0129 plusmn 0047e 0170 plusmn 0028bc 0145 plusmn 0009cd 0129 plusmn 0047e

Ag + HES-100 0214 plusmn 0007b 0209 plusmn 0011c 1224 plusmn 0120b 0195 plusmn 0052b 0143 plusmn 0005cd 1148 plusmn 0159bc

Ag + HES-150 0122 plusmn 0002d 0181 plusmn 0016c 1096 plusmn 0087bc 0114 plusmn 0002d 0126 plusmn 0010cd 1061 plusmn 0045c

Ag + HES-200 0225 plusmn 0031b 0145 plusmn 0010cd 1040 plusmn 0212c 0136 plusmn 0018cd 0096 plusmn 0005cd 1215 plusmn 0029bc

119865 value 7474 58274 175673 28478 58978 386209119875 value 00000 00000 00000 00000 00000 00000

the survival of the mice beyond twenty days from infection[39] Regarding body weight the current study shows nosignificant difference in body liver spleen and intestineweights These findings are in accordance also with ourprevious in vivo study [39] which also showed no significantchanges (119875 gt 005) in body or intestine weights between theexperimental groups

The recorded nonsignificant elevation in spleen weightof both infected and HES-treated groups may be due toAeromonas LPSwhich caused the releasing of secretory prod-ucts from the activated circulating leukocytes and vascularendothelial cells for example TNF-120572 and free radicals TNF-120572 activates a variety of tissue cells to release interleukin 8 (1L-8) 1L-8 enhanced the adhesion of leukocytes to endothelium


B

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DR AZIZ CD14 1442013018 DR AZIZ CD14 1442013002 DR AZIZ CD14 1442013009

(d)

Figure 3 Representative dot plots of FACS analysis showing changes in Mean Fluorescence Index (MFI) of CD3+ CD4+ and CD8+lymphocytes and CD14+ monocytes in intestinal infiltrating cells in different groups control group (C) bacteria-infected group (B) andbacteria treated with hesperidin group (HES-treated group) Data reported as Mean Fluorescence Index (MFI) plusmn standard deviation (SD)Values of the same parameter not sharing common superscripts denote significant differences


and induced leukocytic degranulation and oxygen radicalrelease which causes endothelial cell necrosis [47] Alsoreleased free radicals may react around the blood vesselsof the liver and develop hepatic injury by forming anotherradical peroxynitrite [48]

On activation by different antigens the phagocytic cellsfrom infected animals produced significantly higher ROSthan those from noninfected animals indicating the involve-ment of immune T cells Previous data has shown thatthe bacterial LPS caused an increase in reactive nitrogenintermediates (RNI) reactive oxygen species (ROS) and theirphagocytic index production Excessive ROS could directlylead to cell damage and tissue injury by targeting variousbiomacromolecules such as proteins lipids and DNA [4950] The higher phagocytic activity shown here may bedue to LPS-induced degranulation in macrophages but likeallergens it also stimulates the de novo synthesis and releaseof cytokines in these cells Several Aeromonas infections areknown to stimulate the robust host production of nitrite oxideradicals (NO) and ROS leading to the loss of mitochondrialmembrane potential and apoptosis [51]

Other reasons for the elevation in the phagocytic indexand ROS production recorded in the present study may bedue to aerolysin or cytotoxic enterotoxin (Act) secretionsfrom A hydrophila infection or the release of extracellularproteins Aerolysin binds to cell surface structures andoligomerizes forming channels that result in cell lysis [52]Act is the most potent virulence factor in A hydrophilastrains serving to bind and stimulate infiltration of phago-cytic cells for example monocytes and macrophages andinduce the release of ROS [53 54] Recently Act has beenshown to recruit neutrophils in inflammatory diseases [55ndash58] and to upregulate macrophage inflammatory proteins invitro [59] On the other hand the data from the present studyclearly shows that HES treatment significantly reduced theelevation in ROS production that had been provoked by Ahydrophila infectionThe antioxidant efficacy of HES may beattributed to its ability to inhibit ROS generation includinghydroxyl radical [60] and scavenging peroxynitrite radicals[61]

The significant increase in CD14 bearing cells as a resultof A hydrophila infection may be due to the release ofLPS which may in turn induce responses by interactingwith a soluble binding protein in serum that then bindswith CD14 [62] Also LPS activate macrophages throughCD14 [63] CD14 is a multifunctional high-affinity patternrecognition receptor for bacterial endotoxins LPS and otherbacterial wall components [20 64] CD14 binding of LPS isassociated with a strong IL-12 response by antigen presentingcells [65 66] and IL-12 is regarded as an obligatory signalfor the maturation of naive T cells into Th1 cells [65]Proliferation of mucosal lymphocytes natural killer cellsand macrophages is stimulated by IL-12 [67 68] Li et al[69 70] predicted that potent downregulation of IL-2R120573maybe a key immunosuppressive strategy of A hydrophila tofacilitate successful infection of the skin mucosal surfaceRecently it was reported that subjects with allergic asthmahave increased expression of CD14 after LPS inhalation[71] The current study demonstrates that HES treatment

downregulates CD14 expression on infiltrated cells in theintestinal tissues of A hydrophila-infected mice and this maythen reduce the inflammatory response caused by infectionin such tissues

The CD4+CD8+ ratio is a reflection of immune systemhealth FACS assay showed that A hydrophila infectiondramatically decreased the percentage of CD4+CD8+ cells inintestinal tissues On the other hand the CD4+CD8+ ratio inthe HES-treated group was significantly (119875 lt 0001) elevatedafter four weeks of treatment indicating the progressivedevelopment of CD4+ cells Previously Lee et al [72] in thecontext of a study on asthma showed that effects of HES onlymphocyte subsets in lungs and bronchoalveolar lavage fluid(BALF) were characterised by an increase in the number ofCD4+ helper T cells and a reduction in CD8+ cells

The highly significant elevation in adhesion moleculesobserved after stimulation with Aeromonas LPS or A-ECPmay be due to their direct effects in altering and disruptingthe actin cytoskeleton of targeted cells so as to gain entryto andor manipulate cellular immunity [2 73] These dis-ruptions can themselves often lead to cell death at sites ofinfection [74] In particular A hydrophila infection rapidlyaltered a number of potentially critical lectins chemokinesinterleukins and othermucosal factors in amanner predictedto enhance its ability to adhere to and invade the host tissues[70] Bacterial LPS and inflammatory cytokines includingTNF-120572 IL-1 and IFN-120574 stimulate ICAM-1 and VCAMmRNA accumulation and cell surface expression althoughthis mechanism is thought to promote tissue inflammation[75] The upregulation of the gene expression of adhesionmolecules in microvascular endothelial cells is an importantstep for the migration and accumulation of leukocytes atthe site of inflammation which play a critical role in organdamage during sepsis [23 76] Our data shows that HESdownmodulates expression of E-selectin and ICAM-1 onboth HUVECs and RAW macrophage These results are inagreement with the findings of Nizamutdinova et al [24] whofound that HES suppresses ICAM-1 and VCAM-1 expressionin TNF-120572-treated HUVECs These effects were caused bythe inhibition of PI3 KAkt and PKC signaling pathwaysHES has also been reported to reduce the expression of IL-8 TNF120572 IL-1120573 IL-6 IL-12 ICAM-1 and VCAM-1 in thecase of acute lung inflammation induced by LPS in vivo [36]Moreover it has been shown that pretreatment with HEScould suppress infection-induced endotoxic shock in miceand reduce bacterial numbers during infection [77] Alsothe recorded amerolative effects of HES may result from theinflux of neutrophils into the inflamed area phagocytizingthe bacteria and digesting them This serves to activatedifferent host defence mechanisms to both reduce bacterialnumbers and counteract endotoxic shock [39]

The effect of HES on the expression of E-selectin andICAM-1 is dose dependent since 150 120583M of HES downregu-lated expressions of both E-selectin and ICAM-1 in compar-ison with 100 and 200120583M HES The molecular mechanismsby whichHES attenuates expression of E-selectin and ICAM-1 are unclear and need further investigation Previous studieshave however suggested that several flavonoids includingHES interact selectively with the mitogen-activated protein


(MAP) kinase signalling pathway The extracellular signal-regulated kinase (ERK) phosphorylation was involved inTNF-120572-induced ICAM-1 expression and PI3 KAkt and pro-tein kinase C (PKC) was involved in TNF-120572-inducedVCAM-1 expression [24 78] HES can reduce TNF-120572-inducedVCAM-1 expression through the regulation of the Akt andPKC pathway that is it inhibits the adhesion of monocytesto endothelium [24] In addition the systemic administrationof HES produced a marked reduction in the phosphorylationstate of extracellular signal-regulated kinases 12 (ERK 12) inthe cerebral cortex cerebellum and hippocampus [79]

In conclusion the results of the present study indicate thatHES as one of natural flavonoids effectively suppressed ROSproduction the phagocytic index expression of E-selectinand ICAM-1 induced by A-LPS and A-ECP stimulationThese findings predict that HES treatment may effectivelysuppress cytokine networking and alter the adherence ofstimulated phagocytic cells to endothelial barrier cells duringinflammation In addition the present study provides strongsupport for the anti-inflammatory activities of hesperidin

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This study was funded by Deanship of High studies andResearch Affairs Taif University Taif Saudi Arabia (Projectno 1-432-1243)

References

[1] A H Yousr S Napis G R A Rusul and R Son ldquoDetectionof aerolysin and hemolysin genes in Aeromonas spp isolatedfrom environmental and shellfish sources by polymerase chainreactionrdquo ASEAN Food Journal vol 14 no 2 pp 115ndash122 2007

[2] I-Y Nam and K Joh ldquoRapid detection of virulence factorsof Aeromonas isolated from a trout farm by hexaplex-PCRrdquoJournal of Microbiology vol 45 no 4 pp 297ndash304 2007

[3] F G van der Goot F Pattus M Parker and J T BuckleyldquoThe cytolytic toxin aerolysin from the soluble form to thetransmembrane channelrdquo Toxicology vol 87 no 1ndash3 pp 19ndash281994

[4] R Bucker S M Krug R Rosenthal et al ldquoAerolysin fromAeromonas hydrophila perturbs tight junction integrity and celllesion repair in intestinal epithelial HT-29B6 cellsrdquo Journal ofInfectious Diseases vol 204 no 8 pp 1283ndash1292 2011

[5] C Vuong S Kocianova J M Voyich et al ldquoA crucial role forexopolysaccharide modification in bacterial biofilm formationimmune evasion and virulencerdquo Journal of Biological Chem-istry vol 279 no 52 pp 54881ndash54886 2004

[6] H Rohde S Frankenberger U Zahringer andDMack ldquoStruc-ture function and contribution of polysaccharide intercellularadhesin (PIA) to Staphylococcus epidermidis biofilm formationand pathogenesis of biomaterial-associated infectionsrdquo Euro-pean Journal of Cell Biology vol 89 no 1 pp 103ndash111 2010

[7] C Li B Beck B Su et al ldquoEarly mucosal responses inblue catfish (Ictalurus furcatus) skin to Aeromonas hydrophila

infectionrdquo Fish and Shellfish Immunology vol 34 no 3 pp 920ndash928 2013

[8] J M Janda and S L Abbott ldquoThe genus Aeromonas taxonomypathogenicity and infectionrdquo Clinical Microbiology Reviewsvol 23 no 1 pp 35ndash73 2010

[9] A Turska-Szewczuk B Lindner I Komaniecka et al ldquoStruc-tural and immunochemical studies of the lipopolysaccharidefrom the fish pathogen Aeromonas bestiarum strain K296serotype O18rdquoMarine Drugs vol 11 no 4 pp 1235ndash1255 2013

[10] A Haziot S Chen E Ferrero M G Low R Silber andS M Goyert ldquoThe monocyte differentiation antigen CD14is anchored to the cell membrane by a phosphatidylinositollinkagerdquo Journal of Immunology vol 141 no 2 pp 547ndash5521988

[11] R J Ulevitch and P S Tobias ldquoReceptor-dependent mecha-nisms of cell stimulation by bacterial endotoxinrdquoAnnual Reviewof Immunology vol 13 pp 437ndash457 1995

[12] A Heinzmann H Dietrich S-P Jerkic T Kurz and K ADeichmann ldquoPromoter polymorphisms of the CD14 gene arenot associated with bronchial asthma in Caucasian childrenrdquoEuropean Journal of Immunogenetics vol 30 no 5 pp 345ndash3482003

[13] S Guerra I C Lohman M Halonen F D Martinez and AL Wright ldquoReduced interferon 120574 production and soluble CD14levels in early life predict recurrent wheezing by 1 year of agerdquoTheAmerican Journal of Respiratory and Critical Care Medicinevol 169 no 1 pp 70ndash76 2004

[14] M Kabesch K Hasemann V Schickinger et al ldquoA promoterpolymorphism in the CD14 gene is associated with elevatedlevels of soluble CD14 but not with IgE or atopic diseasesrdquoAllergy vol 59 no 5 pp 520ndash525 2004

[15] C Bieli W Eder R Frei et al ldquoA polymorphism in CD14modifies the effect of farm milk consumption on allergicdiseases and CD14 gene expressionrdquo Journal of Allergy andClinical Immunology vol 120 no 6 pp 1308ndash1315 2007

[16] P G Holt P D Sly and B Bjorksten ldquoAtopic versus infectiousdiseases in childhood a question of balancerdquo Pediatric Allergyand Immunology vol 8 no 2 pp 53ndash58 1997

[17] M Baldini I C LohmanMHalonen R P Erickson P GHoltand F D Martinez ldquoA polymorphism in the 51015840 flanking regionof the CD14 gene is associated with circulating soluble CD14levels and with total serum immunoglobulin Erdquo The AmericanJournal of Respiratory Cell and Molecular Biology vol 20 no 5pp 976ndash983 1999

[18] D Vercelli M Baldini D Stern I C Lohman M Halonenand F Martinez ldquoCD14 a bridge between innate immunity andadaptive IgE responsesrdquo Journal of Endotoxin Research vol 7no 1 pp 45ndash48 2001

[19] M-A Kedda F Lose D Duffy E Bell P J Thompson andJ Upham ldquoThe CD14 C-159T polymorphism is not associatedwith asthma or asthma severity in an Australian adult popula-tionrdquoThorax vol 60 no 3 pp 211ndash214 2005

[20] E M Klaassen B E Thonissen G van Eys et al ldquoA systematicreview of CD14 and toll-like receptors in relation to asthma inCaucasian childrenrdquo Allergy Asthma and Clinical Immunologyvol 9 no 1 article 10 2013

[21] M S Tonetti M A Imboden L Gerber N P Lang J Laissueand C Mueller ldquoLocalized expression of mRNA for phagocyte-specific chemotactic cytokines in human periodontal infec-tionsrdquo Infection and Immunity vol 62 no 9 pp 4005ndash40141994

[22] R P Darveau C M Belton R A Reife and R J LamontldquoLocal chemokine paralysis a novel pathogenic mechanism for


Porphyromonas gingivalisrdquo Infection and Immunity vol 66 no4 pp 1660ndash1665 1998

[23] G Fildissis K Venetsanou P Myrianthefs S Karatzas V Zid-ianakis and G Baltopoulos ldquoWhole blood pro-inflammatorycytokines and adhesion molecules post-lipopolysaccharidesexposure in hyperbaric conditionsrdquo European Cytokine Net-work vol 15 no 3 pp 217ndash221 2004

[24] I T Nizamutdinova J J Jeong GH Xu et al ldquoHesperidin hes-peridin methyl chalone and phellopterin from Poncirus trifoli-ata (Rutaceae) differentially regulate the expression of adhesionmolecules in tumor necrosis factor-120572-stimulated human umbil-ical vein endothelial cellsrdquo International Immunopharmacologyvol 8 no 5 pp 670ndash678 2008

[25] K Pradeep S H Park and K C Ko ldquoHesperidin a flavano-glycone protects against 120574-irradiation induced hepatocellulardamage and oxidative stress in Sprague-Dawley ratsrdquo EuropeanJournal of Pharmacology vol 587 no 1ndash3 pp 273ndash280 2008

[26] A M Mahmoud M B Ashour A Abdel-Moneim et alldquoHesperidin and naringin attenuate hyperglycemia-mediatedoxidative stress and proinflammatory cytokine production inhigh fat fedstreptozotocin-induced type 2 diabetic ratsrdquo Jour-nal of Diabetes and Its Complications vol 26 no 6 pp 483ndash4902012

[27] M J Tunon M V Garcıa-Mediavilla S Sanchez-Camposand J Gonzalez-Gallego ldquoPotential of flavonoids as anti-inflammatory agents modulation of pro-inflammatory geneexpression and signal transduction pathwaysrdquo Current DrugMetabolism vol 10 no 3 pp 256ndash271 2009

[28] J Kakadiya M Shah and N J Shah ldquoEffect of nobivololon serum diabetic marker and lipid profile in normal andstreptozotocin-nicotinamide induced diabetic ratsrdquo ResearchJournal of Pharmaceutical Biological and Chemical Sciences vol1 no 2 pp 329ndash334 2010

[29] Y-J Choi J-S Kang J H Y Park Y-J Lee J-S Choi and Y-HKang ldquoPolyphenolic flavonoids differ in their antiapoptotic effi-cacy in hydrogen peroxide-treated human vascular endothelialcellsrdquo Journal of Nutrition vol 133 no 4 pp 985ndash991 2003

[30] K Sakata Y Hirose Z Qiao T Tanaka and H Mori ldquoInhibi-tion of inducible isoforms of cyclooxygenase and nitric oxidesynthase by flavonoid hesperidin in mouse macrophage celllinerdquo Cancer Letters vol 199 no 2 pp 139ndash145 2003

[31] S S Raza M M Khan A Ahmad et al ldquoHesperidinameliorates functional and histological outcome and reducesneuroinflammation in experimental strokerdquo Brain Researchvol 1420 pp 93ndash105 2011

[32] K A Youdim M S Dobbie G Kuhnle A R Proteggente NJ Abbott and C Rice-Evans ldquoInteraction between flavonoidsand the blood-brain barrier in vitro studiesrdquo Journal of Neuro-chemistry vol 85 no 1 pp 180ndash192 2003

[33] D Wei X Ci X Chu M Wei S Hua and X Deng ldquoHes-peridin suppresses ovalbumin-induced airway inflammation inamouse allergic asthmamodelrdquo Inflammation vol 35 no 1 pp114ndash121 2012

[34] I-Y Choi S-J Kim H-J Jeong et al ldquoHesperidin inhibitsexpression of hypoxia inducible factor-1 alpha and inflam-matory cytokine production from mast cellsrdquo Molecular andCellular Biochemistry vol 305 no 1-2 pp 153ndash161 2007

[35] S Rizza R Muniyappa M Iantorno et al ldquoCitrus polyphenolhesperidin stimulates production of nitric oxide in endothe-lial cells while improving endothelial function and reducinginflammatory markers in patients with metabolic syndromerdquoJournal of Clinical Endocrinology and Metabolism vol 96 no5 pp E782ndashE792 2011

[36] C-C Yeh S-J Kao C-C Lin S-D Wang C-J Liu and S-T Kao ldquoThe immunomodulation of endotoxin-induced acutelung injury by hesperidin in vivo and in vitrordquo Life Sciences vol80 no 20 pp 1821ndash1831 2007

[37] O Westphal and J K Jann ldquoBacterial lipopolysaccharidesextraction with phenol-water and further applications of theprocedurerdquo Carbohydrate Chemistry vol 5 pp 83ndash91 1965

[38] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[39] A S Abuelsaad I Mohamed G Allam et al ldquoAntimicrobialand immunomodulating activities of hesperidin and ellagic acidagainst diarrheicAeromonas hydrophila in amurinemodelrdquo LifeSciences vol 93 no 20 pp 714ndash722 2013

[40] G A W Rook J Steele S Umar and H M Dockrell ldquoA simplemethod for the solubilisation of reduced NBT and its use as acolorimetric assay for activation of human macrophages by 120574-interferonrdquo Journal of Immunological Methods vol 82 no 1 pp161ndash167 1985

[41] A K Siwicki ldquoImmunostimulating influence of levamisole onnonspecific immunity in carp (Cyprinus carpio)rdquo Developmen-tal and Comparative Immunology vol 13 no 1 pp 87ndash91 1989

[42] A Jacobson C Yan Q Gao et al ldquoAging enhances pressure-induced arterial superoxide formationrdquo The American Journalof Physiology Heart and Circulatory Physiology vol 293 no 3pp H1344ndashH1350 2007

[43] G Badr H Ebaid M Mohany and A S Abuelsaad ldquoMod-ulation of immune cell proliferation and chemotaxis towardsCC chemokine ligand (CCL)-21 and CXC chemokine ligand(CXCL)-12 in undenatured whey protein-treated micerdquo Journalof Nutritional Biochemistry 2012

[44] C Neu A Sedlag C Bayer et al ldquoCD14-dependent monocyteisolation enhances phagocytosis of listeria monocytogenes byproinflammatory GM-CSF-derived macrophagesrdquo PLoS ONEvol 8 no 6 Article ID e66898 2013

[45] S Takami S Yamashita S Kihara et al ldquoLipoprotein(a)enhances the expression of intercellular adhesion molecule-1 incultured human umbilical vein endothelial cellsrdquo Circulationvol 97 no 8 pp 721ndash728 1998

[46] N Leitinger J Huber C Rizza et al ldquoThe isoprostane 8-iso-PGF(2alpha) stimulates endothelial cells to bind monocytesdifferences from thromboxane-mediated endothelial activa-tionrdquoThe FASEB Journal vol 15 no 7 pp 1254ndash1256 2001

[47] M Ogata T Matsui T Kita and A Shigematsu ldquoCar-rageenan primes leukocytes to enhance lipopolysaccharide-induced tumor necrosis factor alpha productionrdquo Infection andImmunity vol 67 no 7 pp 3284ndash3289 1999

[48] A Morikawa Y Kato T Sugiyama et al ldquoRole of nitricoxide in lipopolysaccharide-induced hepatic injury in D-galactosamine-sensitized mice as an experimental endotoxicshock modelrdquo Infection and Immunity vol 67 no 3 pp 1018ndash1024 1999

[49] A S Abu-El-Saad ldquoImmunomodulating effect of inosi-tol hexaphosphate against Aeromonas hydrophila-endotoxinrdquoImmunobiology vol 212 no 3 pp 179ndash192 2007

[50] W Che N Lerner-Marmarosh Q Huang et al ldquoInsulin-likegrowth factor-1 enhances inflammatory responses in endothe-lial cells role of Gab1 and MEKK3 in TNF-120572-induced c-Jun and NF-120581B activation and adhesion molecule expressionrdquoCirculation Research vol 90 no 11 pp 1222ndash1230 2002

[51] S Krzyminska A Tanska and A Kaznowski ldquoAeromonasspp induce apoptosis of epithelial cells through an oxidant-dependent activation of the mitochondrial pathwayrdquo Journal ofMedical Microbiology vol 60 no 7 pp 889ndash898 2011


[52] S P Howard and J T Buckley ldquoActivation of the hole-forming toxin aerolysin by extracellular processingrdquo Journal ofBacteriology vol 163 no 1 pp 336ndash340 1985

[53] J Sha E V Kozlova and A K Chopra ldquoRole of variousenterotoxins in Aeromonas hydrophila-induced gastroenteritisgeneration of enterotoxin gene-deficient mutants and evalua-tion of their enterotoxic activityrdquo Infection and Immunity vol70 no 4 pp 1924ndash1935 2002

[54] A K Chopra X-J Xu D Ribardo et al ldquoThe cytotoxicenterotoxin of Aeromonas hydrophila induces proinflammatorycytokine production and activates arachidonic acidmetabolismin macrophagesrdquo Infection and Immunity vol 68 no 5 pp2808ndash2818 2000

[55] L Feng Y Xia T Yoshimura and C B Wilson ldquoModulationof neutrophil influx in glomerulonephritis in the rat withanti-macrophage inflammatory protein-2 (MIP-2) antibodyrdquoJournal of Clinical Investigation vol 95 no 3 pp 1009ndash10171995

[56] R A Skidgel X-P Gao V Brovkovych et al ldquoNitric oxidestimulates macrophage inflammatory protein-2 expression insepsisrdquo Journal of Immunology vol 169 no 4 pp 2093ndash21012002

[57] Y Ohtsuka J Lee D S Stamm and I R Sanderson ldquoMIP-2secreted by epithelial cells increases neutrophil and lymphocyterecruitment in the mouse intestinerdquoGut vol 49 no 4 pp 526ndash533 2001

[58] D J Schrier R C Schimmer C M Flory D K-L Tung andP AWard ldquoRole of chemokines and cytokines in a reactivationmodel of arthritis in rats induced by injectionwith streptococcalcell wallsrdquo Journal of Leukocyte Biology vol 63 no 3 pp 359ndash363 1998

[59] C L Galindo J Sha D A Ribardo A A Fadl L Pillai and AK Chopra ldquoIdentification of Aeromonas hydrophila cytotoxicenterotoxin-induced genes in macrophages using microarraysrdquoJournal of Biological Chemistry vol 278 no 41 pp 40198ndash402122003

[60] A J Hyun J J Mee Y K Ji Y C Hae and S C JaeldquoInhibitory activity of flavonoids from Prunus davidiana andother flavonoids on total ROS and hydroxyl radical generationrdquoArchives of Pharmacal Research vol 26 no 10 pp 809ndash8152003

[61] J Y Kim K J Jung J S Choi and H Y Chung ldquoHesperetin apotent antioxidant against peroxynitriterdquo Free Radical Researchvol 38 no 7 pp 761ndash769 2004

[62] S D Wright R A Ramos P S Tobias R J Ulevitch and J CMathison ldquoCD14 a receptor for complexes of lipopolysaccha-ride (LPS) and LPS binding proteinrdquo Science vol 249 no 4975pp 1431ndash1433 1990

[63] D Gupta Q Wang C Vinson and R Dziarski ldquoBacterial pep-tidoglycan induces CD14-dependent activation of transcriptionfactors CREBATF and AP-1rdquo Journal of Biological Chemistryvol 274 no 20 pp 14012ndash14020 1999

[64] D N Cook D S Pisetsky and D A Schwartz ldquoToll-like recep-tors in the pathogenesis of human diseaserdquoNature Immunologyvol 5 no 10 pp 975ndash979 2004

[65] S E Macatonia N A Hosken M Litton et al ldquoDendritic cellsproduce IL-12 and direct the development of Th1 cells fromnaive CD4+ T cellsrdquo Journal of Immunology vol 154 no 10 pp5071ndash5079 1995

[66] M G Cleveland J D Gorham T L Murphy E Tuoma-nen and K M Murphy ldquoLipoteichoic acid preparations ofgram-positive bacteria induce interleukin-12 through a CD14-dependent pathwayrdquo Infection and Immunity vol 64 no 6 pp1906ndash1912 1996

[67] M A Burchill J Yang K B Vang and M A Farrar ldquoInterleu-kin-2 receptor signaling in regulatory T cell development andhomeostasisrdquo Immunology Letters vol 114 no 1 pp 1ndash8 2007

[68] C J Secombes T Wang and S Bird ldquoThe interleukins of fishrdquoDevelopmental and Comparative Immunology vol 35 no 12 pp1336ndash1345 2011

[69] C Li RWang B Su et al ldquoEvasion of mucosal defenses duringAeromonas hydrophila infection of channel catfish (Ictaluruspunctatus) skinrdquoDevelopmental and Comparative Immunologyvol 39 no 4 pp 447ndash455 2013

[70] C Li Y Zhang R Wang et al ldquoRNA-seq analysis of mucosalimmune responses reveals signatures of intestinal barrier dis-ruption and pathogen entry following Edwardsiella ictaluriinfection in channel catfish Ictalurus punctatusrdquo Fish andShellfish Immunology vol 32 no 5 pp 816ndash827 2012

[71] N Alexis M Eldridge W Reed P Bromberg and D B PedenldquoCD14-dependent airway neutrophil response to inhaled LPSrole of atopyrdquo Journal of Allergy and Clinical Immunology vol107 no 1 pp 31ndash35 2001

[72] Y-C Lee S-H Kim and B-K Kim ldquoAntiasthmatic effects ofhesperidin a potential Th2 cytokine antagonist in a mousemodel of allergic asthmardquoMediators of Inflammation vol 2011Article ID 485402 12 pages 2011

[73] P Boquet and E Lemichez ldquoBacterial virulence factors tar-geting Rho GTPases parasitism or symbiosisrdquo Trends in CellBiology vol 13 no 5 pp 238ndash246 2003

[74] G Suarez B K Khajanchi J C Sierra et al ldquoActin cross-linkingdomain of Aeromonas hydrophila repeat in toxin A (RtxA)induces host cell rounding and apoptosisrdquo Gene vol 506 no2 pp 369ndash376 2012

[75] TM Carlos and JM Harlan ldquoLeukocyte-endothelial adhesionmoleculesrdquo Blood vol 84 no 7 pp 2068ndash2101 1994

[76] R Wu Y Xu X Song and X Meng ldquoGene expression ofadhesion molecules in pulmonary and hepatic microvascularendothelial cells during sepsisrdquo Chinese Journal of Traumatol-ogy vol 5 no 3 pp 146ndash150 2002

[77] K Kawaguchi S-I Kikuchi R Hasunuma H Maruyama TYoshikawa and Y Kumazawa ldquoA citrus flavonoid hesperidinsuppresses infection-induced endotoxin shock in micerdquo Biolog-ical and Pharmaceutical Bulletin vol 27 no 5 pp 679ndash6832004

[78] J S Hwa L Mun H J Kim et al ldquoGenipin selectivelyinhibits TNF-120572-activated VCAM-1 but not ICAM-1 expressionby upregulation of PPAR-120574 in human endothelial cellsrdquo KoreanJournal of Physiology and Pharmacology vol 15 no 3 pp 157ndash162 2011

[79] M C Martınez S P Fernandez L M Loscalzo et al ldquoHes-peridin a flavonoid glycoside with sedative effect decreasesbrain pERK12 levels in micerdquo Pharmacology Biochemistry andBehavior vol 92 no 2 pp 291ndash296 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


between CD14 and its role in the polarisation of T lympho-cytes intoTh1 andTh2 subsets [16ndash20]

Classical immunoregulatory tissues control and deter-mine the success of critical early steps in pathogenesis includ-ing microbe adhesion entry and replication [7] Even whenmucosal tissues are healthy they are bathed in low levels ofE-selectin intercellular adhesion molecule 1 (ICAM-1) andinterleukin 8 (IL-8) Of these IL-8 forms a gradient of expres-sion that is greatest near the bacteriaepithelial cell interface[21 22] E-selectin meanwhile is a membrane glycoproteinand is expressed by endothelial cells in order to mediatethe adhesion of leukocytes It is upregulated rapidly duringinflammation resulting in increased leukocyte-endothelialcell adhesion [23] Adhesion molecules play importantroles in cellular interactions during inflammatory responsesExpression of ICAM-1 for example plays an important rolein the adhesion of monocytes to endothelial cells [24]

Regarding flavonoids these have metal chelating freeradical scavenging properties such as neutralization of thesinglet oxygen and superoxide and inhibition of the hydro-gen peroxide-induced lipid peroxidation (LPO) [25 26]Flavonoids inhibit the expression of isoforms of cyclooxy-genase inducible nitric oxide synthase and lipooxygenasewhich are responsible for the production of NO prostanoidsand leukotrienes as well as inflammatory mediators such ascytokines chemokines or adhesion molecules [27]

Hesperidin (HES) is a flavanone glycoside commonlyfound in the diet in citrus fruits or citrus fruit derivedproducts [26 28] The anti-inflammatory effects of HES havebeen characterized in vitro in both rodent and human celllines [29 30]The scavenging effect of free radicals associatedwithHES has been evidenced by different neurochemical andneurobehavioral parameters with HES treatment appearingto reduce expression of proinflammatory mediators likeinducible nitric oxide synthase (iNOS) TNF-120572 and IL-1120573[31 32] Recently HES has been shown to exhibit pronouncedimmunological activities serving to inhibit inflammatory cellinfiltration and mucus hypersecretion in a murine model ofasthma [33] In addition HES counteracted the upregulationof proinflammatory cytokines such as the expression of TNF-120572 and IL-1120573 in cerebral ischemia [31 34 35] as well as IL-8IL-6 IL-12 and vascular cell adhesion molecule 1 (VCAM-1)in the case of acute lung inflammation induced by LPS in vivo[36]

The aim of the present study was to investigate theantiadhesion and anti-inflammatory role of HES in the caseof gastrointestinal Aeromonas infection in a murine model

2 Materials and Methods21 Bacteria and Growth Conditions A standard A hydroph-ila strain (ATCC catalogue number 7966) was kindlyprovided by the Fish Department Faculty of VeterinaryCairo University Giza EgyptThe bacteriumwasmaintainedand subcultured three times before the experiments Briefly100 120583L ofA hydrophilawas inoculated into 150mL of a liquidpeptone broth (Oxoid) and incubated for 30∘C for 24 h withcontinuous shaking at 250 rpm The harvested bacteria werecentrifuged at 6000 g for 10min and the dried pellet was

suspended twice in phosphate-buffered saline (PBS) to thefinal dose of 2 times 108 CFUmL

211 Preparation of A hydrophila Lipopolysaccharides (A-LPS) LPS was prepared as described by Westphal and Jann[37] Briefly the bacteriawere inoculated in 250mL of a LuriaBertani (LB) broth and incubated for 24 h at 30∘C on a shakerat 250 rpm The culture was then centrifuged at 10000 rpmfor 10min at 4∘C resuspended in 166mL of TAE buffer(40mM Tris-acetate pH85 2mM EDTA) and then mixedwith 332mL alkaline solution (containing 3 g of SDS 06 gof Trizma (Sigma) and 160mL of 2N NaOH in 1000mL ofwater) The suspension was heated at 55 to 60∘C for 70minand then mixed with phenol and chloroform in the ratio of1 1 (VV) The mixture was spun at 10 000 rpm for 10min at4∘C and the supernatant obtained was mixed with 332mLof water and 83mL of 3M sodium acetate buffer (pH 52)LPS was precipitated by adding twice the volume of ethanolThe precipitate was dissolved in 332mL of 50mM Tris-HClpH 80 (Sigma) and 100mM sodium acetate mixed well andwas then reprecipitated with twice the volume of ethanolThecombined water extract was dialyzed for 2ndash4 days againstdistilled water and then freeze-dried

212 Preparation of A hydrophila Extracellular Proteins (A-ECP) The bacterial isolate was grown overnight in 5mLLBbroth for preculturing 100120583L of this culture suspension(inoculum) was added to 50mLLB broth and incubatedovernight at 37∘C at a shaker speed of 200 rpm The culturesuspension was harvested at 5000 rpm at 4∘C for 15min Thesupernatant was precipitated by the addition of 10 (wv)trichloroacetic acidwith overnight incubation at 4∘C Furthercentrifugation at 11000 rpm for 20minutes resulted in a pelletcontaining extracellular proteins which was suspended in50 120583L of 1M Tris-HCl buffer (pH 8) and dialyzed overnightagainst the same buffer The freeze-dried protein content wasdetermined as described by Lowry et al [38] The purifiedprotein was ascertained as endotoxin-free with the limulusamebocyte lysate (LAL) test

22 Animals MaleMF1 albinomice (7-8weeks old weighing20ndash25 g King Fahd Specialist Medical Centre Jeddah KSA)were used in the experiments and housed in a barrier roomunder standard conditions The animals were kept in wire-mesh polycarbonate cages with autoclaved bedding wereacclimatized to laboratory conditions (12 h dark 12 h lightcycles 240 plusmn 10∘C) and had free access to food and waterad libitum The food containers were refilled daily with freshstandard diet and were fitted with bars to reduce losses Rou-tine clinical observations and body weight were measuredregularly throughout the experiments Animal use and thecare protocol were approved by the Research Ethics Commit-tee College of Medicine Taif University Saudi Arabia

23 Natural Products Hesperidin (HES) used in this studywas of analytical grade and purchased from Sigma ChemicalCo (St Louis Missouri USA) and dissolved in 1 dimethylsulphoxide (DMSO) immediately before use





=


(1)


2


2




2



















3 Results



2









A

AA

0

3

6

9

12

15

18

21

24

27

30


(a)

A

A

A

0

02

04

06

08

1

12

14

16

18

2


(b)

A

A

A

0

005

01

015

02

025

03

035

04


(c)

AA

A

0

05

1

15

2

25

3

35

4

45


(d)



4 Discussion



00

01

02

03

04

05

06

07

08

09

10

11

12

13

Phag

ocyt

osis

()

B

Alowastlowast

Blowastlowast

Control group

HES-treated group

B-infected group

(a)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

nM N

BTm

g pr

otei

n tis

sues

30min

C

Alowastlowastlowast

Blowastlowastlowast

Control group

HES-treated group

B-infected group

(b)












119865 value 7474 58274 175673 28478 58978 386209119875 value 00000 00000 00000 00000 00000 00000




B

A A

0102030405060708090

100

Control group


HES-treated group

CD3-

MFI

CD3

CD3

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

FL1-H FL1-H FL1-H

R5 R6R4 R5 R5R4

group group

B-infected group

group



(a)

B B

A

0102030405060708090

100110

CD4-

MFI

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD4

CD4

FL1-H FL1-H FL1-H

R2R1 R2R1 R2R1



(b)

B

A

C

02468

101214161820

CD8

-MFI

SSC-

H

0

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD8

CD8

FL2-H FL2-HFL2-H

R3 R4 R3 R4 R3 R4

200

400

600

800



(c)

B

A

B

0

10

20

30

40

50

60

70

80

Control group

B-infected group

HES-treated group

CD14

-MFI

Cou

nts

0

40

80

120

160

200

100

101

102

103

104

100

101

102

103

104

100

101

102

103

CD14

CD14

M1M1M1

FL1-H FL1-H FL1-H



(d)
















Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















=


(1)


2


2




2



















3 Results



2









A

AA

0

3

6

9

12

15

18

21

24

27

30


(a)

A

A

A

0

02

04

06

08

1

12

14

16

18

2


(b)

A

A

A

0

005

01

015

02

025

03

035

04


(c)

AA

A

0

05

1

15

2

25

3

35

4

45


(d)



4 Discussion



00

01

02

03

04

05

06

07

08

09

10

11

12

13

Phag

ocyt

osis

()

B

Alowastlowast

Blowastlowast

Control group

HES-treated group

B-infected group

(a)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

nM N

BTm

g pr

otei

n tis

sues

30min

C

Alowastlowastlowast

Blowastlowastlowast

Control group

HES-treated group

B-infected group

(b)












119865 value 7474 58274 175673 28478 58978 386209119875 value 00000 00000 00000 00000 00000 00000




B

A A

0102030405060708090

100

Control group


HES-treated group

CD3-

MFI

CD3

CD3

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

FL1-H FL1-H FL1-H

R5 R6R4 R5 R5R4

group group

B-infected group

group



(a)

B B

A

0102030405060708090

100110

CD4-

MFI

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD4

CD4

FL1-H FL1-H FL1-H

R2R1 R2R1 R2R1



(b)

B

A

C

02468

101214161820

CD8

-MFI

SSC-

H

0

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD8

CD8

FL2-H FL2-HFL2-H

R3 R4 R3 R4 R3 R4

200

400

600

800



(c)

B

A

B

0

10

20

30

40

50

60

70

80

Control group

B-infected group

HES-treated group

CD14

-MFI

Cou

nts

0

40

80

120

160

200

100

101

102

103

104

100

101

102

103

104

100

101

102

103

CD14

CD14

M1M1M1

FL1-H FL1-H FL1-H



(d)
















Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results



2









A

AA

0

3

6

9

12

15

18

21

24

27

30


(a)

A

A

A

0

02

04

06

08

1

12

14

16

18

2


(b)

A

A

A

0

005

01

015

02

025

03

035

04


(c)

AA

A

0

05

1

15

2

25

3

35

4

45


(d)



4 Discussion



00

01

02

03

04

05

06

07

08

09

10

11

12

13

Phag

ocyt

osis

()

B

Alowastlowast

Blowastlowast

Control group

HES-treated group

B-infected group

(a)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

nM N

BTm

g pr

otei

n tis

sues

30min

C

Alowastlowastlowast

Blowastlowastlowast

Control group

HES-treated group

B-infected group

(b)












119865 value 7474 58274 175673 28478 58978 386209119875 value 00000 00000 00000 00000 00000 00000




B

A A

0102030405060708090

100

Control group


HES-treated group

CD3-

MFI

CD3

CD3

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

FL1-H FL1-H FL1-H

R5 R6R4 R5 R5R4

group group

B-infected group

group



(a)

B B

A

0102030405060708090

100110

CD4-

MFI

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD4

CD4

FL1-H FL1-H FL1-H

R2R1 R2R1 R2R1



(b)

B

A

C

02468

101214161820

CD8

-MFI

SSC-

H

0

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD8

CD8

FL2-H FL2-HFL2-H

R3 R4 R3 R4 R3 R4

200

400

600

800



(c)

B

A

B

0

10

20

30

40

50

60

70

80

Control group

B-infected group

HES-treated group

CD14

-MFI

Cou

nts

0

40

80

120

160

200

100

101

102

103

104

100

101

102

103

104

100

101

102

103

CD14

CD14

M1M1M1

FL1-H FL1-H FL1-H



(d)
















Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A

AA

0

3

6

9

12

15

18

21

24

27

30


(a)

A

A

A

0

02

04

06

08

1

12

14

16

18

2


(b)

A

A

A

0

005

01

015

02

025

03

035

04


(c)

AA

A

0

05

1

15

2

25

3

35

4

45


(d)



4 Discussion



00

01

02

03

04

05

06

07

08

09

10

11

12

13

Phag

ocyt

osis

()

B

Alowastlowast

Blowastlowast

Control group

HES-treated group

B-infected group

(a)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

nM N

BTm

g pr

otei

n tis

sues

30min

C

Alowastlowastlowast

Blowastlowastlowast

Control group

HES-treated group

B-infected group

(b)












119865 value 7474 58274 175673 28478 58978 386209119875 value 00000 00000 00000 00000 00000 00000




B

A A

0102030405060708090

100

Control group


HES-treated group

CD3-

MFI

CD3

CD3

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

FL1-H FL1-H FL1-H

R5 R6R4 R5 R5R4

group group

B-infected group

group



(a)

B B

A

0102030405060708090

100110

CD4-

MFI

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD4

CD4

FL1-H FL1-H FL1-H

R2R1 R2R1 R2R1



(b)

B

A

C

02468

101214161820

CD8

-MFI

SSC-

H

0

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD8

CD8

FL2-H FL2-HFL2-H

R3 R4 R3 R4 R3 R4

200

400

600

800



(c)

B

A

B

0

10

20

30

40

50

60

70

80

Control group

B-infected group

HES-treated group

CD14

-MFI

Cou

nts

0

40

80

120

160

200

100

101

102

103

104

100

101

102

103

104

100

101

102

103

CD14

CD14

M1M1M1

FL1-H FL1-H FL1-H



(d)
















Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

01

02

03

04

05

06

07

08

09

10

11

12

13

Phag

ocyt

osis

()

B

Alowastlowast

Blowastlowast

Control group

HES-treated group

B-infected group

(a)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

nM N

BTm

g pr

otei

n tis

sues

30min

C

Alowastlowastlowast

Blowastlowastlowast

Control group

HES-treated group

B-infected group

(b)












119865 value 7474 58274 175673 28478 58978 386209119875 value 00000 00000 00000 00000 00000 00000




B

A A

0102030405060708090

100

Control group


HES-treated group

CD3-

MFI

CD3

CD3

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

FL1-H FL1-H FL1-H

R5 R6R4 R5 R5R4

group group

B-infected group

group



(a)

B B

A

0102030405060708090

100110

CD4-

MFI

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD4

CD4

FL1-H FL1-H FL1-H

R2R1 R2R1 R2R1



(b)

B

A

C

02468

101214161820

CD8

-MFI

SSC-

H

0

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD8

CD8

FL2-H FL2-HFL2-H

R3 R4 R3 R4 R3 R4

200

400

600

800



(c)

B

A

B

0

10

20

30

40

50

60

70

80

Control group

B-infected group

HES-treated group

CD14

-MFI

Cou

nts

0

40

80

120

160

200

100

101

102

103

104

100

101

102

103

104

100

101

102

103

CD14

CD14

M1M1M1

FL1-H FL1-H FL1-H



(d)
















Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















B

A A

0102030405060708090

100

Control group


HES-treated group

CD3-

MFI

CD3

CD3

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

FL1-H FL1-H FL1-H

R5 R6R4 R5 R5R4

group group

B-infected group

group



(a)

B B

A

0102030405060708090

100110

CD4-

MFI

SSC-

H

0

200

400

600

800

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD4

CD4

FL1-H FL1-H FL1-H

R2R1 R2R1 R2R1



(b)

B

A

C

02468

101214161820

CD8

-MFI

SSC-

H

0

1000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

CD8

CD8

FL2-H FL2-HFL2-H

R3 R4 R3 R4 R3 R4

200

400

600

800



(c)

B

A

B

0

10

20

30

40

50

60

70

80

Control group

B-infected group

HES-treated group

CD14

-MFI

Cou

nts

0

40

80

120

160

200

100

101

102

103

104

100

101

102

103

104

100

101

102

103

CD14

CD14

M1M1M1

FL1-H FL1-H FL1-H



(d)
















Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Acknowledgment


References

























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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