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Clinical Immunology (2011) 138, 41–49

Status of bacterial colonization, Toll-like receptorexpression and nuclear factor-kappa B activation innormal and diseased human liversRashmi Singha, Jay Bullardb, Mamta Kalra a,1, Senait Assefaa, Anil K. Kaul c,Krystal Vonfeldt a, Stephen C. Stromd, Robert S. Conrada,Harvey L. Sharpe, Rashmi Kaul a,⁎

a Department of Biochemistry and Microbiology, Oklahoma State University, Center for Health Sciences, Tulsa, Oklahoma, USAb Department of Forensic Sciences, Oklahoma State University, Center for Health Sciences, Tulsa, Oklahoma, USAc Department of Obstetrics and Gynecology, Oklahoma State University, Center for Health Sciences, Tulsa, Oklahoma, USAd Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USAe Department of Pediatric Gastroenterology, University School of Minnesota, Minneapolis, MN, USA

Received 12 September 2010; accepted with revision 14 September 2010Available online 12 October 2010

⁎ Corresponding author. Dept of BioOklahoma State University Center for17th St., Tulsa, Oklahoma, USA, 74107

E-mail address: rashmi.kaul10@oks1 Current affiliation: Laureate Institu

Oklahoma, USA.

1521-6616/$ – see front matter © 201doi:10.1016/j.clim.2010.09.006

KEYWORDSBacterial translocation;Toll-like receptors;Nuclear factor-kappa B;Liver infection;Liver cirrhosis;Immune homeostasis

Abstract Epidemiological data on bacterial translocation (BT), colonization and inflammationin normal human livers is lacking. In this study we investigated the status of bacterialcolonization and inflammation in the normal, cirrhotic primary biliary cirrhosis (PBC), andnonalcoholic steatohepatitis (NASH) human liver tissues. Comparatively normal livers showedincreased bacterial colonization than PBC and NASH. We analyzed mRNA levels of Toll-likereceptors (TLR) 2 and TLR4, and protein levels of TLR4. Phosphorylated IKKα (pIKKα) proteinestimation served as a marker for nuclear factor-kappa B (NF-κB) activation. In spite of theincreased bacterial colonization in normal liver tissues, lower levels of TLR2/4 mRNA and TLR4

and pIKKα proteins were found compared to PBC and NASH indicating the maintenance ofsuppressed inflammation and immune tolerance in normal livers. To our knowledge, this is thefirst clinical evidence showing suppressed inflammation despite bacterial colonization in normalhuman livers thus maintaining liver immune homeostasis.© 2010 Elsevier Inc. All rights reserved.

chemistry and Microbiology,Health Sciences, 1111 West. Fax: +1 918 561 5798.tate.edu (R. Kaul).te of Brain Research, Tulsa,

0 Elsevier Inc. All rights reserv

ed

1. Introduction

Healthy gastrointestinal microbial community is essential tomaintain gut immune homeostasis. In a healthy intestinal tractthe microbiota and the gut-associated immune system share afine and dynamic homeostatic equilibrium [1]. Patternrecognition receptors (PRR) such as TLRs are part of innateimmune system that function as the sensors of microbial

.

Table 1 Age gender and bacterial culture results of theclinical tissue samples.

Normal PBC NASH

Explant liver tissuescultured (n)

14 9 6

Sex (F/M) 9/5 9/0 5/1Mean age±SEM (years) 56±2.62 56±2.04 53±5.07% Culture positive tissues 86% (12/14) 56% (5/9) 50% (3/6)

The table represents the gender, age characteristics of the studygroups and percentage positivity for bacterial culture in eachstudy group.

42 R. Singh et al.

infection and are critical for initiation of protective inflam-matory immune responses against the invading pathogens [2].TLRs respond to stimulation by bacterial ligands such aslipopeptides, lipoteichoic acid, flagellin, peptidoglycan, lipo-polysaccharide (LPS), and DNA [2]. Animal studies have shownthat activation of TLRs by intestinal commensal microflora iscritical for the protection against gut injury and associatedmortality [3,4]. Under normal steady-state conditions, theintestinal bacteria and their components are recognized byTLRs, for example LPS from gramnegative bacteria is detectedby TLR4 and lipoproteins, and the peripheral membraneproteins in gram positive bacteria are detected by TLR2 [2].These interactions play a significant role in themaintenance ofthe intestinal epithelial homeostasis and enable the immunesystem to shift the inflammatory responses in favor of the host[3,4].

BT is a spontaneous process defined as passage of viableindigenous bacteria from the intestinal tract through theepithelial mucosa to the mesenteric lymph nodes, systemiccirculation, and other internal organs such as spleen, liverand lungs [5–8]. Bacterial invasion to the extra-intestinalsites depends on the effectiveness of the host innate andadaptive immunity [4–9]. Blood circulation from theintestine to the liver is rich in bacterial components,environmental toxins, and food antigens. Liver is the firstline of defense against systemic infection as it serves as afilter between the digestive tract and rest of the body. Liveris considered as a local sink for LPS and the main site for itsclearance, thus playing a key role in maintenance of innateimmune defenses [10]. It quickly responds to the potentialpathogen attacks by secreting pattern recognition receptors(PRRs) such as TLRs and the complement components [10].Liver cells express a variety of TLRs [11–13]. TLR4 protein isdetected on all the types of liver cells and is likely to beinvolved in uptake and clearance of endotoxins [13]. TLR-mediated signals are involved in the production of pro- andanti-inflammatory cytokines, and generation of oxidativestress and these TLR-signals have been implicated in anumber of chronic liver diseases [12,13]. BT of the gut flora issuggested to play an important role in the pathogenesis andcomplications associated with liver cirrhosis [14]. In normalrats and healthy humans, portal as well as systemic LPS levelsremains undetectable, whereas in the patients with cirrhoticlivers and in the experimental models of hepatic fibrosis, LPSlevels are elevated [15–18]. Liberation of LPS and otherbacterial components in the blood stream leads to up-regulation of TLR4 that may induce inflammation and BT inthe liver [13,19]. Compromise in the intestinal barrierfunction and increased intestinal permeability leads to BT[5,6,20], however detailed mechanisms of BT into the extra-intestinal sites such as liver are not completely understood.BT from intestines into various organs of the body and itsimpact on immune activation have been extensively studied.However, most of these studies have either been conductedin animals or are ex vivo investigations. Thus, at present,information available on BT from human studies is quitelimited. Further, epidemiological data on BT and bacterialcolonization in normal and diseased human livers is com-pletely lacking. Therefore, the role of BT in normal anddiseased human livers and generation of innate immuneresponses in the liver against the invading pathogens needdetailed investigation.

TLR4 and TLR2 activation by LPS and other microbialproducts leads to activation of NF-κB that comprises a criticalstep in which phosphorylation of I kappa Bs (IκB) by the I kappaB kinase (IKK) complex leads to nuclear translocation of NF-κBheterodimer; this further induces transcription of the targetgenes leading to production of the inflammatory cytokines[21,22]. We hypothesize, that in the normal liver underhealthy steady-state conditions a homeostatic balance mayprevail in between the colonizing bacterial population and theinnate immune responses similar to that observed in theintestine. Thus, recognition of the translocating intestinalmicroflora by TLRs in the normal liver may restrict excessiveinflammation in response to pathogen associated molecularpatterns (PAMPs) such as LPS or lipoteichoic acid via regulatingactivation of TLRs and NF-κB pathway.

In the present study, we investigated the status ofbacterial colonization in the normal and diseased humanlivers and further determined the status of inflammation bystudying TLR2 and TLR4 expression and NF-κB activation inthese livers.

2. Materials and methods

2.1. Human explant liver tissues

Explanted liver tissues were obtained from NIH Liver TissueProcurement and Distribution System (LTPADS), University ofMinnesota. Tissues were aseptically collected under theInstitutional Review Board of guidelines of the University ofMinnesota and University of Pittsburgh. The research projectwas conducted at OSU—Center for Health Sciences under therequired Institutional Review Board of guideline regulation.Twenty nine liver specimens were subjected for analysisincluding 14 deceased donor livers (Table 1). Liver specimenswere stored under aseptic conditions at −80 °C until use.

2.2. Bacterial isolation and identification

Standard laboratory procedures were carried out forbacterial isolation from explant liver tissues. Briefly, frozenliver tissues (100–120 mg) were aseptically cut and homog-enized in 950 μl of Brain Heart Infusion (BHI) broth usingOmni Tip homogenizer (Omni International, Marietta GAUSA). Each tissue homogenate (50 μl) was plated in duplicateon trypticase soy agar supplemented with 5% sheep blood

43Status of bacterial colonization, Toll-like receptor expression and NF-κB activation

(TSA-BA) and incubated for 48 h at 37 °C for growth ofaerobes and/or facultative anaerobes. The remaining of eachhomogenate was incubated with shaking at 37 °C for 24 h forculture enrichment, following which 50 μl was plated induplicate on TSA-BA and incubated for 48 h at 37 °C. BHIbroth (50 μl) was spread on TSA-BA and incubated for 48 h at37 °C as negative control. Growth of bacterial colonies onculture plates following 24 to 48 h of incubation wasconsidered positive and absence of any bacterial growth wasconsidered negative for bacterial colonization by aerobic andfacultative anaerobic bacteria. Colony characteristics for eachbacterial colony were recorded and gram staining wasperformed. Pure culture of each isolated bacterial colonywas obtained by sub-culturing on TSA-BA plates. Selectedbacterial isolates were identified using standard methods.Briefly, bacterial isolates from each tissue were randomlyselected on the basis of colony morphology, hemolyticproperties and gram staining and identified using an APIsystem kit (bioMeriux).

2.3. RNA extraction and real-time quantitativeRT-PCR

Total RNA was isolated from liver tissues using TRIzol reagent(Invitrogen Carlsbad, CA, USA), followed by RNA purificationusing RNeasy columns (Qiagen Maryland, USA) and DNase Itreatment (InvitrogenCarlsbad CAUSA) in accordancewith themanufacturer's instructions. Purified RNA was quantitated byNanodrop (ND-1000 V3.3.1, Thermo Scientific, USA) and theirintegrity was checked by denaturing agarose gel electropho-resis. Liver samples with intact RNA profile (7 normal, 7 PBCand 5 NASH) were selected for determining TLR2 and TLR4mRNA levels using real-time RT-PCR. Total RNA (1 μg) wasreverse transcribed using High capacity cDNA Reverse Tran-scription Kit (Applied Biosystems, Foster City CA USA). TLR2and TLR4mRNA levels were quantified on an ABI StepOne Real-TimePCRSystem (Applied Biosystems Foster CityCAUSA) usingPCR master mix (TaqMan Universal PCR Master Mix) andspecific inventoried TaqMan gene expression assays includingHPRT1 (humanhypoxanthine phosphoribosyl transferase) as anendogenous control. Standard TaqMan protocol was followedfor PCR and reactions were performed in triplicate for eachsample. Expression analysis was performed using StepOnesoftware v2.0 and the mean expression value of the targetgenes for replicates was calculated and expressed as cyclethreshold (Ct) for each sample. The levels of TLR2 and TLR4mRNAwere normalized with respect to that of HPRT1 for eachsample using the ΔCt method. The relative differences in geneexpression among study groups were determined usingcomparative Ct (ΔΔCt) method and fold expression wascalculated using the formula 2−ΔΔCt. Results are presented asfold TLR4 and TLR2 mRNA levels in the diseases compared tothat in the normal liver tissues.

2.4. Protein extraction and western blot analysis

Among the clinical samples for study, 7 normal, 9 PBC and 6NASH liver tissues (150–300 mg) were homogenized in liquidnitrogen and lysed in tissue lysis buffer (50 mM Tris–Tris–Hcl,(pH8.0), 400 mM KCl, 10 mM EDTA, 2 mM PMSF, 1 μg/mlaprotinin and 1 μg/ml leupeptin). Tissue lysates were

centrifuged at 30,000×g for 1 h at 4 °C and supernatantswere collected and stored at −80 °C. Protein concentration ofthe tissue lysates was determined using BCA method (Pierce,Rockford IL USA). Tissue lysates (50 μg) were size-fractionatedby gel electrophoresis on 4–12% pre-casted Bis–Tris Gels(Invitrogen) and then transferred electrophoretically onto0.2 μmnitrocellulosemembranes.Membraneswere blocked in5% non fat dry milk in TBST for 2 h at room temperature andthen incubated with rabbit anti-TLR4 (eBiosciences), rabbitanti-total IKKα, or anti-pIKKα/β, (Cell Signaling) overnight at4 °C. Following washing membranes were incubated withalkaline phosphatase (AP) labeled anti-rabbit secondaryantibody (Sigma) and analyzed using chemiluminescencedetection system (Lumi-Phos WB; Pierce). Equivalent proteinloading and transfer efficiency were verified by staining for β-actin using rabbit anti-β-actin antibody (Sigma). Images weredigitally capturedusing Alpha Innotech Instrumentation (AlphaInnotech Corp) and chemiluminescence signals were quanti-fied using Image J software program (NIH Image, Scion Corp,Frederick MD).

2.5. Statistical analysis

Statistical analysis was performed using GraphPad Prismversion 4.02 (GraphPad Software Inc., San Diego, CA).Quantitative real-time RT-PCR and western blot data arepresented as arithmetic mean±SEM and were compared usingone way ANOVA. A P value of 0.05 or less was consideredstatistically significant.

3. Results

3.1. Higher incidence of bacterial colonizationobserved in normals than in PBC and NASH livertissues

Bacterial colonization for aerobic bacteria in the liver tissuesis summarized in Table 1. The normal and NASH study groupincluded subjects from both the genders and the PBC studygroup included only female subjects. Age range of the threestudy groups was comparable (Table 1). The normal as wellas diseased liver tissues were found to be colonized withbacterial flora (Tables 1 and 2). Normal liver tissues hadhigher percent positivity for bacterial colonization than PBCand NASH tissues (Table 1). Bacterial colonies appeared in 12of the 14 normal (N1–N10, N13 and N14), 5 of the 9 PBC(PBC1–5) and 3 of the 6 NASH (NA1–3) liver tissues. Results ofthe bacterial isolates identified in these liver tissues arepresented in Table 2. A wide range of aerobic bacteria wasisolated from all the liver tissues with the predominance(81%) of gram positive bacteria.

Staphylococcus spp. was themost common bacterial isolateidentified from normal and PBC liver tissues in our study.Pathogenic strains including Staphylococcus aureus wereisolated from three normals and one of the PBC liver tissues;Staphylococcus epidermis was isolated from two PBC livertissues and Staphylococcus hemolyticus was isolated from oneof the PBC liver tissues (Table 2). Bacterial flora from thenormal donor liver tissues includedmostly Staphylococcus spp.and some opportunistic pathogens like Arcanobaterium

Table 2 Summary of results of bacterial identification.

Liver specimens Identified Eubacteria

Normal (N)N1 Arcanobacterium hemolyticumN2 Staphylococcus aureus

Brevundimonas vesicularisN3 Staphylococcus aureusN4 Staphylococcus aureus

Streptococcus pneumoniaeCellulomonas/Micrococcus spp.

N5 Staphylococcus hominisSerratia rubidaea

N6 Staphylococcus chromogenesN7 Staphylococcus sciuriN8 Staphylococcus hylicusN9 Lactococcus lactis spp. lactisN10 Streptococcus mitis

Escherichia coliStaphylococcus aureus

N13 Listeria spp.Stapphylococcus captitis

N14 Rothia dentocariosa

Primary biliary cirrhosis (PBC)PBC1 Nesseria meningitidis

Staphylococcus aureusPBC2 Cellulomonas/Micrococcus spp.

Haemophilus parainfluenzaStaphylococcus hemolyticus

PBC3 Brevibacterium/Corynebacterium spp.Staphylococcus captitisStaphylococcus epidermisSphingomonas paucimobilisListeria grayi

PBC4 Staphylococcus sciuriPBC5 Staphylococcus epidermis

NASH (NA)NA1 Cellulomonas/Micrococcus spp.NA2 Nesseria cinereaNA3 Micrococcus spp.

Enterococcus faecalis

The table lists bacterial isolates obtained from normal, PBC andNASH liver explants.

44 R. Singh et al.

hemolyticum, Brevundimonas vesicularis, and Lactococuslactis spp. lactis. Escherichia coli spp. was isolated from onenormal liver tissue (Table 2). The PBC liver tissues had morediverse isolates and included pathogenic strains like Nesseriameningitidis, Haemophilus parainfluenza, S. hemolyticus,S. epidermis and S. aureus (Table 2). Cellulomonas/Micrococ-cus spp., Enterococcus faecalis and Nesseria cinerea wereisolated from the NASH liver tissues (Table 2). The identifiedbacterial flora from culture positive liver tissues representsonly the cultivable microorganisms colonizing these livertissues. Further, the bacterial culture negativity of some ofthe liver tissues in our study samples may not necessarilysuggest sterility of these livers.

3.2. Lower TLR2 and TLR4 mRNA levels observed innormals than in PBC and NASH liver tissues

TLR2 mRNA levels in normals were found to be lower whencompared to that in PBC and NASH liver tissues (Fig. 1A).Significantly lower levels of TLR4 mRNA were observed innormal liver tissues compared to that in PBC and NASH livertissues (Pb0.05), (Fig. 1B). Among the diseased tissues, TLR2mRNA expression was higher in the PBC than in NASH livers(Fig. 1A). In contrast, TLR4 mRNA expression was signifi-cantly higher in NASH than in the PBC liver tissues (Pb0.05),(Fig. 1B). Overall, TLR2 mRNA levels were observed to behigher than TLR4 mRNA levels in all the liver tissues in thisstudy (Fig. 1C). Similar to TLR4 mRNA levels, TLR4 proteinlevels were also found to be significantly higher in the PBCand NASH than in the normal liver tissues (Pb0.05) (Figs. 2Aand B). The protein levels of TLR4 were not found to besignificantly different between the NASH and PBC livertissues (Fig. 2B). We did not find any significant correlationbetween TLR4 mRNA and protein levels in the normal anddiseased liver tissues.

3.3. Lower protein levels of pIKKα observed innormals than in PBC and NASH liver tissues

Activation and translocation of NF-κB following TLR activa-tion are dependent on phosphorylation of IKKα proteins. Wedetermined the protein levels of pIKKα in normal, PBC andNASH liver tissues via quantitative western blot analysis. Inaccordance with the TLR2 and TLR4 expression, the pIKKαprotein levels in normals were also lower when compared toPBC and NASH liver tissues (Figs. 3A and B), however pIKKαprotein levels among the PBC and NASH liver tissues werecomparable.

4. Discussion

The commensal microflora that colonizes the gastrointesti-nal (GI) tract has a significant impact on healthy as well asdiseased state in humans [23]. Disruption of homeostaticbalance between the host and the resident microflora is nowunderstood to be a part of microbial pathogenesis in varioussusceptible hosts such as patients with HIV infection,inflammatory bowel disease, liver cirrhosis or graft vs. hostdisease in transplant recipients [24–26]. The present studywas conducted to investigate the bacterial colonization andthe status of inflammation in normal and diseased humanlivers.

In this study, we identified high yield of viable aerobicbacterial isolates from normal as well as diseased human livertissues that indicates translocation of bacteria occurring inboth normal and diseased livers. The identified microflorafrom normal liver tissues had higher percentage of grampositive bacteria predominantly Staphylococcus spp. and alsopresence of various opportunistic pathogens. Human colon iscolonized with plethora of intestinal microflora comprisingaerobic as well as facultative and obligate anaerobic bacteria.It has been earlier reported that liver is continuously exposedto translocating intestinal bacteria in healthy as well asdiseased states [5,6,27]. Our observations about aerobic

Figure 2 Protein levels of TLR4 in normals, PBC and NASHexplant liver tissues analyzed by western blotting. TLR4 proteinlevels are relative to β-actin and their values represented asmean±SEM. The error bars represent data from 7 normals, 9 PBCand 6 NASH liver tissues. A) TLR4 protein was detected by westernblotting in protein extracts of liver tissues using antibodies specificfor TLR4. A loading control detecting β-actin is shown. B) TLR4protein levels in normals, PBC and NASH liver tissues.

Figure 1 TLR2 and TLR4 mRNA fold expression in normal, PBCand NASH explant liver tissues evaluated by quantitative real-timeRT-PCR using ΔΔCt method. TLR2 and TLR4 mRNA levels werenormalized with HPRT expression in each sample. Fold TLR2 andTLR4 mRNA levels in PBC and NASH with respect to normal tissuesare represented. Expression values are mean fold mRNA expres-sion±SEM and the error bars represent data from 7 normals, 9 PBCand 6 NASH liver tissues. A) TLR2 mRNA levels in normals, PBC andNASH liver tissues. B) TLR4 mRNA levels in normals, PBC and NASHliver tissues. C) TLR2 vs. TLR4 mRNA levels in normals, PBC andNASH liver tissues. Mean relative expression (2−ΔCt) values of TLR2and TLR4 mRNA were used to calculate TLR2 vs. TLR4 mRNAexpression ratio in each of the study groups.

45Status of bacterial colonization, Toll-like receptor expression and NF-κB activation

bacterial translocation in the liver tissues is supported byprevious reports stating that the number of anaerobescolonizing the gut is higher than the aerobic population, andthe translocating bacteria include mostly aerobes [12,28].Study comparing the translocation rate of various indigenous

bacteria from the GI tract to the mesenteric lymph node ingnotobiotic mice found that gram negative enteric bacillitranslocated in large numbers, gram positive bacteria translo-cated at intermediate levels and obligate anaerobic bacteriatranslocated at a very low levels [29]. Therefore, consistentwith these reports, in our study, we were able to isolate onlyaerobes but no anaerobes from the liver tissues. We shouldremember that these results are based on our cultivablemethods aswe did not use additionalmethods to confirm thesefindings. However, there are several reports in the literaturethat support our findings. Studies in animal models havereported differences in the ability of intestinal bacteria totranslocate to extra-intestinal sites. E. coli, Klebsiellapneumonia, enterococci and streptococci were found to bevery proficient in translocation and are cause of majority of

Figure 3 Protein levels of pIKKα in normals, PBC and NASH livertissues analyzed by western blotting. The pIKKα protein levels arerelative to total IKKα normalized to β-actin and values arerepresented as mean±SEM. The error bars represent data from 7normals, 9 PBC and 6 NASH liver tissues. A) Total and pIKKα weredetected by western blotting in protein extracts of liver tissuesusing antibodies specific for total and pIKKα. A loading controldetectingβ-actin is shown. B) The pIKKα protein levels in normals,PBC and NASH liver tissues.

46 R. Singh et al.

infections in liver cirrhosis patients [12]. E. coli, Proteus, andEnterobacter were found to translocate more efficiently fromthe GI tract than do other bacteria [29]. Further, in anotherstudy, E. faecalis and not E. coli was found to translocate inlarger numbers to the liver [30]. We isolated E. faecalis, E. coliand Streptococcus spp. from the normal liver tissues; howevergram positives were the predominant bacterial populationisolated in our study (Table 2). These bacteria colonizing theliver may induce or serve as co-factors in the host diseasepathogenesis and suppress the colonization of probiotics.Thus, the clinical implications of the colonization of variousmicrobes that translocate to the liver can be huge in diseasedas well as normal/healthy conditions.

The presence of bacterial colonization in normal donororgans has been suggested to induce infection in transplantrecipients [31]. Ruiz et al. reported 63.1% incidence ofmicrobial graft colonization among lung donors that included48.3% gram positive cocci with predominance of Staphylo-coccus spp., 35.8% gram negative bacilli and 15.8% fungi andincidence of bacterial infection was found in the lungtransplant recipient with the same bacterial species culturedfrom the donor tissue [32]. Gram positive Staphylococci andEnterococci were the predominant isolates from the bile ofliver transplant recipients suffering from bacteremia[33,34]. In another study, incidence of 42 gram positiveblood stream infection by Staphylococci (including S. aureus)and Enterococci was found in 205 liver transplant recipients[35]. Infection by S. aureus in transplant recipients results inpoor transplant outcome [32–35]. Infection with methicillin-resistance S. aureus (MRSA) has also been frequentlyreported in living-donor as well as deceased donor andliver transplant recipients [36–38]. Listeria monocytogenesand Streptococcus salivarius have been found to be theetiological agents of bacteremia, spontaneous peritonitisand in occurrence of irritable bowel syndrome in some livertransplant recipients [39–41]. Presence of gram positivebacteria in donor organs (as observed in our and alsoreported in other transplant outcome studies) directlysuggests the colonized donor organs to be the potentialsource of infectious agents in transplant recipients [42–44].

Similar to our observations in the normal livers, bacteriaisolated from diseased liver tissues of PBC in our studyconsisted predominantly of gram positive bacteria thatincluded opportunistic as well as some pathogenic bacteria.Tanaka et al. have demonstrated the presence of bacteria inexplant PBC liver specimens collected at the time oftransplantation [45]. Hiramatsu et al. have also demonstrat-ed the presence of bacteria in bile of PBC patients withpredominance (75%) of gram positive bacteria [46]. Grampositive bacterial cell wall component lipoteichoic acid(LTA) was detected at sites of inflammation around damagedbile ducts in PBC patients as well as in PBCmouse model, thusstrongly suggesting a role of bacterial infection in etiopatho-genesis of PBC [47]. An in vivo infection study withHelicobacter sp. conducted in experimental animal modelshas indicated the possibility of bacterial translocation from thestomach and intestine into the liver [48]. The translocatingbacteria may survive intracellularly within the liver macro-phages and trigger bile duct damage contributing to thedevelopment of PBC [48]. Similarly small intestinal bacterialovergrowth has been associated with NASH etiopathogenesis[49–51]. An in vivo study using rat model of NASH reportedincreased serumendotoxin levels andmalfunction of intestinalmucosal barrier suggesting increased bacterial translocationduring progression of NASH in these experimental rats [52].Isolation of bacteria fromhumanNASH tissues in our study froma small sample population also does support the studiesassociating BT with NASH.

BT has been suggested to be a common phenomenon innormal as well as diseased states [5,6,53,54]. However, atpresent, we have a limited understanding of the fate oftranslocating bacteria and their interaction with the immunesystem in the liver under healthy or diseased state. Localenvironment within the liver guides the immune responsesagainst PAMPs which is orchestrated to maintain the balance

47Status of bacterial colonization, Toll-like receptor expression and NF-κB activation

between tolerance and host defense. Regulation of TLR4appears to be a major target for maintenance of the immunetolerance in the liver [55]. LPS is a major component of gramnegative bacterial cell wall that activates TLR4 mediatedinnate immune responses in the liver. TLR4 has a central rolein activation of Kupffer cells that are the resident hepaticmacrophages. TLR4 activation may engage other cellularadaptor proteins leading to the activation of transcriptionfactors such as nuclear factor NF-κB and AP-1 that furtherinduce cytokine genes expression [41,42,44]. Liver damagemay either result from inability of Kupffer cells to properlyrecognize and eliminate danger molecules or from theirinability to halt inflammation. Under inflammatory condi-tions in the liver prolonged activation of TLRs by PAMPs (suchas LPS) or DAMPS (damage associated molecular patternssuch as mitochondrial DNA released from damaging cells)may lead to break in tolerance that would exaggerate thepro-inflammatory responses that have damaging effects asobserved in the diseased liver tissues [13].

In our study, we found reduced expression of TLR2 andTLR4 expression and NF-κB activation in normals than thediseased livers. Increased expression of TLR2, TLR4 andhigher pIKKα levels in PBC and NASH indicate break intolerance in these liver tissues that may have contributed toBT. We isolated some of the highly pathogenic bacteria fromPBC tissues including Nesseria meningitis, H. parainfluenzaand Listeria grayi. Furthermore, TLR2 expression was foundto be higher than TLR4 in all the liver tissues under study.Especially in PBC tissues, TLR2 expression was higher and thiscould be due to the prevalence of gram positive bacteria thatwe found in these tissues. TLR2 is required for signaling inresponse to a number of gram positive microbial stimuli.Indeed, significantly increased expression on peripheral bloodmononuclear cells (PBMCs) of TLR2 but not of TLR4 hasrecently been demonstrated in patients with cirrhosis but noovert infection [56]. Also, increased TLR4 expression in PBCdisease etiology has been widely speculated [45,57–59].Notably, among the three study groups, the NASH liver tissueshad significantly high TLR4 expression at mRNA level. LPS thatactivates TLR4 mediated innate immune responses contribut-ed to the development of NASH in an experimental mousemodel [12,19]. Also, in a recent study, increased intestinalpermeability as well as increased small intestinal bacterialovergrowth was found to be positively correlating withnonalcoholic fatty liver disease (NAFLD) in humans [60] thussupporting our observations. Further, it is possible that themechanisms that negatively regulate TLR-induced immuneresponses in the liver may be inefficient during these diseasedstates [61,62].

The major source of bacteria and bacterially derivedPAMPs into the liver is the intestinal microbiota. It is not yetclearly understood if controlled BT from the intestine toextra-intestinal sites is a part of normal physiology tosensitize the immune system or is related to host factorssuch as genetics, diet, age or immune status and thus needsto be further investigated in detail. A controlled immuneresponsiveness towards colonized bacteria would play animportant role in educating the liver immune cells andkeeping their immune suppression activated to provideprotection against invasion by potential pathogens withoutdamaging the liver. However, bacterial communities residingin the liver may become a potential source of infection and

inflammation when the immune homeostasis is breachedcontributing to the disease pathogenesis.

To our knowledge, this study serves as the first clinicalevidence showing bacterial colonization in the normal humanlivers comparable to the diseased cirrhotic livers. Lowactivation of TLR4 in the normal livers colonized withbacteria highlights the importance of bacterial colonizationand ongoing immune suppression in the normal liver and itsprobable role in orchestrating liver immune homeostasis.TLR4 as well as NF-κB activation found in the PBC and NASHindicates inflammation leading to break in LPS tolerance andthus disruption in the liver immune homeostasis.

Future work will be focused on better understanding ofthe unique mechanisms involved in defining the fine balancebetween host tolerance and immune responses againstmicrobial flora in the normal and diseased livers. Furtherstudies are needed to understand the contribution ofdifferent TLRs in the recognition of microbial flora andactivation of downstream transcription factors during im-mune homeostasis in the liver cells. These studies will lead tothe development of new therapeutic targets for regulatingactivation of TLRs in various chronic liver diseases.

Acknowledgments

This study was supported by Cancer Sucks, Bixby Oklahoma,Oklahoma State University—Center for Health SciencesIntramural Research Award to RK, and Osteopathic MedicineResearch Association Award to medical student KV. Livertissues were provided through the NIH Liver Tissue Procure-ment and Distribution System (DK92310).

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