Hindawi Publishing Corporation Journal of Immunology Research Volume 2016, Article ID 1958650, 12 pages http://dx.doi.org/10.1155/2016/1958650 Review Article Molecular Mechanisms of Induction of Tolerant and Tolerogenic Intestinal Dendritic Cells in Mice AlexSteimleandJulia-StefanieFrick UniversityofTu¨bingen,InstituteofMedicalMicrobiologyandHygiene,Elfriede-Aulhorn-Strasse6,72076Tu¨bingen,Germany CorrespondenceshouldbeaddressedtoJulia-StefanieFrick;[email protected] Received22October2015;Revised6January2016;Accepted17January2016 AcademicEditor:SilviaBeatrizBoscardin Copyright©2016A.SteimleandJ.-S.Frick.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly cited. Howdoesthehostmanagetotolerateitsownintestinalmicrobiota?Asimplequestionleadingtocomplicatedanswers.Inorderto maintainbalancedimmuneresponsesintheintestine,thehostimmunesystemmusttoleratecommensalbacteriainthegutwhile ithastosimultaneouslykeeptheabilitytofightpathogensandtoclearinfections.Ifthistenderequilibriumisdisturbed,severe chronicinflammatoryreactionscanresult.Tolerogenicintestinaldendriticcellsfulfilacrucialroleinbalancingimmuneresponses andthereforecreatinghomeostaticconditionsandpreventingfromuncontrolledinflammation.Althoughseveraldendriticcell subsetshavealreadybeencharacterizedtoplayapivotalroleinthisprocess,lessisknownaboutdefinitemolecularmechanisms ofhowintestinaldendriticcellsareconvertedintotolerogenicones.Herewereviewhowgutcommensalbacteriainteractwith intestinaldendriticcellsandwhythisbacteria-hostcellinteractioniscrucialforinductionofdendriticcelltoleranceintheintestine. Hereby,differentcommensalbacteriacanhavedistincteffectsonthephenotypeofintestinaldendriticcellsandtheseeffectsare mainlymediatedbyimpactingtoll-likereceptorsignallingindendriticcells. 1.Introduction from these findings for the treatmentof chronic inflamma- torydisordersofthegut:inflammatoryboweldiseases. The mammalian intestinal immune system has to rise to different challenges. On the one hand, it has to tolerate 2.IntestinalDendriticCells:Subsetsand the intestinal microbiota consisting of commensal bacteria, BiologicalFunctions fungi,andothermicrobes,therebyprofitingfrombeneficial bacterial metabolites and other advantages. On the other Dendritic cells (DCs) comprise a heterogeneous leukocyte hand, pathogen induced infections of the intestine have to population of different developmental origin and with dis- be cleared without spacious damage of the intestinal tissue. tinctsurfacemarkersandbiologicalfunctions.DCsoriginate Since a loss of tolerance to the own microbiota causes from blood monocytes or a common DC progenitor in the chronic inflammation of the gut, efficient sensing of the bone marrow under steady-state conditions. The differen- intestinalhomeostasisiscrucialtoavoidpathophysiological tiation into DCs relies on local presence of GM-CSF [1]. immune responses. In this context, intestinal tolerogenic DCsingeneralareutterlyspecializedantigenpresentingcells dendritic cells play a crucial role as key mediators for the (APCs)whichareabletoinduceavarietyofdifferentimmune maintenance of the intestinal homeostasis. While the main responses.Theyarethemostimportantcelltypeconnecting question “how does the host manage to tolerate its own theinnateimmunesystemwithadaptiveimmuneresponses intestinal microbiota?” is pretty simple, the answer is not [2].DCspatrolalmostalllymphoidandnonlymphoidorgans trivial. andmeldpropertiesoftheinnateandadaptiveimmunityand Here,wewanttofocuson(1)themolecularmechanisms thereforelinkthesetwomechanisticallydistinctbranchesof thatmightcontributetotheinductionoftolerogenicDCsin the immune system [3]. Furthermore, DCs play a pivotal theintestineand(2)thepotentialclinicalapplicationsarising role in mediating a protective adaptive immunity against 2 JournalofImmunologyResearch pathogens while maintaining immune tolerance to self- theirbiologicalfunctionandcellularoriginthantheirsurface antigens. Their crucial role for mediating self-tolerance is markerexpression[11,12].Wewilladaptthissystem,butwe confirmed by the observation that DC depletion leads to a will focus on DC subsets located in the intestine and add lossofself-toleranceandresultsinmyeloidinflammationand latestfindingsondifferentsurfacemarkerexpressionamong theinductionofautoimmuneprocesses[4]. these subsets [13]. In general, DCs derive from common Thegut-associatedlymphoidtissue(GALT)isthelargest dendritic cell progenitors (CDP) which, in turn, develop immune organ of the body. The GALT has to ensure that fromhematopoieticprecursorcells.CDPsmaydifferentiate thereisadynamicbalancebetweenprotectiveimmunityby intoeitherpreplasmacytoidDCs(pre-pDCs)orprecommon fighting pathogens and regulatory mechanisms to prevent DCs (pre-cDCs) precursor cells [20, 21]. Murine pDCs are autoimmunity [5]. Since the GALT is constantly exposed characterized by PDCA1 expression and their development to large amounts of luminal antigens like food metabolites, is dependent on the transcription factors BATF3 [22], ID2 foreignpathogens,andcommensalmicrobes,thisbalancehas [23], NFIL3 [24], E2-2 [25], and IRF8 [22]. Murine cDCs tobewelladjustedinordertocreatehomeostaticconditions commonly express XCR1 and SIRP𝛼 [26] and need RelB intheintestine.Dendriticcellsareherebythekeyplayersfor [27],RPBJ[28],andIRF4[29]fordifferentiation.Intestinal maintainingintestinalhomeostasis[6].Theyarespreadout murinecDCsadditionallyexpressCD103andcanbefurther intheconnectivetissueunderlyingtheepitheliallayerofthe subdivided into two ontogenetically different populations, gut[7]. dependent on their surface expression of CD11b [30]. IRF4 + isneededfortheCD11b lineageoftheseCD103-expressing or CD103-nonexpressing conventional DCs [29, 31, 32]. 2.1.MorphologicalDifferencesbetweenDCsandMacrophages + − (MΦ) in the Murine Intestine. DCs belong to the group CD103 CD11b build up cDC subset 1 (cDC1) whereas + + CD103 CD11b formsubgroupcDC2(seeFigure1).Oneof of mononuclear phagocytes (MPs) with macrophages (MΦ) being another cell type belonging to this group. themostimportanteventsforthemaintenanceofintestinal Discrimination between DCs on one hand and MΦ on the homeostasisistheinductionofregulatoryTcells(Tregs)(see below).BesidesTGF-𝛽,Tregformationisdependentonthe other hand is still a matter of ongoing debate. However, concerningintestinalDCsandMΦ,certainsurfacemarkers presenceofretinoicacid(RA)thatisproducedbydendritic cells [33, 34]. But only DCs possessing retinaldehyde dehy- andtranscriptionfactorshavebeenreportedtobeuniquely drogenases(ALDHs)canconvertvitaminA-derivedretinol expressed by only one of these two groups. In the murine toRA.Therefore,ALDHisacrucialenzymeforasubsequent intestine, surface proteins which are exclusively expressed inductionofTregsandthuspromotionofintestinaltolerance byDCsareCD103[8–10],CD26,andCD272[9]. However, + andhomeostasis.ItwasnotclearwhichCD103 DCsubset CD103 is not expressed from every DC subset (see below) + + isresponsibleforTreginduction,sincebothCD103 CD11b [11–13]. A DC specific transcription factor is Zbtb46 [13]. + − andCD103 CD11b DCscanproduceRAandinduceTregs The only MPs in the murine intestine that express the in vitro [35, 36]. Meanwhile, it could be demonstrated proteins CD14, MerTK [9, 14], F4/80, and CD64 [15] are + + − intestinal MΦ. The widely used surface markers for DC- that each CD103 DC subset (CD11b versus CD11b ) can be subdivided in an ALDH-expressing and a non-ALDH- macrophage discrimination, CD11c and MHC-II, are not + + useful to distinguish murine intestinal DCs from MΦ, expressing subset [35]. Therefore, both CD11b CD103 and − + CD11b CD103 DCsareabletoinduceaRA-mediatedTreg sincebothproteinscan be expressed inDCormacrophage formation.Thiswas initiallydemonstratedin skin-draining subpopulations [13, 15–19]. The expression of CD11b and MHC-II varies among DC and MΦ subpopulations [13]. lymphnodes[35],butJanelsinsetal.confirmedthepresence + + +/− Therefore,theproteinexpressionpatternofmurineintestinal of CD103 CD11b ALDH DCs also in the murine cLP + DCs under steady state conditions can be summarized as [37].BothCD103 DCsubsetstogethermonitortheluminal CD11c+CD103+/−CD11b+/−MHC-II+CD26+CD272+Zbtb46+ environmentintheintestine.NotonlyareCD103+DCsable CD14−MerTK−F4/80−CD64−,whilethephenotypeofintesti- to induce Treg-mediated immune tolerance in the intestine nal murine MΦ is CD11c+/−CD103−CD11b+/−MHC-II+/− buttheyarealsoabletopromoteTh17differentiationofna¨ıve CD26−CD272−Zbtb46−CD14+MerTK+F4/80+CD64+. An- Tcells.Th17cellscontributetothemanifestationofautoim- + + other distinctive feature between DCs and MΦ is the munediseases[38,39]andCD103 CD11b seemtobemore − migratoryandproliferationbehaviour.Ingeneral,intestinal efficientinTh17promotionthantheirCD11b counterparts DCsareshort-lived,proliferatingmigratorycellswhileMΦ [36, 40]. It can be assumed that ALDH−CD103+CD11b+/− aretissueresident,long-lived,andnonproliferating[13]. mightpromotethisTh17immuneresponse,butfinalevidence ismissinguntilnow. + 2.2.DCSubpopulationsintheIntestine. Asmentionedabove, Concerning their distribution in the intestine, CD103 − dendritic cells do not comprise a homogenous cell popu- CD11b DCs are prominent in the colonic lamina propria + + lation. Different ways to distinguish one DC from another (cLP), while CD103 CD11b DCs are mostly found in the + + are published and popular. The most prominent approach LP of the small intestine [29]. Additionally, CD103 CD11b to differentiate between distinguishable DC subsets is to DCsfromthemesentericlymphnodes(MLN)alsoexpress focus on different expression of surface proteins, especially ALDH,whichis,surprisingly,abdicablefortheinductionof CD103 and CD11b [12, 13]. However, Guilliams and van de TregssincealossofALDHactivityintheMLNdidnotaffect Laar have recently proposed to distinguish DCs rather by Treginduction[29].Thismightsupportthehypothesisthat JournalofImmunologyResearch 3 CDP Pre-pDC Pre-cDC BINDAF2TILF33 RRPelBBJ E2-2 IRF8 SIRP𝛼 IRF4 IRF4 pDC PDCA1+ CD103+CD11b− cDC1 CD103+CD11b+ cDC2 CD103−CD11b+ DC ALDH+ ALDH− ALDH+ ALDH− CCR2+ CCR2− CD103+CD11b− cDC1 CD103+CD11b− cDC1 CD103+CD11b+ cDC2 CD103+CD11b+ cDC2 CD103−CD11b+ DCCD103−CD11b+ DC RA RA Treg Th17? Treg Th17? Th17 Figure1:Ontogeneticdevelopmentofintestinaldendriticcellssubpopulations.AdaptedfromGuilliamsetal.[35]andexpandedbyfindings fromScottetal.[13].Seetextfordetails.Commondendriticcellprogenitor(CDP),preplasmacytoiddendriticcell(pre-pDC),precommon dendriticcell(pre-cDC),plasmacytoiddendriticcell(pDC),commondendriticcell(cDC),aldehydedehydrogenase(ALDH),regulatoryT cell(Treg),retinoicacid(RA),andT-helper17cell(Th17).Arrowswithsolidlinesrepresentpublisheddata,andarrowswithbrokenlines representspeculativehypotheseswithmissingfinalevidence. ALDHactivityismoreimportantatotherintestinalsiteslike Anotherspecificproteinthatisexpressedexclusivelyby thelaminapropriaofthesmallorlargeintestineforlaterTreg intestinalDCsandnotbyintestinalMΦornonintestinalDCs inductionafterDCmigration. isSIRP𝛼[42,43].Itseemstobeessentialforthegenerationof Recently, Scott et al. discovered an additional CD103- CD103+CD11b+ sincealossoffunctionofSIRP𝛼resultsina negativeDCpopulationinthemurineintestine[13].Thereis decreaseofthisDCpopulationintheintestine,accompanied + + − + aCD11c MHC-II CD103 CD11b cellpopulation,ofwhich by markedly reduced induction of Th17 immune responses about 15% provide features of DCs like Zbtb64, CD26, understeady-stateandinflammatoryconditions[42]. and CD272 expression; they respond to Flt3L; they are In general, it is important to keep in mind that CD103 migratorycellsandlackmacrophagemarkerslikeF4/80and expression on DCs is not a marker for universal tolero- CD64 [13, 41]. They could be shown to be derived from genicity, since (1) even CD103+ DCs can fail to induce + committedpre-DCsasareCD103 mucosalDCs[8].These tolerogenicityifALDHisnotexpressedand(2)atolerogenic − CD103 DCscanbefurthersubdividedintotwofunctionally environment can be established even in the absence of distinct subpopulations dependent on their CCR2 expres- CD103+DCs[44]. + + + sion. CCR2 CD103 CD11b DCs are more efficient in Th17 induction than their CCR-negative counterparts and a loss 2.3. Locations of Intestinal DCs. The murine intestine is a + − + of the CCR2 CD103 CD11b DCs leads to a defective Th17 multifarious habitat for DCs where distinct sites harbour responseandthereforefailstoclearaCitrobacterinfectionin differentDCsubsets.AcommonfeatureofintestinalDCsdis- vivo. tinguishingthemfromDCsfromothernonintestinaltissues 4 JournalofImmunologyResearch is the expression of CD103, with the already mentioned role in maintaining intestinal homeostasis since Traf6−/− exceptionofCCR2+/−CD103−CD11b+DCs,especiallyDCsin mice fail to maintain intestinal homeostasis mediated by thesmallintestine(SI)andPeyer’sPatches(PP)inmesenteric a reduction of Tregs and an increase of T-helper 2 (Th2) lymph nodes (MLN) and, with minor occurrence, in the cells,finallyresultinginamicrobiotacomposition-dependent colonic lamina propria (cLP) [8, 29, 41, 43, 45]. DCs from inductionofcolonicinflammation[55]. nonlymph node tissues remain some days at their inherent sitebeforemigratingtoneighbouringdraininglymphnodes 5.TheDifferentMaturationPhenotypesof [35,46]. DendriticCells 3.AntigenSensingandSamplingby Thecapabilityofinitiatinganimmuneresponsedependson IntestinalDendriticCells thecurrentDCmaturationstate.Usually,antigenencounter results in rapid DC maturation which is characterized Invading microorganisms are recognized by pattern recog- by efficient endocytosis and antigen processing. Further- nition receptors (PRRs) on the DC surface. PRRs include more, upregulation of MHC-II and T cell costimulatory toll-like receptors (TLRs), retinoic acid-inducible gene I- moleculeslikeCD40,CD80,andCD86enhancedexpression like receptors (RLRs), and nucleotide-binding oligomeriza- ofchemokinereceptorsandthesecretionofproinflammatory tion domain-like receptors (NLRs) [47, 48]. PRRs recog- cytokines like IL-1𝛽, IL-6, TNF𝛼, and IL-12 are part of nizepathogen-associatedmolecularpatterns(PAMPs)[49]. DC maturation. These events influence and activate other PAMPscompriseaheterogeneousclassofdifferentantigens, cellular components of an induced immune response like that is, surface components of bacteria. One of the most MΦ,neutrophils,andespeciallyTcells[56]. prominentPAMPswhichusuallyinducesDCmaturationis lipopolysaccharide(LPS),anintegralcellsurfacecomponent 5.1. Mature DCs (mDCs). Induction of DC maturation is ofallGram-negativebacteria.Usually,dendriticcellsunderlie accompanied by a loss of the capacity to take up and the intestinal epithelium and therefore the connection to process antigen [57]. However, they literally develop into the colonic lumen is restricted. However, there are three professional antigen presenting cells (APCs) indicated by prominent ways how intestinal dendritic cells can sample powerfulantigenpresentationtona¨ıveTcells[2],aswellasby luminalantigens:(1)withparticipationofgobletcellswhich theirabilitytomigratetosecondarylymphaticorganswhere deliversolubleandpreferablylowmolecularweightantigen theypresentantigenstoTcells. toneighbouringDCs[50],(2)withthesupportofCX3CR1+ phagocytotic cells which can actively capture antigen fol- lowedbytransporttoneighbouringDCsviatightjunctions 5.2. Immature DCs (iDCs). Immature DCs (iDCs) express [51], and (3) a direct sampling by DCs that extend their lowamountsofMHC-IIandTcellcostimulatorymolecules. dendritestowardsthelumenestablishingadirectconnection They tend to promote T cell anergy and to generate Tregs, tothecoloniclumen[52]. with both effects being crucial for intestinal homeostasis [58]. iDCs furthermore express high levels of PRRs with whichtheymediatetherecognitionofpotentialantigensand 4.IntestinalDendriticCellsand thereforetheirendocytosis[57]. theGutMicrobiota CD103+ DCs are reported to sample mainly bacteria [52] 5.3. Semimature DCs (smDCs) and Tolerant DCs. The in contrast to CX3CR1+ MΦ which also capture soluble definition of a semimature DC phenotype is less distinct. proteins and fungi [51, 53]. This illustrates the relevance The most important property of smDCs uniting different of the bacterial microbiota composition for intestinal DCs. definitions is the inability to induce a proinflammatory InteractionofDCswiththegutmicrobiotacanoccurdirectly Th1 or Th17 response and to be nonresponsive, or in other bysamplingbacterialantigenorbyinteractionwithbacterial words “tolerant,” towards subsequent maturation stimuli metabolic products like short chain fatty acids (SCFAs). [59,60]withthelatterbeingthecriterionthatmediatesthe SCFAs like butyrate can interact with the DC receptor tolerogenic functions of smDCs [60]. DC semimaturation GPR109AwhichfinallyleadstoanIL-10mediatedinduction leadstoacertainexpressionofTcellactivationandacytokine ofTregs[54].Sincenotallgutcommensalbacteriaproduce secretionpatternthatisdistinctfromtheonesofimmature SCFAs,amicrobiotashiftleadingtodysbiosiscanprofoundly and mature DCs. The definite phenotype varies from one affect immunologicalmechanisms in the intestine. Toll-like semimaturation inducing strategy to another. SmDCs receptor (TLR) signalling in DCs also seems to be crucial that are generated by treating immature DCs with TNF𝛼 for the maintenance of intestinal homeostasis. Different display a phenotype that can be summarized as CD11c+ bacterial components bind to distinct TLRs on the surface MHCIIhiCD86hiCD80hiCD40loCD54+CD205hiCD25hiTNFlo of DCs resulting in the activation of intracellular signalling IL-12p40loIL-10lo[61].Inductionofsemimaturationvialow- cascades which leads to DC maturation or semimaturation dose LPS and subsequent dexamethasone treatment results (see below) accompanied by secretion of pro- or anti- in CD14+CD1aloCD80hiCD86hiMHCIIhiIL-10hiTNFlo DCs inflammatory cytokines. The TLT adaptor molecule TNF- [62]. We use a Gram-negative gut commensal, Bacteroides receptorassociatedfactor6(TRAF6)seemstoplayapivotal vulgatus, to induce semimaturation and define the smDC JournalofImmunologyResearch 5 Table1:Phenotypesofsemimaturedendriticcellsdependentonsemimaturationinducingagent.LPS(lipopolysaccharide),Dex(dexameth- asone),andPSA(polysaccharideA);highexpression(hi),lowexpression(lo),intermediateexpression(int),andnotdetermined(n.d.). Semimaturationinducingagent MHC-II CD40 CD80 CD86 IL-10 IL-6 TNF𝛼 IL-12 Source B.vulgatus int lo lo lo n.d. int lo lo [59] TNF𝛼 hi lo hi hi lo n.d. lo lo [61] LPS+Dex hi n.d. hi hi hi n.d. lo n.d. [62] B.fragilisPSA int n.d. n.d. n.d. hi n.d. n.d. n.d. [66] ATP+LPS hi lo hi hi hi n.d. lo lo [120] E.multilocularis lo n.d. n.d. lo int lo n.d. lo [121] LowdoseLPS int lo lo lo n.d. int lo lo [64] 𝛼-1Antitrypsin int lo n.d. int hi lo n.d. n.d. [65] phenotype as CD11c+MHCIIintCD40loCD80loCD86lo 6.TheRoleofDendriticCellsinInductionof TNF𝛼loIL-12loIL-6int[59].Besidesthesestrategies,DCsem- InflammatoryBowelDisease(IBD) imaturation can be induced by treating immature DCs with ATP and LPS [63], low dose Salmonella LPS [64], 𝛼-1 The development of inflammatory bowel diseases (IBD) antitrypsin[65],BacteroidesfragilisPSA[66],orEchinococcus withitstwomajorrepresentativesCrohn’sdisease(CD)and multiloculariscellaggregates[67].Theresultingphenotypes ulcerativecolitis(UC)isassociatedwith(1)aninappropriate concerning the most important immunomodulatory mole- immuneresponsetonormallybenignstimulilikecommensal culesaresummarizedinTable1. microbes, (2) an inefficient clearance of microbes leading to a continuous stimulation of the immune system, or (3) 5.4. Tolerogenic DCs. While mature DCs (mDCs) promote failingto turnfromanadequateproinflammatoryresponse efficientinductionofinflammatoryimmuneresponses,iDCs toinflammationresolvinganti-inflammatoryimmunereac- and smDCs fail to do so. They rather have the property tions[70].Inthiscontext,thecompositionoftheintestinal to actively prevent from inflammatory reactions and are microbiotaisdecisivefortheonsetofcolonicinflammation therefore also termed tolerogenic DCs (tolDCs). The term in most mouse models of experimental colitis [71] and “tolerogenic” includes one, several, or all of the following intestinal DCs are crucial for driving immune responses in features DCs must provide to be considered “tolerogenic”: either a proinflammatory or a rather homeostatic direction (1) the induction of unresponsiveness of T cells, (2) active [72].Forexample,Il10−/−micedevelopchroniccolitiswhich induction of Tregs, (3) inhibition of proinflammatory T resultsfromtheabsenceofsuppressionofMyD88-dependent cell responses, and (4) promotion of T cell apoptosis or T commensal-inducedinflammationbyIL-10[73]. cell anergy [6]. In this context, the interplay between the Understeady-stateconditions,circulatingLy6Chimono- intestinal epithelial cells and the host immune system is of cytes are repopulated into tolerogenic F4/80lowCD103+ essentialimportance. CD11c+ LP DCs, which contribute to homeostasis by sup- Moregenerally,regulatoryortolerogenicDCskeeptheir portingtolerogenicfunctions[16].Ontheotherhand,under abilitytopresentantigens,butatthesametimetheydown- inflammatory conditions during colitis, Ly6Chi monocytes regulatetheexpressionofTcellcostimulatorymoleculesand develop into CD103−CX3CR1intCD11b+ LP DCs, which proinflammatorycytokinesbutinturnupregulateinhibitory mediateinflammationduringcolitis[16]. molecules like PD-L1, CD95L, or IDO as well as anti- inflammatorycytokinessuchasTGF-𝛽andIL-10[68].Fur- The tolerogenic functions of intestinal DCs are mainly thermore,theyareresistanttoasecondmaturationinducing mediated by the induction of regulatory T cells (Tregs). signal[68].Importantly,DCsalsoinfluencethedifferentia- As a characteristic feature, Tregs express the transcrip- tionofna¨ıveTcellsintoTh1,Th2,Th17,orTregcells,mostly tion factor forkhead box P3 (Foxp3) [74]. Induction of + + + due to supplying a certain cytokine environment [69]. In CD4 CD25 Foxp3 Tregs is essential for intestinal home- a healthy individual, the presence of tolDCs is important ostasis [75] and a loss of Tregs leads to a fatal multiple- and a loss of tolDCs can result in the development of AID organ-associatedautoimmunedisease[76].Tregsareusually [4]. Semimature DCs are potent tolerant and tolerogenic convertedintheperipheralimmunesystemwiththehelpof + DCs since they fulfil many to all of the above-mentioned CD103 dendriticcells[77]whereuponthisTreginductionis criteria,dependentontheagentwithwhichsemimaturation dependentonthepresenceofTGF-𝛽andretinoicacid(RA) is induced. As already mentioned before, the main char- [78]. acteristic that makes semimature DCs tolerogenic is their However,duringcolitis,CD103−CXCR1intCD11b+DCs, unresponsiveness(tolerance)towardssubsequentmaturating although also present under steady-state conditions, mas- stimuli[59,64,65]. sivelyinfiltratethecolonicLPandmediateproinflammatory 6 JournalofImmunologyResearch Treg Intestinal epithelial cell TGF𝛽 CD47 IL-10 IL-10 LPS SIRP𝛼 2 MyD88 TLR-4 wnt 1 JAK-2 ITRRAAKF46 PI3K 4 IL-10 TGF𝛽 IRAK1 MHC II P STAT3 𝛽-catenin 5 mTOR STAT3 H+ PSA P IL-10 p38𝛼 V1 TLR-2 P 6 V0 7 + H 3 𝛽-catenin TGF𝛽/ALDH1A2 Lysosome Gadd45𝛼 NF𝜅B p50 IFN𝛽/IL-𝛽/IL-18 p38 Intestinal Nucleus NF𝜅B p50 dendritic cell Proinflammatory cytokines Activation Inhibition Figure2:Possiblemolecularmechanismsoftoleranceinductioninintestinaldendriticcells.Thewhitenumbersinblackcirclesrefertothe numberingofregulationmechanismsinthetext.Seetextfordetails. immuneresponsesbyproducingIL-12,IL-23,iNOS,andTNF intestinalDCs.Allproposedmechanismsaresummarizedin [16]. Figure2. (1)Cell-to-Cell-ContactandSTAT3Signaling.Epithelialcells 7.PossibleMolecular Mechanismsof oftheintestineexpressthesurfaceproteinCD47whichcan DCToleranceInductionintheIntestine directly interact with signal regulatory protein 𝛼 (SIRP𝛼) expressedonthesurfaceofDCswhichunderlietheintestinal Lessisknownaboutdefinedmechanismsoftoleranceinduc- epithelialcelllayer.Thisprotein-protein-interactionhasbeen tioninintestinalDCs.However,knowledgeabouttolerance showntoresultinajanuskinase-2(JAK-2)dependentsignal induction mechanisms of other DC subsets or of in vitro transducer and activator of transcription 3 (STAT3) activa- generatedDCscanbetransferredtointestinalDCstoexplain tion downstream of SIRP𝛼 in DCs [79]. STAT3 activation, howtheymanagetotolerateluminalbacterialorfoodanti- in turn, leads to enhanced IL-10 secretion from DCs and gensandthereforepreventfromuncontrolledinflammatory therefore promotes tolerogenic properties in the intestinal reactions.Here,wewanttopresentlatestresearchresultsand environment[79].STAT3haslongbeenknownasacrucial discuss how and if these findings can be assigned also to negativeregulatorofimmunity.DisruptionofSTAT3leadsto JournalofImmunologyResearch 7 alossofTcelltoleranceinmiceandefficientSTAT3signaling indoleaminedioxygenases(IDOs)anddecreasedexpression isassociatedwiththeimmatureDCphenotype,generalIL- of proinflammatory cytokines like IFN𝛽, IL-1𝛽, and IL-18 10 secretion, and tolerance induction [80]. Therefore, not [91]. These implications for p50 in the induction of tolDCs only does DC alone seem to be important for homeostasis aresupportedbythefindingthatp50-deficientDCsareweak + maintenance but also the “teamwork” with neighbouring inducersofaFoxp3 Tregdifferentiation[92].Formationof epithelialcellsseemstocontributetotolerancemechanisms. p50-p50 homodimers contributes to LPS tolerance in MΦ [93]andp50expressioninimmatureDCsiscrucialtoprevent (2) IL-10 as a Central Cytokine for Intestinal Homeostasis. fromautoreactiveTcells[91]. Interleukin-10isakeyinhibitorycytokineinTcellactivation and a mediator of intestinal homeostasis [81]. It is secreted (4) 𝛽-Catenin Promotes DC Tolerogenicity. 𝛽-catenin is a byTcells,Bcells,andmostmyeloid-derivedcells[82].Mice transcription factor and part of the wnt signalling pathway. lacking functional IL-10 or its IL-10R receptor counterpart It could be demonstrated that this signalling pathway with spontaneouslydevelopsevereintestinalinflammation. the subsequent release of 𝛽-catenin into the nucleus results Supporting the idea of IL-10 being a crucial mediator in the induction of tolDCs [94]. Gene expression profiles for intestinal homeostasis [83]. Also humans with defec- ofintestinalLPDCsrevealedthatthissignallingpathwayis tive mutations in the genes encoding for IL-10 or IL- decisivefortheDCtobecomeeithermatureortolerogenic. 10R develop a severe form of enterocolitis within the first 𝛽-catenin translocation into the DC nucleus resulted in months after birth [84]. These observations made IL-10 a the expression of various tolerance-associated factors like promising therapeutic candidate in order to treat chronic retinoicacid-metabolizingenzymes,IL-10andTGF-𝛽[94]. inflammatory conditions of the intestine. However, results were not convincing since, in mice as well as in humans, (5)PreventionofV-ATPaseDomainAssemblyInducesTolero- + IL-10 administration did not ameliorate the inflammatory genic DCs. Vacuolar (H )-ATPases (V-ATPases) are ATP- conditions [85]. IL-10 not only affects T cell responses but drivenprotonpumps.Theyarecomposedoftwodomains:a can also provide autocrine and paracrine effects on DCs. peripheralV1domainandmembrane-embeddedV0domain Since DCs express the IL-10 receptor (IL10R), IL-10 can [95].V-ATPasesareinvolvedinacidificationoflysosomesby bind to IL10R resulting in a negative regulation of myeloid shufflingprotonsfromthecytosolintothelysosomiclumen differentiationprimaryresponse88(MyD88)signalinginside [96]. The pH value of lysosomes is a crucial regulator for DCs.MyD88isanadaptormoleculeofTLRsandisrequired theefficiencyofantigenprocessingsincelysosomalproteases for downstream TLR signalling. IL-10/IL10R interaction being involved in antigen proteolysis require acidic envi- mediates this negative regulation by a downregulation of ronments [95]. The most important mechanism to regulate interleukin 1 receptor associated kinase 4 (IRAK4) on the lysosomalacidificationistocontroltheassemblyofthetwo proteinlevelwithoutalteringIRAK4genetranscriptionrates V-ATPase domains which is a required event for forming [86].ItalsoleadstodissociationofMyD88fromTLRsand a functional proton pump. It is known that activation of subsequently promotes proteasomal degradation of IRAK1, TLRs promotes domain assembly and therefore supports IRAK4, and TRAF6, therefore silencing MyD88-dependent DC maturation [96]. Domain assembly seems to be a PI-3 TLR signalling [86]. However, this is just the case if LPS as kinaseandmTORmediatedeventsinceinhibitorysubstances a TLR4 ligand is present at the same time to induce TLR forbothmoleculescouldblockV-ATPasedomainassembly signaling. IL-10 silencing of MyD88 signaling seems to be andthereforepreventfromDCmaturationandpromotethe crucial for the maintenance of intestinal homeostasis since IL-10−/− micefailtodevelopintestinalinflammationifthese inductionofatolerogenicphenotype[95].Also,stimulation of integrins and E-cadherins by cluster disruption of DC micesimultaneouslylackMyD88[73].Asaconsequence,the prevents from domain assembly and supports induction of cytokineenvironmentdoesalsoaffectDCsintheirabilityto atolerogenicphenotype[96,97]. inducetolerancemechanisms. (3) NF𝜅B Signalling as a Mediator for Tolerogenicity. A key (6)p38𝛼ExpressionInfluencesExpressionofALDH1A2.MAP kinases like ERK, JNK, and p38𝛼 form central pathways regulator for DC maturation and inflammatory reactions in general is NF𝜅B [87, 88]. NF𝜅B family members do that are activated by innate immune signals like PAMPs [98,99]andexcessiveactivationofMAPkinasesarereported not only have an activating potential for the induction of proinflammatory cytokines. Two NF𝜅B proteins, p50 and to be associated with many autoimmune and inflammatory p52,havebeenassociatedwithtranscriptionrepressionfunc- diseases [99]. However, the MAP kinase p38𝛼 provides a tions and therefore induction of tolerance [89, 90]. Both dichotomicrole.Besidesbeinginvolvedinpromotingproin- proteins lack the carboxyterminal transactivation domain flammatory responses, its activity seems also to be crucial andcanforminhibitoryhomodimercomplexesthatprevent for the induction of a tolerogenic phenotype in intestinal from transcription of proinflammatory genes. NF𝜅B p50 CD103+ DCs. In these DCs, p38𝛼 is constitutively active has been shown to promote a tolerogenic DC phenotype and this activity is crucial for the expression of TGF-𝛽 and by negatively affecting DC survival and their capacity to aldehyde dehydrogenase 1A2 (ALDH1A2), the latter being efficiently activate T cells [91]. Accumulation of p50 in involvedinmetabolizingretinoicacid(RA).TGF-𝛽andRA the nucleus of tolerogenic DCs can be accompanied by are involved in Treg generation and therefore promote gut enhancedexpressionoftolerance-promotingmoleculeslike homingpropertiesofTcells[99]. 8 JournalofImmunologyResearch (7)Gadd45𝛼-MediatedTLR2SignallingContributestoTolero- DCs.Aswehavedemonstrated,differentcommensalbacteria genic Features of Intestinal DCs. An abundant bacterial gut can have opposite effects on DC maturation. This makes commensal,B.fragilis,isabletoprotectfromtheinductionof thecompositionofthemicrobiotadecisiveonwhetherDCs EAEandexperimentalcolitisandincreasestheproportions mediatetolerogenicorinflammatoryLPS-triggeredimmune + + of CD103 CD11c DCs [100, 101]. It could be demonstrated responses. that this effect is mediated by polysaccharide A (PSA), an immunomodulatorycomponentpresentinoutermembrane 8.PerspectivesforClinicalApproachesfor vesicles derived from B. fragilis bacteria [66]. PSA pro- theTreatmentofIBDUsingTolerantand motesimmunologicaltolerancebyinducingIL-10producing Foxp3+Tregsandprotectsanimalsfromexperimentalcolitis TolerogenicDendriticCells [102]. The PSA caused induction of tolDCs is dependent In order to be suitable as an administrable therapeutic, on TLR2 and growth arrest and DNA-damage-inducible 45 𝛼 (Gadd45𝛼), since Gadd45𝛼-deficient DCs are unable tolerogenicDCs(tolDCs)havetobegeneratedinvitro.One efficient way to induce tolDCs is coincubating them with to mediate PSA-induced protection of experimental colitis [66]. Gadd45𝛼 itself inhibits an alternative way of MAPK apoptoticcells.PhagocytosisofapoptoticcellsthroughDCs p38-mediated signalling [103] and PSA-containing outer results in production of TGF-𝛽 which in turn contributes membranevesiclesleadtoupregulationofGadd45𝛼[66]. to immune tolerance. Apoptotic cell-treated DCs efficiently + + converted na¨ıve CD4 T cells into Foxp3 Tregs [108, 109]. Taken together, all of the mentioned molecular mech- In general, apoptotic cell induced tolDCs are important for anisms of tolerance induction in DCs are potentially able inductionofimmunetolerance[110,111].Theyprovideupreg- to take place in the intestine, either through participation ulation of Galectin-1 and CD205 [112], two molecules that of neighbouring intestinal epithelial cells or through direct interactionofDCswithluminalcontent.Concerningluminal facilitate the manifestation of immune tolerance [113, 114]. content, bacteria and their PAMPs could promote all of At the same time, apoptotic cell treated DCs downregulate the potential mechanisms via interaction with host pattern Gr-1andB-220[112],twomoleculestriggeringinflammatory PRRs, especially TLRs. We identified apathogenic Gram- responses. These DCs furthermore downregulate the tran- negative commensal strains, namely, Bacteroides vulgatus scription factor ROR𝛾t which is the decisive transcription mpkandEscherichiacolimpk,mediatingcompletelycontrary factor for Th17 differentiation. Not only does treatment effectsonDCmaturationand,inconsequence,theprogress with apoptotic cells lead to induction of tolDCs but also of experimental colitis in mice [104, 105]. As mentioned treatment with herbal coumarins [115] and the macrocyclic above,B.vulgatusinteractionwithimmatureDCsconverted antibioticrapamycin[116]leadstotolDCinduction.Invitro themintoatolerantandtolerogenicsemimaturephenotype generated tolDCs have already been successfully used for characterized by low to intermediate expression of MHC- the treatment of autoimmune disorders in animal models II,CD40,CD80,andCD86,almostabsentsecretionratesof andperipheraltolerancecouldberestoredbyadministrating TNF𝛼andIL-12p70,andremarkableIL-6secretion[59].As tolerogenicDCs[117].Approachestotreatautoimmunetype acharacteristicoftolerantDC,thisphenotypecouldnotbe 1diabetesinamousemodelusingnonobesediabetic(NOD) overcome with a subsequent maturating bacterial stimulus micewithtolerogenicDCswereverysuccessful[112].Todo orbyCD40ligation[106].E.colimpkstimulation,however, so, apoptotic islet cells were used to induce DC mediated resultedinefficientDCmaturation.Asaconsequence,E.coli tolerance against own islet cells [118]. All these applications mpkcolonizationinexperimentalmousecolitisusingIl2−/− leadtothequestioniftransferoftolerogenicdendriticcells mice resulted in colonic inflammation, a feature that could would also be an approach to treat IBD and, if yes, which bepreventedbysimultaneousB.vulgatusmpkcolonization method to induce DC tolerance would be the method of [104, 105]. We could prove that both bacteria differentially choice. A published approach for the treatment of Crohn’s alterthephenotypeofdendriticcellsnotonlyinvitrobutalso disease patients is in vitro generation of DCs followed by invivointhecolonicLPviaadjustedbacterialcolonization pulsingwithdexamethasone,proinflammatorycytokinesIL- of the gut [105]. In this context, feeding B. vulgatus always 6,IL-1𝛽,andTNF𝛼,andPGE2[119].Concerningourfindings induced tolerant and tolerogenic DC in the colonic LP. In that a certain gut commensal, B. vulgatus mpk, efficiently anotherstudyusingdistinctE.colibacteriathatjustdifferin induces tolerant and tolerogenic DCs in vitro as well as in the structure of their cell surface LPS, we could prove that vivo [59, 104, 105], we would recommend using host gut theLPSstructurealonedecidesifLPDCsareconvertedinto commensalbacteriaforinvitrotolDCgeneration.Inorderto amaturephenotypeandthereforepromoteinflammationor providemorepotentialluminalantigenspresentedbyMHC- if they are converted into tolerogenic semimature DCs and IIoftolDCs,adefinedmixtureofcommensalbacteriacould thusmaintainintestinalhomeostasis[107].AsLPSprimarily beused.Thiswouldenlargetheamountofantigenicpeptides signals via TLR4, tolerance induction mechanisms where againstwhichtolerancewouldbeinduced. NF𝜅B p50, Gadd45𝛼, MyD88-signaling, 𝛽-catenin, and/or V-ATPase domain assembly are involved could be possible. 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