Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-γ and RelA

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1 Commensl neroic gut cteri ttenute inflmmtion y regulting nucler-cytoplsmic shuttling of PPAR-γ nd RelA Denise Kelly 1, Jmie I Cmpell 1, Timothy P King 1, George Grnt 1, Emmelie A Jnsson 2, Alistir G P Coutts 1, Sven Pettersson 2 & Shun Conwy 1 The humn gut microflor is importnt in regulting host inflmmtory responses nd in mintining immune homeostsis. The cellulr nd moleculr ses of these ctions re unknown. Here we descrie unique nti-inflmmtory mechnism, ctivted y nonpthogenic cteri, tht selectively ntgonizes trnscription fctor NF-κB. Bcteroides thetiotomicron trgets trnscriptionlly ctive NF-κB suunit RelA, enhncing its nucler export through mechnism independent of nucler export receptor Crm-1. Peroxisome prolifertor ctivted receptor-γ (PPAR-γ), in complex with nucler RelA, lso undergoes nucleocytoplsmic redistriution in response to B. thetiotomicron. A decrese in PPAR-γ olishes oth the nucler export of RelA nd the nti-inflmmtory ctivity of B. thetiotomicron. This PPAR-γ-dependent nti-inflmmtory mechnism defines new cellulr trgets for therpeutic drug design nd interventions for the tretment of chronic inflmmtion. The humn gut contins diverse popultion of nonpthogenic, commensl cteri tht contriute to gstrointestinl helth nd disese. Considerle clinicl nd experimentl evidence links immune responses directed ginst the norml cteril flor to the pthogenesis of humn inflmmtory owel disese 1 4. Prdoxiclly, nonpthogenic gut cteri re lso thought to contriute to immune homeostsis y ltering microil lnce or y specificlly intercting with the gut immune system, mechnisms purported to underlie the therpeutic sis for cteril proiosis in inflmmtory owel disese 2,5,6. Although the impliction of these clims for gut helth is potentilly very importnt, the cellulr nd moleculr mechnisms y which individul memers of the commensl flor contriute to immune homeostsis hve not een elucidted. Study of these mechnisms could underpin new therpies for inflmmtory gut disorders. In terms of trget sites, intestinl epithelil cells provide the first point of contct for cteri within the gut lumen; they lso interfce nd segregte the gut immune system nd therefore hve pivotl function in cteri-host communiction. By virtue of recognition receptors expressed on picl nd/or solterl surfces of epithelil cells 7, cteril moieties common to commensl nd pthogenic cteri ind nd ctivte signling cscdes tht cn trigger proinflmmtory gene trnscription. Although the presence of virulence fctors on pthogenic cteri mplifies inflmmtory immune responses, the moleculr sis of hyporeponsiveness to commensl cteri is not fully pprecited. Regultory mechnisms driven y CD4 + regultory T cells in the lmin propri re importnt in mintining tolernce to enteric cteri 8. However, it is likely tht other eqully importnt mechnisms controlling innte immunity nd inflmmtion operte t the level of epithelil cells. Tht nonpthogenic gut cteri contriute to this process is lso reltively new ide 9. Given the complex mechnisms defining host-pthogen dpttion, the possiility lso exists tht homologous systems evolved mongst the commensl cteril species tht reside permnently within the gstrointestinl trct nd tht such dpttions ctively contriute to immunologicl tolernce nd homeostsis within the helthy gut. Here we descrie n ntiinflmmtory mechnism ctivted y Bcteroides thetiotomicron, prevlent neroe of the humn intestine; this cterium ttenutes proinflmmtory cytokine expression y promoting nucler export of NF-κB suunit RelA, through PPAR-γ-dependent pthwy. This mechnism highlights new cellulr trgets nd pproches for therpeutic intervention in the tretment of inflmmtory disese. RESULTS B. thetiotomicron ttenutes gut inflmmtion The commensl microflor, in ddition to driving the expnsion of lymphoid tissues in the gut, contriute to mny other functions, including coloniztion resistnce. Tht they lso ctively regulte locl immune responses within the gut is lso distinct possiility. We therefore hypothesized tht commensl cteri influence immunologicl outcome y modulting the innte immune response mounted y 1 Gut Immunology Group, Rowett Reserch Institute, Greenurn Rod, Bucksurn, Aerdeen AB21 9SB, Scotlnd, UK. 2 Microiology & Tumor Biology Center, Division of Moleculr Pthology, Krolinsk Institute, S Stockholm, Sweden. Correspondence should e ddressed to D.K. (D.Kelly@rri.sri.c.uk). Pulished online 21 Decemer 2003; doi: /ni VOLUME 5 NUMBER 1 JANUARY 2004 NATURE IMMUNOLOGY

2 g h c d e Figure 1 B. thetiotomicron specificlly ttenutes inflmmtory responses in Cco-2 cells nd in vivo. () Cco-2 cells were incuted for 2 h with medium (1); S. enteritidis (2); S. enteritidis nd B. thetiotomicron (3); or B. thetiotomicron (4) nd cytokine expression ws determined y RNA lot: Tnf, TNF; Il8, IL-8; Cxcl2, MIP-2α, Tgf1, TGF-β; Ptgs2, cyclooxygense 2; Gpd, GAPDH. () Cco-2 cells were incuted for 2 h with medium (1); cteri or lignds s indicted ove ech gel (2); cteri or lignds s indicted ove ech gel, ut including B. thetiotomicron (3); or B. thetiotomicron (4). IL-8 expression ws determined y PCR. (c) IEC-6 cells were incuted with cteri s descried in nd the expression of TNF ws determined y RNA lot. (d) Cco-2 cells were incuted for 2 h with medium (1); S. enteritidis (2); S. enteritidis nd B. vulgtus (3); or B. vulgtus (4) nd cytokine expression (left mrgin) ws determined y RNA lot. (e) Trnsepithelil migrtion of PMN cells through Cco-2 monolyer, s determined y MPO ssy. Tretments 1 4 were s descried in. ***, P < (f) In vivo confirmtion of the nti-inflmmtory effect of B. thetiotomicron, s determined y MPO ssy of rt ilel mucos. Tretments 1 4 were s descried in. All experiments were repeted minimum of three times. **, P < (g) Histology of ilel tissue from rt infected with S. enteritidis. (h) Histology of ilel tissue from rt infected with S. enteritidis nd B. thetiotomicron. Scle rs, 50 µm. intestinl epithelil cells. To investigte this, we exmined the effect of B. thetiotomicron, prevlent commensl neroic cterium of the humn gut microflor, on the cute inflmmtory response triggered in intestinl cells y exposure to pthogenic Slmonell enteric serovr Enteritidis. Using cdna microrry technology, we identified severl inflmmtory genes whose induction fter exposure of Cco-2 cells to S. enteritidis ws modulted y the presence of B. thetiotomicron. Genes relevnt to inflmmtion nd showing lrge responses included those encoding tumor necrosis fctor (TNF), interleukin 8 (IL-8) nd cyclooxygense 2. We confirmed these gene expression chnges y RNA hyridiztion, semiquntittive PCR (Fig. 1,) nd rel-time PCR 10. The nti-inflmmtory ctivity of B. thetiotomicron could lso e demonstrted with other intestinl cells, including IEC-6 cells (Fig. 1c) nd T84 cells (dt not shown). The ttenuting effect seemed to e specific to B. thetiotomicron, s relted erotolernt strin, B. vulgtus, did not produce similr effects on these proinflmmtory cytokines (Fig. 1d). Induction of IL-8 in Cco-2 cells y other inflmmtory meditors, including IL-1α, IL-1β, TNF, phorol 12-myristte 13-cette (PMA), lipopolyscchride (LPS), purified flgellin nd enterohemorrhgic Escherichi coli 0157:H7 demonstrted tht B. thetiotomicron hd specific ntgonistic effect on the IL-8 expression induced y PMA, flgellin nd E. coli 0157:H7, wheres the ctivtion induced y IL-1α nd IL-1β ws unffected (Fig. 1 nd dt not shown). LPS nd TNF did not induce IL-8 expression in Cco-2 cells. The lck of n effect of B. thetiotomicron on the expression of IL-8 induced y IL-1α nd IL-1β indicted tht lthough the cterium ttenuted inflmmtory cytokine expression induced y severl stimuli, it ws not universlly effective, indicting either conditionl regultion or elements of specificity in its mode of ction. We estlished the physiologicl relevnce of the nti-inflmmtory effect of B. thetiotomicron with n in vitro model of inflmmtion sed on polymorphonucler leukocyte (PMN) recruitment, monitored through myeloperoxidse (MPO) detection (Fig. 1e). The vrile effect of S. enteritidis nd B. thetiotomicron cteri on PMN recruitment f could e correlted with the secretion of IL-8 protein, chemotctic fctor produced y epithelil cells tht is known to e essentil for oth PMN recruitment nd trnsepithelil migrtion 11,12. Consistent with the gene expression dt (Fig. 1), S. enteritidis induced 227 ± 33.1 pg/ml of IL-8 protein in culture superntnts over 4 h, wheres fter coculture in the presence of B. thetiotomicron, the resultnt concentrtion ws significntly lower, t 104 ± 9.2 pg/ml (P < 0.001). We lso demonstrted the efficcy of this nti-inflmmtory ctivity with miniml flor rts, in which the norml microil flor were mintined t low levels y housing rt pups in isoltor conditions. Pups were dosed orlly with B. thetiotomicron, nd successful coloniztion ws confirmed y specific mplifiction of cteril DNA from rt jejunl nd ilel tissues nd y conventionl culture-sed microiologicl nlysis (dt not shown). Rts orlly infected with S. enteritidis nd B. thetiotomicron hd significntly ttenuted MPO concentrtions compred with tht of rts chllenged with S. enteritidis without therpeutic dministrtion of B. thetiotomicron (Fig. 1f). Consistent with these dt, we found sustntil histologicl disruption of the villus nd crypt structure concomitnt with extensive cellulr infiltrte in the lmin propri of rts infected with S. enteritidis (Fig. 1g). In contrst, lthough some cellulr infiltrte ws present in the rts treted with S. enteritidis nd B. thetiotomicron, their intestinl structure ws lrgely preserved (Fig. 1h). The sustntil nti-inflmmtory effect derived from in vivo coloniztion with this neroic cterium provided n importnt physiologicl confirmtion of the responses seen in our Cco-2 cell model system. B. thetiotomicron ttenutes nucler RelA NF-κB is nucler fctor involved in the trnscriptionl regultion of inflmmtory genes. Severl signl trnsduction cscdes, including those generted in response to cteril infection, trigger nucler trnsloction of NF-κB s sequel to the phosphoryltion, uiquitintion nd proteosome-medited degrdtion of the inhiitory IκB proteins 13,14. Consistent with this, we found tht exposure of Cco-2 cells to S. enteritidis for 2 h enhnced the nucler trnsloction of the NF-κB NATURE IMMUNOLOGY VOLUME 5 NUMBER 1 JANUARY

3 d Figure 2 B. thetiotomicron ttenutes inflmmtion y enhncing nucler export of NF-κB RelA protein in Cco-2 cells. () Immunofluorescence microscopy of RelA (top row) nd IκBα (ottom row). Tretments were for 2 h: medium lone (1); S. enteritidis (2); S. enteritidis nd B. thetiotomicron (3); or B. thetiotomicron (4). Scle rs, 25 µm. () Nonshifted nd supershifted EMSAs of Cco-2 nucler extrcts with consensus NF-κB oligonucleotide nd RelA ntiody. Tretments were s descried in (times, elow photos). (c) Nonshifted nd supershifted EMSAs of Cco-2 nucler extrcts with consensus AP-1 oligonucleotide nd JunD ntiody. Tretments 1 4 were s descried in, for 2 h. (d) Immunolot of immunoprecipitted IκBα nd piκbα from Cco-2 cells. Tretments 1 4 were s descried in (times, ove lots). (e) RNA hyridiztion of Nfki (IκBα) in Cco-2 cells. Tretments 1 4 were s descried in. (f) Immunolot of immunoprecipitted IκBα protein. Tretments 1 4 were s descried in. Dt re representtive of t lest three independent experiments. e c f suunit RelA (Fig. 2, top row). Gel-shift nd supershift nlyses showed tht RelA ws the min nucler sucomponent of NF-κB fter incution of Cco-2 cells with slmonell. When Cco-2 cells were coincuted with slmonell nd B. thetiotomicron, similr mount of nucler import of RelA ws pprent t 30 nd 60 min (Fig. 2). However, unlike the cells treted with Slmonell lone, B. thetiotomicron induced nucler export of RelA, with considerly reduced mounts in the nucleus nd minly cytoplsmic redistriution t 2 h (Fig. 2, nd dt not shown). Given our erlier finding tht B. thetiotomicron did not ttenute the IL-1α- or IL-1β-medited induction of IL-8, we investigted if this difference ws ecuse of differentil effects on the specificlly ctivted NF-κB suunits. B. thetiotomicron hd profound effects on ll stimuli tht propgted their inflmmtory response through sustined ctivtion of RelA (S. enteritidis, E. coli, PMA nd flgellin) y reducing nucler RelA ccumultion. IL-1α nd IL-1β, however, produced only trnsient ctivtion of RelA evident t 1 h ut negligile t 2 h fter stimultion (dt not shown). As ws evident from the time course studies, the B. thetiotomicron effect on nucler RelA ws only pprent t times fter 1 h of exposure to this cterium (Fig. 2), when the induction of RelA y IL-1α nd IL-1β hd susided. Susequent supershift ssys demonstrted tht the effect of B. thetiotomicron seemed to e highly specific for NF-κB RelA, s other nucler trnscription fctors including ctivtor protein 1 (AP-1), Figure 3 The nucler export of RelA induced y B. thetiotomicron is independent of the Crm-1 pthwy. Cco-2 cells were incuted for 2 h with medium (1); S. enteritidis (2); S. enteritidis nd B. thetiotomicron (3); or B. thetiotomicron (4) with (+ LMB) or without (No LMB) LMB. The cellulr distriution of immunofluorescently detected RelA ws determined. Nuc., nucler; Cyt., cytoplsmic. Dt re representtive of t lest three independent experiments ± s.d. ***, P < VOLUME 5 NUMBER 1 JANUARY 2004 NATURE IMMUNOLOGY

4 c shown here to e composed minly of JunD, were nerly unffected fter 2 h of incution, when nucler trnsloction of RelA hd occurred (Fig. 2,c nd dt not shown). The trnscription fctor requirements of certin inflmmtory genes my explin their differentil susceptiility to the ttenuting effects of B. thetiotomicron. Het-inctivted B. thetiotomicron, culture superntnt nd conditioned culture medi derived from Cco-2 cells exposed to B. thetiotomicron lcked this ttenuting ctivity (dt not shown), indicting tht cteril-epithelil cell contct ws required. Moreover, the viility nd growth of S. enteritidis nd B. thetiotomicron cteril strins were unffected fter coculture with intestinl epithelil cells (dt not shown). Also, oth epithelil ttchment nd invsion of S. enteritidis were unffected y the presence of B. thetiotomicron (dt not shown) nd hence the oserved effect of B. thetiotomicron on inflmmtory cscdes cnnot e scried to differences in S. enteritidis recognition or ctivtion of epithelil cell surfce receptors. We noted oth phosphoryltion nd degrdtion of the NF-κB inhiitor IκBα fter exposure to S. enteritidis in the presence or sence of B. thetiotomicron (Fig. 2d). There were no differences in the kinetics of IκBα phosphoryltion or degrdtion, indicting tht the regultion of NF-κB is downstrem of IκBα ctivtion nd therefore is not due to the inhiition of IκBα kinse (IKK)ctivity, s shown for pthogens 15. After 2 h of exposure to the cteri, the totl mounts of IκBα mrna (Fig. 2e) nd protein (Fig. 2, ottom row, nd f) were enhnced, indictive of trnscriptionlly ctive NF-κB 16,17. Inhiition of uiquitintion nd degrdtion of the IκBα protein hs een postulted s eing n importnt mechnism of immune regultion wherey commensl cteri prevent or limit inflmmtion in the gut 9. However, s shown here, the nti-inflmmtory ctivity of B. thetiotomicron, prevlent humn gut neroe, cnnot e explined y this mechnism. Export of nucler RelA independent of Crm-1 We postulted tht the S. enteritidis induced RelA-medited trnscription, which ws initited in the presence nd sence of B. thetiotomicron, ws reduced fter 2 h of coculture with B. thetiotomicron ecuse of precocious nucler clernce of ctivted NF-κB RelA. Direct cetyltion nd decetyltion of RelA influences oth its durtion of ction nd nucler export 18. Furthermore, trnsforming growth fctor-β (TGF-β), Figure 4 B. thetiotomicron induces cellulr shuttling of PPAR-γ in Cco-2 cells. () Immunolot of PPAR-γ. Cco-2 cells were incuted for 2 h with medium (1); S. enteritidis (2); S. enteritidis nd B. thetiotomicron (3); or B. thetiotomicron. After incutions, cells were seprted into nucler nd cytoplsmic extrcts, or detergent-solule (D. solule) or detergent-insolule (D. insolule) frctions, nd were lotted for PPAR-γ. () Immunofluorescence microscopy of PPAR-γ in Cco-2 cells. Tretments were s descried in. (c) Time course of PPAR-γ nucler trnsloction. Immunofluorescence microscopy of PPAR-γ in Cco-2 cells treted with S. enteritidis (top row) or S. enteritidis nd B. thetiotomicron (ottom row) for the times indicted elow the photos. Scle rs, 25 µm. Dt re representtive of t lest three independent experiments. n nti-inflmmtory cytokine tht lso inhiits NF-κB-medited trnscription, specificlly modultes histone cetyltion 19. To investigte the possile function of cetyltion in our system, we used the specific histone decetylse inhiitor trichosttin A (TSA) to enhnce nucler cetyltion in Cco-2 cells (dt not shown). Although nucler cetyltion nd the S. enteritidis induced IL-8 expression were sustntilly incresed, the ttenuting effects medited y B. thetiotomicron were unffected y TSA tretment (dt not shown). To chrcterize the nucler export mechnism further, we then investigted whether the B. thetiotomicron induced export of RelA occurred through Crm-1 (exportin 1), the only pthwy defined so fr for the nucler export of NF-κB IκBα complexes 20. We chieved inhiition of this receptor pthwy with the nucler export inhiitor leptomycin B (LMB) 21. LMB did not influence the ttenuting effect of B. thetiotomicron on IL-8 expression, s determined y rel-time PCR (dt not shown). The nucler export of RelA induced y B. thetiotomicron ws not locked y LMB tretment (Fig. 3) nd therefore ws independent of the Crm-1 pthwy, indicting different moleculr mechnism of export. B. thetiotomicron induces nucler export of PPAR-γ PPARs nd their lignds hve een identified s modultors of inflmmtion The modes of ction of the PPARs in this context hve not een fully defined, ut oth receptor-dependent nd receptor-independent mechnisms hve een documented Becuse PPAR-γ is potentilly importnt protein in the regultion of inflmmtory cscdes, we investigted the effects of B. thetiotomicron on its expression nd locliztion in Cco-2 cells. Immunolot nd immunocytochemicl nlyses showed tht chllenge with S. enteritidis induced nucler ccumultion of PPAR-γ in Cco-2 cells (Fig. 4,). In contrst, PPAR-γ redistriuted to the cytosol fter coculture with S. enteritidis nd B. thetiotomicron (Fig. 4,). PPAR-γ induced y slmonell ws mostly detergent insolule, indicting prole cytoskeletl ssocition. However, PPAR-γ ws present in pproximtely equl mounts in the detergent-solule nd detergent-insolule extrcts of Cco-2 cells cocultured with slmonell nd B. thetiotomicron, nd ws mostly in the detergent frction in cells incuted with B. thetiotomicron lone (Fig. 4). This indictes differentil regultion of the iochemicl or iologicl properties of PPAR-γ y these cteri. Further investigtion of the time course of nucler trnsloction of PPAR-γ NATURE IMMUNOLOGY VOLUME 5 NUMBER 1 JANUARY

5 d c Figure 5 PPAR-γ, ut not the dominnt negtive PPAR-γ, intercts with ctive RelA nd fcilittes its nucler export. () Immunolot of RelA derived from immunoprecipittion with PPAR-γ grose ntiodies in Cco-2 cells treted for 2 h with medium (1); S. enteritidis (2); S. enteritidis nd B. thetiotomicron (3); or B. thetiotomicron. Right, control with secondry (2 ) ntiody lone. B, lnk (no cellulr protein). () Immunodetection of in vitro trnslted RelA fter incution with reticulocyte lyste only (1); reticulocyte lyste nd in vitro trnslted wild-type (WT) PPAR-γ (2); reticulocyte lyste nd in vitro trnslted dominnt negtive PPAR-γ (3) nd immunoprecipittion with ntiody to PPAR-γ. (c) NF-κB luciferse reporter ssy. Cco-2 cells were cotrnsfected with n NF-κB luciferse reporter construct nd construct for the expression of the dominnt negtive (DN) PPAR-γ or wild-type (WT) PPAR-γ. Tretments were s descried in for 2 h; cteri were then removed nd cells were cultured for further 6 h; luciferse expression ws determined nd dt were normlized to the slmonell vlues to give percentge stimultion indices. (d) Cco-2 cells were trnsfected with YFP-RelA chimeric construct nd CFP wild-type (WT) PPAR-γ construct (top row) or CFP dominnt negtive (DN) PPAR-γ construct (ottom row). Then, 2 d fter trnsfection, cells were incuted with S. enteritidis nd B. thetiotomicron nd the dul locliztion of RelA nd PPAR-γ ws exmined y fluorescence microscopy with specific filter sets. fter exposure to cteri showed notle ccumultion within 30 min with ll tretments (Fig. 4c). This ws sustined in cells cultured with S. enteritidis during the 2-hour culture period (Fig. 4c, top row), wheres in cells cocultured with B. thetiotomicron, clernce from the nucleus ws evident y 60 min nd completed y 2 h (Fig. 4c, ottom row). Notly, the nucleocytoplsmic shuttling of PPAR-γ induced in response to coculture of Cco-2 cells with S. enteritidis nd B. thetiotomicron mimicked tht of RelA. Furthermore, s with RelA (Fig. 3), the nucler export of PPAR-γ ws not locked y LMB tretment (dt not shown). These oservtions therefore cnnot e ttriuted to export through the nucler receptor Crm-1. The dt re comptile with previous findings for other nucler receptors 29 ut clerly denote different mechnism for the nucler export of RelA. We postulted tht the nucler export nd cytosolic locliztion of PPAR-γ nd RelA induced y B. thetiotomicron ws due to the formtion of stle protein complex, possily involving dditionl fctors. B. thetiotomicron induces ssocition of PPAR-γ nd RelA PPAR-γ nd NF-κB cn form complex in solution Thus, we investigted whether direct ssocitions etween PPAR-γ nd RelA represented possile mechnism to explin their common cytoplsmic locliztion in intestinl cells exposed to S. enteritidis nd B. thetiotomicron. Immunoprecipittion of PPAR-γ from cells treted with S. enteritidis in the presence of B. thetiotomicron resulted in the copurifiction of RelA (Fig. 5). Although the functionl domins for oth the interction etween RelA nd PPAR-γ nd potentilly for their nucler export were unknown, we ssessed dominnt negtive PPAR-γ with two mino cid replcements in its C-terminl lignd-inding domin 32 for RelA-inding ctivity. We resoned tht ecuse this dominnt negtive PPAR-γ shows impired relese or recruitment of corepressor nd coctivtor proteins 32, its interctions with other proteins might lso e impired. In support of this, when we tested the dominnt negtive PPAR-γ receptor 32 in immunoprecipittion experiments with in vitro trnslted proteins, we found no cossocition with RelA (Fig. 5). In contrst, wild-type PPAR-γ cossocited with RelA, consistent with the immunoprecipittion experiments in Cco-2 cells. Both the DNA-inding nd the C-terminl domins of the PPAR-γ protein re RelA-intercting regions 33. Hence, in ddition to identifying iologiclly importnt moleculr interctions etween PPAR-γ nd RelA, our dt indicte the functionl importnce of the C-terminl domin within the PPAR-γ protein s potentil RelA-intercting domin. To investigte whether the interction of PPAR-γ nd RelA fter exposure to B. thetiotomicron led to the inhiition of RelA-induced trnscription, we cotrnsfected cells with dominnt negtive PPAR-γ nd NF-κB luciferse reporter construct. The slmonell-medited induction of luciferse ws not notly different in cells trnsfected with dominnt negtive PPAR-γ or wild-type PPAR-γ. In cells trnsfected with wild-type PPAR-γ nd dominnt negtive PPAR-γ, S. enteritidis induced sustntil reporter gene ctivity (Fig. 5c). In cells trnsfected with wild-type PPAR-γ, the previously identified ttenution effect of B. thetiotomicron ws mintined, ut in cells trnsfected with dominnt negtive PPAR-γ this ws no longer evident (Fig. 5c). As the dominnt negtive PPAR-γ did not ind RelA in our in vitro trnsltion nd immunoprecipittion experiments, we postulted tht lthough the dominnt negtive PPAR-γ ws unlikely to reduce or limit the interction etween RelA nd the ntive PPAR-γ in the Cco-2 cells, the overexpressed dominnt negtive PPAR-γ my hve interfered with the export of this complex, possily y inding to or interfering with s-yet-unidentified proteins essentil for the export of RelA nd PPAR-γ. PPAR-γ-dependent nucler export of RelA We investigted the importnce of PPAR-γ in reltion to nucler export of RelA fter exposure to B. thetiotomicron with chimeric fluorescent protein constructs of wild-type nd dominnt negtive PPAR-γ linked to the C-terminl domin of cyn fluorescent protein (CFP) or chimeric RelA linked to yellow fluorescent protein (YFP) 34. Much of the expressed PPAR-γ protein ws loclized within the nuclei of trnsfected Cco-2 (nd Hel) cells, wheres the RelA ws minly cytoplsmic. In Cco-2 cells trnsfected with wild-type CFP-PPAR-γ nd YFP-RelA nd treted with S. enteritidis, the min locliztion of these two proteins ws 108 VOLUME 5 NUMBER 1 JANUARY 2004 NATURE IMMUNOLOGY

6 The distriution of CFP PPAR-γ nd YFP-RelA ws very similr in the nuclei of trnsfected Cco-2 nd Hel cells fter culture in the presence of S. enteritidis nd B. thetiotomicron (Fig. 5d nd dt not shown). This common distriution my indicte tht interction etween PPAR-γ nd RelA occurs within the nucler comprtment. Figure 6 RNA interference of PPAR-γ. () Cco-2 cells were trnsfected with YFP-RelA nd CFP PPAR-γ constructs nd PPAR-γ-RNAi construct (top row) or negtive control RNAi construct (ottom row). Trnsfected cells were suject to fluorescence microscopy with specific YFP (left) nd CFP (right) filter sets. () Immunolots of Cco-2 cells trnsfected with YFP-RelA nd CFP PPAR-γ constructs nd PPAR-γ-RNAi or negtive control RNAi construct; extrcts were immunostined with ntiodies specific for PPAR-γ nd RelA. nucler, ut there ws evidence of punctte cytosolic leling nd colocliztion of PPAR-γ nd RelA fter coincution with B. thetiotomicron (Fig. 5d, top row). In cells trnsfected with dominnt negtive CFP PPAR-γ, this leling ws sent from the cytosolic comprtment (Fig. 5d, ottom row), indicting tht nucler export of the dominnt negtive PPAR-γ nd RelA in response to B. thetiotomicron ws impired. Hence, PPAR-γ seems to e essentil for oth nucler export nd the cytosolic distriution of RelA induced in intestinl cells y B. thetiotomicron. Our dt indicte tht conditionlly regulted proteins like PPAR-γ cn govern the distriution nd function of RelA, nlogous to previously reported mechnism involving IκBα 35,36. Decrese in PPAR-γ olishes nti-inflmmtory mechnism To support our evidence of the PPAR-γ-medited nucler export of RelA, we used RNA interference (RNAi) 37,38 to decrese the mount of PPAR-γ. To llow for rpid nd roust nlysis of the RNAi effect, we monitored the inhiition of n engineered CFP PPAR-γ chimeric construct in trnsiently trnsfected Cco-2 cells cotrnsfected with YFP-RelA chimeric construct s control. This design provided the most ccurte method for the determintion of RNAimedited effects on PPAR-γ mounts exclusively in the trnsfected popultion of cells. As shown in dully trnsfected cells, we noted considerly decrese in CFP PPAR-γ protein with no effect on the mounts of YFP-RelA (Fig. 6). This ws lso demonstrted with immunodetection (Fig. 6). When we repeted these experiments, including coculture with S. enteritidis nd B. thetiotomicron, cells trnsfected with the PPARγ-specific RNAi construct showed nucler locliztion of RelA t 2 h, which ws in contrst to ll of the djcent, non-rnai-trnsfected cells, which showed cytosolic distriution of RelA (Fig. 7, top row). The negtive control RNAi construct hd no effect on RelA, with trnsfected cells showing minly cytosolic distriution (Fig. 7, ottom row). To estlish tht decrese in PPAR-γ ffected specific NF-κBmedited gene expression, we mesured IL-8 mrna y rel-time PCR fter incution with S. enteritidis (Fig. 7)or purified S. enteritidis flgellin (Fig. 7c) in the presence nd sence of B. thetiotomicron. As in the previous studies with dominnt negtive PPAR-γ nd NF-κBluciferse, trnsfection with PPAR-γ RNAi constructs hd no effect on lignd-induced IL-8 concentrtions (Fig. 7,c). The decrese in PPAR- Figure 7 PPAR-γ RNAi olishes the nucler export of RelA nd the trnscriptionl inhiition of IL-8 in Cco-2 cells treted with S. enteritidis nd B. thetiotomicron. () Cco-2 cells were dully trnsfected with pecfp-c1 (CFP only; control vector) nd PPAR-γ RNAi construct (top row) or negtive control RNAi construct (ottom row) nd were incuted for 2 h with S. enteritidis nd B. thetiotomicron. Trnsfected cells were detected y CFP fluorescence (left) nd RelA distriution ws identified y immunofluorescence (right). Arrows indicte trnsfected cells. Dt re representtive of t lest three independent experiments. () Cco-2 cells were trnsfected with pecfp-c1 nd PPAR-γ RNAi construct or negtive control RNAi construct. Trnsfected cells were incuted for 2 h with medium lone (1); S. enteritidis (2); S. enteritidis nd B. thetiotomicron (3); or B. thetiotomicron lone (4). Expression of IL-8 ws determined y rel-time PCR. Dt re normlized to the slmonell response nd re representtive of one of t lest three independent experiments. (c) Cco-2 cells c d were trnsfected s in () nd incuted for 2 h with medium lone (1); S. enteritidis recominnt flgellin (2); S. enteritidis flgellin with B. thetiotomicron (3); or B. thetiotomicron lone (4). Expression of IL-8 ws determined y rel-time PCR. Dt re normlized to the S. enteritidis flgellin response nd re representtive of one of t lest three independent experiments. (d) Cells were treted with S. enteritidis nd B. thetiotomicron (1) or S. enteritidis flgellin nd B. thetiotomicron (2) nd IL-8 expression ws determined. Dt re normlized vlues for the B. thetiotomicron inhiition of IL-8 mrna in the presence of either RNAi PPAR-γ or the RNAi negtive control. Dt re representtive of t lest three experiments for S. enteritidis nd two experiments for flgellin ± s.d. **, P < 0.01, ***, P < In ech experiment, trnsfection efficiencies were determined y monitoring the percentge of CFP-expressing cells reltive to cells leled with 4,6-dimidino-2-phenylindole. NATURE IMMUNOLOGY VOLUME 5 NUMBER 1 JANUARY

7 γ y RNAi resulted in significnt reduction in the inhiitory effect of B. thetiotomicron on IL-8 expression (slmonell, 9.1 ± 2.1%; flgellin, 30.5 ± 8.2%; Fig. 7d). We ttriuted this reduction exclusively to the trnsfected cell popultion tht showed reduced PPAR-γ levels. For these experiments, routine trnsfection efficiencies were in the rnge of 18 26% nd hence correction of the dt to ssume totl cell trnsfection would result in loss of the inhiitory effect of B. thetiotomicron equivlent to tht seen in dominnt negtive PPARγ NF-κB luciferse experiments. Overll, these studies show tht B. thetiotomicron ttenutes inflmmtory responses in intestinl epithelil cells y enhncing the nucler export of the NF-κB RelA suunit through PPAR-γ-dependent mechnism. DISCUSSION The helthy gut mintins physiologicl level of inflmmtion in response to the estlished cteril flor. However, the presence of threshold numer of pthogenic cteri is sufficient to ctivte trnscriptionl fctors, including NF-κB, which trigger proinflmmtory gene expression, inducing oth innte nd dptive mechnisms of defense 39. Although such mucosl responses re essentil in comting gut infections, the intervention of regultory systems tht quench inflmmtion nd restore immune homeostsis is n solute requirement to prevent progression from the cute to the chronic stte. In this context, the nti-inflmmtory cytokines IL-10 (ref. 40) nd TGF-β 19,41 derived from epithelil nd regultory T cells re recognized to e importnt. Gut cteri, typiclly pthogenic species, prevent or limit the inflmmtory response y trgeting NF-κB or, more specificlly, y inhiiting its ctivtion 9,15. We found tht B. thetiotomicron lso cted on NF-κB ut tht its mode of ction ws distinct. B. thetiotomicron trgeted trnscriptionlly ctive RelA nd induced precocious nucler clernce, therey limiting the durtion of NF-κB ction. The physiologicl effect of this nti-inflmmtory cterium ws mesurle in vivo nd, s prevlent constituent of the norml humn gut microflor, its contriution to immune homeostsis nd innte nd dptive mechnisms of defense is likely to e importnt. In ddition to the effect on RelA, oth pthogenic slmonell nd nonpthogenic cteroides induced PPAR-γ protein expression, ut its cellulr locliztion ws differentilly modulted. PPAR-γ protein ws nucler in the presence of S. enteritidis ut cytoplsmic in the presence of B. thetiotomicron. We noted tht the kinetics of the nucleocytoplsmic export of PPAR-γ induced y B. thetiotomicron were reminiscent of those of RelA, indicting possile ssocition of these proteins during nucler export. Consistent with this supposition, oth RNAi reduction of constitutive PPAR-γ nd trnsfection with dominnt negtive PPAR-γ constructs prevented nucler export of RelA, therey confirming the importnce of the B. thetiotomicron medited effects on PPAR-γ in determining the nucleocytoplsmic distriution of RelA. The RelA tht remined in the nucleus during these studies retined trnscriptionl ctivity, demonstrted y oth NF-κB reporter ssys nd the induction of IL-8 mrna, indicting tht PPAR-γdependent export of RelA ws essentil in ttenuting the slmonell-medited proinflmmtory responses. Immunoprecipittion of in vitro trnslted proteins nd epithelil cell extrcts demonstrted tht RelA nd PPAR-γ were cple of physicl ssocition nd tht B. thetiotomicron could trigger this interction. In vitro imges lso indicted tht such n ssocition might form in the nucleus. Although this is the first description to our knowledge of nucler PPAR-γ NF-κB complex in epithelil cells, the formtion of similr complex tht limits the trnscriptionl ctivity of the PPAR-γ protein ws descried in ST2 mesenchyml cells 33. In vitro studies demonstrted tht dominnt negtive PPAR-γ did not ssocite with RelA, nd lthough dditionl experiments re required to further clrify the mechnism of ssocition of these proteins in our system, oth the DNA-inding nd the C- terminl lignd-inding domin regions of PPAR-γ protein hve een linked to NF-κB inding 33. It is lso possile tht RelA nd PPAR-γ re components of much lrger complex, s other proteins normlly ssocited with PPAR-γ, such s PPAR-γ coctivtor-1, cn physiclly interct with RelA 33. Our dt provide evidence for the IκBα- nd Crm-1-independent export of RelA 14, s LMB, specific inhiitor of Crm-1, did not ffect B. thetiotomicron induced export of the PPAR-γ RelA complex from the nucleus. This indicted n lterntive nucler export pthwy, perhps similr to tht used y the thyroid receptor 29. Consistent with the nucler export function of PPAR-γ, the dominnt negtive PPAR-γ locked RelA export from the nuclei of Cco-2 cells exposed to B. thetiotomicron, indicting tht the dominnt negtive PPAR-γ interfered with the nucler export of the endogenous receptor. The precise mechnism for how PPAR-γ fcilitted the nucler export of RelA remins to e elucidted, nd will require further explortion. Collectively, however, these dt provide insight into iologiclly importnt protein interctions nd previously unknown nucler export mechnisms tht divert ctivted trnscription fctors to other cellulr locliztions, therey limiting gene trnscription. The nucler export of NFκB PPAR-γ complexes, in ddition to limiting inflmmtory cscdes, my lso underpin other physiologicl functions for these proteins within the cell. Our findings identify previously unknown nti-inflmmtory mechnism involving PPAR-γ nd NF-κB nd provide new insights into moleculr mechnisms in epithelil cells tht contriute to immune homeostsis. They lso extend our understnding of the evolutionry dpttion of commensl cteri within gut ecosystems nd emphsize the potentil for identifying cterilly derived mechnisms nd/or ioctive molecules with immune-modulting functions. The identifiction of such fctors hs recently ecome more possile with the puliction of the genomes of commensl cteri, including B. thetiotomicron 42. The exploittion of such findings my led to the development of new therpeutics for humn inflmmtory owel disese nd other inflmmtory conditions. METHODS S. enteritidis nd B. thetiotomicron coculture models. Cco-2 nd Hel cells were routinely cultured in 35-mm culture dishes. Bsed on optimized dose-response nd time-course studies, typicl experiments used cells incuted for 2 or 4 h with the following four tretments: medium lone; S. enteritidis; S. enteritidis plus B. thetiotomicron; or B. thetiotomicron. Bcteri were then removed y extensive wshing. Alterntive incutions included the following cteri nd/or lignds, where pproprite: E. coli 0157:H7 ( ), B. vulgtus ( ), PMA (300 ng/ml), IL-1α nd IL-1β (20 ng/ml), TNF (20 ng/ml) nd LMB (20 nm). Trnsepithelil migrtion of PMN cells through Cco-2 cell monolyer grown on trnswells ws determined y MPO ssy 43. Cco-2 cells were incuted with cteri for 2 h nd thoroughly wshed, nd fresh medium ws pplied. Therefter, the Cco-2 cells were incuted for further 2 h efore cells nd medi derived from the picl comprtment were soluilized in PBS contining Triton X-100 (1%), nd MPO concentrtions were determined. In vivo rt study. Newly wened (21-dy-old) miniml flor rts (fed on norml lortory diets) were split into two groups nd dditionlly fed neroiclly prepred jelly (0.5 g/d) or neroiclly prepred jelly contining B. thetiotomicron for 19 d. Hlf of the rts in ech group were then orlly 110 VOLUME 5 NUMBER 1 JANUARY 2004 NATURE IMMUNOLOGY

8 chllenged with S. enteritidis. MPO ws mesured in isolted ilel mucos t 6 d fter S. enteritidis infection. Experiments were undertken t lest three times with similr results. Histology. Rt ilel smples were fixed for h t 20 C in prformldehyde (4%) in 0.1 M phosphte uffer, ph 7.3. Tissues were susequently wshed in phosphte uffer, dehydrted in n ethnol series nd emedded in Historesin (Leic). Sections 1 µm in thickness were stined with toluidine lue, nd representtive imges were otined with Zeiss Axioskop microscope equipped with n AxioCm digitl cmer. Cytokine nlyses. Cytokines in Cco-2 cells were nlyzed with microrry (Atls cytokine-receptor rry; Clontech), RNA hyridiztions nd rel-time nd semiquntittive PCR. Totl RNA or mrna ws isolted, cdna ws produced nd experiments were done with stndrd conditions. IL-8 protein concentrtions were determined y enzyme-linked immunosorent ssy. PCR nlysis. IL-8 mrna ws estimted y semiquntittive nd/or rel-time PCR. For semiquntittive studies, reverse trnscription of totl RNA ws done followed y tril PCR to determine the cycle rnge for the liner mplifiction of IL-8 nd glycerldehyde phosphodehydrogense (GAPDH) sequences in seprte rections. Experiments were then done for the pproprite numer of cycles to ensure tht ll smples were within the liner rnge for ech product. Products were resolved on 1% TAE grose gel (0.5 µg/ml of ethidium romide) nd nd intensities were determined y Quntity One softwre (Bio-Rd). IL-8 vlues were normlized reltive to GAPDH vlues. Rel-time PCR ws done with predeveloped ssy regents for humn IL-8 nd GAPDH s descried y the mnufcturer (Applied Biosystems). Smples were run on n ABI Prism 7700 nd nlyzed with stndrd methodologies. Anlyses of NF-κB nd AP-1. Nucler extrcts of Cco-2 cells were nlyzed y electrophoretic moility-shift ssy (EMSA). The proes used were doulestrnded 32 P-leled oligonucleotides contining the consensus inding sequences for NF-κB (5 -AGTTGAGGGGACTTTCCCAGGC-3 ) or AP-1 (5 -CGCTTGATGAGTCAGCCGGAA-3 ). Products were seprted y electrophoresis nd visulized y utordiogrphy. EMSA supershifts were done with specific NF-κB nd AP-1 suunit ntiodies (RelA, sc-109x; JunD, sc-74x; Snt Cruz Biotechnology). The effects of B. thetiotomicron on NF-κB signling were determined y EMSA, immunolotting (RelA sc-109; IκBα, sc-371 nd sc-371ac; piκbα, sc-8404; Snt Cruz Biotechnology), NF-κB luciferse reporter ssys nd trget gene expression. Luciferse expression ws determined with the Stedy-Glo Luciferse ssy system (Promeg). Immunoprecipittion experiments. After experimentl tretments, Cco-2 cells were wshed nd soluilized in PBS contining Igepl CA-630 (1%), sodium deoxycholte (0.5%), SDS (0.1%) nd protese inhiitor cocktil (Sigm) nd were incuted with grose conjugtes s descried y the Snt Cruz Biotechnology immunoprecipittion nd immunolot protocols. The following ntiody-grose conjugtes were used: RelA, sc-372ac; PPAR-γ sc-7273ac or sc-1984; nd IκBα, sc-371ac. Immunofluorescence nlysis. After experimentl tretments, Cco-2 nd Hel cells were fixed in prformldehyde (4%) nd permeilized in 0.2% Triton X- 100 in PBS. Cells were incuted for 1 h t room temperture with primry ntiodies (1 µg/ml) in PBS (RelA, sc-109; IκBα, sc-371; nd PPAR-γ, sc-7196 nd sc-1984) contining 1% serum from the species in which the secondry ntiody ws rised. Secondry ntiodies (1 µg/ml) were either Alex Fluor donkey ntiody to got or Alex Fluor got ntiody to rit IgG (Moleculr Proes), where pproprite. Leled cells were mounted with Vectorshield (Vector) nd exmined on Zeiss Axioskop 50 widefield fluorescence microscope or on Bio- Rd Rdince 2100 lser-scnning microscope. Representtive digitl imges were imported into Adoe Photoshop 6.0 for construction of dul-color overlys. In LMB experiments, digitl imges were otined with stndrd conditions of illumintion nd exposure on Zeiss Axiocm cmer ttched to Zeiss Axioskop fluorescence microscope. Digitl cptures were converted to 8-it gryscle imges nd men pixel vlues were recorded with Ntionl Institutes of Helth Imge J softwre ( for representtive res of nuclei nd cytoplsm from 100 cells. Immunofluorescence microscopy nd immunolotting. Cco-2 cells were cotrnsfected with constructs encoding YFP-RelA nd CFP-PPAR-γ, nd either the PPAR-γ RNAi or the negtive control psilencer 3.0-H1 plsmid, t rtio of 1:1:2, with lipofectmine 2000 (Invitrogen). Cells were cultured for 48 h efore eing exmined on Zeiss AxioVert fluorescence microscope with specific filters for the identifiction of YFP nd CFP. All cell imges were otined with equl exposure times (4,000 ms). After eing photogrphed, cells were wshed with PBS nd collected in SDS-PAGE gel loding uffer, resolved on 4 12% grdient polycrylmide gel nd lotted onto polyvinyldifluoride memrne. PPAR-γ nd RelA were identified with specific primry ntiodies (PPAR-γ, sc-7273; RelA, sc-109; Snt Cruz Biotechnology). Construction nd expression of fluorescence-tgged PPAR-γ. The coding sequence of PPAR-γ nd the dominnt negtive mutnt of PPAR-γ 32 were modified y PCR mplifiction with Pfu DNA polymerse to dd n XhoI recognition sequence t the 5 end nd ScII sequence t the 3 end of the products. Products were digested with these restriction enzymes nd cloned into pecfp-c1 nd peyfp-c1 (Promeg). Successful construction ws verified y DNA sequencing. Humn RelA cloned into peyfp-c1 ws s reported 34. Cco-2 or Hel cells were grown to 90% confluence nd trnsfected with stndrd lipofectmine-medited methods (Invitrogen). After 48 h, cteril incutions were undertken s descried ove; cells were fixed for 30 min t room temperture in prformldehyde (4%) in 0.1 M sodium phosphte uffer, ph 7.4, wshed in PBS nd exmined on Zeiss Axioskop 50 widefield fluorescence microscope equipped with custom filters for CFP nd YFP. Representtive digitl imges were imported into Adoe Photoshop 6.0 for construction of dul-color overlys. In some experiments, trnsfected cells were lso suject to immunofluorescence s descried ove. Preprtion of PPAR-γ RNAi constructs. Constructs to provide RNAi of humn PPAR-γ were engineered with the psilencer system (Amion). After initil testing of five constructs, one ws selected tht showed the most roust RNAi effect on PPAR-γ. This construct contined the humn PPAR-γ sequence from nucleotides (5 -GCCCTTCACTACTGTTGAC-3 ) in the plsmid psilencer 3.0-H1. A negtive control psilencer 3.0-H1 plsmid contining 19 nucleotides with no known identity to ny humn sequence ws provided with the system. ACKNOWLEDGMENTS We thnk E.T. Logn, K.E Grden, D.J. Frser-Pitt, D.L. Wilson nd J.C. Mrtin for technicl support. We lso thnk V.K. Chtterjee (Addenrooke s Hospitl, Cmridge, UK) nd J.A. Schmid (University of Vienn, Austri) for providing the PPAR-γ nd YFP-RelA clones for this work. Supported y SEERAD (Scottish Executive for Environmentl nd Rurl Affirs Deprtment). COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Received 16 July; ccepted 29 Octoer 2003 Pulished online t 1. Srtor, R.B. The influence of norml microil flor on the development of chronic inflmmtion. Res. Immunol. 148, (1997). 2. Remcken, B.J. et l. Non-pthogenic E. coli versus meslzine for the trement of ulcertive colitis: rndomised tril. Lncet 354, (1999) 3. Kuhn, R., Lohler, J., Rennick, D., Rjewsky, K. & Muller, W. Interleukin-10 deficient mice develop chronic enterocolitis. Cell 75, (1993). 4. Powrie, F., Corre-Oliveir, R., Muze, S. & Coffmn, R.L. Regultory interctions etween CD45RB high nd CD45RB low CD4 + T cells re importnt for the lnce etween protective nd pthogenic T-cell immunity. J. Exp. Med. 179, (1994). 5. Cmpieri, M. & Gionchetti, P. Proiotics in inflmmtory owel disese: new insight to pthogenesis or possile therpeutic lterntive. Gstroenterol. 116, (1999). 6. Mrteu, P.R., de Vrese, M., Cellier, C.J. & Schrezenmeir, J. Protection from gstrointestinl diseses with the use of proiotics. Am. J. Clin. Nutr. 73, 430S 436S (2001). 7. Crio, E. et l. Lipopolyscchride ctivtes distinct signling pthwys in intestinl epithelil cell lines expressing Toll-like receptors. J. Immunol. 164, (2000). 8. Cong, Y., Wever, C.T., Lzeny, A. & Elson, C.O. Bcteril-rective T regultory cells inhiit pthogenic immune responses to the enteric flor. J. Immunol. 169, (2002). NATURE IMMUNOLOGY VOLUME 5 NUMBER 1 JANUARY

9 9. Neish, A.S. et l. Prokryotic regultion of epithelil responses y inhiition of IκB-α uiquitintion. Science 289, (2000). 10. Kelly, D. & Conwy, S. Genomics t work: the glol gene response to enteric cteri. Gut 49, (2001). 11. McCormick, B.A., Colgn, S.P., Delp-Archer, C., Miller, S.I. & Mdr, J.L. Slmonell typhimurium ttchment to humn intestinl epithelil monolyers: trnscellulr signlling to suepithelil neutrophils. J. Cell Biol. 123, (1993). 12. Hng, L. et l. Mcrophge inflmmtory protein-2 is required for neutrophil pssge cross the epithelil rrier of the infected urinry trct. J. Immunol. 162, (1999). 13. Ghosh, S., My, M.J. & Kopp, E.B. NF-κB nd Rel proteins: evolutionrily conserved meditors of immune responses. Annu. Rev. Immunol. 16, (1998). 14. My, M.J. & Ghosh, S. Signl trnsduction through NF-κB. Immunol. Tody 19, (1998). 15. Schesser, K. et l. The YopJ locus is required for Yersini-medited inhiition of NFκB ctivtion nd cytokine expression: YopJ contins eukryotic SH2-like domin tht is essentil for its repressive ctivity. Mol. Microiol. 28, (1998). 16. Cheng, Q. et l. NF-κB suunit-specific regultion of the IκBα promoter. J. Biol. Chem. 269, (1994). 17. Chio, P.J., Miymoto, S. & Verm, I.M. Autoregultion of IκBα ctivity. Proc. Ntl. Acd. Sci. USA 91, (1994). 18. Hller, D. et l. TGFβ-1 inhiits non-pthogenic Grm negtive cteri-induced NF-κB recruitment to the IL-6 gene promoter in intestinl epithelil cells through modultion of histone cetyltion. J. Biol. Chem. 278, (2003). 19. Chen, L-f., Fischle, W., Verdin, E. & Greene, W.C. Durtion of NF-κB ction regulted y reversile cetyltion. Science 293, (2001). 20. Hung, T.T., Kudo, N., Yoshid, M. & Miymoto, S. A nucler export signl in the N- terminl regultory domin of IκBα controls cytoplsmic loclistion of inctive NFκB/IκBα complexes. Proc. Ntl. Acd. Sci. USA 97, (2000). 21. Kudo, H. et l. Leptomycin B inctivtes CRM1/exportin 1 y covlent modifiction t cysteine residue in the centrl conserved region. Proc. Ntl. Acd. Sci. USA 96, (1999). 22. Su, C.G. et l. A novel therpy for colitis utilizing PPAR-γ lignds to inhiit the epithelil inflmmtory response. J. Clin. Invest. 104, (1999). 23. Nkjim, A. et l. Endogenous PPARγ medites nti-inflmmtory ctivity in murine ischemi-reperfusion injury. Gstroenterol. 120, (2001). 24. Wng, N. et l. Constitutive ctivtion of peroxisome prolifertor-ctivted receptorγ suppresses pro-inflmmtory dhesion molecules in humn vsculr endothelil cells. J. Biol. Chem. 277, (2002). 25. Ktym, K. et l. A novel PPARγ gene therpy to control inflmmtion ssocited with inflmmtory owel disese in murine model. Gstroenterol. 124, (2003). 26. Chwl, K. et l. PPAR-γ dependent nd independent effects on mcrophge-gene expression in lipid metolism nd inflmmtion. Nt. Med. 7, (2001). 27. Rossi, A. et l. Anti-inflmmtory cyclopentenone prostglndins re direct inhiitors of IκB kinse. Nture 403, (2000). 28. Strus, D.S. et l. 15-Deoxy- 12,14 -prostglndin J 2 inhiits multiple steps in the NF-κB signling pthwy. PNAS, 97, (2000). 29. Bunn, C.F. et l. Nucleocytoplsmic shuttling of the thyroid hormone receptor α. Mol. Endocrinol. 15, (2001). 30. Ricote, M., Hung, J.T., Welch, J.S. & Glss, C.K. The peroxisome prolifertor-ctivted receptorγ (PPARγ) s regultor of monocyte/mcrophge function. J. Leukoc. Biol. 66, (1999). 31. Chung, S.W. et l. Oxidized low density lipoprotein inhiits interleukin-12 production in lipopolyscchride-ctivted mouse mcrophges vi direct interctions etween peroxisome prolifertor-ctivted receptor-γ nd nucler fctor-κb. J. Biol. Chem. 275, (2000). 32. Gurnell, M. et l. A dominnt-negtive peroxisome prolifertor-ctivted receptor γ (PPARγ) mutnt is constitutive repressor nd inhiits PPARγ-medited dipogenesis. J. Biol. Chem. 275, (2000). 33. Suzw et l. Cytokines suppress dipogenesis nd PPARγ function through the TAK1/TAB1/NIK cscde. Nture Cell Biol. 5, (2003). 34. Schmid, J.A. et l. Dynmics of NF-κB nd IκBα studied with green fluorescent protein (GFP) fusion proteins. Investigtion of GFP-p65 inding to DNA y fluorescence resonnce energy trnsfer. J. Biol. Chem. 275, (2000). 35. Ghosh, S. & Krin, M. Missing pieces in the NF-κB puzzle. Cell 109, S81 S96 (2002). 36. Tm, W.F. & Sen, R. IκB fmily memers function y different mechnisms. J. Biol. Chem. 276, (2001). 37. Elshir, S.M. et l. Duplexes of 21-nucleotide RNAs medite RNA interference in cultured mmmlin cells. Nture 411, (2001). 38. Sui, G. et l. A DNA vector-sed RNAi technology to suppress gene expression in mmmlin cells. Proc. Ntl. Acd. Sci. USA 99, (2002). 39. Gewirtz, A.T., Nvs, T.A., Lyons, S., Godowski, P.J. & Mdr, J.L. Bcteril flgellin ctivtes solterlly expressed TLR5 to induce epithelil proinflmmtory gene expression. J. Immunol. 167, (2001). 40. De Winter, H. et l. Regultion of mucosl immune responses y interleukin 10 produced y intestinl epithelil cells in mice. Gstroenterol. 122, (2002). 41. Di Leo, V., Yng, P.C., Berin, M.C. & Perdue, M.H. Fctors regulting the effect of IL- 4 on intestinl epithelil rrier function. Int. Arch. Allergy Immunol. 129, (2002). 42. Xu, J. et l. A genomic view of the humn-bcteroides thetiotomicron symosis. Science 299, (2003). 43. Prkos, C.A., Delp, C., Arnout, M.A. & Mdr, J.L. Neutrophil migrtion cross cultured intestinl epithelium. Dependence on CD11/CD18-medited event nd enhnced efficiency in physiologicl direction. J. Clin. Invest. 88, (1991). 112 VOLUME 5 NUMBER 1 JANUARY 2004 NATURE IMMUNOLOGY