Innate and Adaptive Immunity Interact to Quench Microbiome Flagellar Motility in the Gut

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1 Article Innte nd Adptive Immunity Interct to Quench Microbiome Flgellr Motility in the Gut Tyler C. Cullender, 1,2 Benoit Chssing, 3 Anders Jnzon, 1,2 Krithik Kumr, 1,2 Ctherine E. Muller, 4 Jeffrey J. Werner, 5,7 Lrgus T. Angenent, 5 M. Elizbeth Bell, 1,2 Anthony G. Hy, 1 Dniel A. Peterson, 6 Jens Wlter, 4 Mtm Vijy-Kumr, 3,8 Andrew T. Gewirtz, 3 nd Ruth E. Ley 1,2, * 1 Deprtment of Microbiology, Cornell University, Ithc, NY 14853, USA 2 Deprtment of Moleculr Biology nd Genetics, Cornell University, Ithc, NY 14853, USA 3 Center for Inflmmtion, Immunity nd Infection, Georgi Stte University, Atlnt, GA 30303, USA 4 Deprtment of Food Science nd Technology, University of Nebrsk, Lincoln, NE 68583, USA 5 Deprtment of Biologicl nd Environmentl Engineering, Cornell University, Ithc, NY 14853, USA 6 Deprtment of Pthology, Johns Hopkins University, Bltimore, MD 21287, USA 7 Present ddress: Deprtment of Chemistry, SUNY Cortlnd, Cortlnd, NY 13045, USA 8 Present ddress: Deprtment of Nutritionl Sciences, The Pennsylvni Stte University, University Prk, PA 16802, USA *Correspondence: rel222@cornell.edu SUMMARY Gut mucosl brrier brekdown nd inflmmtion hve been ssocited with high levels of flgellin, the principl bcteril flgellr protein. Although severl gut commensls cn produce flgell, flgellin levels re low in the helthy gut, suggesting the existence of control mechnisms. We find tht mice lcking the flgellin receptor Toll-like receptor 5 (TLR5) exhibit profound loss of flgellin-specific immunoglobulins (Igs) despite higher totl Ig levels in the gut. Ribotyping of IgA-coted cecl microbiot showed Proteobcteri evding ntibody coting in the TLR5 / gut. A diversity of microbiome members overexpressed flgellr genes in the TLR5 / host. Proteobcteri nd Firmicutes penetrted smll intestinl villi, nd flgellted bcteri breched the colonic mucosl brrier. In vitro, flgellin-specific Ig inhibited bcteril motility nd downregulted flgellr gene expression. Thus, innte-immunitydirected development of flgellin-specific dptive immune responses cn modulte the microbiome s production of flgell in three-wy interction tht helps to mintin mucosl brrier integrity nd homeostsis. INTRODUCTION The humn gut contins trillion bcteril cells, which in the helthy stte reside in the lumen nd on the outside of the mucosl brrier seprting host cells from gut contents. The breching of this brrier by microbil cells cn led to inflmmtion nd tissue dmge. The dult humn is estimted to secrete 3 6 g of immunoglobulin A (IgA) into the gut dily (Delcroix et l., 1982), nd this IgA cots lrge frction of the resident microbes (vn der Wij et l., 1996), thereby stving off dmging inflmmtory responses (Slim nd Söderholm, 2011; Turner, 2009). IgA s role in brrier defense is generlly ssumed to be immune exclusion, in which the IgA binds microbil surfce ntigens nd promotes the gglutintion of microbil cells nd their entrpment in mucus nd physicl clernce (Hooper nd Mcpherson, 2010; Mntis et l., 2011). In this view, bcteri re lrgely pssive objects tht re trpped; however, their bility to lter surfce ntigen presenttion rises the possibility tht they my ctively prticipte in ntibody binding nd brrier defense. A few bcteri hve been shown to modulte the degree of IgA binding by hlting production of the inducing ntigen (Lönnermrk et l., 2012; Mntis et l., 2011). Although most studies hve been conducted with pthogens, behviorl response to IgA coting hs lso been observed in commensl gut bcterium. Bcteroides thetiotomicron ws monossocited to germfree RAG1 / mice producing single ntibody rised ginst one of the bcterium s cpsulr polyscchrides; its response to this ntibody milieu ws to downregulte the epitope s expression (Peterson et l., 2007). If wide diversity of microbiot responds to IgA binding by ltering the gene expression of surfce epitopes, this collective behvior could hve significnt role in how IgA intercts with bcteri in host brrier defense. Mucosl brrier brekdown nd inflmmtion in the gut hve been ssocited with high levels of flgellin, the principl protein comprising bcteril flgell (McCole nd Brrett, 2003; Snders, 2005). A wide diversity of gut commensls, including members of the phyl Firmicutes nd Proteobcteri, though not Bcteroidetes, hve the cpcity to produce flgell (Lozupone et l., 2012). As these re the dominnt phyl in the humn gut, motility-relted genes re redily recovered in helthy gut metgenomes (Kurokw et l., 2007; Turnbugh et l., 2006). But despite the gut microbiome s genomiclly encoded cpcity to produce flgell, levels of flgellin protein re low in the helthy gut (Verberkmoes et l., 2009), suggesting tht some control occurs to render commensl gut bcteri generlly nonmotile. Here, we investigte the reltionships between innte nd dptive immunity nd the production of flgell by complex microbiot s well s the importnce of this three-wy interction in host brrier defense. We used mice deficient in Toll-like receptor Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc. 571

2 Totl IgA (ug/g) Flgellin equivlent (ng/g) A C (TLR5) to determine the impct of nti-flgellin ntibodies upon the composition, gene expression, nd locliztion of the microbiot. Although trditionlly thought component of the innte system, TLR5 cts s both specific sensor in the innte immune system nd s its own djuvnt (Letrn et l., 2011). Loss of innte immune recognition of flgellin is specificlly ssocited with reduced levels of nti-flgelllin ntibodies (Gewirtz et l., 2006; Snders et l., 2006). Thus, the TLR5 / mouse model is useful for sking how deficiency in specific suite of ntibodies (i.e., ginst flgellin) impcts the microbiot nd brrier defense. Our results indicte tht nti-flgellin ntibodies induce the downregultion of flgellr motility genes by wide vriety of gut bcteri. In the bsence of these specific ntibodies, the host fils to contin the microbiot, which brech the mucosl brrier. RESULTS b WT TLR5-/- MyD88-/- DSS b WT TLR5-/- MyD88-/- DSS c c Anti-flgellin IgA (ng/g) B Con-R RAG1-/- WT +SFB RAG1-/- +SFB Reduced Levels of Anti-Flgellin Antibodies in TLR5 / Gut We mesured ntibody levels in cecum nd stool by ELISA. As expected, TLR5 / mice exhibited lower levels of nti-flgellin IgA in their cec nd fecl pellets compred to wild-type (WT) mice, despite higher level of totl IgA constnt over time (Figures 1A nd 1B, see Figure S1 for IgG ptterns). To ssess if the impired production of nti-flgellin ntibodies ws relted D b 0 WT TLR5-/- MyD88-/- DSS c monocolonized Figure 1. Gut Flgellin Lod Is Inversely Proportionl to Anti- Flgellin IgA Levels (A C) We nlyzed totl IgA levels (A), nti-flgellin IgA levels (nti-flgellin IgG equivlents) (B), nd flgellin (Slmonell Typhimurium flgellin equivlents) lod (C) for WT, TLR5 /, MyD88 /, nd DSS-treted WT B57BL/6 mice. (D) Flgellin mounts for conventionlly rised RAG1 / mice (Con-R), nd WT nd RAG1 / mice monocolonized with SFB (+SFB). The y xis lbel for (C) lso pplies to (D). Columns represent mens ± SEM. n = 8 mice per group; lowercse letters next to the brs indicte significnce; brs with different letters indicte significntly different mens t p < 0.05 using two-tiled t test corrected for multiple comprisons. Relted to Figure S1. to loss of TLR5 signling or to inflmmtion, which hs been reported for TLR5 / intestines (Vijy-Kumr et l., 2010), we lso mesured totl nd nti-flgellin IgA nd IgG in MyD88 / mice tht hve impired, but not completely eliminted, TLR5 signling (i.e., prtil vi TIR-domin-contining dpterinducing interferon-b [TRIF]; Choi et l., 2010) s well s WT mice with intct TLR5 signling but treted with dextrn sulfte sodium (DSS) to induce n inflmed stte. Levels of nti-flgellin IgA nd IgG levels were intermedite in MyD88 / mice (Figures 1B nd S1). In contrst, DSS-treted WT mice displyed WT levels of nti-flgellin ntibodies (Figure 1B). These results re consistent with loss of TLR5 signling leding to reduced ntiflgellin IgA production irrespective of inflmmtion. High Levels of Flgellin in the TLR5 / Gut We ssessed levels of bioctive flgellin in the cec nd stool of mice using TLR5 reporter cell ssy stndrdized to flgellin from the proteobcterium Slmonell Typhimurium. Flgellin bioctivity ws significntly higher in the cec of TLR5 / mice compred to WT mice (Figures 1C nd S1A). For both WT nd TLR5 / mice, cecl nd fecl levels were equivlent nd firly constnt over time, s were the corresponding nti-flgellin ntibody levels (Figures S1D nd S1E). We observed tht flgellin bioctivity levels were high in cec of TLR5 / mice housed t four different institutions (Figure S1F). The rtio of bioctive flgellin in TLR5 / to WT mice ws equivlent whether or not smples were boiled, indicting tht interference by immunoglobulins or other proteins were not the cuse of low flgellin levels in the WT mice (see Supplementl Experimentl Procedures). Elevted levels of flgellin bioctivity were lso present in MyD88 / mice, but not in DSS-treted WT mice (Figure 1C), suggesting n ssocition with impired TLR5 signling, but not inflmmtion lone. To ensure tht the elevted levels of flgellin bioctivity in the TLR5 / gut were relted to loss of ntibodies rther thn nother consequence of impired TLR5 signling, we lso mesured flgellin bioctivity in conventionlly rised RAG1 / mice, which re brodly deficient in ll spects of dptive immunity due to inbility to rerrnge Ig genes. As predicted from the lck of ntibodies in the gut, conventionlly rised RAG1 / mice lso exhibited high levels of flgellin in the gut (Figure 1D). RAG1 / nd MyD88 / mice hve been reported to hrbor n expnded popultion of segmented filmentous bcteri (SFB) when these re present in the microbiome (Lrsson et l., 2012; Suzuki et l., 2004), nd SFB re reported to genomiclly encode flgell (Prksh et l., 2011). However, the cecl contents derived from RAG1 / mice monocolonized with SFBs did not stimulte TLR5 signling (Figure 1D). A Wide Diversity of Commensl Microbes Express Flgell-Relted Genes in the TLR5 / Gut Using combined shotgun mettrnscriptomics nd metgenomics pproch, we identified diversity of bcteril species tht upregulted flgell-relted gene expression in the TLR5 / cecum. We designed custom probes to selectively remove rrna from totl cecl RNA (verge percentge of nonribosoml RNA ws 53.6% ± 6.3%) prior to cdna synthesis nd Illumin sequencing (Stewrt et l., 2010), nd we shotgun sequenced the cdna s well s bulk community DNA obtined from the 572 Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc.

3 Figure 2. Flgell Motility Genes Are Upregulted in the TLR5 / Microbiome Despite Similr Encoding Cpcity Mettrnscriptome nd metgenome nlyses for TLR5 / nd WT cecl microbiomes. Left pnel, mettrnscriptomes: cdnas ssigned to the 18 most significnt COG ctegories were normlized nd hierrchiclly clustered. Functionl ctegories re indicted to the left of the pnels; flgell-ssocited gene functions re highlighted in red. Dendrogrm (bottom) depicts the uncentered correltion similrity metric. Correltion coefficients re represented by color rnging from blue ( 53 depletion) to yellow (53 enrichment). Right pnel, metgenomes: metgenomic reds from the sme 18 COG ctegories were identiclly processed, with the exception tht correltion coefficients re n order of mgnitude less ( ). Relted to Figure S2 nd Tble S1. cecum (Tble S1). Unsupervised hierrchicl clustering of smples bsed on the enrichment or depletion of conserved orthologous groups (COGs) of genes segregted the mettrnscriptomes, but not the metgenomes, by host genotype (Figure 2). A similr clustering of genotypes bsed on the txonomic ssignments of the sequence reds lso filed to differentite the genotypes (Figure S2). These results indicte tht the species composition nd the functionl cpcities of the microbiomes were similr between TLR5 / nd WT, but the gene expression ptterns differed substntilly (Figure 2). Three of the six COGs upregulted in TLR5 / microbiomes were motility relted. Txonomic ssignments of the gene trnscripts indicted tht the mjority of the sequence reds mpping to flgell-relted genes in the TLR5 / microbiome belonged to commensl members of the Firmicutes phylum (Figure 3). These included commensl gener such s Roseburi, Eubcterium, nd Clostridium. In ddition, subset of the flgell-relted trnscripts mpped to Proteobcteri (e.g., Desulfovibrio spp.; Figure 3). In ddition to n enrichment in flgellr motility in TLR5 / mice, COG nlysis in the TLR5 / mettrnscriptome indicted depletion in crbohydrte metbolism (Figure 2). Flgell of Gut Commensl Bcteri Cn Stimulte TLR5 Flgellin produced by different bcteril species cn differ substntilly in structure nd bility to stimulte TLR5 (Andersen-Nissen et l., 2005; Smith et l., 2003), but there is little informtion vilble on the bility of flgellin produced by gut commensl microbes to stimulte TLR5. We purified flgellins from cultured gut bcteri nd tested their bility to stimulte TLR5. We found tht flgell hrvested from severl species of gut commensl Firmicutes (Clostridium scindens, C. rmosum, C. boltee, C. brtletti, Roseburi inulivorns, R. intestinlis) could stimulte TLR5, s could severl members of the Proteobcteri (Providenci sturtii, Citrobcter mlonticus, S. Typhimurium) (Figure S3A, Tble S2). In ccord with these observtions, multiple sequence lignment constructed with flgellin gene peptide sequences from diverse bcteri, including gut commensl Firmicutes, indicted tht they contin the mino cid residues previously shown to be necessry for TLR5 stimultion (Figure S3B) (Andersen-Nissen et l., 2005). Levels of E. coli Flgellin in Trissocited Gnotobiotic Mouse Model Are Decoupled from Its Popultion Size To ssess how bcteril community structure ws relted to levels of flgellin bioctivity in the gut, we used simplified gnotobiotic system in which we could control the composition of the microbiot nd quntify its popultion levels. Germfree RAG1 / nd WT mice were colonized with one motile (E. coli MG1655) nd two nonmotile (Bifidobcterium dolescentis FST-1, Bcteroides thetiotomicron VPI-5482) species. We found tht levels of E. coli flgellin in the cec of trissocited RAG1 / mice were significntly greter thn those in WT mice (Figure 4A). Colony-forming unit (cfu) counts nd denturing grdient gel electrophoresis nlysis indicted tht the three species reched similr popultion levels in both mouse genotypes (Figures 4B nd S4). Thus, the high flgellin lod in the TLR5 / mouse gut occurred despite highly similr microbil community composition nd structure. IgA Cots Different Suite of Gut Bcteri in TLR5 / Compred to WT Mice To evlute how n IgA repertoire depleted in nti-flgellin Igs intercts with complex bcteril popultions, we ssessed the proportion of bcteril biomss coted by IgA in TLR5 / nd WT by sorting cecl bcteri into IgA-coted (IgA + ) nd uncoted (IgA ) pools using fluorescence-ctivted cell sorting (FACS) (vn der Wij et l., 1996). We found tht the proportion of IgA + bcteri ws similr for TLR5 / nd WT mice (Figure 5A; 21.7% ± 5.9% versus 18.8% ± 4.2%, n = 4/group, n.s.). When incubted with bcteril biomss previously unexposed to IgA (derived from RAG1 / mice), IgA from both genotypes lso bound bcteri equivlently (55.7% ± 7.8% nd 50.1% ± 7.8% of sorted cells, respectively, n = 4/group, n.s.). We next sked if the differing TLR5 / nd WT IgA repertoires result in different suite of bcteri coted with IgA. After sorting IgA + nd IgA microbil cells derived from cec of TLR5 / Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc. 573

4 Figure 3. Mettrnscriptomic Reds Annotted s Flgellin Are Phylogeneticlly Diverse Txonomic ssignments of flgellin gene trnscripts re shown to the species level, with bundnce of reds corresponding to the key t the bottom. Hierrchicl clssifiction nd the Subsystems dtbse in MG-RAST were used to nnotte function. We isolted reds with n nnottion of flgellin nd ssigned txonomy using BLASTX with defult rguments. Note tht Bcteroides pectinophilus is Firmicute bsed on 16S rrna gene sequence nlysis but misclssified in the phylum Bcteroidetes in the MG- RAST dtbse. Relted to Figure S3, which shows stimultion of TLR5 by flgellins from different species, nd to Tble S2. nd WT mice, we extrcted DNA from the two pools nd pyrosequenced 16S rrna gene sequence mplicons using bcteri-specific primers. We repeted this for MyD88 / nd DSS-treted WT mice s controls to nlyze the effects of TLR5 signling nd inflmmtion, respectively. Compred to WT levels, TLR5 / mice were ssocited with n overcoting of Firmicutes by IgA nd n undercoting of Proteobcteri (Figure 5A), even though the totl sorted microbiot were similr in phylum composition for WT nd TLR5 / mice (Figure S5). Ptterns of bcteril coting were similr for TLR5 / nd MyD88 / mice, nd those for DSS-treted mice were similr to WT (with the exception of Bcteroidetes), which points gin to role for TLR5 signling rther thn inflmmtion in driving these ptterns. At the genus level, the frequencies of specific tx in the IgA + nd IgA popultions were generlly equivlent within WT or TLR5 / gut microbiomes (Figure 5B). This is consistent with lck of selection by IgA for specific tx. However, some tx devited from this neutrl pttern, with differences between genotypes (Figure 5C). Members of the Bcteroidetes phylum were consistently enriched in the IgA + popultions in both WT nd TLR5 / mice. In the TLR5 / mouse lone, overcoted gener included Desulfovibrio nd Lctobcillus. Members of the Helicobcter genus were lso undercoted in the TLR5 / host (Figure 5C). Anti-Flgellin Antibodies Cn Be Cross-Rective for the Flgell of Different Bcteri Since flgell from phylogeneticlly diverse bcteril species stimulte TLR5 due to conserved regions of the flgellin, we resoned tht ntibodies rised ginst flgell from one species could hve the potentil to be cross-rective with flgell from other unrelted species. Indeed, we observed cross rectivity of nti-flgellin IgG rised ginst single flgellin types (R. hominis Fl1 nd R. hominis Fl2) for flgell hrvested from different bcteril species (Proteobcteri: P. sturtii, S. Typhimurium, Citrobcter mlonticus; Firmcutes: R. inulinivorns, R. intestinlis, C. scindens, C. rmosum, C. brtletii; Figure S6). Anti-Flgellin Antibodies Immobilize Bcteri In Vitro We next conducted in vitro ssys to ssess how nti-flgellin ntibodies could impct bcteril motility. Anti-flgellin ntibodies totlly inhibited E. coli motility, wheres nti-lipopolyscchride (LPS) ntibodies only prtilly inhibited motility (Figure 6A). Furthermore, we pplied two nti-flgellin ntibody tretments ( FliC, rised ginst flgellin from Slmonell, nd Fl2, n ntiserum rised ginst R. hominis flgellin FL2) nd control ntibody (nti-mouse IgG) to liquid cultures of R. intestinlis nd Clostridium rmosum. Movies of R. intestinlis nd C. rmosum cultures with nd without ntibody show qulittively fewer ctively moving rods nd less movement overll with both ntiflgellin ntibody tretments ( edu/hndle/1813/31547). Anti-Flgellin Antibodies Induce Downregultion of Flgell Genes in E. coli To verify tht ntibodies could impct the production of flgell directly, we constructed flgellin gene (FliC) reporter E. coli K12 strin with GFP fused to the FliC gene. Monitoring of GFP levels over time indicted tht the ddition of nti-lps ntibodies 574 Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc.

5 Figure 4. The Popultion Sizes for Three Species of Bcteri Colonizing Gnotobiotic WT nd RAG1 / Mice Are Equivlent Despite Elevted Flgellin Production in RAG1 / Mice (A) Flgellin lod (Slmonell Typhimurium flgellin equivlents mesured by cell reporter ssy) in cec of gnotobiotic WT nd RAG1 / mice colonized with E. coli (Ec), Bcteroides thetiotomicron (Bt), nd Bifidobcterium dolescentis (B). (B) cfu counts of bcteri cultured from the cec. Brs re mens ± SEM; *p < 0.001; two-tiled t test; n.s., nonsignificnt. Relted to Figure S4. hd no effect on E. coli flgell gene expression, wheres ntiflgellin ntibodies reduced the expression of flgellin significntly within 2 hr of ddition (Figure 6B). Flgellted Bcteri Brech the Mucosl Brrier in the TLR5 / Mouse Smll nd Lrge Intestines Using fluorescence in situ hybridiztion (FISH) with fluorescently lbeled oligonucleotide probe (EUB338) specific to bcteri in generl, we observed bcteril cells penetrting the crypts nd the villi in the smll intestine of TLR5 / (Figure 7A). Using FISH probes specific for members of the g-proteobcteri nd Firmicutes (GAM42 nd mix of LGC354, LGC354b, nd LGC354c, respectively), we identified members of both groups mking up the smll clusters of bcteri penetrting the villi (Figure 7B). No bcteri were observed penetrting villi of WT smll intestines (Figure 7C). In the lrge intestine, lbeling of flgellin using fluorescent ntibody showed tht flgellted bcteril cells were present in the inner mucus lyer in the TLR5 /, but not WT intestine, resulting in direct contct between bcteri nd epithelil cells (Figures 7D, 7E, nd S7). DISCUSSION Our results suggest tht TLR5-medited innte immune recognition of flgellin is necessry for genertion of mucosl ntibodies directed ginst flgell nd tht such ntibodies ply key role in mnging the microbiot. In the bsence of this innte-dptive signling pthwy, bcteril motility goes unchecked nd results in brech of the mucosl brrier, which is ssocited with gret risk of developing chronic inflmmtion. Thus, while commensl bcteri cn dynmiclly respond to mucosl ntibodies nd void clernce, the norml mintennce of flgellin-directed immune pressure helps contin the microbiot nd promote helth of the host. The high flgellin bioctivity nd low nti-flgellin ntibody sttus of the TLR5 / microbiome suggests tht the high flgellin bioctivity fils to drive commensurte nti-flgellin ntibody response in the TLR5 / host. In the WT mouse, mucosl IgA induction hs been shown to function s stepwise response to bcteril lod (Hpfelmeier et l., 2010). A proportionl ntibody:ntigen reltionship likely pplies to ntigens tht re constitutively expressed, such s LPS, but not to optionl ntigens such s flgell. It is likely tht the TLR-medited feedbck between ntibody nd ntigen described here is unique to TLR5, since lignds to other TLRs re constitutively expressed. The mettrnscriptome dt indicte tht mny different types of gut commensl bcteri upregulted their flgellr motility genes in the TLR5 / hosts, even though the underlying species composition nd functionl gene profiles of the microbiome were similr for TLR5 / nd WT mice. Flgell hve been shown to promote dherence of tx such s Clostridium difficile in the gut nd to id coloniztion (Tsteyre et l., 2001). Whether flgell perform similr roles for commensl bcteri remins to be explored. Regrdless of the role of flgell in the gut, our results indicte tht the immune environment is key regultor of their production. We showed tht in vitro, two species of commensl Firmicutes were redily immobilized by nti-flgell ntibodies nd tht the ddition of nti-flgellin ntibodies to E. coli grown in vitro induced downregultion of its flgellin gene within few hours of ddition. Studies in pure culture (using E. coli nd Slmonell) hve previously shown tht ntiflgell ntibodies cn inhibit motility (Forbes et l., 2012; Suo et l., 2009); our results extend these findings to phylogeneticlly diverse vriety of commensl microbes comprising the microbiome. Anti-flgell ntibodies function directly by immobilizing bcteri, but our results show tht they lso function indirectly by inducing downregultion of flgellin gene expression. When LPS ntibodies were dded to E. coli cultures, we observed less inhibition of motility compred to nti-flgellin ntibodies nd no chnge in flgellin gene expression. This rises the question of how nti-flgellin ntibodies induce downregultion of flgellin gene expression. One possibility is tht the totl immobiliztion is sensed, nd bcteri respond with downregultion of the flgellr genes. Alterntively, nti-flgellin immunoglobulins my ct s signl to downregulte the flgellin genes. We note tht lthough our in vitro experiments utilized flgellin-specific IgG, the sme responses cn be expected with IgA. Our results corroborte erlier findings tht TLR5 cn recognize the flgellins from wide rnge of species nd support the notion tht ntibodies ginst flgellins cn be cross-rective cross bcteril species. Although highly specific flgellr ntigens hve been used to differentite Enterobcteril serotypes for century, serologicl cross-rectivity ginst flgellr ntigens hs lso been reported cross species (Dtt et l., 2008; Ibrhim et l., 1985; Kovch et l., 2011) nd cross serovrs nd genotypes (Bisws et l., 2010; Sh et l., 2007). Conserved epitopes re unmsked when the flgellum filment is depolymerized (Ibrhim et l., 1985), which is lso required for TLR5 ctivtion (Smith et l., 2003). The TLR5-binding site of flgellin is conserved cross phyl (Andersen-Nissen et l., 2005; Erridge et l., 2010) such tht ntibodies tht bind this region of flgellin re likely to be cross-rective cross species. Phse vrition of flgellr ntigens hs been described for species of Proteobcteri (vn der Woude nd Bäumler, 2004). Other ntigens, such s polyscchrides, cn evde ntibody Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc. 575

6 Figure 5. Effect of TLR5 Signling on IgA Coting of Bcteril Popultions (A) Box plots represent the rtio of IgA-coted:noncoted reds for ech OTU within the specified phyl, such tht vlue = 1 mens the OTUs within phyl re coted or noncoted with IgA t equl frequency. An open circle represents n outlier. n = 5 8 mice/group. *p < 0.05, two-tiled t test; n.s., nonsignificnt. (B) The frequency of IgA-coted cells is displyed for the 25 most bundnt OTUs in either TLR5 / or wild-type mice. The two most bundnt OTUs for ech genotype re indicted by their consensus txonomy. (C) OTUs with significnt Cook s distnce (nd therefore outliers from the trend of equl frequencies of IgA-coting nd noncoting) re ctegorized s hving high IgA coting or low IgA coting for both TLR5 / nd WT mice. The most specific consensus linege is shown. Relted to Figure S Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc.

7 Figure 6. The Addition of Anti-Flgellin Antibody Represses Bcteril Motility (A) Motility pltes contining 0.3% gr were stbinoculted with E. coli with or without the ddition of ntibody (nti-flgellin IgG). Pltes were incubted t 37 C for 14 hr. Red circles highlight the extent of bcteril movement through the gr. (B) GFP FliC-reporter E. coli PHL628 treted with ntibodies or glucose. Mens ± SEM for rtios of normlized GFP fluorescence:od re plotted, n = 3/group. Relted to Figure S6. recognition by phse vrition (Peterson et l., 2007), so bcteri my use phse vrition in flgellins to similrly evde ntibody recognition. However, the region of flgellin tht is phse vrible is not the sme s the conserved regions tht ctivte TLR5, nd if the ntibodies recognize this region, then phse vrition is not likely to ffect ntibody binding. The loss of TLR5 signling ws lso ssocited with n ltered profile of IgA-coted microbiot. If IgA coted the vrious species of bcteri in the gut indiscrimintely, we would expect 1:1 rtio of IgA-coted nd uncoted cells. This is lrgely wht we observed in both WT nd TLR5 / mice for the Bcteroidetes phylum, whose representtives in the gut do not possess genes for the production of flgell. For the Fimicutes nd Proteobcteri phyl, which include flgellted gut species, the ptterns were opposite, with TLR5 / generlly undercoting Proteobcteri nd overcoting Firmicutes compred to WT. Comprisons with DSS-treted WT nd MyD88 / mice indicted tht these ptterns were due to loss of TLR5 / signling rther thn inflmmtion per se. These ptterns suggest tht deficiency in nti-flgellin ntibodies is compensted by lternte ntibodies for the Firmicutes, but not for the Proteobcteri. Despite the high levels of ntibody coting of the Firmicutes in the TLR5 / host, we nevertheless observed brrier brech by Firmicutes in the TLR5 / smll intestine. Thus, consistent with our in vitro observtion tht nti-lps ntibodies did not inhibit E. coli s motility, these lternte ntibodies pper to be insufficient to contin the microbiot. The hypermotile phenotype of the TLR5 / host microbiome is form of dysbiosis, one tht is unrelted to microbil community composition. We hd previously reported n ltered microbiot for TLR5 / mice compred to WT bsed on 16S rrna gene sequence profiles (Crvlho et l., 2012; Vijy-Kumr et l., 2010). Bsed on these nlyses, the min differences between the TLR5 / nd WT microbiomes included higher between-mouse vrition in diversity nd greter voltility (reduced stbility) in microbiot over time. Levels of Proteobcteri were prticulrly voltile in the TLR5 / nd slightly elevted compred to WT mouse gut (Crvlho et l., 2012). In contrst to the TLR5 / microbiome s temporl voltility, we found tht the levels of flgellin nd nti-flgellin ntibodies were stble over time. The notion tht the highflgellin microbiome phenotype is robust to underlying community diversity is further supported by our observtions tht TLR5 / mice from severl different fcilities, nd stemming from two independent derivtions (Flvell versus Akir), ll hd high flgellin lods. It is incresingly cler tht the species composition of mouse gut microbiots cn differ between fcilities nd even between cges within fcility. Together, these results indicte tht microbiomes cn vry widely in species content but still show high levels of flgell production in the right immune milieu. Our experiment with the trissocited gnotobiotic RAG1 / nd WT mice, in which community composition nd structure were identicl for both genotypes nd only the E. coli could produce flgell, clerly indicted tht the levels of flgell were independent of E. coli s popultion size. Wht is interesting to note is tht the production of flgell did not pper to impose fitness cost on E. coli in this highly simplified model. Flgell re considered to be energeticlly expensive, nd glucose induces downregultion of these genes. Thus, other fctors besides energy expenditure likely limited E. coli popultion levels in this system. Expression of flgell genes is one mechnism tht cn led to the observed higher levels of flgellin in the TLR5 / gut; nother is selection for motile over nonmotile strins within popultions. Previous work hs shown tht motility in E. coli MG1655 is selected ginst in the mouse gut, lthough portion of the cells retins motility (Guger et l., 2007). Monocoloniztion of E. coli into WT nd MyD88 / germfree mice hs been shown to result in selection for nonmotile mutnts within 10 dys of inocultion in both mouse genotypes (De Pepe et l., 2011; Girud et l., 2008). If ntibodies ginst flgell re importnt in this selection Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc. 577

8 Figure 7. Flgellted Bcteri Penetrte Smll Intestinl Villi of TLR5 / Mice nd Brech the Lrge Intestinl Mucous Brrier (A) Fluorescent in situ hybridiztion (FISH) using the universl probe EUB338 (green) shows clusters of bcteri penetrting the intestinl villi of the TLR5 / smll intestine. (B) FISH using Proteobcteri-specific (green) nd Firmicute-specific (ornge) probes shows members of both phyl penetrting the intestinl villi of the TLR5 / smll intestine. Green nd ornge overly shows s white. (C) WT smll intestinl villi re consistently free of bcteri. (D nd E) Flgellin imged using immunohistochemistry with fluorescently lbeled ntiflgellin ntibody (green) in TLR5 / nd WT lrge intestines. Arrows in (D) highlight exmples of flgellted bcteril rods in the s lyer of the mucus of TLR5 /. The outer mucus lyer in the WT (E) is free of bcteril cells nd lso ppers to contin less flgellin thn tht in TLR5 /. The letter s indictes the inner, thicker lyer of the mucus, nd the letter o indictes the outer lyer where bcteri re normlly found. For ll pnels, the scle br is 50 mm, nd sections re counterstined with Hoechst (blue) to visulize host tissue. Relted to Figure S7. In (D) nd (E), host tissue is on the left nd mucus is on the right. process, inocultion of E. coli into germfree RAG1 / or TLR5 / mice should fil to select nonmotile mutnts. Roseburi cecicol monossocited in gnotobiotic WT mice hs lso been shown to lose motility, but only in monossocition not when ssocited with four other species (Stnton nd Svge, 1984). Thus, motility is likely importnt for bcteril fitness in the context of microbe-microbe interctions in the complex microbiome. This might explin why so mny different kinds of bcteri mintin flgell genes in their genomes despite the host selection ginst flgellr motility. How, when, nd where flgell re essentil for bcteril fitness in the mmmlin gut remins to be explored. We propose tht greter flgellr motility leds directly to brrier brech. Imging of bcteri in the TLR5 / gut showed penetrtion of intestinl villi in the smll intestine, which we did not observe in the WT mice. Consistent with the mettrnscriptome dt indicting tht members of both Firmicutes nd Proteobcteri upregulted their flgell genes, we observed both phyl in the smll intestinl villi of TLR5 / mice. We lso observed flgellted rods in the normlly bcteri-free thicker (s, surfce) mucus lyer of the lrge intestine, clerly djcent to host epithelil cells. Although the probes re not likely to detect ll flgellted species, we nevertheless observed qulittively strk difference between the genotypes. Proteobcteril brech of the s lyer hs been previously reported for the TLR5 / mouse (Crvlho et l., 2012), nd in the MyD88 / mouse (Vishnv et l., 2011), nd ppers to be ssocited with inflmmtion (Johnsson et l., 2013). Thus, in the bsence of TLR5 signling, lrge bcteril lod, much of which cn be flgellted, comes into contct with host tissues in both the smll nd lrge intestines nd likely prticiptes in perpetrting inflmmtion. In summry, we suggest tht nti-flgellin ntibodies re key to mucosl brrier protection nd homeostsis. Our findings further suggest tht mucosl ntibodies quench motility by immobilizing flgellted bcteri nd lso by inducing the downregultion of motility-relted genes. Thus, we propose tht mucosl immunoglobulins function physiclly on flgell to bind them, s well s signling molecule, to lter gut microbil gene expression nd ultimtely control bcteril behvior nd locliztion. EXPERIMENTAL PROCEDURES Experimentl procedures re outlined in brief here. For more detils, see the Supplementl Informtion. All niml experiments were pproved by the locl IACUCs. All mice used in this study were C57BL/6 strin nd were housed t different fcilities. The trissocited gnotobiotic mice were mintined t the University of Nebrsk Lincoln, nd their popultions were monitored by plte counts nd by denturing grdient gel electrophoresis (DGGE; see Supplementl Experimentl Procedures). Bcteril strins used in the study re listed in Tble S2. Mesurement of Antibodies Using ELISA We mesured totl IgA, totl IgG, flgellin-specific IgA, nd flgellin-specific IgG in fecl nd cecl smples by ELISA s previously described (Sitrmn et l., 2005). Detils of the ELISA, including the sndwich ELISA used for the flgellin-specifc IgA mesures, re in the Supplementl Experimentl Procedures. Flgellin Quntifiction We quntified flgellin using HEK-Blue htlr5 cells (Invivogen). Between 10 nd 100 mg of either cecl or fecl mteril were diluted by homogenizing for 10 s using Mini-Bedbeter-24 without the ddition of beds (BioSpec), nd the smples were centrifuged t 8,000 3 g for 2 min, followed by seril dilutions. After hr of incubtion, we pplied cell culture superntnt to HEK-Blue Detection medium (Invivogen) nd mesured lkline phosphtse ctivity t 620 nm every 30 min for 3 hr on Synergy H1 multiple detection microplte reder (Biotek). Results re representtive of one repet of the experiment, nd both experiments included one technicl replicte per smple. We used purified Slmonell Typhimurium strin flgellin (Enzo Life Sciences) in stndrd curves. As control for TLR5 specificity, we included one dditionl replicte per smple, per plte, to which we dded nti-htlr5 neutrlizing ntibody (5 mg/ml; Invivogen). 578 Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc.

9 Flgellin Purifiction nd Production Flgell were shered using 80 ml of ech culture (strins listed in Tble S2)by vortex gittion. Stphylococcus epidermidis ws used s n flgellte control. Cultures were centrifuged t 2,754 3 g for 30 min t 4 C, nd superntnts were centrifuged gin t 2,754 3 g for 1.5 hr t 4 C. The resulting pellets were resuspended in PBS nd centrifuged once more t 3,442 3 g for 30 min t 4 C. To reduce endotoxin levels in the flgellr purifiction of Grm-negtive bcteri (Citrobcter mlonticus nd Providenci sturtii), we detoxified using polymyxin B with Detoxi-Gel AffinityPck Pre-Pcked Columns (Thermo Scientific). Recombinnt Fl1 from R. hominis ws produced by cloning the fl1 gene with His tg into n E. coli expression vector (see Supplementl Experimentl Procedures for detils). Western Blot Purified flgellin or cell pellets (R. hominis nd E. coli cell pellets with recombinnt Fl1, or superntnt from those cells) were prepred by dding Lemmli smple buffer with b-mercptoethnol nd boiled for 10 min. Proteins were seprted on 10% SDS-PAGE gels, trnsferred to nitrocellulose membrne, nd blocked for 1 hr in TBS solution contining 5% milk. The membrne ws probed overnight t 4 C with primry ntibodies nti-fl2 nd nti-fl1 (Covlb). Mettrnscriptomic Anlysis We enriched for nonribosoml RNA using modifiction of technique previously described (Stewrt et l., 2010). We divided cecl contents in hlf nd extrcted bulk RNA nd bulk DNA in prllel using the PowerSoil RNA Isoltion Kit nd Powersoil DNA Isoltion Kit, respectively, s described by the mnufcturer (MO BIO Lbortories). We generted smple-specific ribonucleotide probes trgeting bcteril 16S nd 23S rrna genes by PCR mplifying these gene sequences from bulk DNA using the universl primers 27F nd 1492R for 16S nd 189F nd 2490R for 23S. We seprtely converted 16S nd 23S rrna gene sequence mplicons to biotinylted ntisense rrna probes, which hybridized to complementry rrna molecules in the totl RNA smple. Next, we converted rrna-subtrcted smples to double-strnded cdna, mplified vi in vitro trnscription, nd converted bck to double-strnded cdna. We used the lrge subunit (LSU) nd smll subunit (SSU) reference dtbses from SILVA ( to seprte Illumin reds with >70% similrity to dtbse rrna sequence. Using this pproch, we identified 40.2% of the reds s ribosoml nd removed them from downstrem nlysis. We used metgenomics RAST (MG-RAST; Meyer et l., 2008) with the defult qulity filtering. COG reltive bundnce dt for protein-coding reds were summrized using MG-RAST (e vlue < 10 5 ; identity [ID] > 50%; length > 20 mino cids []). We uploded functionl ssignments to Cluster 3.0 nd centered nd normlized counts before hierrchicl clustering using the uncentered correltion similrity metric. To ssign txonomy to sequences nnotted s flgellin, we used hierrchicl clssifiction nd the Subsystems dtbse in MG- RAST to nnotte function using defult cutoff prmeters. We isolted reds with n nnottion of flgellin nd ssigned txonomy using BLASTX (Altschul et l., 1990). Metgenomic Anlysis DNAs were sequenced t the Core Lbortories Center t Cornell University on the Roche 454 FLX pltform nd t the DNA Sequencing Lb (Columbi University Medicl Center) using the Illumin HiSeq 2000 pltform. We used MG-RAST (Meyer et l., 2008) with the defult qulity filtering nd without identicl red derepliction. Txonomy ssignments (lowest common ncestor, LCA) nd COG reltive bundnce dt for protein-coding reds were summrized using MG-RAST. Sorting of IgA-Coted Bcteri Cecl preprtions were incubted with nd without FITC-lbeled got F(b 0 ) 2 nti-mouse IgA nd propidium iodide (PI). We performed flow cytometry with BD Biosciences FACSAri high-speed flow cytometer/cell sorting using n rgon lser operting t 15 mw nd 488 nm. We used stndrd ELITE softwre comprising the Immuno-4 progrm to determine the percentge of stined events. The discrimintor ws set on PI fluorescence s specific probe for bcteri. We nlyzed portion of ech smple incubted with PBS (bckground fluorescence) nd portion incubted with FITC-lbeled got F(b 0 ) 2 nti-mouse IgA. Both mesurements were performed with 10,000 events. We collected minimum of 500,000 cells of ech ctegory (IgA bound nd nonbound). 16S Gene Sequence Anlysis For qulity filtering of 16S rrna gene sequence dt, we discrded sequences <200 bp or >1,000 bp s well s those contining >0 primer mismtches, uncorrectble brcodes, >0 mbiguous bses, or homopolymer runs in excess of 6 bses using the open source softwre pckge Quntittive Insights into Microbil Ecology (QIIME) (Cporso et l., 2010). We checked sequences for chimers (UCHIME) nd ssigned them to opertionl txonomic units (OTUs) using Otupipe (Edgr et l., 2011) with 97% threshold of pirwise identity nd then clssified them txonomiclly using the Greengenes reference dtbse (McDonld et l., 2012). We rrified smples to 7,000 reds per smple, clculted the rtio of IgA + :IgA for ech OTU, nd clculted Cook s distnce in R to find OTUs with IgA coting rtios tht diverged significntly from the men (Ferrri nd Cribri-Neto, 2004). Observtions of Bcteril Motility Clostridium rmosum nd Roseburi intestinlis were cultured for 48 hr in M2GSC medium in strictly nerobic conditions t 37 C. Then, 10 ml of culture ws mixed with either 1 ml of nti-flgellin ntibody, nti-mouse ntibody s specificity control, or wter nd pplied to glss slide nd seled with glss coverslip nd crylic selnt. Slide preprtions were completely dried nd seled before exiting the nerobic chmber. Time-lpse microscopy ws performed on slides 30 min fter the ddition of ntibody with MetMorph imging system. We mde time series of the cultures with spcing of 0.3 s over the course of 35 frmes. The videos re nmed s follows: bcteril species, ntibody used (or wter in the cse of control), nd the mount of time elpsed between slide preprtion nd imging (30 min). The ntibodies used were either nti-flic (monoclonl ntibody mde ginst E. coli flgellin), nti-fl2 (rbbit ntiserum mde ginst Roseburi hominis flgellin), or polyclonl nti-mouse ntibody (used s control, nonflgellin-specific ntibody). Detils of the plte motility ssys re in the Supplementl Experimentl Procedures. Monitoring Flgellin Expression of E. coli Bioreporter Detils on the construction of the reporter strin re in the Supplementl Experimentl Procedures. We grew E. coli in miniml slts medium (MSM) contining 0.2% glucose (s n inhibitor of flgellin expression) nd mpicillin (15 mg/l) t 37 C to n opticl density 600 (OD 600 ) of 0.8 ± 0.2. We then diluted the culture 1:100 in MSM contining either 0.2% glucose or 0.2% csmino cids. We divided cultures into 100 ml liquots in 96-well cler, flt-bottom pltes (CoStr) nd incubted t 37 C with periodic shking on Synergy H1 multiple detection microplte reder (Biotek). OD 600 nd fluorescence of the flic promoter fusion (Abs/Em: 488/509 nm) were mesured every 30 min over the course of 15 hr. We dded ntibodies fter 2 hr of growth t concentrtion of (5 mg/ml) (Forbes et l., 2008). The fluorescence vlues were normlized to growth. Results re representtive of three repets of the experiment, ech of which included one technicl replicte per smple. Microscopy Smples of intestine were preserved in Crnoy s solution nd prffin embedded for sectioning. FISH for bcteril groups ws performed using flueorescently lbeled oligonucleotide probes specific for ll bcteri, or specific subgroups (see Supplementl Experimentl Procedures). Flgellin nd IgA were visulized using fluorescently lbeled ntibodies (FITC-conjugted nti-flgellin IgG nd nti-iga IgA). Sttistics We performed sttisticl nlysis nd liner regression nlysis in Microsoft Excel 2011, SttPlus 2009, R version , nd IBM SPSS Sttistics. Sttisticl tests were one-wy ANOVA, unpired Student s t test, or generlized mixed liner model, s indicted. Significnce level ws p < 0.05 unless otherwise indicted. All multiple comprison testing ws ccounted for with Bonferroni correction. Cell Host & Microbe 14, , November 13, 2013 ª2013 Elsevier Inc. 579

10 ACCESSION NUMBERS 16S rrna gene sequence dt re deposited under Study ID 1891 in the QIIME-DB dtbse (qiime.org). Trnscriptome nd metgenome dt re deposited under project nmes TLR5KO_Trnscritptome, TLR5KO_Metgenome (Illumin dt), nd TLR5KO_454_Metgenome (454 dt) in the MG-RAST dtbse (metgenomics.nl.gov). SUPPLEMENTAL INFORMATION Supplementl Informtion includes Supplementl Experimentl Procedures, seven figures, nd two tbles nd cn be found with this rticle online t ACKNOWLEDGMENTS We thnk S. McSorley, D. Hckm, F. Crvlho, nd L. Junker. This work ws supported by NIH grnts R01DK nd DP2OD nd Fellowships to R.E.L. from The Hrtwell Foundtion, the Arnold nd Mbel Beckmn Foundtion, nd the Lucile nd Dvid Pckrd Foundtion. The Cornell University cytometry core is supported in prt by the ESSCF, NYS-DOH, Contrct # Received: June 1, 2013 Revised: August 23, 2013 Accepted: September 26, 2013 Published: November 13, 2013 REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.W., nd Lipmn, D.J. (1990). 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