Presence of SFB correlates with the presence of Th17 cells

We next examined the representation of Candidatus Arthromitus in Th17 cell-sufficient and Th17 cell-deficient mice. Arthromitus is an unofficial candidate genus name for the group of so-called segmented filamentous bacteria (Snel et al., 1995). SFB are yet to be cultured, commensal, gram-positive, anaerobic, spore-forming bacteria that are resident in the terminal ileum under steady state conditions (Davis and Savage, 1974). SFB have a characteristic long filamentous morphology, are comprised of multiple segments with well-defined septa, and often span the length of several villi. They colonize the gastrointestinal tract of mice at weaning time and adhere tightly to epithelial cells (Koopman et al., 1987). SFB are present in a many vertebrate species, including rodents (Davis and Savage, 1974), fish, chicken, dogs, and primates (Klaasen et al., 1993a; Ley et al., 2008). A phylogenetic tree based on an alignment of the available SFB 16S rRNA gene sequences according to their sequence origin is presented in Figure S3. SFB are known to actively interact with the immune system (Klaasen et al., 1993b). Colonization of germ-free animals with SFB leads to stimulation of secretory IgA (SIgA) production and recruitment of intraepithelial lymphocytes (IELs) to the gut (Talham et al., 1999; Umesaki et al., 1999). Mice lacking the activation-induced cytidine deaminase (AID) required for antibody diversification had outgrowth of SFB in their small intestine (Suzuki et al., 2004).

We validated the abundance of SFB in the gut of Taconic and Jackson B6 mice by quantitative real-time PCR (qPCR) for 16S rDNA sequences. SFB were present in fecal material from cecum as well as small and large intestine of Taconic B6 mice, but could not be detected in Jackson B6 mice (Figure 2A and data not shown). Scanning electron microscopy revealed a thick network of SFB present in the terminal ileum of 6-8 week old Taconic B6 mice (Figure 2B). In contrast we could not detect any bacteria with SFB morphology in age- and sex-matched Jackson B6 mice, even after equilibration of housing conditions and diet (Figure 2B). Despite similar numbers of total bacteria in the feces of both strains, only non-SFB bacteria were evident in the terminal ileum of mice from Jackson Laboratory (Figure S4). Transmission electron microscopy confirmed typical SFB morphology with well-defined segments in tight contact with the epithelial cells of ileum from Taconic but not Jackson B6 mice (Figure 2B). To confirm that SFB can be horizontally transferred, we co-housed female mice obtained from the two sources and observed Th17 cells in the lamina propria of Jackson B6 mice within 10 days ((Ivanov et al., 2008) and Figure S5). qPCR analysis of fecal material and microscopy of terminal ileum confirmed the appearance of SFB in the co-housed Jackson B6 mice (Figure 2C and 2D).

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Figure 2

Segmented Filamentous Bacteria (SFB) in the intestinal tract of Th17 cell-sufficient and Th17 cell-deficient mice

A. Quantitative PCR (qPCR) analysis of SFB and total bacterial (EUB) 16S rRNA genes in mouse feces from Taconic (Tac) and Jackson (Jax) B6 mice. Genomic DNA was isolated from combined fecal pellets from 4 animals from each strain. The experiment was repeated numerous times with similar results.

B. Scanning (SEM) and transmission (TEM) electron microscopy of terminal ileum of 8 week-old Jackson (Jax) and Taconic (Tac) C57BL/6 mice housed under similar conditions and diet for at least one week. Note the presence of long filamentous bacteria with SFB morphology in Taconic, but not Jackson mice

C. qPCR analysis for SFB presence in Jackson B6 mice after 14 days of co-housing with Taconic B6 mice (Jax-Coh). Genomic DNA was isolated from pooled feces from 3-4 mice per group.

D. SFB colonization of terminal ileum of Jackson B6 mice after 14 days of co-housing with Taconic B6 mice (Jax-Coh). Toluidine-blue sections were prepared from 0.5 cm piece of the terminal ileum as described in Methods and examined by light microscopy. Adherent bacteria with SFB morphology were counted in 4-5 sections from each sample. Each column represents a separate animal.

SFB specifically induce Th17 cells in the intestinal lamina propria

To test if SFB are sufficient to induce Th17 cells, we colonized germ-free (GF) Swiss-Webster mice with fecal material obtained from mice mono-colonized with SFB (SFB-mono mice) (Umesaki et al., 1995) and examined lamina propria CD4⁺ T cells for Th17 cell differentiation 10 days later. Non-colonized control GF mice housed under separate but similar conditions had no Th17 cells (Figure 3A). In contrast, SFB colonization induced robust accumulation of Th17 cells in both the SI and LI LP (Figure 3A and Figure S6). SFB induced production of both IL-22 and IL-17 in CD4⁺ T cells (Figure 3A and 3B). The effect of SFB on Th17 cell differentiation was similar in Swiss-Webster and IQI GF mice housed at different institutions (Figure 3B and 3C). Moreover, the effect of SFB on inducing IL-17 production in LP T cells is bacterial species specific, because colonization with Bacteroides species as well as with a defined mix of Clostridium species, which are closely related to SFB, did not induce Th17 cells in GF mice (Figure 3C). Finally, SFB had no effect on IFN-γ production, indicating that they specifically influence Th17 and not Th1 cell differentiation (Figure 3D). Colonization of GF mice with SFB restored RORγt⁺ T cells to the levels observed in mice kept under SPF conditions (Figure 3E). By contrast, the number of RORγt⁺ non-T cells, which include lymphoid tissue inducer-like cells and NK-like cells, was similar in GF mice, SFB-mono mice, and mice kept in SPF conditions (Figure 3E), and there was no significant difference in IL-17 and IL-22 production by these cells (Figure S7). Notably, SFB colonization and induction of Th17 cells did not reverse the elevated proportion of Foxp3⁺ cells among the CD4⁺ T cells in the SI LP and the peritoneal cavity of GF mice. (Figure S8).

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Figure 3

SFB specifically induce Th17 cell differentiation in germ-free mice

A-B. 6-week old Swiss-Webster (SW) germ-free mice (GF) were colonized with SFB (GF+SFB) as described in Methods and small intestinal lamina propria lymphocytes (SI LPL) were isolated 10 days later. Representative plots in (A) and combined data in (B) of IL-17 and IL-22 expression in TCRβ⁺CD4⁺ LPL. Data are from one of three separate experiments with similar results. Error bars (SD). SPF – mice raised under conventional specific pathogen free conditions. Each circle in (B) represents a separate animal

C-D. IL-17 (C) and IFNγ (D) expression in TCRβ⁺CD4⁺ SI LPL from mice colonized with different commensal bacteria. IQI germ-free (GF) mice were colonized with 16 strains of Bacteroidaceae (B. mix), SFB, 46 strains of Clostridium sp mixture (Clost. mix), or microbiota from conventionally raised mice (SPF). Intracellular cytokine production in SI LP CD4 T cells was analyzed 3 weeks later by flow cytometry. Circles represent separate animals

E. SFB colonization induces RORγt expression only in CD4⁺ T cells. RORγt expression in total SI LPL (top panels) and RORγt and Foxp3 expression in TCRβ⁺CD4⁺ SI LPL (bottom panels) in GF mice, GF mice colonized with SFB (GF+SFB) and conventionally raised mice (SPF).

To determine whether SFB can also induce Th17 cell differentiation in conventionally raised mice, we introduced fecal material from SFB-mono mice by oral gavage into 6 week-old Jackson B6 mice and analyzed colonization and cytokine production in the SI LP. By 10 days, SFB were detected by scanning electron microscopy in the terminal ileum (Figure 4A) and by qPCR in the feces (data not shown), and robust Th17 cell differentiation was observed in the SI LP (Figures 4B and 4C). In contrast, control untreated Jackson B6 mice or Jackson B6 mice gavaged with bacterial suspensions from their littermates did not show an increase in Th17 cells (Figure 4B and 4C). Similarly, introduction of Jackson microbiota into GF animals did not induce Th17 cells, unless the microbiota were supplemented with SFB (Figure 4D and 4E). Th17 cell induction by SFB was also demonstrated by the expression of a number of Th17 cell effector cytokine mRNAs, including those for IL-17 and IL-21 (Figure 4F). We therefore conclude that SFB are members of the commensal microbiota that specifically induce the accumulation of Th17 cells in the SI LP.

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Figure 4

SFB induce Th17 cell differentiation upon colonization of Jackson C57BL/6 mice

A. Colonization of the terminal ileum by SFB 10 days after transfer of fecal homogenates from SFB-mono mice into Jackson B6 mice

B-C. IL-17 and IL-22 expression in TCRβ⁺CD4⁺ SI LPL in Jackson B6 mice colonized with SFB (Jax+SFB) compared to controls (Jax). Data from one of two experiments

D-E. Jackson microbiota induces Th17 cells only when complemented with SFB. Germ-free IQI mice were colonized with SFB, Jackson microbiota isolated from fecal pellets by itself (Jax), or a mixture of both (Jax + SFB). Th17 cell proportions were analyzed in the LP 3 weeks later by flow cytometry. Plots in (D) gated on total lymphocytes. Data in (E) represent percentage of IL-17⁺ cells in the CD4⁺ gate

F. RT-PCR for Th17 cell effector cytokines in total RNA from terminal ileum of the mice in (E)

SFB induce an immune response program in the gut

To identify specific effects of SFB, we compared the gene expression profiles in the terminal ileum of Swiss-Webster GF mice before and after colonization with SFB and in Jackson B6 mice before and after co-housing with Taconic B6 animals. Colonization of GF mice with SFB induced at least a two-fold change in expression of 253 genes while co-housing of Jackson B6 mice with Taconic B6 mice induced a similar change in 470 genes (Figure 5A). More importantly, there was a high degree of overlap between the two groups, with expression of 131 genes affected by both treatments. We could therefore distinguish three groups of genetic profiles. Group 1 includes genes whose expression was affected only in Jackson mice by co-housing, but was not statistically different after SFB colonization. This group most likely includes genes whose expression is influenced by microbiota other than SFB that differs between the mice from the different vendors, as well as strain-specific changes. Group 2 consists of genes whose expression only changed in GF mice upon colonization with SFB, but not in Jackson B6 mice following co-housing. A subset of these genes is expected to reflect changes induced in GF animals upon general intestinal colonization with bacteria. Group 3 includes the genes with expression differences following both SFB colonization and co-housing with Taconic mice (Figure 5A) and thus contains genes specifically induced by SFB and associated with Th17 cell induction.

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Figure 5

Transcriptional programs induced by SFB colonization

A. Venn diagrams showing the overlap between genes affected by either SFB colonization of SW GF mice only (Group 2), introduction of Taconic microbiota into Jackson B6 mice by co-housing (Group 1), or both (Group 3). Total RNA was prepared from terminal ileum of the corresponding mice after 10 days of colonization and Affymetrix gene chip analysis was performed as described in Methods.

B. Heat-map analysis of the three groups in (A). Each line represents a single Affymetrix probe and each column a single mouse. Green, probes that were at least 2 fold down-regulated. Red, probes that were at least 2 fold up-regulated.

C. Top up-regulated genes in Group 3 in (A) arranged by fold change in GF+SFB mice.

D. Biological processes specifically induced by SFB (genes in Group 3 in (A)). Gene ontology analysis was performed as described in Methods.

E. Changes in anti-microbial peptide related genes upon SFB colonization and Th17 cell induction by co-housing. Each column represents an individual mouse.

F. RT-PCR analysis of selected genes, induced by SFB colonization. IQI GF mice (GF) were colonized with fecal homogenates from SFB-mono mice (SFB), Jackson B6 mice (Jackson), or a mixture of both (Jackson+SFB). Total RNA from terminal ileum was prepared 3 weeks later and RT-PCR performed as described in Methods.

SFB exerted an inductive effect in the host, which was demonstrated by the finding that most (>70%) of the genes in Group 3 were up-regulated after SFB colonization (Figure 5B). By comparison, most genes in Group 1 (>70%) were down-regulated, which suggests that the rest of the Taconic microbiota has a suppressive effect that may possibly restrain the inductive effect of SFB (Figure 5B). Group 2, on the other hand, consisted of roughly equal numbers of up-regulated and down-regulated genes.

To evaluate changes specifically associated with Th17 cell-inducing SFB, we next concentrated on the genes in Group 3. A list of the top up-regulated genes is presented in Figure 5C. A GO Biological Pathway analysis of up-regulated genes in Group 3 showed that immune system pathways were among the programs most significantly induced by SFB (Figure 5D) and raised the possibility that at least some of the observed gene expression changes were mediated by Th17 cells or their effector cytokines. Because IL-17 and IL-22 have been associated with induction of anti-microbial peptides (AMP) (Curtis and Way, 2009; Kolls et al., 2008; Zheng et al., 2008), we compared the induction of AMP-related genes in our arrays. Multiple AMP genes were induced specifically by colonization with SFB, consistent with an up-regulated Th17 cell response (Figure 5E). Upregulation of Th17 cell-associated genes (Il17, Il21, Ccr6, Nos2) and AMPs (Reg3g) following SFB colonization was confirmed by quantitative RT-PCR (Figures ​(Figures4F4F and ​and5F5F).

Serum Amyloid A is induced by SFB colonization and influences Th17 cell differentiation

The top up-regulated transcript upon SFB colonization of GF mice encoded an isoform of SAA – Saa1, a member of the family of acute-phase response proteins induced during infection, tissue damage, or inflammatory disease (Uhlar and Whitehead, 1999). This transcript was also up-regulated upon co-housing of Jackson B6 mice with Taconic B6 animals (Figure 5C). Transcripts for the other SAA isoforms, Saa2 and Saa3, were also among the most highly up-regulated genes upon colonization with SFB or co-housing (Figure 5C).

Real-time PCR confirmed that all three SAA isoforms were induced in the terminal ileum of GF mice upon colonization with SFB or SFB + Jackson microbiota, but not by Jackson microbiota alone (Figure 6A). Recent studies have demonstrated that SAA may act as a cytokine that induces IL-8, TNFα, and IL-1β in neutrophils and IL-23 in monocytes (Furlaneto and Campa, 2000; He et al., 2006). We therefore investigated the effect of SAA on Th17 cell differentiation in vitro. Addition of recombinant SAA to co-cultures of naïve CD4⁺ T cells and LP DCs induced a Th17 cell differentiation program in a concentration-dependent manner, including Th17 cell effector cytokines and RORγt (Figure 6B). In addition, SAA induced production of IL-17 in CD4⁺ splenic OT-II T cells co-cultured with LP DCs in vitro (Figure S9). Addition of SAA to cultures containing only T cells, without DCs, did not induce Th17 cell cytokines (Figure 6B) and SAA induced production of IL-6 and IL-23 by LP DCs in vitro (Figure S10). We conclude that SFB colonization results in the production of SAA, which in turn acts on gut DCs to stimulate a Th17 cell-inducing environment.

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Figure 6

SFB colonization induces SAA expression that influences Th17 differentiation

A. Relative mRNA expression levels of SAA1-3 genes by real-time RT-PCR in the terminal ileum of IQI GF mice (GF) colonized with fecal homogenates from SFB-mono mice (SFB), Jackson B6 mice (Jackson), or a mixture of both (Jackson+SFB).

B. Splenic naïve CD4⁺ T cells were cocultured with or without LP CD11c⁺ cells in the presence of an anti-CD3 antibody with the indicated concentration of recombinant Apo-SAA for 4 days. T cells were collected, restimulated with PMA and ionomycin, and real-time RT-PCR performed. Results were normalized to expression of GAPDH mRNA. The data are representative of four independent experiments with similar results.

SFB and Th17 cell-mediated protection from pathogenic microorganisms

The identification of SFB as Th17 cell inducers in the intestine may have important implications for a better understanding of how components of the commensal microbiota contribute to host protection from microbial pathogens. It is well known that treatment with broad-spectrum antibiotics can result in outgrowth of intestinal pathogens, such as vancomycin-resistant Enterococcus (VRE) or Clostridium difficile, resulting in severe colitis. SFB colonization has been suggested to reduce replication in rabbits of enteropathogenic Escherichia coli (EPEC) and in rats of Salmonella enteritidis (Garland et al., 1982; Heczko et al., 2000). In mice, Th17 cell effector cytokines, such as IL-17 and IL-22, as well as IL-23, which is required for Th17 cell function, have been proposed to play protective roles in infections with Salmonella and Citrobacter rodentium (Curtis and Way, 2009). We found that colonization with SFB reduced the capacity of orally inoculated C. rodentium to grow and/or invade colonic tissue. Although we cannot at this point formally demonstrate that this protection is a direct result of Th17 cell induction, our data, taken together with results of recent studies (Kolls et al., 2008; Zheng et al., 2008), strongly suggest that SFB-induced Th17 cytokines, particularly IL-22, limit the growth of C. rodentium, at least in part through production of AMP’s such as RegIIIγ. While IL-22, IL-23, and RegIIIγ are required for host survival after C. rodentium infection (Mangan et al., 2006; Zheng et al., 2008), mice lacking SFB and Th17 cells survive despite increased bacterial growth. This may be because intestinal γδT cells, CD4⁺CD3⁻ lymphoid tissue inducer (LTi)-like cells, and NK22 cells that also produce Th17 cytokines are present even in the absence of SFB and other microbiota. Contribution of these cells to SFB-independent anti-microbial defense may hence protect the host from lethal outgrowth of the pathogenic bacteria. Our results are also consistent with the report that a vancomycin-sensitive component of the commensal microbiota induces RegIIIγ in the mouse small intestine, thus reducing colonization by VRE and enhancing killing of the pathogen (Brandl et al., 2008). Future studies will be required to determine if vancomycin-sensitive SFB enhance mucosal protection from pathogenic VRE and other bacteria through the up-regulation of Th17 cells and anti-microbial peptides. Such studies will further test the hypothesis that specific commensal microbiota, by regulating the host immune system rather than by direct microbial competition, enhance protection from potentially harmful microbes.

PhyloChip Analysis

Six week old Jackson B6 and Taconic B6 mice were purchased from the corresponding vendor and housed for 3 weeks in separate microisolator cages at the NYUSOM animal facility to equilibrate housing conditions, including bedding and diet. Sample collection, processing and PhyloChip analysis are described in detail in the Supplemental section.

16S rRNA Gene Quantitative PCR Analysis

Bacterial genomic DNA was isolated from fecal pellets as described in the Supplemental section. Quantitative PCR analysis was carried out as described in (Barman et al., 2008). Primer sequences for SFB and bacterial 16S rRNA genes as well as PCR conditions were as described in (Barman et al., 2008). For SFB, relative quantity was calculated by the ΔCt method and normalized by the presence of total bacteria (EUB primers), dilution and weight of the sample and presented as relative fold change to an external sample. Typical Ct values for SFB were ~20 cycles and for EUB ~11 cycles. Samples that were negative after 40 cycles were considered “not detected” (n.d.).

Gene Expression Analysis

RNA was prepared from terminal ileum as described (Ivanov et al., 2008). For microarray analysis, RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX10. Significant genes were selected based on p values smaller than 0.05 and fold change greater than 2. For enrichment analysis of biological process ontology, probe lists were analyzed in DAVID (Dennis et al., 2003; Huang da et al., 2009) and processes were selected based on p values smaller than 0.01.

Real-time RT-PCR (Q-PCR)

cDNAs were synthesized from RNA samples prepared with a RNeasy Mini Kit (QIAGEN) using M-MLV Reverse Transcriptase (Promega). Real-time RT-PCR was performed using the ABI 7300 real time PCR system. Serial dilutions of a standard were included for each gene to generate a standard curve and allow calculation of the input amount of cDNA for each gene. Values were then normalized by the amount of GAPDH in each sample. Primer sequences are reported in the Supplemental section.

Co-housing and Microbiota Reconstitution

Co-housing and microbiota reconstitutions were performed as described before (Ivanov et al., 2008). For inoculation of germ-free mice with SFB, fecal pellets were collected from SFB-monocolonized mice using sterilized test tubes in the vinyl-isolator and were preserved frozen under dry ice until immediately before oral administration. SFB colonizations were performed by oral gavage with 300-400 μl of suspension obtained by homogenizing the fecal pellets from SFB-mono donor mice in water. Control mice were gavaged with water or homogenates prepared from their own feces.

Cell Isolation and Flow Cytometry

Lamina propria lymphocyte (LPL) isolation and intracellular cytokine staining were performed as described before (Ivanov et al., 2008). Naive CD4⁺ T cells were purified from spleens using a CD4⁺CD62L⁺ T cell isolation kit II (Miltenyi Biotec; purity 95%). Anti-mouse RORγ monoclonal antibody conjugated to PE was purchased from eBioscience.

In vitro T-cell Differentiation

Naïve CD4⁺ T cells were cultured in 24-well plates at 2 × 10⁵ cells/well for 4 days with MACS-purified LP CD11c⁺ cells (1 × 10⁵/ well) and 1 μg/ml anti-CD3 antibody (BD Biosciences) in the presence or absence of recombinant human Apo-SAA (Peprotech). The cultured cells were harvested and restimulated with PMA and ionomycin for 3 h before analysis.

Electron Microscopy

Electron micrscopy was performed on 0.5-1 cm pieces from terminal ileum (immediately proximal to the ileal-cecal junction). Tissue processing for EM is described in the Supplemental section. The analysis was performed with a Zeiss Supra 55 FESEM.

We thank members of the Littman laboratory for valuable discussions, Takeshi Egawa and Homer Boushey for their contribution to establishing the collaborative study, and Junichi Nishimura for technical assistance. We also thank Feng-Xia (Alice) Liang and Eric Roth from the NYU imaging core facility for performing transmission electron microscopy and for preparing samples for SEM. We also thank Jiri Zavadil and Agnes Viale and the genomic core facilities of NYU and Memorial Sloan Ketterring Cancer Center, respectively, for performing the array studies. We thank the staff at the Yakult Central Institute for gnotobiotic handling of the mice. The work was supported by Crohn’s and Colitis Foundation of America (I.I.I.) and Cancer Research Institute (N.M.) fellowships and by the Howard Hughes Medical Institute (D.R.L.), the Helen and Martin Kimmel Center for Biology and Medicine (D.R.L.), NIH grant AI33856 (D.R.L.), Grants-in-Aid for Scientific Research from PRESTO, the Ministry of Education, Culture, Sports, Science and Technology (K.H.), Senri Life Science Foundation and the Naito Foundation (K.H.). Part of this work was performed under the auspices of the US Department of Energy by the University of California, Lawrence Berkeley National Laboratory, under contract DE-AC02-05CH11231.