Inflammatory bowel disease comprises a group of heterogeneous diseases characterized by chronic and relapsing mucosal inflammation. Alterations in microbiota composition have been proposed to contribute to disease development, but no uniform signatures have yet been identified. Here, we compare the ability of a diverse set of microbial communities to exacerbate intestinal inflammation after chemical damage to the intestinal barrier. Strikingly, genetically identical wild-type mice differing only in their microbiota composition varied strongly in their colitis susceptibility. Transfer of distinct colitogenic communities in gene-deficient mice revealed that they triggered disease via opposing pathways either independent or dependent on adaptive immunity, specifically requiring antigen-specific CD4 + T cells. Our data provide evidence for the concept that microbial communities may alter disease susceptibility via different immune pathways despite eventually resulting in similar host pathology. This suggests a potential benefit for personalizing IBD therapies according to patient-specific microbiota signatures.

In the present study, we have characterized the susceptibility of mouse lines differing only in their microbiota composition toward DSS colitis. Besides the dysbiotic community (DysN6) from Nlrp6 −/− mice, interestingly, also certain but not all SPF communities demonstrated the ability to cause severe intestinal inflammation in immunocompetent mice. Strikingly, mice displayed different inflammatory responses depending on their intestinal microbiota composition, either characterized by infiltration of neutrophils or the presence of proinflammatory CD4 + T cells. By utilizing gene-deficient mice and antibody-mediated depletion of T cell subsets, we demonstrated that the DysN6 community, but not another colitogenic community, depends on CD4 + T cells to exacerbate DSS colitis severity. Our data identify that specific interactions between colitogenic communities and host immune pathways drive colitis development via distinct mechanisms.

The acute dextran sulfate sodium (DSS) colitis model of human UC is considered to be largely dependent on innate immunity (). We previously demonstrated that the dysbiotic microbiota of Nlrp6 inflammasome-deficient mice has the ability to directly enhance DSS colitis severity, but the effector mechanism remained unknown (). Notably, a recent study identified that specific metabolites of this dysbiotic community actively modulate innate immune signaling and, subsequently, the host-microbiota interface (). Subsequently, similar dysbiotic communities with the ability to modulate the severity of DSS colitis have been described in other gene-deficient mice (). However, it remains to be examined whether different colitogenic communities trigger intestinal pathologies via shared or distinct immune pathways. This knowledge could potentially explain the variable roles that have been suggested for various immune effectors and pathways for IBD pathogenesis.

Notably, various human disease conditions have been associated with imbalances in the composition of the gut microbiota, so-called dysbiosis; however, whether these changes contribute directly to the development of the disease or reflect an altered physiology of the host remains debated in many instances (). In various mouse models of IBD, the microbiota and, in some cases, specific members have been shown to influence disease outcome (). Examples of IBD mouse models that lack disease development in the absence of any microbiota are the Il10model of colitis and the TNFmodel of ileitis (). Furthermore, disease development in these models is impaired or delayed under specific pathogen-free (SPF) conditions compared with conventional housing conditions, which potentially contain pathogenic bacteria, demonstrating that particular microbiota members or distinct communities only present in conventionally housed mice modulate disease onset (). Specifically, Enterobacteriaceae in TbetRag2mice () as well as Bacteroides spp. (), Helicobacter spp. (), and Bilophila wadsworthia () in Il10have been shown to enhance intestinal inflammation.

Inflammatory bowel disease (IBD) consists of a complex group of incurable inflammatory disorders comprising Crohn’s disease (CD) and ulcerative colitis (UC). Although the etiopathogenesis of IBD development is not fully understood, numerous studies support the hypothesis of IBD as a pathological immune response against microbial and environmental antigens in genetically predisposed individuals (). The relative contribution of innate and adaptive immune cells and various cytokines to the development of IBD has been controversially debated (). Nonetheless, an imbalanced interaction between the host immune system and gut microbiota is thought to play a pivotal role in disease manifestation and maintenance ().

Although the DysN6 and SPF-2 communities both trigger severe colitis in the host, the mechanisms of pathogenesis are completely opposing. Consequently, we wanted to understand whether triggering of innate or adaptive immunity by the SPF-2 or DysN6 community dominate over each other when cotransferring them into SPF-1 recipients. Analysis of microbiota composition in recipient mice after cohousing of SPF-1 recipient as well as SPF-2 and DysN6 donor mice showed that the resulting community largely resembled the cDysN6 community ( Figure 7 C). Accordingly, the host gene expression signatures in cDysN6+SPF-2 mice were similar to the ones observed in cDysN6 mice, including upregulation of genes associated with T cell, B cell, cytokine, and chemokine signaling as well as upregulation of Cd4 ( Figure 7 D; Figures S7 E and S7F). Moreover, mice with cDysN6+SPF-2 displayed high weight loss, intestinal inflammation, colon shortening, and mortality compared with SPF-1 mice ( Figure 7 E; Figures S7 G and S7H). Strikingly, the cDysN6+SPF-2 community failed to induce severe colitis in CD4mice ( Figure 7 F; data not shown). These results demonstrate that the DysN6 community and its pathogenesis mechanism (i.e., the induction of pathogenic antigen-specific CD4T cell responses) dominate over SPF-2-induced changes during colitis induction.

To investigate whether recognition of microbial antigens by CD4T cells is required for exacerbation of colitis in DysN6 mice, DSS colitis was induced in OTII transgenic mice colonized with the SPF-1, cSPF-2, or cDysN6 communities ( Figure S7 A). Strikingly, cDysN6 OTII mice did not display exacerbation of DSS colitis severity, as indicated by the lack of DysN6 transfer-induced changes in body weight loss, intestinal inflammation, and colon shortening ( Figures 7 A and 7B ; Figure S7 B). In contrast, cSPF-2 OTII mice were characterized by similar weight loss compared with cSPF-2 WT mice ( Figures S7 C and S7D). This shows that antigen specificity of CD4T cells is required for modulation of disease severity by the DysN6 but not SPF-2 community.

(C–E) SPF-1 WT mice were cohoused with DysN6, SPF-2, or both DysN6 and SPF-2 donor mice, respectively. Shown is β-diversity analysis (PCoA) of fecal microbiota (C) and pathway analysis based on GO terms of genes significantly upregulated (2-fold), as determined by RNA-seq in cDysN6+SPF-2 compared with SPF-1 mice (D). Also shown are body weight loss and survival after induction of DSS colitis (E).

To further investigate the ability of the DysN6 to drive T cell-mediated intestinal inflammation, we transferred CD45RB(high)Foxp3-CD4T cells from IL-17AIFN-γFoxP3triple reporter mice into SPF-1, cSPF-2, and cDysN6 Rag2mice. After 2 weeks, when mice differed only mildly in their weight loss ( Figure 6 H), we already observed higher intestinal inflammation, as quantified by colonoscopy in cDysN6 compared with SPF-1 and cSPF-2 recipients ( Figure 6 I). cDysN6 mice displayed an enhanced colon weight to length ratio and cellular infiltration ( Figures 6 J and 6K). Specifically, IFN-γCD4T cells numbers were significantly increased ( Figure S6 G). Although the numbers of IL-17Aand double cytokine-producing T cells were also significantly enhanced in cDysN6-colonized mice, their total numbers were much lower than those of IFN-γCD4T cells ( Figure S6 G). Taken together, this demonstrates that DysN6 induces pathogenic CD4T cells producing high levels of proinflammatory cytokines. Moreover, these microbiota-induced cells are essential to drive disease in two distinct colitis models.

Consequently, we extended our immunophenotyping and analyzed the production of proinflammatory cytokines in CD4T cells before and during DSS colitis. We initially focused on IFN-γ and IL-17 and, hence, isolated colonic LPLs (cLPLs) from SPF-1, cSPF-2, and cDysN6 IL-17AIFN-γFoxP3triple reporter mice allowing the in situ monitoring of cytokine production (). Transfer of the DysN6 but not SPF-2 community resulted in enhanced numbers of IL-17A and IFN-γ single and IL-17A/IFN-γ double cytokine-producing CD4T cells already before induction of DSS colitis ( Figure 6 F). After induction of DSS colitis, enhanced numbers of cytokine-producing CD4T cells were observed in mice with both colitogenic communities ( Figure 6 F). In addition to monitoring cytokine production in situ, we isolated cLPLs from SPF-1, cSPF-2, and cDysN6 mice before and after induction of DSS colitis and stimulated them with αCD3 and αCD28 to quantify cytokine production from T cells. Strikingly, T cells from cDysN6 mice produced larger amounts of IL-17A and IFN-γ than T cells from SPF-1 and cSPF-2 mice ( Figure 6 G), both during the steady state and colitis. Notably, TNF-α production after restimulation of T cells was highest during colitis in mice colonized with SPF-2 ( Figure 6 G).

To investigate whether pathogenic CD4T cells are required during DysN6-enhanced DSS colitis, we treated SPF-1 and cDysN6 WT mice during DSS colitis with an isotype control antibody or depleting antibodies against CD4 or CD8, respectively. Depletion of CD4-expressing cells, but not CD8-expressing cells, resulted in failure of the DysN6 community to exacerbate DSS colitis ( Figure 6 E), highlighting that CD4T cells are required during the development of DysN6-enhanced colitis.

CD4but also CD8T cells contribute to different aspects of intestinal homeostasis and inflammation (). Hence, we compared DSS colitis severity in SPF-1 and cDysN6 CD4and CD8mice. Fecal microbiota of mice clustered again according to SPF-1 and cDysN6 but not according to genotype ( Figures S6 A and S6B). After DSS induction, CD8but not CD4mice showed enhanced weight loss and colitis severity after DysN6 transfer, comparable with WT mice ( Figures 6 A and 6B ; Figure S6 C). Furthermore, analysis of intestinal inflammation using histology and quantification of colon shortening ( Figures 6 C and 6D) in SPF-1 and cDysN6 WT and CD4mice corroborated that CD4but not CD8T cells are required for DysN6-induced exacerbation of disease. To evaluate whether CD4T cells contribute to enhanced colitis severity by the colitogenic SPF-2 microbiota, we introduced the SPF-2 community into SPF-1 WT and CD4mice. Analysis of fecal microbiota of mice confirmed clustering according to microbiota and not by genotype ( Figure S6 D). Deficiency in CD4T cells did not affect the transfer of heightened disease severity ( Figure S6 E), further supporting that SPF-2 drives colitis severity irrespective of T cells.

(H–K) T cell transfer colitis was induced by injecting CD4 + Foxp3 − CD45RB(high) T cells into SPF-1, cDysN6, or cSPF-2 Rag2 −/− recipients. Body weight was measured after T cell transfer (H). Shown is the colonoscopy severity score on day 14 after transfer (I). Colon weight/length ratio (J) and total numbers of CD4 + cells in cLPLs on day 16 after injection were monitored by FACS (K). n = 7–14 mice/group.

(G) cLPLs were isolated from SPF-1, cDysN6, and cSPF-2 mice during the steady state and on day 5 after DSS colitis induction and restimulated with α-CD3/CD28 for 3 days. Cytokine levels were measured from supernatant. n = 5 mice/group.

(B–D) SPF-1 WT and CD4 −/− mice were cohoused with DysN6 donor mice, and DSS colitis was induced. Body weight (B) during DSS colitis as well as colon shortening (C) and intestinal inflammation (D) on day 5 of DSS colitis were monitored. Shown are representative pictures of H&E-stained colon sections. Scale bars represent ∼50 μm (D). n = 5–20 mice/group.

To investigate which type of pathogenic adaptive immune responses contribute to disease exacerbation after colonization with the DysN6 community, we decided to compare the severity of DSS colitis in WT as well as B or T cell-deficient mice under SPF-1 and cDysN6 conditions. To assure comparable microbiota composition in WT and gene-deficient mice at baseline, all gene-deficient mouse lines were initially rederived into SPF-1 conditions using embryo transfer. To then generate experimental cohorts of WT and gene-deficient mice, the DysN6 microbiota was transferred into SPF-1 recipients using FT or cohousing, and the composition of the fecal microbiota was recorded before induction of disease. To investigate an involvement of T and B cells, we studied SPF-1 and cDysN6 Tcrbdand muMTmice, respectively. Comparison of microbiota composition and multi-variate analysis before the start of DSS colitis revealed that mice clustered according to SPF-1 and cDysN6 communities, with genotype contributing only little (i.e., 3%) to differences in microbiome composition ( Figures S5 A and S5B). Despite a similar transfer of DysN6 into WT and Tcrbdmice, strikingly, no difference in the severity of DSS colitis was observed between SPF-1 and cDysN6 Tcrbdmice, as indicated by similar weight loss, unlike in WT mice, which showed microbiota-modulated disease severity ( Figure 5 A). An involvement of T cells in transferring exacerbated disease severity was further corroborated by analyzing intestinal inflammation using histology ( Figure 5 B) and endoscopy ( Figure S5 C) as well as quantifying colon shortening ( Figure S5 D) of WT and Tcrbdmice. In contrast to WT mice, deficiency in T cells resulted in no detectable differences in these parameters between SPF-1 and cDysN6 Tcrbdmice. Transfer of the DysN6 community into SPF-1 muMTmice resulted in exacerbation of DSS colitis severity, as indicated by significantly enhanced weight loss, colon shortening, and heightened intestinal inflammation compared with SPF-1 muMTmice, suggesting limited involvement of B cells in colitis exacerbation ( Figures 5 C and 5D; Figure S5 D). To investigate whether T cells are also required for disease exacerbation by the colitogenic SPF-2 microbiota, we introduced the SPF-2 community into SPF-1 WT, Tcrbd, and muMTmice. After confirming that the fecal microbiota of mice clustered according to their microbial communities and not by genotype ( Figure S5 E and S5F), we induced DSS colitis. As expected from the results with Rag2mice, deficiency in B or T cells alone did not affect the transfer of heightened disease severity by cSPF-2 ( Figure S5 G). γδ T cells have been implicated in colonic tissue repair (); hence, we next characterized DSS colitis severity in SPF-1 and cDysN6 Tcrdmice. Notably, characterization of fecal microbiota demonstrated that mice clustered according to SPF-1 and cDysN6 microbiota ( Figure S5 H). Similar to what we observed in WT mice, transfer of DysN6 microbiota induced in Tcrdmice enhanced weight loss, colon shortening, and heightened intestinal inflammation ( Figures 5 E and 5F; Figure S5 I). From these results we concluded that T cells are essential for DysN6- but not SPF-2-induced exacerbation of disease. Specifically, our data suggest that modulation of αβ T cells by members of the DysN6 community is important. Finally, we exclude a major contribution of B cells to DysN6-mediated colitis.

(A–F) SPF-1 WT and SPF-1 gene-deficient mice were cohoused with a DysN6 donor. Body weight was monitored upon induction of DSS colitis (A, C, and E). Histological analysis of the distal colon was performed 5 days after induction of DSS colitis (B, D, and F). Shown are representative pictures of H&E-stained colon sections. Scale bars represent ∼50 μm (B, D, and F).

Despite similar disease severity in DysN6 and SPF-2 mice upon DSS colitis induction, our initial results corroborated the hypothesis that distinct colitogenic communities contribute to disease development via different pathways. To further compare intestinal inflammation induced in cDysN6 compared with cSPF-2 mice, the presence of cytokines and chemokines was measured in tissue homogenates on day 7 of DSS colitis. The levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-17A were significantly higher in the distal colon of both cDysN6 and cSPF-2 mice compared with SPF-1 mice ( Figure S4 A). Compared with SPF-1 and cDysN6, colitis induced in cSPF-2 mice was distinctively characterized by higher levels of interferon γ (IFN-γ), IL-22, and tumor necrosis factor alpha (TNF-α) as well as lower levels of IL-18, mainly in the distal colon ( Figure S4 A). No changes were observed in IL-2, IL-4, IL-5, IL-10, and IL-13 between the three microbiota communities (data not shown). In line with our previous observations (), higher levels of the chemokine CCL5 were detected in the proximal colon of cDysN6 mice compared with SPF-1 and cSPF-2 mice ( Figure S4 B). In contrast, several other chemokines, including LIX and KC, which recruit and activate neutrophils, along with MIP-1a and MIP-1b, were significantly increased during colitis induced by cSPF-2 ( Figure S4 B). In parallel, we analyzed lamina propria leukocytes (LPLs) from colonic tissue by flow cytometry to identify whether distinct immune cell subsets are associated with disease induced by SPF-1, cDysN6, and cSPF-2 communities. Indeed, 2-fold increased numbers of CD45cells were observed in cDysN6 WT mice compared with SPF-1 and cSPF-2 WT mice both before and 5 days after induction of DSS colitis ( Figure 4 A). In line with the enhanced levels of neutrophil-attracting chemokines, colitis in cSPF-2 mice was associated with a specific increase in the relative abundance and total number of neutrophils ( Figures 4 B–4D). However, all SPF-1-, cSPF-2-, and cDysN6-colonized mice did not demonstrate any significant difference in disease outcome while being treated with antibody against Ly6G compared with the isotype control (data not shown). This might indicate a complex interaction among different components of the innate immune system to enhance microbiota-mediated colitis severity. Despite similar frequencies of immune cell subsets of the adaptive immune system ( Figures S4 C and S4D), significant increases in the numbers of B220B cells and CD3T cells were observed before and after induction of DSS colitis in cDysN6 mice ( Figures 4 E and 4F). Increases in the numbers of CD4and CD8T cells, but not γδ T cells, contributed to this difference ( Figure 4 F). During, but not before DSS colitis, a higher frequency of CD4T cells in the colon of cDysN6 and cSPF-2 mice displayed an activated phenotype ( Figure S4 D). Notably, the absolute numbers of activated CD4T cells were only increased in cDysN6 mice, both before and after induction of DSS colitis ( Figure 4 F). These analyses show that two colitogenic communities trigger distinct inflammatory immune pathways—i.e., enhanced neutrophil recruitment and pathogenic adaptive immune cell responses—during DSS colitis.

(E and F) Analysis of adaptive immune cells upon DSS induction. Representative FACS plots show CD4 and CD8 frequencies gated on CD3 + cells and frequencies of naive and activated CD4 + T cells during the steady state (E). Also shown are total numbers of the indicated immune cell subsets on day 0 and day 5 of DSS (F).

Because transfer of the colitogenic SPF-2 community, unlike the DysN6 community, did not trigger large changes in the host transcriptome in the intestine, we hypothesized that the mere presence of the SPF-2 community may be sufficient to trigger more severe colitis upon damage to the intestinal barrier. Therefore, we assessed disease severity in SPF-1 mice that received FT of the SPF-2 or DysN6 community 2 or 28 days prior to disease induction, respectively. Despite minor but detectable differences in communities of mice receiving FT for 2 or 28 days ( Figures 3 A and 3C ; Figure S3 A), brief colonization with the SPF-2 microbiota was sufficient to transfer exacerbated disease severity that was comparable with the result following extended colonization ( Figure 3 B). In contrast, brief colonization with the DysN6 microbiota did not transfer heightened disease susceptibility ( Figure 3 D). This inability of the DysN6 microbiota to transfer colitis severity potentially results from incomplete microbiota transfer, a requirement for extended immunomodulation or priming of adaptive immune responses. Comparison of the communities in mice receiving the DysN6 FT for 2 or 28 days by linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed very minute differences ( Figure 3 E), including a higher abundance of SFB as well as Odoribacteriaceae 28 days after the transfer. Notably, despite successful transfer of colitis severity, communities differed stronger in the case of SPF-2 FT ( Figure 3 F). Interestingly, similar to the DysN6 FT, SFB and Odoribacteriaceae displayed higher abundances 28 days after SPF-2 transfer. This suggests that these bacteria may not be involved in modulating DSS colitis severity. Next, to test whether DysN6 requires priming of adaptive immunity, we compared the severity of DSS colitis between Rag2mice harboring either the SPF-1, cSPF-2, or cDysN6 communities. Strikingly, unlike in WT mice, cDysN6 could not enhance colitis severity in Rag2mice, as indicated by similar weight loss ( Figure 3 G) and colon length ( Figure S3 D) between Rag2mice with SPF-1 and cDysN6. In contrast, cSPF-2 also induced severe colitis in Rag2mice, as indicated by increased weight loss and mortality ( Figure 3 H; Figure S3 E). Importantly, we confirmed comparable transfer of the donor communities into WT and Rag2mice ( Figures S3 B and S3C). We used permutational multivariate analysis of variance (ADONIS) (), considering the variables “genotype,” “microbiota,” and “cage” to evaluate their relative contribution to variability within the groups ( Figures S3 B and S3C). This analysis revealed that genotype contributed only 3% of variability, whereas microbiota contributed around 60%. Together, these data demonstrate that extended immunomodulation and priming of adaptive immunity by DysN6, but not SPF-2, are required to exacerbate colitis severity.

Induction of DSS colitis has been shown to alter the composition of the intestinal microbiota (). To identify whether a shared group of commensals alters their abundance during DSS colitis in SPF-1 mice as well as in cSPF-2 and cDysN6 mice, we compared their fecal microbial communities before and after induction of DSS colitis (day 5). Strikingly, β-diversity analysis (PCoA) as well as an analysis of relative abundances of different bacterial families revealed minor differences between the two time points for each community, respectively ( Figure 2 E; Figure S2 D). Minor alterations included an increase in Verrucomicrobiaceae in cSPF-2 and an increase in abundance of some Bacteroidaceae in cDysN6 ( Figure 2 F; Figure S2 D), but no unified changes were observed between the cSPF-2 and cDysN6 communities despite a similar induction of colitis at this time point. Hence, we hypothesized that colitogenic communities already modulate host immunity before disease induction, which, in turn, results in enhancement of colitis severity. Thus, global gene expression in colonic tissues of mice harboring either SPF-1, cSPF-2, or cDysN6 was compared using RNA sequencing (RNA-seq). Interestingly, SPF-1 and cSPF-2 mice clustered together and separately from cDysN6 mice with a distinct gene expression signature ( Figure 2 G; Figure S2 E). Specifically, pathway enrichment analysis showed that many upregulated genes in cDysN6 were involved in T cell and B cell signaling as well as cytokine and chemokine signaling ( Figure 2 H). In contrast, despite the fact that a similar colitis severity outcome was observed in cDysN6 mice, SPF-2 colonization of SPF-1 mice did not result in significant alterations in the host transcriptome ( Figure 2 G; Figure S2 E). These data together suggest that alteration of the SPF-1 community by colonizing it with colitogenic SPF-2 or DysN6 triggers a very different response at the host transcriptional level.

Next we investigated whether the degree of colitis severity was also transferable between SPF mice with variable DSS colitis susceptibility, similar to what has been observed for Nlrp6 inflammasome-deficient mice (DysN6) (). Therefore, we performed cohousing experiments of mice featuring mild colitis (SPF-1) with mice having high colitis severity (SPF-2 and DysN6). Cohousing for 4 weeks resulted in reshaping of the microbiota in SPF-1 mice cohoused with SPF-2 mice (SPF-1 + SPF-2) and DysN6 mice (SPF-1 + DysN6) compared with SPF-1 control mice, respectively ( Figure 2 A). Moreover, cohousing also transferred colitis susceptibility ( Figure 2 B; Figure S2 A). Because SPF-1 + SPF-2 and SPF-1 + DysN6 mice behaved like SPF-2 and DysN6 mice, we refer to them hereafter as cSPF-2 and cDysN6 (cohoused SPF-2 or DysN6), respectively. A similar transfer of colitis severity was also achieved by cohousing SPF-2 with SPF-6 mice ( Figure S2 C) and after fecal transplantation (FT) from SPF-2 and DysN6 mice into SPF-1 mice (data not shown). Increased colitis severity in cSPF-2 and cDysN6 mice was also illustrated by enhanced colon shortening and corroborated by histological characterization of tissue damage as well as endoscopy ( Figures 2 C and 2D; Figure S2 B). These data demonstrate that distinct types of microbial communities are able to alter the host’s susceptibility to DSS colitis even in already colonized immunocompetent recipients.

Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test (B) and nonparametric Kruskal-Wallis test (C and D):p < 0.05,p < 0.01,p < 0.001,p < 0.0001. See also Figure S2

(G and H) RNA-seq analysis from total colonic tissue of WT mice colonized with SPF-1, cSPF-2, or cDysN6. The heatmap shows quantification of RNA reads (G). Also shown is a pathway analysis based on gene ontology (GO) terms of genes significantly upregulated (2-fold) in cDysN6 mice compared with SPF-1 (H). n = 4 mice/group.

(E and F) 16S rRNA sequencing of fecal microbiota from WT SPF-1, cSPF-2, and cDysN6 on day 0 and day 5 of DSS colitis. Shown are analysis of β-diversity (PCoA) (E) and analysis of differentially abundant microbial families in cDysN6 and cSPF-2 mice on day 0 and day 5 of DSS by LEfSe (Kruskal-Wallis test, p < 0.05, LDA 4.0) (F). n = 8–12 mice/group.

(B–D) Acute DSS colitis was induced, and the weight of microbiota recipient mice was monitored for 10 days (B). Colon length was measured 5 days after induction of DSS colitis. Shown is a representative image of excised colons (C). Histological analysis of distal colon was performed 5 days after induction of DSS colitis (D). Representative pictures of H&E-stained colon sections are shown. The scale bars represent ∼50 μm. n = 5–16 mice/group.

To exclude the effect of genetic drift in inbred mice from different sources, we performed cohousing experiments with microbiota donor and germ-free recipient mice. We focused on SPF-1 (low susceptibility, higher Firmicutes), SPF-2 (high susceptibility, higher Bacteroides), and DysN6 mice (high susceptibility, higher Bacteroides, and higher Proteobacteria) representing the different colitis outcomes and microbiota compositions. Transfer of the donor microbiota into germ-free (GF) recipient (exGF) mice was confirmed by 16S rRNA gene sequencing ( Figure 1 E). Upon induction of DSS colitis, exGF mice phenocopied the respective donor mice, supporting that the differences in colitis severity were dependent on the microbiota ( Figure 1 E). Similar microbiota-driven phenotypes were confirmed for the SPF-5 and SPF-6 communities (data not shown). These data demonstrate that distinct types of microbial communities that are stably maintained in wild-type (WT) mice are able to alter the host’s susceptibility to DSS colitis.

Distinct differences in microbiota composition between isogenic mice from commercial vendors—e.g., the presence of segmented filamentous bacteria (SFB)—have been found to influence the outcome of disease models in mice (). To investigate whether C57BL/6N mice differ in their susceptibility to intestinal inflammation after chemically induced damage to the intestinal barrier, we induced DSS colitis in SPF mouse lines obtained from vendors or bred in-house ( Figure 1 A; Table S1 ). The severity of disease was compared within lines of SPF mice and with previously described dysbiotic Nlrp6mice that were obtained from the original vivarium and subsequently bred in our animal facility without rederivation ( Figure 1 B; Figure S1 A;). SPF-1, SPF-5, and SPF-6 mice were characterized by mild colitis with moderate weight loss and no mortality, but SPF-2, SPF-3, and SPF-4 mice as well as dysbiotic Nlrp6mice developed a similar severe colitis with profound loss of body mass and mortality ( Figure 1 B; Figure S1 A). Colitis severity in each representative isogenic mouse line from different commercial or in-house sources (SPF-1, SPF-2, SPF-4, SPF-6, and DysN6) was also illustrated by measuring colon shortening and supported by histological characterization of tissue damage ( Figures S1 C and S1D). Next we investigated fecal microbiota composition before induction of DSS colitis using 16S rRNA gene sequencing. Analysis of β diversity using principle coordinates analysis (PCoA) showed that mice with mild colitis severity (SPF-1, SPF-5, and SPF-6) clustered separately from mice featuring a high severity of colitis (SPF-2, SPF-3, SPF-4, and DysN6). We noted a high similarity between SPF-2, SPF-3 (both from different barriers of the same vendor), and SPF-4 mice as well as between SPF-5 and SPF-6 mice (both from different barriers of the same vendor), respectively, whereas SPF-1 and DysN6 mice clustered distinctly ( Figure 1 C). A more detailed analysis revealed that species richness (Chao index) was lower in SPF-1 mice but that the complexity of the community structure (Shannon index) was not significantly different between mouse lines ( Figure S1 B). Global changes in the composition of microbiota have been associated with IBD (), such as a decrease in the level of resident Firmicutes and/or Bacteroides and an overabundance of Proteobacteria (). We observed a significant expansion of Bacteroides over Firmicutes in colitogenic SPF-2, SPF-3, SPF-4, and DysN6 mice compared with SPF-1, SPF-5, and SPF-6 mice ( Figure 1 D). Overgrowth in Proteobacteria was highest in DysN6 mice, followed by SPF-2, SPF-3, SPF-4, and SPF-5 mice, and was mostly absent in SPF-1 and SPF-6 mice ( Figure 1 D; Table S2 ).

(E) Germ-free C57BL/6N mice were cohoused with donor SPF WT (SPF-1, SPF-2) and Nlrp6 −/− (DysN6) mice, followed by induction of DSS colitis. Shown is analysis of β-diversity (PCoA) before and disease severity (body weight and survival) upon induction of DSS colitis. n = 7–8 mice/group.

(C and D) Analysis of fecal microbiota composition of the mice described in (A) before DSS colitis induction using 16S rRNA sequencing. Shown is analysis of β-diversity (PCoA) (C) and the ratio of relative abundances between Firmicutes to Bacteroides and Firmicutes to Proteobacteria (D). n = 15–33 mice/group.

Discussion

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Flavell R.A. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. + T cells in DysN6 mice, hinting toward a potential involvement of these cells in intestinal pathogenesis. The hypothesis of adaptive immune cells being involved in DysN6 mice was further corroborated by the observation that extended colonization with the DysN6 but not SPF-2 community was required to transfer disease susceptibility. Subsequently, we evaluated the effect of the transfer of the two colitogenic communities in mice lacking specific subsets of adaptive immune cells. For these comparisons we employed WT and gene-deficient mice that were embryo-transferred into our vivarium using SPF-1 foster mothers, resulting in a standardized microbiota (E.J.C.G, unpublished data). Moreover, we included cohousing of WT and gene-deficient mice to further reduce microbiota variability within experiments and documented, for all experiments, microbiota composition using 16S rRNA gene sequencing. Using this carefully controlled approach, we observed significant increases in IL-17A and IFN-γ secretion by CD4+ T cells during DysN6- and SPF-2 driven colitis. Notably, this is in line with an association of CD4+ T cells and proinflammatory cytokines, including IL-17, IFN-γ, and IL-23, with human IBD ( Kaser et al., 2010 Kaser A.

Zeissig S.

Blumberg R.S. Inflammatory bowel disease. + T cells are only essential to mediate the exacerbation of DSS colitis in DysN6 but not SPF-2 mice. In contrast, despite measurable CD4+ T cell activation during DSS colitis, SPF-2 modulated disease severity independent of adaptive immune cells. T cell receptor (TCR)-mediated recognition of cognate antigens is required for proper T cell function, and recognition of microbial antigens has been suggested to significantly contribute to the development of colitis ( Feng et al., 2010 Feng T.

Wang L.

Schoeb T.R.

Elson C.O.

Cong Y. Microbiota innate stimulation is a prerequisite for T cell spontaneous proliferation and induction of experimental colitis. + T cells. The presence of in vivo cytokine-secreting CD4+ T cells before induction of DSS colitis in DysN6 mice suggests that colonic CD4+ T cells already recognize cognate microbial antigens during this phase, similar to what has been observed for SFB-specific CD4+ T cells in the small intestine ( Yang et al., 2014 Yang Y.

Torchinsky M.B.

Gobert M.

Xiong H.

Xu M.

Linehan J.L.

Alonzo F.

Ng C.

Chen A.

Lin X.

et al. Focused specificity of intestinal TH17 cells towards commensal bacterial antigens. + T cells during colitis resulted in failure to transfer enhanced colitis susceptibility. This demonstrated that, to enhance colitis, modulation of the mucosal barrier by CD4+ T cells in the steady state was not sufficient and, rather, required the presence and, presumably, the effector functions of CD4+ T cells during colitis. The distinct property of the DysN6 community to prime and activate pathogenic CD4+ T cell responses was further corroborated using a model for CD4+ T cell-mediated colitis. Specifically, transfer of CD4+ T cells in Rag2−/− mice harboring the DysN6 but not the SPF-2 microbiota enhanced intestinal inflammation and cytokine production by CD4+ T cells. Whether these different communities also cause different disease susceptibility or pathogenesis via shared or distinct pathways in other inbred mouse strains or IBD models such as the Il10−/− model of colitis and the TNFdeltaARE model of ileitis, remains to be tested ( Keubler et al., 2015 Keubler L.M.

Buettner M.

Häger C.

Bleich A. A Multihit Model: Colitis Lessons from the Interleukin-10-deficient Mouse. Schaubeck et al., 2016 Schaubeck M.

Clavel T.

Calasan J.

Lagkouvardos I.

Haange S.B.

Jehmlich N.

Basic M.

Dupont A.

Hornef M.

Von Bergen M.

et al. Dysbiotic gut microbiota causes transmissible Crohn’s disease-like ileitis independent of failure in antimicrobial defence. A common feature in IBD, particularly in UC, is impairment of the intestinal barrier, resulting in enhanced exposure to luminal microbes. By employing a mouse model of damage to the intestinal barrier, DSS colitis, we demonstrate that isogenic SPF mice with differences in microbiome composition feature altered susceptibility to intestinal inflammation. Specifically, we noted that transfer of colitogenic communities into mice relatively resistant to induction of DSS colitis is sufficient to alter disease susceptibility even in immunocompetent mice. Upon induction of disease, the DysN6 community as well as the SPF-2 community induced severe colitis compared with the relatively resistant SPF-1 community, but the mechanisms of pathogenesis differed strongly. Inflammation in SPF-2 mice was characterized by high levels of TNF-α and neutrophil-attracting chemokines coinciding with significant higher infiltration of neutrophils into the inflamed tissue. In line with previous findings, DysN6 mice featured higher levels of the chemokine CCL5, known to attract innate and adaptive immune cells carrying CCR1, CCR3, CCR4, and CCR5 (). Here we identified high infiltration of activated CD4T cells in DysN6 mice, hinting toward a potential involvement of these cells in intestinal pathogenesis. The hypothesis of adaptive immune cells being involved in DysN6 mice was further corroborated by the observation that extended colonization with the DysN6 but not SPF-2 community was required to transfer disease susceptibility. Subsequently, we evaluated the effect of the transfer of the two colitogenic communities in mice lacking specific subsets of adaptive immune cells. For these comparisons we employed WT and gene-deficient mice that were embryo-transferred into our vivarium using SPF-1 foster mothers, resulting in a standardized microbiota (E.J.C.G, unpublished data). Moreover, we included cohousing of WT and gene-deficient mice to further reduce microbiota variability within experiments and documented, for all experiments, microbiota composition using 16S rRNA gene sequencing. Using this carefully controlled approach, we observed significant increases in IL-17A and IFN-γ secretion by CD4T cells during DysN6- and SPF-2 driven colitis. Notably, this is in line with an association of CD4T cells and proinflammatory cytokines, including IL-17, IFN-γ, and IL-23, with human IBD (). Strikingly, our experiments demonstrated that CD4T cells are only essential to mediate the exacerbation of DSS colitis in DysN6 but not SPF-2 mice. In contrast, despite measurable CD4T cell activation during DSS colitis, SPF-2 modulated disease severity independent of adaptive immune cells. T cell receptor (TCR)-mediated recognition of cognate antigens is required for proper T cell function, and recognition of microbial antigens has been suggested to significantly contribute to the development of colitis (). Using OTII transgenic mice, we could show that DysN6-driven but not SPF-2-driven colitis development strongly depended on the presence of antigen-specific CD4T cells. The presence of in vivo cytokine-secreting CD4T cells before induction of DSS colitis in DysN6 mice suggests that colonic CD4T cells already recognize cognate microbial antigens during this phase, similar to what has been observed for SFB-specific CD4T cells in the small intestine (). Importantly, antibody-mediated depletion of CD4T cells during colitis resulted in failure to transfer enhanced colitis susceptibility. This demonstrated that, to enhance colitis, modulation of the mucosal barrier by CD4T cells in the steady state was not sufficient and, rather, required the presence and, presumably, the effector functions of CD4T cells during colitis. The distinct property of the DysN6 community to prime and activate pathogenic CD4T cell responses was further corroborated using a model for CD4T cell-mediated colitis. Specifically, transfer of CD4T cells in Rag2mice harboring the DysN6 but not the SPF-2 microbiota enhanced intestinal inflammation and cytokine production by CD4T cells. Whether these different communities also cause different disease susceptibility or pathogenesis via shared or distinct pathways in other inbred mouse strains or IBD models such as the Il10model of colitis and the TNFmodel of ileitis, remains to be tested (). This shows that colitogenic communities exert their pathogenic effects in the same disease model by opposing mechanisms.

Ivanov et al., 2009 Ivanov I.I.I.I.

Atarashi K.

Manel N.

Brodie E.L.

Shima T.

Karaoz U.

Wei D.

Goldfarb K.C.

Santee C.A.

Lynch S.V.S.

et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. + T cells can be excluded. Similarly, members of the genus Prevotella, previously found to be enriched in the colitogenic microbiota of Nlrp6−/− mice ( Elinav et al., 2011 Elinav E.

Strowig T.

Kau A.L.

Henao-Mejia J.

Thaiss C.A.

Booth C.J.

Peaper D.R.

Bertin J.

Eisenbarth S.C.

Gordon J.I.

Flavell R.A. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. −/− mice in cooperation with other members of the microbiota ( Keubler et al., 2015 Keubler L.M.

Buettner M.

Häger C.

Bleich A. A Multihit Model: Colitis Lessons from the Interleukin-10-deficient Mouse. Petersen and Round, 2014 Petersen C.

Round J.L. Defining dysbiosis and its influence on host immunity and disease. Frank et al., 2007 Frank D.N.

St Amand A.L.

Feldman R.A.

Boedeker E.C.

Harpaz N.

Pace N.R. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Couturier-Maillard et al., 2013 Couturier-Maillard A.

Secher T.

Rehman A.

Normand S.

De Arcangelis A.

Haesler R.

Huot L.

Grandjean T.

Bressenot A.

Delanoye-Crespin A.

et al. NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer. Hu et al., 2015 Hu S.

Peng L.

Kwak Y.T.

Tekippe E.M.

Pasare C.

Malter J.S.

Hooper L.V.

Zaki M.H. The DNA Sensor AIM2 Maintains Intestinal Homeostasis via Regulation of Epithelial Antimicrobial Host Defense. Roberts et al., 2014 Roberts M.E.

Bishop J.L.

Fan X.

Beer J.L.

Kum W.W.S.

Krebs D.L.

Huang M.

Gill N.

Priatel J.J.

Finlay B.B.

Harder K.W. Lyn deficiency leads to increased microbiota-dependent intestinal inflammation and susceptibility to enteric pathogens. Detailed characterization of the colitogenic communities using 16S rRNA gene sequencing revealed the varying presence of potential pathobionts such as SFB, Prevotella spp., Helicobacter spp., Enterobacteriaceae, and Verrucomicrobiaceae in DysN6 and SPF-2 mice. SFB have been shown to modulate intestinal T cell immunity and systemic autoimmunity (). However, based on their presence in both SPF-2 and DysN6 mice, a role in driving the differential requirement for CD4T cells can be excluded. Similarly, members of the genus Prevotella, previously found to be enriched in the colitogenic microbiota of Nlrp6mice (), were present in both colitogenic communities, indicating that they are not involved in regulating the different pathogenicity modes. Helicobacteraceae have been demonstrated to induce the development of colitis in Il10mice in cooperation with other members of the microbiota (). Although Helicobacteraceae were absent in SPF-2 mice, DysN6 mice harbored different members of this family, including H. typhlonius, H. rodentium, and H. muridarum, but did not harbor H. hepaticus. Finally, both Enterobacteriaceae and Verrucomicrobiaceae, specifically Akkermansia muciphilia, bloomed during induction of DSS colitis in SPF-2 mice, but it is being debated whether expansion during disease suggests a contribution to disease development or, rather, a consequence of the ability to utilize inflammation-induced metabolites. In contrast to the “one microbe one disease” model, the concept of dysbiosis, an imbalance of the community, has been proposed for microbiome-mediated modulation of diseases (). One characteristic of dysbiotic communities, including those in IBD patients, has been suggested to be an imbalance between Bacteroides, Firmicutes, and Proteobacteria, with an overexpansion of Bacteroides and Proteobacteria over Firmicutes (). Lowered Firmicutes/Bacteroides ratios were noted in all colitogenic communities, including SPF-2 and DysN6, whereas the ratios between Firmicutes and Proteobacteria (F/P) was not consistently different between susceptible and resistant groups. Notably, the F/P ratio was the lowest in DysN6 mice, and according to our data, this is associated with a distinct mode of pathogenicity. Whether, in the cases of the SPF-2 and DysN6 community, specific pathobionts or a general dysbiosis are responsible for driving distinct pathogenicity requires further investigation because dysbiotic communities have also been reported in other gene-deficient mouse lines ().

−/− mice with a similar microbiome compared with what has been reported previously ( Elinav et al., 2011 Elinav E.

Strowig T.

Kau A.L.

Henao-Mejia J.

Thaiss C.A.

Booth C.J.

Peaper D.R.

Bertin J.

Eisenbarth S.C.

Gordon J.I.

Flavell R.A. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. −/− mice raised under SPF conditions did not differ in their composition, suggesting that the development of dysbiotic communities reflects a complex interplay between genetic and environmental factors ( Mamantopoulos et al., 2017 Mamantopoulos M.

Ronchi F.

Van Hauwermeiren F.

Vieira-Silva S.

Yilmaz B.

Martens L.

Saeys Y.

Drexler S.K.

Yazdi A.S.

Raes J.

et al. Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition. Finally, it remains debated how these dysbiotic communities arise in gene-deficient mice and how similar they are in regard to their composition in different vivariums, taking into account that a large variability in the composition and function of microbial ecosystems in WT mice already exist. In this study, we employed Nlrp6mice with a similar microbiome compared with what has been reported previously (). However, it remains to be tested whether the pathological mechanism of dysbiotic communities occurring in unrelated lines of Nlrp6 inflammasome-deficient mice causes exacerbated pathology via the same or different mechanisms or does not cause any pathology at all. Along these lines, a recent study has suggested that the intestinal microbiomes of WT and Nlrp6mice raised under SPF conditions did not differ in their composition, suggesting that the development of dysbiotic communities reflects a complex interplay between genetic and environmental factors ().

In summary, our data show how distinct microbial communities drive the development of intestinal inflammation in immunocompetent hosts by modulating opposing arms of the immune system. Our study suggests the concept that triggering of different immune pathways by microbial communities can alter disease susceptibility, eventually resulting in similar host pathophysiology. This implies that personalized immunomodulatory treatment according to distinct microbial signatures may be beneficial for IBD patients.