ABSTRACT Tumor necrosis factor-like cytokine 1A (_TL1A_, _TNFSF15_) is implicated in inflammatory bowel disease (IBD), modulating the location and severity of intestinal inflammation and

fibrosis. _TL1A_ expression is increased in inflamed gut mucosa and associated with fibrostenosing Crohn’s disease. Tl1a-overexpression in mice lead to spontaneous ileitis, and exacerbated

induced proximal colitis and fibrosis. IBD is associated with shifts in the gut microbiome, but the effect of differing microbial populations and their interaction with _TL1A_ on fibrosis

has not been investigated. We demonstrate that the pro-fibrotic and inflammatory phenotype resulting from Tl1a-overexpression is abrogated in the absence of resident microbiota. To evaluate

activation in Tl1a-Tg mice. Notably, there was reduced fibroblast migration and activation under GF conditions compared to native conditions. We then identified several candidate organisms

that correlated directly with increased fibrosis in reconstituted mice and showed that these organisms directly impact fibroblast function in vitro. Thus, Tl1a-mediated intestinal fibrosis

and fibroblast activation are dependent on specific microbial populations. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS

DIRECT SIGNALING OF TL1A-DR3 ON FIBROBLASTS INDUCES INTESTINAL FIBROSIS IN VIVO Article Open access 23 October 2020 DIFFERENT FIBROBLAST SUBTYPES PROPEL SPATIALLY DEFINED ILEAL INFLAMMATION

THROUGH TNFR1 SIGNALLING IN MURINE ILEITIS Article Open access 28 March 2025 TOLL-LIKE RECEPTOR 4 REGULATES INTESTINAL FIBROSIS VIA CYTOKINE EXPRESSION AND EPITHELIAL-MESENCHYMAL TRANSITION

Article Open access 16 November 2020 INTRODUCTION Tl1a (a protein encoded by _TNFSF15_) is a member of the tumor necrosis factor (TNF) superfamily that binds to death domain receptor 3

(DR3), expressed on a variety of cell types including immune cells, epithelial cells, and fibroblasts.1,2,3,4,5 Modulating an array of immune responses, Tl1a can be produced by endothelial

cells in response to IL-1β and TNFα, by macrophages and dendritic cells in response to Toll-like receptor stimulation, as well as in some lymphoid lineage cells.2,3,6,7,8,9 A _TNFSF15_

haplotype is associated with higher Tl1a production, increased risk of CD, intestinal fibrostenosis, and greater need for surgery.10,11,12,13 In mice, constitutive Tl1a expression-induced

increased collagen deposition in the colon without detectable histologic colitis, as well as increased collagen deposition in the ileum with spontaneous ileitis.14,15,16,17 Under colitogenic

conditions induced by chronic DSS treatment or adoptive T-cell transfer, there was increased collagen deposition with fibrostenotic lesions in the gut that caused intestinal obstruction in

the Tl1a-Tg mice.12 These results support the role of Tl1a in fibrogenesis that can lead to fibrostenosis in the setting of chronic inflammation, which is a common complication of CD leading

to resection. The intestinal microbiome has been linked with many inflammatory diseases including IBD.18,19,20,21 Although previous studies have found alterations in various bacterial taxa

in IBD patients, and a recent study found an association with a fibrostenotic disease cohort, none have correlated-specific microbes with degree of fibrosis and fibroblast phenotype.22 Most

of these studies have been largely associative without the ability (by design) to prove causality. The question remains, therefore, whether altered microbiota associated with IBD contribute

to the disease phenotype or are its consequence. In rodent models, comparison of experimental IBD models under GF conditions have yielded disparate results with development of colitis in

most spontaneous genetically engineered models dependent on resident microbiota, or uniquely, potentiated DSS-induced colitis in GF mice.23,24,25 No animal experiments have evaluated the

contribution of the microbiome to fibrosis in the context of IBD. In this study, we show that the native murine fecal microbiota is required for optimal Tl1a-dependent fibroblast activation

and transformation into myofibroblasts. Moreover, we provide evidence that the intestinal fibrotic phenotype requires specific microbial cues provided by mouse microbiota from an SPF

facility but absent in human feces from a healthy donor. Our analysis further identified several candidate organisms that correlate directly with degree of fibrosis in reconstituted hosts

and impact fibroblasts in vitro. To our knowledge, this is the first study to establish a potential causal role for the microbiome in intestinal fibrosis, fibroblast activation, and

function. RESULTS THE INTESTINAL MICROBIOME IS REQUIRED FOR TL1A-ENHANCED INTESTINAL INFLAMMATION, COLLAGEN DEPOSITION AND FIBROBLAST MIGRATION In agreement with our prior results, Tl1a-Tg

mice raised under SPF microbial conditions display significant spontaneous ileitis, as evident by increased histopathological scoring under H&E, with over a twofold increase in average

histopathology compared with wild-type mice (Fig. 1a).16 No histologically apparent cecal inflammation was observed (Fig. 1b). Despite this, Tl1a-Tg mice demonstrate increased cecal collagen

deposition (Fig. 1d), underscoring the importance of TL1A as a mediator of fibrosis that can act independently of its pro-inflammatory effects. As the microbiome is relevant to inflammation

in several diseases, we evaluated the effects of GF conditions on Tl1a-mediated intestinal inflammation. The absence of a microbiome abrogated the spontaneous ileitis induced by

Tl1a-overexpression, as there were no significant differences in ileal histopathology between GF Tl1a-Tg and GF WT mice (Fig. 1a). We next evaluated if the fibrosis observed in Tl1a-Tg mice

were dependent upon the microbiome. The absence of microbes significantly reduced ileal and cecal collagen deposition in GF Tl1a-Tg mice compared with microbiome intact Tl1a-Tg mice (Fig. 

1c, d). No difference was observed in WT mice. As we observed notable changes in fibrosis between Tl1a-Tg and WT mice under native SPF conditions that were abrogated under GF conditions, we

sought to determine the impact of Tl1a-overexpression and resident microbiota on fibroblast phenotype. Colonic fibroblasts isolated from Tl1a-Tg mice raised under native microbial conditions

displayed significantly increased migratory capacity after simulated wounding compared with those from WT mice (Fig. 2a, b). Tl1a-overexpression also increased fibroblast adhesion, which

was unchanged in GF mice (Supplementary Figure 1). The enhanced rate of fibroblast gap-closure observed with Tl1a-overexpression under native conditions was eliminated under GF conditions,

consistent with the observed reduction in histological fibrosis (Fig. 2a, b). Interestingly, the absence of microbiome reduced fibroblast migratory capacity even in WT mice, but to a lesser

extent than in Tl1a-Tg mice. These results indicate that the intestinal microbiome is required for Tl1a-mediated intestinal fibrosis and influences fibroblast migratory function. An

important question arising from these results is whether the observed findings are due to direct effects of SPF microbiota and Tl1a (alone or in concert) on fibroblasts themselves, or if the

microbiome and Tl1a in Tl1a-Tg mice affect other non-fibroblast cell types, which then promote fibroblast activation and pro-fibrotic phenotype. Bacterial components and products can induce

fibroblast activation,26 but it is unclear if they can promote fibroblast migration directly. Moreover, the direct effect of bacterial stimulus on fibroblasts in the context of host

Tl1a-overexpression has not been evaluated. We therefore assessed if bacterial products isolated from the cecal luminal washings of native WT SPF mice could promote fibroblast migration

directly, and if this effect was enhanced by host Tl1a-overexpression. WT fibroblasts exposed to native SPF cecal washings demonstrate significantly increased migration compared to those

exposed to cecal washings from GF mice (Fig. 2c). This direct effect of cecal bacterial components was enhanced by host Tl1a-overexpression in Tl1a-Tg mice. Thus, a significant part of the

commensal microbiome’s effect on fibroblast migration seen in Fig. 2a, b may be due to direct effects mediated by bacterial components (or products) on fibroblasts themselves. We next asked

whether this enhanced migratory phenotype in fibroblasts from Tl1a-Tg mice were partly due to direct Tl1a-mediated effects on fibroblasts; namely, can Tl1a promote fibroblast migration

directly and do so in concert with the direct effect of SPF microbiota on fibroblasts seen in Fig. 2c? We have previously demonstrated that fibroblasts express the Tl1a receptor DR3, and

upon treatment with Tl1a in vitro, demonstrate expression of alpha-smooth-muscle actin (indicative of activation of myofibroblasts) and collagen.27 Consequently, fibroblasts treated with

Tl1a in vitro demonstrate expression of alph-smooth-muscle actin (activation to myofibroblasts) and collagen.27 We therefore hypothesized that direct stimulation of fibroblasts with Tl1a

would increase migration. WT fibroblasts treated with Tl1a (and without bacterial components) in vitro displayed significantly increased migration compared with untreated cells, suggesting a

direct effect of Tl1a on fibroblast migration (Fig. 2d). To determine whether Tl1a can enhance the fibroblast migratory response to bacterial components, we conducted the same experiments

as in Fig. 2c in the context of exogenous Tl1a stimulation. WT fibroblasts treated with both Tl1a and SPF cecal bacterial products together demonstrated enhanced migration compared with

those treated solely with the bacterial products (Fig. 2d). These data demonstrate that resident bacteria and Tl1a can both stimulate fibroblasts, and the direct effect of resident bacteria

on fibroblasts is enhanced directly by Tl1a. GAVAGE WITH MURINE BUT NOT HUMAN FECAL MICROBIOTA PROMOTES INTESTINAL INFLAMMATION AND COLLAGEN DEPOSITION IN TL1A TRANSGENIC MICE As these data

demonstrate that the microbiome is required for Tl1a-mediated fibrosis in intestinal regions with and without underlying inflammation, we sought to evaluate if this phenotype is due to the

absence of a unique bacterial population, as opposed to any bacterial colonization per se. We used two distinct microbiota to test the hypothesis that the pro-fibrotic phenotype observed in

Tl1a-Tg mice under native microbial conditions was due to a specific bacterial population adapted to the mouse intestine rather than the presence of any gut bacteria. GF mice were gavaged

with stool collected from wild-type mice housed in SPF or with stool from a healthy human (Hu) donor and evaluated 2 months later. WT mice displayed no increase in intestinal inflammation or

collagen deposition when colonized with either SPF or Hu flora, indicating that in the absence of Tl1a-overexpression the species-specific microbiome does not induce intestinal inflammation

or fibrosis (Fig. 3a–d). Tl1a-transgenic mice colonized with SPF microbiota demonstrated increased collagen deposition in both inflamed ileum and non-inflamed cecum, consistent with

findings in mice under native conditions (Fig. 3a–d, Fig. 1). In contrast, Tl1a-transgenic recipients of Hu microbiota showed no increase in ileal or cecal collagen deposition or

inflammation. Together, these data indicate that Tl1a-mediated intestinal fibrosis is modulated by the composition of the intestinal microbiome and suggest that this phenotype is induced by

microbes selectively contained in the SPF mouse microbiota but missing from the human microbiota. MURINE MICROBIOTA POTENTIATE TL1A-MEDIATED INTESTINAL FIBROBLAST DIFFERENTIATION TO

MYOFIBROBLASTS Fibroblast activation has been shown to occur after bacterial stimulation e.g., with lipopolysaccharide.26 Previously, we showed that there is an increase in the proportion of

intestinal myofibroblasts in Tl1a-Tg mice raised under conventional SPF conditions.27 We investigated whether absence of microbial stimulation (i.e., under GF conditions) impairs fibroblast

differentiation to myofibroblasts. GF Tl1a-Tg mice did not display an increased number or proportion of activated fibroblasts in the cecum compared to GF WT mice (Fig. 4a, b). Colonization

with SPF microbiota-induced intestinal myofibroblasts in both WT and Tl1a-Tg mice relative to GF conditions or colonization with Hu microbiota (Fig. 4a, b). SPF microbiota, but not Hu

microbiota also restored the increased proportion of myofibroblasts in Tl1a-Tg mice compared to WT controls (54.7% vs. 36.7%). Interestingly, GF Tl1a-Tg mice had reduced myofibroblast

proportion compared to GF WT mice, which was not seen in the presence of Hu microbiota (Fig. 4a, b). These results show that fibroblast activation in the cecum induced by Tl1a overexpression

is microbiota-dependent and that microbial composition affects fibroblast differentiation into myofibroblasts. To assess whether Tl1a mediated fibroblast activation in the ileum also

requires the microbiome, we quantitated myofibroblasts in Tl1a-Tg mice under GF conditions. In contrast to the cecum, there was an increased proportion of activated myofibroblasts in the

ileum of GF Tl1a-Tg mice compared with GF WT mice (Fig. 4c, d). We next tested whether the degree of Tl1a-mediated fibroblast activation is affected by the specific microbiome. SPF gavage

increased myofibroblast numbers and proportion in both WT and Tl1a-Tg mice while preserving the relative increase in myofibroblasts in Tl1a-Tg mice (Fig. 4c, d). In contrast, mice colonized

with Hu microbiota had reduced proportion of fibroblast activation compared with GF conditions, suggesting that members of the Hu microbiota may have inhibited fibroblast activation. This

did not result in significant histopathological differences in collagen deposition, however. Taken together, these results suggest greater modulation of Tl1a-mediated fibroblast activation

by the microbiome in the cecum (without concomitant changes in inflammation), for which Tl1a can potentially compensate in the ileum, (and in which there is significant increases in

inflammation). This may reflect distinct microbial communities and mucosal immunity in the ileum, reflected in the significant increase in ileal inflammation in Tl1a-Tg mice. Indeed,

differing microbial composition between the ileum and the cecum has been well-documented in mice and humans, including patients with IBD who show distinct microbiome profiles between subsets

with ileal vs. colonic disease.28 Consistent with this, sequencing of mucosal and luminal microbial communities in the ileum and cecum demonstrated distinct microbial populations in the

ileum vs. the cecum in both Tl1a-Tg and WT mice colonized with SPF microbiota (Supplemental Fig. 2). These data illustrate biogeographic differences in the host-microbe interactions

underlying intestinal collagen deposition. Additionally, our data demonstrated that microbial composition modulates the degree of Tl1a-mediated myofibroblast activation independent of the

intestinal location. FIBROSIS SEVERITY IS ASSOCIATED WITH THE ABUNDANCE OF SPECIFIC MICROBES FOUND IN THE MOUSE SPF MICROBIOME Since SPF and Hu microbiota had differential effects on

collagen deposition and fibroblast activation, we hypothesized that the abundance of specific bacteria would be associated with the degree of fibrosis seen in recipient mice. To evaluate

this, we performed 16S rRNA sequencing to characterize the ileal and cecal microbiome of colonized mice and then employed multivariate models to identify microbes with a statistically

significant positive or negative association with fibrosis score. These microbes were then used to construct co-occurrence/co-exclusion networks with fibrosis severity to identify the

microbes that were directly associated with increased or decreased fibrosis rather than merely having a co-occurrence or co-exclusion relationship with fibrosis-associated microbes. Separate

analyses were performed of the cecum and ileum of humanized and SPF-colonized ex-GF mice (Fig. 5). In the cecum of SPF-colonized mice, which demonstrated significant collagen deposition and

fibroblast activation, we identified several microbes not present in Hu-gavaged mice that clustered tightly with fibrosis (Fig. 5a). This included groups of mucolytic bacteria such as

_Mucispirillum schaedleri_ and _Ruminococcus_. Additionally, _Anaeroplasma_ were also significantly associated with fibrosis in the cecum of SPF-colonized mice. Members of _Oscillospira_ and

_Coprococcus_ were negatively correlated with fibrosis in the cecum (Fig. 5a). In the ileum of SPF-colonized mice, we observed that there were competing sets of microbes associated with

enhanced or reduced fibrosis severity (Fig. 5b). For example, members of the _Streptococcus_ and _Lactobacillus_ genera were found to be positively associated with fibrosis, whereas

_Faecalibacterium prausnitzii_ and members of _Bacteroides_ were negatively associated with fibrosis. Consistent with the absence of histological fibrosis, we observed only negative

correlations between microbial species and fibrosis in both the cecum and ileum of Hu-colonized mice (Fig. 5c, d). DIFFERENTIAL EFFECTS OF BACTERIA POSITIVELY OR NEGATIVELY CORRELATED WITH

FIBROSIS ON IN VITRO FIBROBLAST FUNCTION Next, we determined whether bacterial strains that positively or negatively correlated with cecal fibrosis severity in vivo could alter fibroblast

function directly in in vitro. Cell lysates of _Ruminoccocus_ and _M_. _schaedleri_, two bacterial strains that were positively correlated with the degree of fibrosis, promoted fibroblast

migration and collagen expression compared with negatively correlated _Oscillospira_, which had comparatively less pronounced effects (Fig. 6). These results show that microbes that

positively or negatively correlate with fibrosis in vivo can directly and disparately impact fibroblasts in vitro. Furthermore, one potential causal mechanism is now suggested by which

specific organisms in the gut microbiome mediate fibrosis. DISCUSSION To our knowledge, this is the first study that causally implicates the intestinal microbiome in intestinal fibrosis,

demonstrating that fibrosis requires the presence of resident microbiota and that Tl1a-mediated fibrosis is dependent upon specific bacteria or bacterial consortia. Furthermore, we show that

microbes that positively or negatively correlate with intestinal fibrosis in vivo have direct (and opposing) effects on fibroblast function in vitro. These results also suggest that

microbiome-TL1A interactions may influence the degree and location of intestinal fibrosis in IBD, which has up to now been attributed to the severity of inflammation. Accordingly, no

histologically significant cecal inflammation was observed under SPF microbial conditions but despite this, Tl1a-Tg mice demonstrate increased cecal collagen deposition, underscoring the

importance of TL1A as a modulator of the location and severity of mucosal inflammation, as well as a pro-fibrotic mediator that can act independently of its pro-inflammatory effects. Indeed,

this disjunction between inflammation and fibrosis is clinically significant. While inflammatory disease may be associated with significant fibrotic change, as increased inflammation

perpetuates the cascade of mucosal repair, the frequency of fibrostenosing complications remains significant despite immunosuppressive therapy in CD patients in the form of steroids or

immunomodulators.29 Findings that provide insight into unique pro-fibrotic mediators—whether cytokine- or microbiome-driven (or both)—are highly relevant for clinical disease. This theme

apparent in the histopathological results was mirrored in our results for fibroblast activation, which further underscore the relevance of a pro-fibrotic SPF consortia and Tl1a. Given their

expression of Toll-like receptors, fibroblasts have the capacity to become activated by bacterial products.26 Consistent with this, Tl1a-mediated fibroblast activation required the

microbiome in the cecum (despite no concomitant changes in inflammation). However, in the ileum, (in which there is significant increase in inflammation), Tl1a could partially compensate for

the lack of the microbiome and promote some fibroblast activation. This might point to unique effects of Tl1a on fibroblast activation that are tissue-specific, but may still ultimately

require a specific consortium of organisms in SPF to yield histopathologically evident fibrosis, as Tl1a-Tg mice still had reduced ileal fibrosis under GF conditions and with Humanized

microbiome compared with conventional SPF microbiota (Figs. 1 and 3). The effect of SPF microbiota on ileal fibrosis was not as dramatic as in the cecum. One possible explanation for this

effect is the differing microbial communities that colonize the ileum vs. the cecum. We show that fibrosis correlated much more closely with several organisms in the cecum compared with the

ileum where there were equal positive and negative “pulls” at fibrosis, with a few positively correlated organisms and numerous negatively correlated organisms. The organisms that are

tightly correlated with increased fibrosis in the cecum are less abundant in the ileum. Similarly, the ileum harbors numerous organisms that are negatively correlated with fibrosis compared

with the cecum where there are fewer such organisms. A related potential explanation, is the quantity, structure, and type of mucous seen in the small intestine vs. the large intestine. The

differing mucin composition may account for observed differences in bacterial composition in different regions of the gut.30 The mucin-rich large intestine can harbor anaerobic organisms

with a repertoire of glycosidic enzymes that disassemble complex mucus glycans to be used as a carbon source.31 We identified mucin-degrading bacteria _(M_. _schaedleri_) to correlate with

fibrosis in the cecum. The effect of differing microbial compositions in the cecum vs. ileum on fibrosis may be mediated through bacterial modulation of fibroblast phenotype, since specific

bacteria may have direct and opposing effects on fibroblast, as we demonstrated in vitro. Cecal and ileal colonization with SPF microbiota induced intestinal myofibroblasts in both WT and

Tl1a-Tg mice relative to GF conditions or colonization with Hu microbiota. Notably, however, colonization with SPF microbiota (but not Hu microbiota), in addition to Tl1a-overexpression,

resulted in overall increased proportion of myofibroblasts. Interestingly, GF Tl1a-Tg mice had reduced myofibroblast proportion in the cecum compared to GF WT mice, which was not seen in the

presence of Hu microbiota. This raises the question as to the specific contribution of Tl1a-overexpression vs. microbial changes in previously referenced colitigenic models conducted in

native microbiome-intact mice.12,27 It would, therefore, be important to determine what effects Tl1a-overexpression, independent of any bacterial stimulation, but in the presence of other

mucosal stimulation, such as inflammatory insults due to DSS for example, may have on intestinal fibrosis and inflammation; or the effects that differing microbial populations may impact on

experimental colitis. These results also have novel implications for a microbiome effect on fibroblast function in concert with Tl1a. Consistent with the above noted points regarding the

disconnect between pro-inflammatory and pro-fibrotic stimuli, colonic fibroblasts do not migrate in response to classic pro-inflammatory cytokines such as TNFα or IL-1, but rather require

traditionally “pro-fibrotic” cytokines such as TGFβ to induce migration.32 It is notable that in our migration assay Tl1a significantly and directly increased fibroblast migration compared

with controls, again suggesting a direct contribution of Tl1a to the pro-fibrotic pathway, which may act independently of inflammation. Importantly, our data demonstrate that the gut

microbiota can promote this effect directly in concert with Tl1a. These findings propose novel roles for both Tl1a and SPF microbiota in fibroblast function. In this study, we utilized a

novel correlation of direct changes in fibrosis with specific bacterial abundance in a region-specific manner. In the cecum of SPF-colonized mice, which demonstrated significant collagen

deposition and fibroblast activation, we identified several microbes that clustered tightly with fibrosis. This included _Mucispirillum schaedleri_, a mucous degrading organism that has been

reported to discriminate between colitis and remission33 in a mouse model but has not been linked to fibrosis. _Ruminococcus_ are another group of mucolytic bacteria that have been observed

to be increased in CD in some studies, associated with the stricturing phenotype in a recent pediatric CD study, and contribute to experimental colitis.22,34,35,36 _Ruminococcus_ and _M_.

_schaedleri_ were capable of directly modulating fibroblast function in vitro. Therefore, further studies demonstrating potential causal efficacy of these correlated organisms in vivo are

warranted. It would be interesting to assess in future studies whether _M_. _schaedleri_, or previously identified species of _Ruminococcus_, are present in Tl1a-Tg mice with fibrostenosis

under colitic conditions and in CD patients with the high risk _TNFSF15_ haplotype and stricturing disease. Furthermore, it would be informative to assess mucus structure, mucosal barrier

function, and fibroblast activation in mice and humans with and without these mucous degrading organisms. Finally, _Anaeroplasma_, a genus which has been previously associated with

experimental colitis,37 was also significantly associated with fibrosis in the cecum of SPF-colonized mice. In terms of organisms that were associated with reduced fibrosis in the cecum,

_Coprococcus_, a genus that has been reported to be depleted in patients with CD38 was associated with reduced fibrosis in both, mice reconstituted with SPF microbiota and human microbiota.

_Oscillospira_ have been associated with gut health, and their reduced abundance has been implicated in a variety of diseases including CD.39 Notably, compared with positively correlated

organisms, _Oscillopsira_ mitigated fibroblast function in vitro. In the ileum of SPF-colonized mice, we observed that there were competing sets of microbes associated with either enhanced

or reduced fibrosis severity. It is worth noting that ileal inflammation may have impacted the microbial variations and associations with fibrosis seen, compared with the cecum which had

very tight microbial associations with fibrosis in the context of no significant inflammation. Members of the _Streptococcus_ and _Lactobacillus_ genera were found to be positively

associated with fibrosis in the ileum. This is concordant with recent human data indicating that fecal abundance of these microbes is associated with another fibrotic complication of IBD,

primary sclerosing cholangitis.40 Many organisms that were associated with reduced fibrosis in SPF-colonized mice have previously been observed to be depleted in patients with CD, including

_Faecalibacterium prausnitzii_, which has been well-described to have anti-inflammatory properties.34 Additionally, members of the Lachnospiraceae family, which contains many butyrate

producers that are decreased in CD patients,41 were associated with reduced fibrosis in the ileum of SPF-colonized mice. To our knowledge, this is the first study to link these microbes not

just to protection from inflammation, but also fibrotic disease. Thus, it would be important to determine mechanistically how these short-chain fatty acid producers affect intestinal

fibrosis in addition to inflammation. One possibility is a direct effect of these microbes (or their products) on fibroblast function, as our data suggest. Interestingly, our

microbiome-fibrosis correlation studies also underscore the disjunction between inflammation and fibrosis noted above. Sulfite-reducing bacteria such as _Bilophila_ have been associated with

a pro-inflammatory T helper type 1 immune response and an ability to induce experimental colitis.42 Despite the potential pro-inflammatory effects of such bacteria, we found that

_Bilophila_ correlated with a reduction in fibrosis in the ileum of SPF-colonized mice. Thus, hydrogen sulfide, one of the metabolic products of these bacteria, may have opposing effects on

intestinal inflammation compared with fibrosis.43,44 To our knowledge, this is the first study that causally implicates the intestinal microbiome in intestinal fibrosis, demonstrating that

Tl1a-mediated fibrosis is dependent upon specific bacteria or bacterial consortia and that those bacteria can directly affect fibroblast function. Thus, a focus on TL1A pathways acting in

concert with the microbiome may identify future therapeutic targets for fibrostenosing Crohn’s disease. MATERIALS AND METHODS GNOTOBIOTIC EXPERIMENTS Tl1a-Tg (which have sustained TL1A

expression) and WT mice, both on C57Bl/6 background were re-derived into germ-free status and bred under sterile conditions at the National Gnotobiotic Rodent Resource Center, Chapel Hill,

NC. Tl1a-Tg mice and WT littermates at 2–4 months of age were orally gavaged with 200 µL of a 1:10 suspension of stool from either Cedars-Sinai specific pathogen free (SPF) mice or a healthy

human donor diluted in pre-reduced phosphate-buffered saline. Mice were killed after 2 months of colonization for assessment of intestinal fibrosis and histopathology. Mucosal areas of

collagen deposition identified by Picrosirius red-stained gut sections were quantitated for the relative degree of fibrosis using ImageJ software, as previously described.27 Two animal

pathologists scored H&E stained sections in a blinded manner using previously described histopathological scoring system used in GF experiments.45 FIBROBLAST GAP-CLOSURE ASSAYS Mouse

primary colonic fibroblasts were isolated as previously described.27 Equal numbers of fibroblasts per group (1 × 105 cells) were seeded in 8 chamber slides and cultured for 24–48 h until a

monolayer was formed. A scratch was created with a P200 pipette tip. Cell debris was removed by washing cells with PBS and then cell-culture medium was replaced with time-lapse images taken

every 4 h under an Olympus CK2 microscope at ×100 magnification. The area of the gap between the two migrating fronts of the cells was quantified using ImageJ software and relative percent

area of gap closed at the indicated time points was calculated as (area _t_0 – area _t__x_)/area _t_0. For assays involving supplementation with cecal washings, cecal contents from native

SPF WT and GF mice were released by flushing with 1 ml of distilled deionized water, as previously described.45 The washings were then homogenized by vortexing, and pelleted by

centrifugation. Supernatant were collected, filtered through a 0.22 μm filter, and was added directly to the cells after simulated wound, at a 1:20 dilution (5% volume). In the indicated

assays, mouse recombinant Tl1a (R&D Systems, Minneapolis, MN) was added at a concentration 100 ng/ml for 4 h prior to simulated wound and then maintained during the indicated migration

period. For assays involving the addition of bacterial lysates, _Oscillospira sp_., _Mucispirillum schaedleri_ and _Ruminococcus gnavus_ were cultured anaerobically on chocolate blood agar.

Fresh bacterial colonies were resuspended in sterile PBS and lysed. After simulated wound, 25 µg/ml lysate was added to the culture chamber, as describe previously.42 Cell migration was

assessed after 16 h of incubation. For fibroblast adhesion assays, an equal number of cells were seeded into 24-well plates and allowed to settle for either 20 or 80 min, after which the

wells were washed twice with PBS to remove non-adherent cells. Adherent cells were counted for 5 visual fields/well (representing four quadrants and the center of the well) at ×200

magnification, then averaged. The average number of adherent cells per visual field is then displayed for each well. HISTOLOGICAL MYOFIBROBLAST QUANTIFICATION Fibroblast and myofibroblasts

were quantified by anti-vimentin and anti-α-Smooth Muscle Actin immunofluorescence-stained OCT tissue sections. A total of 4 µm frozen sections were fixed with 10% formalin, blocked in 10%

BSA, 0.1% Triton X-100 TBST, and stained overnight at 4 °C with primary antibodies: rabbit polyclonal anti-αSMA Ab (Abcam, Cambridge, MA) at 1:100 dilution and chicken polyclonal

anti-Vimentin Ab (Abcam, Cambridge, MA) at 1:2000 dilution. Secondary antibody at 1:500 dilution was added for 2 h at room temperature with donkey anti-rabbit IgG-Alexa-fluor-647 and goat

anti-chicken IgY- DyLight 488 (Abcam, Cambridge, MA). Images were captured with Leica TCS spectral microscope. Total numbers and percentage of myofibroblasts (that co-localize fluorescence)

over total vimentin-positive cells per HPF in ileum or cecum were quantitated by two independent investigators. QUANTITATIVE REAL‐TIME PCR ANALYSIS Total RNA was isolated from cultured

fibroblasts using Qiagen RNeasy Micro Kit according to the manufacturer’s protocol. A total of 250 ng of total RNA was used in each RT reaction, with oligo(dT) as primer, using the

Omniscript kit and protocol (Qiagen). Collagen 1a2 and β-actin transcripts were amplified by quantitative real-time RT-PCR with TaqMan probes and primers (ThermoFisher Scientific, Waltham

MA, USA). PCR was done on 1/4 the RT reaction in duplicate as follows: 50 °C for 2 min, 95 °C for 2 min, then 45 cycles at 95 °C for 15 s, and 60 °C for 1 min. Assays were performed

following the predeveloped TaqMan assay reagents protocol for Platinum qPCR mix (Invitrogen Life Technologies) in a Mastercycler Ep realplex2 (Eppendorf). The Mastercycler System Interface

was used to analyze samples. Duplicates differing by less than one cycle were averaged and amount of transcript was analyzed. Replicate Ct values were normalized to replicate reference gene

(β‐actin) Ct values (ΔCt), and relative expression was calculated with respect to the indicated reference sample (ΔΔCt), expressed as percentage of β-actin. MICROBIAL CORRELATION WITH

FIBROSIS Cecal and ileal luminal content was released by flushing with distilled deionized water then the mucosa-associated bacteria were released by DTT treatment according to our published

protocol.45 DNA extraction and sequencing of the 16S ribosomal RNA gene was then performed for luminal and mucosal samples as previously described.46 In brief, bacterial DNA was extracted

using the MO BIO Powersoil kit with bead beating. The V4 region of the 16S gene was amplified and barcoded using 515f/806r primers then 150x2 bp sequencing was performed on an Illumina HiSeq

2500. Raw data were processed in QIIME 1.9.1 and 97% operational taxonomic units (OTUs) were identified by closed reference OTU picking against the Greengenes database.47 OTUs associated

with fibrosis were identified using DESeq2, an algorithm that employs multivariate negative binomial models to identify differentially abundant features.48 Separate analyses were performed

of the cecum and ileum of humanized and SPF-colonized ex-GF mice. The models included fibrosis score by Sirius Red staining and sample type, sex, and TL1a genotype as covariates. Results

were adjusted for multiple hypothesis testing.49 OTUs with adjusted _p_-values <0.05 were inputted into CoNet along with fibrosis scores to generate co-occurrence networks.50 This

analysis involved four metrics (Spearman correlation, Bray Curtis dissimilarity, Jensen Shannon dissimilarity, scaled variance of log ratios) from which a merged _p_-value was obtained by

Fisher’s method and corrected with the Benjamini-Hochberg method. The resulting network was visualized in Cytoscape 3.2.1 (http://cytoscape.org) with an edge-weighted, spring-embedded

layout. STATISTICS Data are presented as dot plots and means with group differences tested using standard methods depending on variables measured: Student’s _t_-test for comparisons between

two groups or Mann–Whitney test for comparisons between two groups requiring non-parametric testing. When indicated, one-way Analysis of Variance (ANOVA) with Tukey’s honestly significant

difference (HSD) test for multiple comparisons was used. In all settings, a _P_-value of <0.05 indicated a statistically significant difference in the parameter being compared. Additional

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of biological association networks using Cytoscape. _F1000Res_. 5, 1519 (2016). Article  Google Scholar  Download references ACKNOWLEDGEMENTS This work is supported by the National

Institutes of Health (NIH) NIH R01 DK056328-16 (N.J., S.R.T., and D.Q.S.), NIH K08 Career Development Award DK093578 (D.Q.S.), NIH T32 DK07180-40 (N.J.), Specialty Training and Advanced

Research (STAR) Program at UCLA (N.J.), The Crohn’s and Colitis Foundation and the F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute (N.J., S.R.T., and D.Q.S.),

5-P30-DK034987 and 5-P40-OD010995 (R.B.S.). AUTHOR INFORMATION Author notes * These authors contributed equally: Noam Jacob, Jonathan P. Jacobs AUTHORS AND AFFILIATIONS * F. Widjaja

Foundation, Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA Noam Jacob, Kotaro Kumagai, Connie W. Y. Ha, Yoshitake Kanazawa,

 Katherine Altmayer, Ariel M. Hamill, Aimee Von Arx, Suzanne Devkota, Kathrin S. Michelsen, Stephan R. Targan & David Q. Shih * Vatche and Tamar Manoukian Division of Digestive Diseases,

Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, 90095, USA Noam Jacob, Jonathan P. Jacobs & Venu Lagishetty * Departments

of Medicine, Microbiology and Immunology and National Gnotobiotic Rodent Resource Center, University of North Carolina, Chapel Hill, NC, 27599, USA R. Balfour Sartor * Department of

Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, 90095, USA Jonathan Braun Authors * Noam Jacob View author

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inPubMed Google Scholar CONTRIBUTIONS N.J., J.J., C.W.Y.H., S.D., J.B., K.S.M., R.B.S., S.R.T., and D.Q.S. designed experiments; N.J., J.J., K.K., V.L., Y.K., K.A., A.M.H., A.V.A.,

C.W.Y.H., and R.B.S. performed experiments and analyzed data; and N.J., J.J., R.B.S., K.S.M., S.R.T., and D.Q.S. wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Noam Jacob or

David Q. Shih. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE: Springer Nature remains neutral with regard

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ARTICLE CITE THIS ARTICLE Jacob, N., Jacobs, J.P., Kumagai, K. _et al._ Inflammation-independent TL1A-mediated intestinal fibrosis is dependent on the gut microbiome. _Mucosal Immunol_ 11,

1466–1476 (2018). https://doi.org/10.1038/s41385-018-0055-y Download citation * Received: 03 October 2017 * Revised: 16 May 2018 * Accepted: 04 June 2018 * Published: 09 July 2018 * Issue

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