Joint inflammation, or spondyloarthritis (SpA), is the most common extra-intestinal manifestation of inflammatory bowel disease (IBD), but the specific role for therapies targeting SpA is not well defined. One of the earliest medications used for the treatment of IBD is sulfasalazine (SAS). SAS is a prodrug composed of two chemical moieties, the anti-inflammatory 5-aminosalicilate and the antibiotic sulfapyridine. The efficacy of SAS in peripheral arthritis is thought to depend on its “antibacterial” properties, however the impact of SAS on the IBD-SpA microbiome and how it may improve extra-intestinal symptoms is unknown. Therefore, our study aims to diagnostically evaluate the role for the fecal microbiome in clinical response to SAS therapy and identify microbial and immunologic therapeutic targets associated with clinical response. We have designed an observational study to longitudinally follow IBD patients with SpA who have a medical indication for SAS therapy. Clinical data and fecal samples from 22 patients were collected before initiation of SAS and at week 12 after initiation of SAS. Eleven IBD-SpA patients were concomitantly enrolled as controls and followed by 12 weeks. Metagenomic sequencing was used to define the effect of SAS on the IBD-SpA fecal microbiome and to evaluate its relationship with improvement in joint symptoms. Mouse models and in vitro assays were used to test the sufficiency of the SAS effect observed in patients. The fecal microbiome of SAS-responders was distinct from that observed in non-responders and 6 pre-treatment microbial markers (including the short chain fatty acid (SCFA) producer Faecalibacterium prausnitzii) predicted SAS-response (AUC = 0.9). Fecal metabolome of SAS responders had lower thymine and higher deoxyuridine compared to non-responders consistent with evidence of a folate trap in response to SAS treatment. SAS therapy in SPF mouse-model of chemically-induced colitis alleviated colitis in GPR 109a-dependent fashion consistent with a synergistic role for SCFA. In vitro assays revealed SAS direct regulation of F. prausnitzii metabolic function and butyrate synthesis. CONCLUSIONS: Collectively, these findings highlight the potential role for microbial diagnostics to improve SAS efficacy, and drug modulation of microbial markers to potentiate therapy for IBD patients with SpA.

Abstract Background Over two million people worldwide suffer from ulcerative colitis (UC). Biologic therapy has significantly improved treatment, but nearly two-thirds of patients attenuate response. Fecal microbiota transplant (FMT) is an emerging therapy for the treatment of UC, but the microbial mechanism responsible for clinical response is poorly understood. Using samples from our pilot FMT study (Jacob V, et al 2017), we aim to define the core transferable microbiota (CTM) in UC patients responsive to FMT therapy and its therapeutic mechanism. Methods IBD disease activity scores were used to define clinical response. Metagenomic sequencing of donor, recipient, and 4 week post-FMT fecal samples was performed to define the CTM and strain level transferability. To define the transferable immune-reactive microbiota (TIM), IgA-seq was also performed on donor and recipient samples. Patient TIM strains were isolated and tested in gnotobiotic mouse models to evaluate their impact on mucosal immunity and colitis. Results We defined a CTM associated with clinical response to FMT. CTM strain tracking confirmed that clinical response correlated with strain transferability. We defined a core TIM by IgA-seq that correlated with clinical response. In humanized mouse models, TIM induced IgA in a T cell independent manner. Colonization of germ-free mice with a core TIM strain, Odoribacter splanchnicus, robustly induced mucosal Th17 and RORgt+/Foxp3+ iTreg cells and reduced the severity of transfer T cell colitis. Our data highlights a core TIM in UC responders to FMT and the mechanistic impact of it in shaping mucosal immunity and guiding the response to UC. This provides a framework for rational selection of TIM for microbial-therapy in IBD.

Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptibe host. Polymorphisms in the TNFSF15 locus link TNF-like ligand 1A (TL1A) with IBD, but the functional impact of TL1A in regulating barrier immunity and intestinal inflammation is not clear. Here, we used cell-specific genetic deletion and gnotobiotic mouse models to evaluate the role for microbial induced TL1A signaling through death receptor 3 (DR3) in group 3 innate lymphoid cell (ILC3) in colitis. Flow cytometry, RNA-seq, and antigen specific proliferation assays are used to examine the mechanistic and functional impact of TL1A signaling in ILC3s. These novel genetic models revealed an essential protective role for TL1A regulation of group 3 innate lymphoid cell (ILC3) production of IL-22 in acute colitis. Intestinal CX3CR1+ macrophage production of TL1A is induced by IBD-associated adherent microbiota and promoted ILC3-dependent mucosal healing in vivo. In addition to regulating IL-22 production, colitis induced DR3-dependent expression of co-stimulatory molecules on MHCII+ ILC3. In contrast to the protective role in acute colitis, TL1A stimulation of ILC3 enabled co-stimulation dependent antigen specific T cell priming and exacerbated chronic T cell-dependent colitis. These dual effects of TL1A in acute and chronic colitis highlight a central role for this pathway in regulating ILC3 contribution to barrier immunity and will help guide TL1A-based diagnostic and therapeutic approaches to IBD.

Inflammatory bowel disease (IBD) results from a dys-regulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1(+) mononuclear phagocytes (MNPs). Using cell-specific genetic deletion models, we identified an essential role for CX3CR1(+)MNP-derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin-22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.

Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16S ribosomal RNA-based analysis (IgA-seq) revealed a selective enrichment in IgA-coated Escherichia coli in patients with Crohn's disease-associated SpA (CD-SpA) compared to CD alone. E. coli isolates from CD-SpA-derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. In comparison to non-AIEC E. coli, colonization of germ-free mice with CD-SpA E. coli isolates induced T helper 17 cell (TH17) mucosal immunity, which required the virulence-associated metabolic enzyme propanediol dehydratase (pduC). Modeling the increase in mucosal and systemic TH17 immunity we observed in CD-SpA patients, colonization of interleukin-10-deficient or K/BxN mice with CD-SpA-derived E. coli lead to more severe colitis or inflammatory arthritis, respectively. Collectively, these data reveal the power of IgA-seq to identify immunoreactive resident pathosymbionts that link mucosal and systemic TH17-dependent inflammation and offer microbial and immunophenotype stratification of CD-SpA that may guide medical and biologic therapy.

Background: Recent trials suggest fecal microbiota transplantation (FMT) with repeated enemas and high-diversity FMT donors is a promising treatment to induce remission in ulcerative colitis. Methods: We designed a prospective, open-label pilot study to assess the safety, clinical efficacy, and microbial engraftment of single FMT delivery by colonoscopy for active ulcerative colitis using a 2-donor fecal microbiota preparation (FMP). Safety and clinical endpoints of response, remission, and mucosal healing at week 4 were assessed. Fecal DNA and rectal biopsies were used to characterize the microbiome and mucosal CD4+ T cells, respectively, before and after FMT. Results: Of the 20 patients enrolled in this study, 7 patients (35%) achieved a clinical response by week 4. Three patients (15%) were in remission at week 4 and 2 of these patients (10%) achieved mucosal healing. Three patients (15%) required escalation of care. No serious adverse events were observed. Microbiome analysis revealed that restricted diversity of recipients pre-FMT was significantly increased by high-diversity 2-donor FMP. The microbiome of recipients post-transplant was more similar to the donor FMP than the pretransplant recipient sample in both responders and nonresponders. Notably, donor composition correlated with clinical response. Mucosal CD4+ T-cell analysis revealed a reduction in both Th1 and regulatory T-cells post-FMT. Conclusions: High-diversity, 2-donor FMP delivery by colonoscopy seems safe and effective in increasing fecal microbial diversity in patients with active ulcerative colitis. Donor composition correlated with clinical response and further characterization of immunological parameters may provide insight into factors influencing clinical outcome.

Background: Colonic lamina propria mononuclear phagocytes (MNPs) expressing the fractalkine receptor CX3CR1 play a critical role in maintaining barrier homeostasis. Recently, we have shown that these MNPs promote mucosal healing in response to microbial signals by supporting group 3 innate lymphoid cell (ILC3) production of IL-22 in both mouse models and human colitis. Mechanistically, we identified a role for CX3CR1+ MNP production of TNF family cytokine TL1A—whose gene, TNFSF15, encodes functional polymorphisms strongly associated with IBD—in regulating ILC3 effector cytokine production; however, the mechanism and physiologic importance of MNP-derived TL1A signaling via DR3 in promoting mucosal healing remain unknown. Methods: CX3CR1-GFP mice were used to evaluate TL1A expression in intestinal MNPs. Gnotobiotic mice and human endoscopic biopsy specimens were used to evaluate the role for microbiota and colitis, respectively, in driving TL1A expression. To examine the physiologic role for the TL1A:DR3 axis in mucosal healing, we generated novel genetic mouse models with targeted TL1A deletion on CX3CR1+ MNP (CX3CR1creER TL1Aflox/flox) and DR3 deletion on ILC3 (RORgtcre DR3flox/flox RAG2−/−). Mice deficient for TL1A or DR3 and their littermate controls were exposed to experimental colitis models, including 2% dextran sodium sulfate (DSS) or Citrobacter rodentium. Lamina propria mononuclear cells and ILC3 effector cytokine production were analyzed ex vivo. Results: Analysis of MNPs ex vivo revealed reduced TL1A expression in germ free mice compared to SPF and colonization with bacteria that adhere to the mucosa was sufficient to induce TL1A expression. Consistent with the microbial-dependence of TL1A production, CD11c+MHCII+CX3CR1+ splenocytes upregulated TL1A in response to LPS stimulation in vitro. Furthermore, increased TL1A expression was seen in CD11c+MHCII+CX3CR1+ LPMCs from mouse models of experimental colitis and IBD patients. During Citrobacter-induced colitis, CX3CR1creER TL1Aflox/flox mice had increased weight loss and greater bacterial burden systemically compared to their littermate controls. Similar to recent studies, we found that DR3-deficient mice exposed to DSS had more severe weight loss, pathology, and reduced survival. This phenotype correlated with reduced ILC3 production of IL-22 and was rescued by recombinant IL-22. Notably, RORgtcre DR3flox/flox RAG2−/− exposed to DSS also had significantly more severe weight loss and pathology suggesting a critical role for DR3 on ILC3. This phenotype correlated with a reduction in colonic ILC3 production of IL-22 and epithelial cell production of anti-microbial peptides. Ex vivo characterization of ILC3 shows that TL1A synergy with IL-23 is independent of IL-1R and MyD88 signaling and not reproduced by related TNFSF (OX40L, TRAIL, or TNF&agr;). To evaluate the signaling pathway for TL1A-mediated synergy, soluble inhibitors revealed a critical role for MAPK signaling—specifically p38 MAPK—in mediating DR3 co-stimulation of IL-23 in ILC3. Conclusions: Collectively, these data highlight a key role for microbial regulation of MNP-derived TL1A in co-stimulating ILC3 effector cytokine production to regulate mucosal healing in vivo. TL1A co-stimulation of ILC3 is robust, selective, and specific. Further mechanistic studies of the microbial components that induce TL1A in MNPs and the critical signaling pathway required for ILC3 co-stimulation will provide therapeutic targets to promote mucosal healing.