Intestinal epithelial expression of human TNF is sufficient to induce small bowel inflammation and sacroiliitis mimicking human Spondyloarthritis.

OBJECTIVES: Gut and joint disease commonly co-occur in spondyloarthritis (SpA). Up to 50% of SpA-patients show signs of subclinical gut inflammation and 10% evolves into inflammatory bowel disease (IBD). However, the mechanisms underlying this gut-joint axis are still unclear. Here we tested the hypothesis whether restricted expression of a pro-inflammatory cytokine in the intestine may trigger onset of combined gut and joint inflammation. METHODS: Intestinal expression of human TNF (hTNF) was achieved by driving hTNF gene expression under control of the rat FAPB2 promoter, creating a new animal model, the TNFgut mice, which expresses hTNF in the proximal intestinal tract. Intestinal-specific TNFgut mice were examined for pathological changes in the intestine and extra-intestinal tissues by means of histology, qPCR and flow cytometry, along with 16S sequencing on stools. RESULTS: Local expression of hTNF in the epithelium of the small intestine induces a pro-inflammatory state of the proximal intestinal tract with epithelial alterations and induction of members of the S100 family, as well as local upregulation of T helper 17 and regulatory T cells, but no obvious signs of dysbiosis. Curiously, TNFgut mice develop sacroiliitis (p< 0.05) in addition to small bowel inflammation (p< 0.05). However, no signs of peripheral arthritis nor enthesitis could be documented. CONCLUSION: Intestinal expression of hTNF is sufficient to initiate a pro-inflammatory cascade culminating in small bowel inflammation and sacroiliitis. Thus, gut-derived cytokines are sufficient to induce spondyloarthritis.

Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF-driven spondyloarthritis.

OBJECTIVES: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+ regulatory T (Treg) cells in a model of Crohn's-like ileitis and concomitant arthritis. METHODS: RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion. RESULTS: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression. CONCLUSIONS: These data point to profound differences in immune-regulation between Crohn's ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

RORγt inhibition ameliorates IL-23 driven experimental psoriatic arthritis by predominantly modulating γδ-T cells.

OBJECTIVE: Divergent therapeutic outcomes on different disease domains have been noted with IL-23 and IL-17A-blockade in PsA. Therefore, elucidating the role of RORγt, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, RORγt inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model. METHODS: We tested the efficacy of a RORγt antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR. RESULTS: RORγt-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, P < 0.001), in line with profound suppression of an enhanced type IL-17 immune signature in PsA-affected tissues. Moreover, inflammation-induced bone loss and bone erosions were reduced (P < 0.05 in calcaneus, P < 0.01 in tibia). Sustained IL-23 overexpression resulted in only mild signs of sacroiliitis. Gamma-delta (γδ)-T cells, the dominant source of T cell-derived IL-17A and IL-22, were expanded during IL-23 overexpression, and together with Th17 cells, clearly countered by RORγt inhibition (P < 0.001). CONCLUSION: RORγt-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by γδ-T cells and Th17 cells.

The Role of the Microbiome in Gut and Joint Inflammation in Psoriatic Arthritis and Spondyloarthritis.

Spondyloarthritis (SpA) encompasses a group of diseases characterized by an inflammatory arthritis involving both joints and entheses. However, extraarticular symptoms constitute a large element of the pathology and should not be underestimated. Microscopic gut inflammation is observed in 50% of patients with SpA and has been linked to disease activity, underscoring the effect of gut inflammation in SpA. In this review, we discuss the influence of gut microbiota on SpA pathogenesis. A change in microbiota composition has been linked to the development of various inflammatory arthritides, and dysbiosis is a potential factor in the pathogenesis of multiple inflammatory diseases. In this context, several groups have reported the modulatory effects of gut microbiota-derived metabolites on the effect of immune cells. The gut mucosa is populated by several types of regulatory T cells, but also some specialized unconventional innate-like T cells. These cells are predominantly found at mucosal and epithelial barrier sites, where they serve an essential role in modulating host-microbial interplay. Apart from the close association between the composition of the microbiota and inflammatory diseases, the therapeutic value of dysbiosis needs further investigation, and the identification of a causal inflammatory pathway between gut dysbiosis and musculoskeletal inflammation could revolutionize the therapeutic approach in SpA.

Sterile triggers drive joint inflammation in TNF- and IL-1ß-dependent mouse arthritis models.

Arthritis is the most common extra-intestinal complication in inflammatory bowel disease (IBD). Conversely, arthritis patients are at risk for developing IBD and often display subclinical gut inflammation. These observations suggest a shared disease etiology, commonly termed "the gut-joint-axis." The clinical association between gut and joint inflammation is further supported by the success of common therapeutic strategies and microbiota dysbiosis in both conditions. Most data, however, support a correlative relationship between gut and joint inflammation, while causative evidence is lacking. Using two independent transgenic mouse arthritis models, either TNF- or IL-1ß dependent, we demonstrate that arthritis develops independently of the microbiota and intestinal inflammation, since both lines develop full-blown articular inflammation under germ-free conditions. In contrast, TNF-driven gut inflammation is fully rescued in germ-free conditions, indicating that the microbiota is driving TNF-induced gut inflammation. Together, our study demonstrates that although common inflammatory pathways may drive both gut and joint inflammation, the molecular triggers initiating such pathways are distinct in these tissues.

Protein citrullination and NET formation do not contribute to the pathology of A20/TNFAIP3 mutant mice.

A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.

Deletion of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 in Mouse T Cells Protects Against Development of Autoimmune Arthritis but Leads to Spontaneous Osteoporosis.

OBJECTIVE: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) plays a crucial role in innate and adaptive immune signaling by modulating the threshold for activation of immune cells, including Treg cells. Therefore, MALT-1 is regarded to be an interesting therapeutic target in several immune-mediated diseases. The goal of this study was to examine the role of MALT-1 in experimental animal models of rheumatoid arthritis (RA). METHODS: MALT-1 activation was assessed by measuring cleavage of the deubiquitinase CYLD in lymphocytes from mice with collagen-induced arthritis (CIA). Furthermore, the impact of MALT-1 deficiency on arthritis was evaluated in Malt1KO mice with CIA or with collagen antibody-induced arthritis (CAIA). T cell-specific MALT-1 deficiency was measured in mice with deletion of T cell-specific MALT-1 (Malt1Tcell KO ), and the time-dependent effects of MALT-1 deficiency were assessed in mice with deletion of tamoxifen-inducible T cell-specific MALT-1 (Malt1iTcell KO ). Bone density was determined in MALT-1-deficient mice using micro-computed tomography and femur-bending tests. Reconstitution of Treg cells was performed using adoptive transfer experiments. RESULTS: MALT-1 activation was observed in the lymphocytes of mice with CIA. T cell-specific MALT-1 deletion in the induction phase of arthritis (incidence of arthritis, 25% in control mice versus 0% in Malt1iTcell KO mice; P < 0.05), but not in the effector phase of arthritis, completely protected mice against the development of CIA. Consistent with this finding, MALT-1 deficiency had no impact on CAIA, an effector phase model of RA. Finally, mice with MALT-1 deficiency showed a spontaneous decrease in bone density (mean ± SEM trabecular thickness, 46.3 ± 0.7 µm in control mice versus 40 ± 1.1 µm in Malt1KO mice; P < 0.001), which was linked to the loss of Treg cells in these mice. CONCLUSION: Overall, these data in murine models of RA highlight MALT-1 as a master regulator of T cell activation, which is relevant to the pathogenesis of autoimmune arthritis. Furthermore, these findings show that MALT-1 deficiency can lead to spontaneous osteoporosis, which is associated with impaired Treg cell numbers.