Rebecca Rogier

Toll-Like Receptor Mediated Modulation of T Cell Response by Commensal Intestinal Microbiota as a Trigger for Autoimmune Arthritis

In autoimmune diseases, a disturbance of the balance between T helper 17 (Th17) and regulatory T cells (Tregs) is often observed. This disturbed balance is also the case in rheumatoid arthritis (RA). Genetic predisposition to RA confers the presence of several polymorphisms mainly regulating activation of T lymphocytes. However, the presence of susceptibility factors is neither necessary nor sufficient to explain the disease development, emphasizing the importance of environmental factors. Multiple studies have shown that commensal gut microbiota is of great influence on immune homeostasis and can trigger the development of autoimmune diseases by favoring induction of Th17 cells over Tregs. However the mechanism by which intestinal microbiota influences the Th cell balance is not completely understood. Here we review the current evidence supporting the involvement of commensal intestinal microbiota in rheumatoid arthritis, along with a potential role of Toll-like receptors (TLRs) in modulating the relevant Th cell responses to trigger autoimmunity. A better understanding of TLR triggering by intestinal microbiota and subsequent T cell activation might offer new perspectives for manipulating the T cell response in RA patients and may lead to the discovery of new therapeutic targets or even preventive measures.

Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis

BackgroundPerturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn−/− mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn−/− mice.ResultsUsing high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn−/− mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn−/− mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn−/− mice. The arthritis phenotype in IL1rn−/− mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn−/− microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis.ConclusionsThese findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.

Alteration of the intestinal microbiome characterizes preclinical inflammatory arthritis in mice and its modulation attenuates established arthritis

Perturbations of the intestinal microbiome have been observed in patients with new-onset and chronic autoimmune inflammatory arthritis. However, it is currently unknown whether these alterations precede the development of arthritis or are rather a consequence of disease. Modulation of intestinal microbiota by oral antibiotics or germ-free condition can prevent arthritis in mice. Yet, the therapeutic potential of modulation of the microbiota after the onset of arthritis is not well characterized. We here show that the intestinal microbial community undergoes marked changes in the preclinical phase of collagen induced arthritis (CIA). The abundance of the phylum Bacteroidetes, specifically families S24-7 and Bacteroidaceae was reduced, whereas Firmicutes and Proteobacteria, such as Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae, were expanded during the immune-priming phase of arthritis. In addition, we found that the abundance of lamina propria Th17, but not Th1, cells is highly correlated with the severity of arthritis. Elimination of the intestinal microbiota during established arthritis specifically reduced intestinal Th17 cells and attenuated arthritis. These effects were associated with reduced serum amyloid A expression in ileum and synovial tissue. Our observations suggest that intestinal microbiota perturbations precede arthritis, and that modulation of the intestinal microbiota after the onset of arthritis may offer therapeutic opportunities.

Involvement of T helper 17 cells in inflammatory arthritis depends on the host intestinal microbiota

Intestinal microbiota are associated with the development of inflammatory arthritis. The aim of this study was to dissect intestinal mucosal immune responses in the preclinical phase of arthritis and determine whether the presence of Th17 cells, beyond involvement of the cytokine interleukin‐17 (IL‐17), is required for arthritis development, and whether the involvement of Th17 cells in arthritis depends on the composition of the host microbiota.

07.04 Partial elimination of intestinal microbiota dampens t helper 17 cell differentiation and established collagen-induced arthritis in mice

Background/objectives Both germ-free condition and administration of oral antibiotics before the onset of arthritis modulate T cell differentiation and attenuate arthritis in mice. However, it is not known whether and how the modulation of intestinal microbiota after the onset of arthritis may influence the disease. Here, we investigated the involvement of commensal intestinal microbiota in the progression of established arthritis in both T cell-dependent and -independent mouse models. Materials and methods Mice with established collagen-induced arthritis (CIA) as well as mice with K/BxN serum-transfer arthritis were treated orally with broad-spectrum antibiotics for one week to partially eliminate intestinal microbiota. 16S rRNA gene high-throughput sequencing was performed to assess the effect of arthritis and antibiotic treatment on the gut microbiota composition. Arthritis was assessed macroscopically and by histology. Differentiation of Th1, Th17 and regulatory T (Treg) cells and production of their prototypic cytokines in intestinal lamina propria and joint-draining lymph nodes were assessed by flow cytometry and Luminex cytokine array. Results Induction of arthritis as such resulted in significant alterations in the gut microbiota. Antibiotic treatment of CIA mice eliminated the majority of the gut microbiota, while the microbiota composition of control mice remained relatively stable. Partial elimination of intestinal microbiota during ongoing CIA specifically suppressed intestinal Th17 cell differentiation without affecting Th1 and Treg cells. Accordingly, production of IL-17, but not IFNγ, IL-4 and IL-10, by lamina propria lymphocytes was significantly diminished in antibiotic-treated mice. Importantly, elimination of intestinal microbiota resulted in suppressed Th17 cell differentiation in joint-draining lymph nodes and reduced the severity of established CIA. In contrast, antibiotic treatment did not influence disease severity in the T cell-independent K/BxN serum-transfer arthritis. Intriguingly, the abundance of intestinal Th17 cells strongly correlated with the severity of arthritis in the CIA mice. However, elimination of intestinal microbiota after disease onset did not affect the development of anti-collagen type II auto-antibodies. Conclusion These observations suggest that modulation of commensal intestinal microbiota during established arthritis specifically suppresses Th17 differentiation and dampens T cell-mediated arthritic processes. Our study supports the notion that inflammatory signals provided by the gut microbiota continue to promote arthritis after its onset.

OP0329 Involvement of t helper 17 cells in inflammatory arthritis depends on the host intestinal microbiota

Background Intestinal microbiota have been associated with psoriatic and rheumatoid arthritis. One of the major effects of microbiota is the induction of mucosal T helper 17 (Th17) cells. We therefore reasoned that the efficiacy of Th17-targeted therapies in arthritis may depend on the host microbiota. Previous studies focused on the role of the cytokine interleukin-17A (IL-17), rather than Th17 cells, by using IL-17 inhibitors or IL-17-deficient mice. Therefore, the role of Th17 cells, which produce multiple pro-inflammatory mediators in addition to IL-17, is not yet fully understood. Objectives The aim of this study was to determine the role of Th17 cells, beyond the cytokine IL-17, in arthritis, and to investigate whether Th17 cells are differentially involved in arthritis depending on the microbiota present. Methods We established conditional Th17-deficient mice, which exhibit a CD4-Cre-induced floxing of a part of the Rorc allele that encodes the Th17 master regulator RORγt. We compared the development of collagen-induced arthritis in Th17-deficient (CD4-Cre+ Rorcflox/flox) and -sufficient (CD4-Cre- Rorcflox/flox) littermate mice, either colonized with known Th17 cell inducers segmented filamentous bacteria (SFB) or harboring the SFB-free Jackson microbiota. The abundance of Th1 and Th17 cells and the production of IL-17, IFNγ and GM-CSF were quantified by flow cytometry and multiplex cytokine assay. Results CD4-Cre+ Rorcflox/flox mice had significantly lower Th17, but similar Th1 cell abundance, in intestinal lamina propria compared with Cre- littermate controls. Surprisingly, the total amount of IL-17A production by all lamina propria cells during arthritis was rather increased in Th17-deficient mice, with CD8+ T cells and Gr1+ neutrophils being the main alternative sources of IL-17. Despite this increased total IL-17 levels, conditional Th17-deficient mice developed a less severe arthritis compared with Th17-sufficient mice when intestinal microbiota comprised SFB. This suggests a role for Th17 cells in inflammatory arthritis distinct from IL-17. Accordingly, synovial inflammation, cartilage destruction and proteoglycan depletion were reduced in SFB-colonized Th17-deficient mice. While the production of IL-17 by joint-draining lymph node cells stimulated with PMA and ionomycin was similar between Th17-sufficient and –deficient mice, cells from the latter group produced significantly less IL-17 upon antigen-specific stimulation with type II collagen. Furthermore, the production of GM-CSF, another Th17 cell-derived cytokine, was significantly lower in the lymph nodes of Th17-deficient mice, an effect associated with the protection against arthritis. Importantly, substitution of the intestinal microbiota with SFB-free Jackson microbiota resulted in the loss of Th17 cell dependency of arthritis as Th17-sufficient and –deficient mice showed similar disease progression under this condition. Conclusions These data suggest that Th17 cells may mediate inflammatory arthritis partly through IL-17-independent mechanisms. Our observations also suggest that the involvement of Th17 cells in arthritis depends on the microbiota subset present in the host. Therefore, a microbiome-guided stratification of rheumatoid or psoriatic arthritis patients might improve the efficacy of Th17 (or IL-17)-targeted therapies. Disclosure of Interest None declared

A1.01 Activation of TLR4 by dysbiotic intestinal microbiota following IL-1 receptor antagonist deficiency triggers TH17-mediated arthritis

Background and objectives Interleukin-1 receptor antagonist deficient (IL-1Ra-/-) mice spontaneously develop a T cell-driven autoimmune arthritis, which depends on the presence of commensal microbiota and Toll-like receptor 4 (TLR4). The aim of this study was to elucidate the role of IL-1 receptor signalling and TLR4 in defining the intestinal microbiota and the associated mucosal and systemic immune response during arthritis. Methods 16S rRNA 454-pyrosequencing meta-genome analysis was used to define intestinal microbial communities in BALB/c wild type (WT), IL-1Ra-/- and IL-1Ra/TLR4 double knock-out (DKO) mice. IL-1Ra-/- mice were treated with antibiotics and re-colonised with segmented filamentous bacteria (SFB). T cell differentiation and cytokine production was assessed in small intestine lamina propria (SI-LP) and draining lymph nodes. Results IL-1Ra-/- mice had a significant reduction in microbial diversity compared to WT mice. Interestingly, the species diversity was restored in IL-1Ra/TLR4 DKO, suggesting that IL-1R-driven skewing of bacterial diversity depends on TLR4. IL-1Ra-/- mice had greatly increased intestinal Th17 levels, which significantly correlated with arthritis scores. Relevance of intestinal microbiota in arthritis was underlined by significant long-term suppression of arthritis by one-week oral treatment with broad-spectrum antibiotics. Re-colonisation of antibiotic-treated IL-1Ra-/- mice by SFB, a potent intestinal Th17 inducer, was sufficient to cause full-blown arthritis. Absence of TLR4 resulted in the sustained reduction of arthritis in IL-1Ra-/- mice. TLR4 is known to play a major role in recognition of Gram-negative bacteria. Interestingly, treatment with tobramycin, specifically eliminating Gram-negative bacteria, significantly reduced arthritis severity. SI-LP mononuclear cells from IL-1Ra-/-TLR4-/- mice ex vivo cultured with PMA and ionomycin produced substantially less IL-17. In addition, production of IL-23, IL-1β and IL-6 upon ex vivo stimulation with intestinal microbial antigens was reduced in the absence of TLR4. This suggests that TLR4 plays a role in microbiota-induced production of cytokines involved in intestinal Th17 differentiation. Conclusions Our data suggest that activation of TLR4 by commensal intestinal microbiota drives arthritis in IL-1Ra-/- mice via intestinal IL-1, IL-23, IL-6production and subsequent Th17 induction. Understanding the mechanisms linking the intestinal T cell response with arthritis may help identifying novel therapeutic targets in rheumatoid arthritis.

A2.07 Antagonisticregulation of IL-17 and GM-CSF during cell development ex vivo and during experimental arthritis

Background and objectives GM-CSF is a pro-inflammatory cytokine suggested to be mainly expressed by the Th17 cell subset. Combination blocking of GM-CSF and the key Th17-cytokine IL-17 has shown to be much more effective in reducing experimental arthritis compared to single cytokine blocking, revealing a potential connexion between the two cytokines. With this study, we aimed to unravel the interplay between GM-CSF and IL-17 during Th17 differentiation ex vivo and in vivo during experimental arthritis. Materials and methods We investigated whether IL-17 and GM-CSF levels were affected in a similar fashion when differentiating naïve murine CD4+ T cells ex vivo under conditions suboptimal for Th17 development. Additionally, we determined the effect of incubation with GM-CSF and IL-17 recombinant proteins and neutralising antibodies on expression of both cytokines during ex vivo Th17 differentiation. Finally, we studied the IL-17/GM-CSF interplay in C57Bl6/J mice with mBSA/IL-1β-induced experimental arthritis treated with anti-GM-CSF or anti-IL-17 antibodies. Results Interestingly, our ex vivo studies showed increased GM-CSF levels after differentiating naïve cells under conditions suboptimal for Th17 development, while IL-17 levels decreased as expected. In line with this ex vivo finding, we observed higher systemic levels of IL-17 and GM-CSF in mice with experimental arthritis after GM-CSF and IL-17 neutralisation respectively. Remarkably, no effects of incubation of naïve CD4+ T cells with IL-17 or GM-CSF recombinant proteins or neutralising antibodies during T cell differentiation were detected. This indicates that the two cytokines do not directly regulate each other’s expression but require interaction with environmental cells for their suppressive actions. Conclusion These data point towards deviating differentiation conditions and indirect antagonistic regulation of IL-17 and GM-CSF expression by CD4+ T cells, and provides further rationale for a combination blocking strategy of IL-17 and GM-CSF in treatment of Rheumatoid Arthritis.

FRI0005 Combination Blocking of IL-22 and IL-17 During Experimental Arthritis Potently Reduces TH17-Driven Disease Progression

Background Rheumatoid arthritis (RA) patients show elevated levels of IL-22 and IL-22-producing T helper cells that correlate to erosive disease, suggesting a role for this cytokine in the pathogenesis of RA. Interestingly, IL-22 is a dual cytokine with pro- and anti-inflammatory properties, and its effects might be regulated by cooperation and crosstalk with IL-17. Objectives The purpose of this study was to elucidate the role of IL-22 in the development of a spontaneous model of experimental arthritis by using IL-1Ra knockout mice. Additionally, we aimed to investigate the therapeutic potential of combined IL-22/IL-17 blocking during experimental arthritis. Methods IL-1Ra-deficient mice develop spontaneous arthritis due to excess IL-1 signaling, and we previously demonstrated the importance of IL-17 and Th17 cells in this model1. To investigate the role of IL-22 in this arthritis model, we compared IL-1Ra-/- x IL-22+/+ mice to IL-1Ra-/- mice lacking IL-22 expression. Paw joint swelling was scored weekly, and mice were sacrificed at the age of fifteen weeks. In addition, arthritic IL-1Ra-/- x IL-22-/- mice were treated with anti-IL-17 antibodies to determine the therapeutic potency of this combined blocking strategy during experimental arthritis. Results IL-1Ra-/- mice that also lack IL-22 showed strongly reduced arthritis development, reaching a disease incidence of only 54% at the age of 15 weeks compared to 93% in IL-1Ra-/- x IL-22+/+ mice. In addition, arthritis severity of the mice that did develop arthritis was significantly reduced by 30.6% in the absence of IL-22. Interestingly, combined blocking of IL-22 and IL-17 using IL-1Ra-/- x IL-22-/- mice treated with neutralizing anti-IL-17 antibodies after the onset of arthritis demonstrated clear additive effects compared to blocking these single cytokines alone, thereby potently reducing progression of this Th17-driven arthritis model. Conclusions These findings suggest that IL-22 plays an important role both in the initiation and augmentation of Th17-dependent experimental arthritis, and that targeting IL-22, especially in combination with IL-17 therefore seems an interesting, potent strategy in RA treatment. References Koenders MI, Devesa I, Marijnissen RJ, Abdollahi-Roodsaz S, Boots AM, Walgreen B, di Padova FE, Nicklin MJ, Joosten LA, van den Berg WB. Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleuking-1 receptor antagonist-deficient mice. Arthritis Rheum. 58(11): 3461-70, 2008. Disclosure of Interest None declared

Toll-like receptor 4-induced interleukin-1 defines the intestinal microbiome and mucosal immune response in arthritis-prone IL-1 receptor antagonist deficient mice

Background Mice deficient in interleukin-1 receptor antagonist (IL-1Ra-/-) spontaneously develop a T cell-driven autoimmune arthritis, which we previously showed to depend on the presence of commensal microbiota. Recent findings suggest alteration of intestinal microbiome in new-onset rheumatoid arthritis (RA). Objectives The aim of this study was to investigate the role of IL-1 receptor signaling and Toll-like receptor (TLR) 2 and TLR4 in defining the intestinal microbiota and the associated mucosal and systemic immune response during arthritis. Methods Multiplex 454 pyrosequencing of fecal bacterial 16S rRNA was used to define intestinal microbial communities in BALB/c wild type (WT), IL-1Ra-/- and IL-1Ra/TLR double knock-out (DKO) mice. For gene sequencing analysis, a customized workflow based on Quantitative Insights Into Microbial Ecology (QIIME version 1.2) was adopted. T cell differentiation was assessed in small intestine lamina propria (SI-LP) and spleen using flow cytometry and gene expression was assessed by qPCR. Results IL-1Ra-/- mice had a significant reduction in microbial diversity compared to WT mice. Both alpha diversity (number of unique taxonomic entities) and phylogenetic diversity (PD) whole tree (based on taxonomic distance) were significantly diminished in IL-1Ra-/- mice. Interestingly, the loss of species diversity was absent in IL-1Ra/TLR4 DKO, but not IL-1Ra/TLR2 DKO mice, suggesting that IL-1R-driven skewing of bacterial diversity depends on TLR4. IL-1Ra-/- mice exhibited significantly increased abundance of the genus Helicobacter and reduced Prevotella (p=0.008 and p =0.004, respectively). Importantly, significant alterations in the genera Xylanibacter, Prevotella, Streptococcus, and Ruminococcus were markedly normalized in TLR4, but not TLR2, deficient mice, identifying a role for TLR4 in IL-1 mediated shifts in microbial community. In line with the relevance of intestinal microbiota in mucosal T cell polarization, IL-1Ra-/- mice had greatly increased Th17 in SI-LP. Interestingly, SI-LP Th17% significantly correlated with arthritis score. Although intestinal mRNA expression of IL-1 itself remained unaltered, IL-23p19 expression was increased. Both IL-1b and IL-23 were significantly diminished in IL-1Ra/TLR4 DKO mice, suggesting a TLR4-mediated regulation. These mucosal responses paralleled systemic response and arthritis development, since Th17% and associated genes such as RORγt were increased in IL-1Ra-/- and reduced in IL-1Ra/TLR4 DKO mice which had less severe arthritis. Conclusions These data suggest a TLR4-mediated regulation of intestinal microbiome, and mucosal and splenic immune responses controlling arthritis severity, potentially through an IL-1 and IL-23 dependent mechanism. Understanding the molecular and cellular mechanisms linking the intestinal T cell response with arthritis may help identifying novel therapeutic targets in RA. Disclosure of Interest None declared