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.
The intestinal microbiome is perturbed in patients with new-onset and chronic autoimmune inflammatory arthritis. Recent studies in mouse models suggest that development and progression of autoimmune arthritis is highly affected by the intestinal microbiome. This makes modulation of the intestinal microbiota an interesting novel approach to suppress inflammatory arthritis. Prebiotics, defined as non-digestible carbohydrates that selectively stimulate the growth and activity of beneficial microorganisms, provide a relatively non-invasive approach to modulate the intestinal microbiota. The aim of this study was to assess the therapeutic potential of dietary supplementation with a prebiotic mixture of 90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (scGOS/lcFOS) in experimental arthritis in mice. We here show that dietary supplementation with scGOS/lcFOS has a pronounced effect on the composition of the fecal microbiota. Interestingly, the genera Enterococcus and Clostridium were markedly decreased by scGOS/lcFOS dietary supplementation. In contrast, the family Lachnospiraceae and the genus Lactobacillus, both associated with healthy microbiota, increased in mice receiving scGOS/lcFOS diet. However, the scGOS/lcFOS induced alterations of the intestinal microbiota did not induce significant effects on the intestinal and systemic T helper cell subsets and were not sufficient to reproducibly suppress arthritis in mice. As expected, we did observe a significant increase in the bone mineral density in mice upon dietary supplementation with scGOS/lcFOS for 8 weeks. Altogether, this study suggests that dietary scGOS/lcFOS supplementation is able to promote presumably healthy gut microbiota and improve bone mineral density, but not inflammation, in arthritis-prone mice.
Background: Intestinal microbiota plays a prominent role in shaping the T cell immune response. Increasing evidence suggests that the gut microbiota is perturbed in patients with RA, and a variety of animal models demonstrated involvement of (mouse) microbiota in arthritis development. This underlines the necessity of understanding whether and how indigenous human NORA-associated microbiota may trigger RA. Objectives: To comprehensively investigate the intestinal mucosa cytokine production and DC, T and B cell responses to human gut microbiota associated with new-onset RA. Methods: We utilized in vitro cultures of mucosal-like DCs (differentiated from bone marrow cells) and primary splenic DCs, as well as ex vivo cultures of healthy human intestinal biopsies, cultured in the presence of heat-killed fecal microbiota from either NORA or control donors. Furthermore, we performed studies in humanized mice carrying intestinal NORA microbiota, to study the effect on immune response during homeostasis and upon joint inflammation during collagen-induced arthritis (CIA). Results: In 24h DC cultures, NORA fecal microbiota more potently induced the expression of co-stimulatory molecules CD40 and CD80, and this enhanced DC maturation was partially mediated through TLR4 as demonstrated using the TLR4 antagonist TAK242. Interestingly, HC and NORA fecal microbiota differentially induced IL-12 and IL-6 production, with significantly enhanced IL-6 and reduced IL-12 secretion by the NORA microbiome. Furthermore, in ex vivo cultures of human ileum biopsies, the production of IL-1 and IL-33, as well as IL-23/Th17 cytokines IL-23, IL-22, and GM-CSF, were significantly increased by NORA-derived microbiome. Interestingly, in the small intestine lamina propria (SILP) of NORA-colonized mice, we observed enhanced Th17 polarization, increased innate GM-CSF expression and higher B cell CD40 and IgA levels during homeostasis. To study whether colonization with HC and NORA microbiota alters arthritis development, humanized mice and controls (mock, autologous, HC and NORA microbiota) were used in a CIA experiment. Macroscopic scoring of the arthritis severity at weekly intervals demonstrated that arthritis severity was significantly enhanced in NORA-colonized mice compared to HC-colonization and mock controls. Conclusion: Our data reveal that NORA microbiota, in addition to the previously described Th17 differentiation, induce higher levels of GM-CSF and B cell IgA in LP and have increased potential to aggravate arthritis through the activation of TLR4. References: [1]Scher et al., eLife 2013; Maeda Y et al., Arthritis & rheumatology 2016; Zhang X et al., Nature medicine 2015; Chen J et al., Genome Med 2016 Disclosure of Interests: Marije Koenders: None declared, Heather Evans-Marin: None declared, Joyce Aarts: None declared, Parvathy Girija: None declared, Rebecca Rogier: None declared, Sergei Koralov: None declared, Julia Manasson: None declared, Peter van der Kraan: None declared, Shahla Abdollahi-Roodsaz: None declared, Jose Scher Consultant of: Novartis, Janssen, UCB, Sanofi.
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.
Altered composition of intestinal microbiota in recent-onset rheumatoid arthritis (RA) and possible efficacy of oral antibiotics suggest a role of intestinal microbiota in RA. This study aimed to investigate the involvement of commensal intestinal microbiota in T cell-dependent experimental arthritis.
Methods
IL-1 receptor antagonist deficient (IL-1Ra-/-) mice spontaneously developing T cell-driven IL-17-dependent autoimmune arthritis were used. Intestinal and systemic T cell differentiation and arthritis development were studied in conventional and germ-free (GF) mice. Contribution of intestinal microbiota was investigated using oral broad-spectrum and selective antibiotic treatments, combined with recolonization by specific microbiota. Multiplex 454 pyrosequencing of V5 and V6 hyper-variable regions of fecal bacterial 16S rRNA was used to identify specific microbiota associated with arthritis.
Results
Compared to wild-type mice, small intestinal lamina propria of IL-1Ra-/- mice contained increased Th17 and to lower extent Th1 percentages, both of which significantly correlated with arthritis severity. Importantly, GF IL-1Ra-/- mice had a marked abrogation of arthritis along with reduced intestinal Th1 and in particular Th17. GF IL-1Ra-/- mice exhibited a notable decrease in IL-1b and IL-17 production by splenocytes upon CD3 and Toll-like receptor stimulations, suggesting abolishment of systemic Th17 response. Relevance of intestinal microbiota was underlined by significant long-term suppression of arthritis by one-week oral treatment with Metronidazole, Neomycin and Ampicillin (each 1g/l). Interestingly, recolonization of antibiotic-treated IL-1Ra-/- mice by segmented filamentous bacteria, previously reported as a prominent intestinal Th17 inducer, was sufficient to cause full-blown arthritis. Selective elimination of Gram-negative bacteria, but not Gram-positive, suppressed arthritis, indicating members of intestinal Gram-negative commensals may drive arthritis. High-throughput pyrosequencing revealed lower microbiota abundance (operational taxonomic units) and reduced species richness and diversity (Chao and Shannon indices, resp.) in arthritic IL-1Ra-/- compared to wild-type mice. The genus Helicobacter, belonging to Gram-negative bacteria, was found associated with arthritis severity (0.0% in wild-type versus 1.1% in arthritic mice). Validation of microbiota alterations and studies on T cell-modulatory and disease-inducing characteristics in GF mice are in progress.
Conclusion
The presence of commensal intestinal microbiota is critical for the development of autoimmune T cell-driven arthritis, probably via shaping the T cell differentiation by Gram-negative bacteria. Understanding the molecular and cellular mechanisms linking the intestinal T cell response with extra-intestinal disease may help identify novel therapeutic targets in RA.
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.
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 duringex 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.
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.
