Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice.

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.

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

BACKGROUND: Perturbation 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. RESULTS: Using 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. CONCLUSIONS: These 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.

S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis.

BACKGROUND: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. METHODS: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. RESULTS: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. CONCLUSIONS: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

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.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis.

OBJECTIVE: Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS: Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS: Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION: These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.