Fraser L. Collins

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction

IntroductionActivation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology.MethodsAntigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays.ResultsIn AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis.ConclusionsThese data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

Lactobacillus reuteri 6475 increases bone density in intact females only under an inflammatory setting

Background & Aims We previously demonstrated that short-term oral administration of the probiotic Lactobacillus reuteri 6475 enhanced bone density in male but not female mice. We also established that L. reuteri 6475 enhanced bone health and prevented bone loss in estrogen-deficient female mice. In this study, we tested whether a mild inflammatory state and/or a long-term treatment with the probiotic was required to promote a positive bone effect in estrogen-sufficient female mice. Methods A mild inflammatory state was induced in female mice by dorsal surgical incision (DSI). Following DSI animals were orally supplemented with L. reuteri or vehicle control for a period of 8 weeks. Gene expression was measured in the intestine and bone marrow by qPCR. Distal femoral bone density and architecture was analyzed by micro-CT. Results We report that 8 weeks after DSI there is a significant increase in the weight of spleen, thymus and visceral (retroperitoneal) fat pads. Expression of intestinal cytokines and tight junction proteins are also altered 8 weeks post-DSI. Interestingly, L. reuteri treatment was found to display both intestinal region- and inflammation-dependent effects. Unexpectedly we identified that 1) L. reuteri treatment increased bone density in females but only in those that underwent DSI and 2) DSI benefited cortical bone parameters. In the bone marrow, dorsal surgery induced CD4+ T cell numbers, a response that was unaffected by L. reuteri treatment, whereas expression of RANKL, OPG and IL-10 were significantly affected by L. reuteri treatment. Conclusion Our data reveals a previously unappreciated effect of a mild surgical procedure causing a long-lasting effect on inflammatory gene expression in the gut and the bone. Additionally, we demonstrate that in intact female mice, the beneficial effect of L. reuteri on bone requires an elevated inflammatory status.

Regulation of Early Cartilage Destruction in Inflammatory Arthritis by Death Receptor 3

To investigate the role of death receptor 3 (DR‐3) and its ligand tumor necrosis factor–like molecule 1A (TL1A) in the early stages of inflammatory arthritis.

Death Receptor 3 (TNFRSF25) Increases Mineral Apposition by Osteoblasts and Region Specific New Bone Formation in the Axial Skeleton of Male DBA/1 Mice

Objectives. Genome wide association studies identified TNFSF member TNF-like protein 1A (TL1A, TNFSF15) as a potential modulator of ankylosing spondylitis (AS). TL1A is the only confirmed TNFSF ligand of death receptor 3 (DR3, TNFRSF25); however, its role in disease pathology is not characterised. We evaluated DR3s role in controlling osteoblast- (OB-) dependent bone formation in vitro and in vivo. Methods. Osteoprogenitor cells and OB were cultured from male DR3-deficient (DR3ko) and wild-type (DR3wt) DBA/1 mice. DR3 and RANKL expression were tested by flow cytometry. Alkaline phosphatase and mineralization were quantified. Osteopontin, osteoprotegerin, and pro MMP-9 were measured by ELISA. A fluorescent probe (BoneTag) was used to measure in vivo mineralization in 10-month-old mice. Results. DR3 was expressed on osteoprogenitors and OB from DR3wt mice. Alkaline phosphatase, osteopontin, and mineral apposition were significantly elevated in DR3wt cultures. Levels of RANKL were comparable whilst osteoprotegerin was significantly increased in DR3wt cultures. In vivo incorporation of BoneTag was significantly lower in the thoracic vertebrae of 10-month-old DR3ko mice. Conclusions. These data identify new roles for DR3 in regulating OB-dependent bone mineral apposition. They potentially begin to explain the atypical pattern of new bone formation observed in the axial skeleton of grouped, aging DBA/1 mice.

Temporal and regional intestinal changes in permeability, tight junction, and cytokine gene expression following ovariectomy‐induced estrogen deficiency

Estrogen deficiency that occurs during menopause is associated with wide‐ranging consequences, including effects on the gastrointestinal system. Although previous studies have implicated a role for estrogen in modulating colonic permeability and inflammatory gene expression, the kinetics of these changes following loss of estrogen and whether they are intestinal region specific are unknown. To test this, we performed sham or ovariectomy (OVX) surgery in BALB/c mice and examined permeability (in vivo and ex vivo) and gene expression changes in the duodenum, jejunum, ileum, and colon at 1, 4, and 8 weeks postsurgery. In vivo permeability, assessed by FITC‐dextran gavage and subsequent measures of serum levels, indicated that OVX significantly increased whole intestinal permeability 1 week postsurgery before returning to sham levels at 4 and 8 weeks. Permeability of individual intestinal sections, measured ex vivo by Ussing chambers, revealed specific regional and temporal responses to OVX, with the most dynamic changes exhibited by the ileum. Analysis of gene expression, by qPCR and by mathematical modeling, revealed an OVX‐specific effect with tight junction and inflammatory gene expression elevated and suppressed with both temporal and regional specificity. Furthermore, ileal and colonic expression of the tight junction protein occludin was found to be significantly correlated with expression of TNFα and IL‐1β. Together, our studies reveal previously unappreciated effects of estrogen deficiency in specific intestinal segments and further demonstrate temporal links between estrogen deficiency, inflammatory genes, and intestinal permeability.

CCL3 and MMP-9 are induced by TL1A during death receptor 3 (TNFRSF25)-dependent osteoclast function and systemic bone loss.

Reduced bone density and secondary osteoporosis, resulting in increased risk of fracture, is a significant complicating factor in the inflammatory arthritides. While the exact etiology of systemic bone loss is not fully elucidated, recent insights into the tumor necrosis factor super family (TNFSF) revealed a potential role for death receptor 3 (DR3/TNFRSF25) and one of its ligands, TNF-like protein 1A (TL1A/TNFSF15). The mechanisms by which DR3/TL1A signalling modulates bone loss are unclear. We investigated the effect of DR3/TL1A signalling upon osteoclast-dependent chemokine and MMP production to unravel novel mechanisms whereby this pathway regulates OC formation and OC-dependent bone resorption. Collagen induced arthritis (CIA) was established in DR3wt and DR3ko mice, joints were sectioned and analysed histologically for bone damage while systemic trabecular bone loss distal to the affected joints was compared by micro-CT. Ablation of DR3 protected DBA/1 mice against the development and progression of CIA. In DR3ko, joints of the ankle and mid-foot were almost free of bone erosions and long bones of mice with CIA were protected against systemic trabecular bone loss. In vitro, expression of DR3 was confirmed on primary human CD14+ osteoclast precursors by flow cytometry. These cells were treated with TL1A in osteoclast differentiation medium and TRAP+ osteoclasts, bone resorption, levels of osteoclast-associated chemokines (CCL3, CCL2 and CXCL8) and MMP-9 measured. TL1A intensified human osteoclast differentiation and bone resorption and increased osteoclast-associated production of CCL3 and MMP-9. Our data reveals the DR3 pathway as an attractive therapeutic target to combat adverse bone pathology associated with inflammatory arthritis. We demonstrate that DR3 is critical in the pathogenesis of murine CIA and associated secondary osteoporosis. Furthermore, we identify a novel mechanism by which the DR3/TL1A pathway directly enhances human OC formation and resorptive activity, controlling expression and activation of CCL3 and MMP-9.

Immunology of Gut-Bone Signaling

In recent years a link between the gastrointestinal tract and bone health has started to gain significant attention. Dysbiosis of the intestinal microbiota has been linked to the pathology of a number of diseases which are associated with bone loss. In addition modulation of the intestinal microbiota with probiotic bacteria has revealed to have both beneficial local and systemic effects. In the present chapter, we discuss the intestinal and bone immune systems, explore how intestinal disease affects the immune system, and examine how these pathologic changes could adversely impact bone health.

Epithelial Barrier Function in Gut-Bone Signaling.

The intestinal epithelial barrier plays an essential role in maintaining host homeostasis. The barrier regulates nutrient absorption as well as prevents the invasion of pathogenic bacteria in the host. It is composed of epithelial cells, tight junctions, and a mucus layer. Several factors, such as cytokines, diet, and diseases, can affect this barrier. These factors have been shown to increase intestinal permeability, inflammation, and translocation of pathogenic bacteria. In addition, dysregulation of the epithelial barrier can result in inflammatory diseases such as inflammatory bowel disease. Our lab and others have also shown that barrier disruption can have systemic effects including bone loss. In this chapter, we will discuss the current literature to understand the link between intestinal barrier and bone. We will discuss how inflammation, aging, dysbiosis, and metabolic diseases can affect intestinal barrier-bone link. In addition, we will highlight the current suggested mechanism between intestinal barrier and bone.

Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice

Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specific bone formation and abnormal bone mineral content. A recent study suggested a role for the complement membrane attack complex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attack complex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture. In vivo bone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cortical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed in female mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis, with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bone cells were studied in isolation, in vitro osteoclastogenesis was significantly increased in male CD59a-deficient mice, although osteoblast formation was not altered. Our data reveal, for the first time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation in male mice results in longer and wider bones, but with less density, which is likely a major contributing factor for their susceptibility to osteoarthritis. These findings increase our understanding of the role of complement regulation in degenerative arthritis.

The Potential of Probiotics as a Therapy for Osteoporosis

Osteoporosis, characterized by low bone mass and micro-architectural deterioration of bone tissue with increased risk of fracture, can be categorized into two forms: primary and secondary, depending on whether it occurs as part of the natural aging process (estrogen deficiency) or as part of disease pathology. In both forms bone loss is due to an imbalance in the bone remodeling process, with resorption/formation skewed more toward bone loss. Recent studies and emerging evidence consistently demonstrate the potential of the intestinal microbiota to modulate bone health. This review discusses the process of bone remodeling and the pathology of osteoporosis and introduces the intestinal microbiota and its potential to influence bone health. In particular, we highlight recent murine studies that examine how probiotic supplementation can both increase bone density in healthy individuals and protect against primary (estrogen deficiency) as well as secondary osteoporosis. Potential mechanisms are described to account for how probiotic treatments could be exerting their beneficial effect on bone health.