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Chronic, low-grade inflammation in osteoarthritis (OA) contributes to symptoms and disease progression. Effective disease-modifying medical OA therapies are lacking, but better understanding inflammatory pathophysiology in OA could lead to transformative therapy. Networks of diverse innate inflammatory danger signals, including complement and alarmins, are activated in OA. Through inflammatory mediators, biomechanical cartilage injury and oxidative stress compromise chondrocyte viability and reprogram viable chondrocytes to hypertrophic differentiation and proinflammatory, and procatabolic responses in mechanistically similar ways. Integral to this reprogramming are certain ‘switching’ pathways in transcriptional signals, other than the well-characterized effects of NFκB and mitogen-activated protein kinase signalling. HIF-2α transcriptional signalling and ZIP8-mediated Zn2+ uptake, with downstream MTF1 transcriptional signalling, have been implicated in chondrocyte reprogramming, but further validation is required. Permissive factors in procatabolic reprogramming of OA chondrocytes by inflammatory mediators also have come to light, including impaired bioenergetics, such as altered mitochondrial function and decreased activity of the bioenergy sensors AMPK and SIRT1. These factors interact with molecular inflammatory responses and proteostasis mechanisms that normally resolve cell stress, such as the unfolded protein response and autophagy. Bioenergy-sensing by AMPK and SIRT1 modulates proteostasis and provides ‘stop signals’ for oxidative stress, inflammatory, and matrix catabolic processes in chondrocytes. The complexity of molecular inflammatory processes in OA, and the involvement of multiple inflammatory mediators in tissue repair responses, raises daunting questions about how to therapeutically target inflammatory processes and macroscopic inflammation in OA. Bioenergy sensing might provide a pragmatic ‘entry point’ for novel strategies to limit OA progression.

Objective. In gout, incompletely defined molecular factors alter recognition of dormant articular and bursal monosodium urate monohydrate (MSU) crystal deposits, thereby inducing self-limiting bouts of characteristically severe neutrophilic inflammation. To define primary determinants of cellular recognition, uptake, and inflammatory responses to MSU crystals, we conducted a study to test the role of Toll-like receptor 2 (TLR-2), TLR-4, and the cytosolic TLR adapter protein myeloid differentiation factor 88 (MyD88), which are centrally involved in innate immune recognition of microbial pathogens.Methods. We isolated bone marrow-derived macrophages (BMDMs) in TLR-2 ؊/؊ , TLR-4 ؊/؊ , MyD88 ؊/؊ , and congenic wild-type mice, and assessed phagocytosis and cytokine expression in response to endotoxin-free MSU crystals under serum-free conditions. MSU crystals also were injected into mouse synovium-like subcutaneous air pouches.Results. TLR-2 ؊/؊ , TLR-4 ؊/؊ , and MyD88 ؊/؊ BMDMs demonstrated impaired uptake of MSU crystals in vitro. MSU crystal-induced production of interleukin-1␤ (IL-1␤), tumor necrosis factor ␣, keratinocyte-derived cytokine/growth-related oncogene ␣, and transforming growth factor ␤1 also were significantly suppressed in TLR-2 ؊/؊ and TLR-4 ؊/؊ BMDMs and were blunted in MyD88 ؊/؊ BMDMs in vitro. Neutrophil influx and local induction of IL-1␤ in subcutaneous air pouches were suppressed 6 hours after injection of MSU crystals in TLR-2 ؊/؊ and TLR-4 ؊/؊ mice and were attenuated in MyD88 ؊/؊ mice.Conclusion. The murine host requires TLR-2, TLR-4, and MyD88 for macrophage activation and development of full-blown neutrophilic, air pouch inflammation in response to MSU crystals. Our findings implicate innate immune cellular recognition of naked MSU crystals by specific TLRs as a major factor in determining the inflammatory potential of MSU crystal deposits and the course of gouty arthritis.

Objective. Interleukin-1␤ (IL-1␤) is a key cytokine linked to the pathogenesis of acute arthritis. Caspase 1, neutrophil elastase, and chymase all process proIL-1␤ to its biologically active form. This study was undertaken to examine the potential contributions of each of these proteases in experimental models of inflammatory arthritis.Methods. Conclusion. The production of IL-1␤ by neutrophils and mast cells is not exclusively dependent on caspase 1, and other proteases can compensate for the loss of caspase 1 in vivo. These pathways might therefore compromise the caspase 1-targeted therapies in neutrophil-predominant arthritis.

Objective. Interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP-activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1␤ and TNF␣.Methods. Expression of AMPK subunits was examined, and AMPK␣ activity was ascertained by the phosphorylation status of AMPK␣ Thr 172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL-1␤ and TNF␣, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPK␣ by small interfering RNA.Results. Normal human knee articular chondrocytes expressed AMPK␣1, ␣2, ␤1, ␤2, and ␥1 subunits. Interleukin-1␤ (IL-1␤), tumor necrosis factor ␣ (TNF␣), and certain other proinflammatory cytokines stimulate chondrocyte responses that promote catabolism of type II collagen and proteoglycans (PGs), thereby compromising cartilage extracellular matrix integrity and tissue homeostasis in osteoarthritis (OA) and inflammatory arthritides (1,2). For example, IL-1␤ and TNF␣ induce expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, induce activation of aggrecanases including ADAMTS-5, and stimulate inducible nitric oxide synthase expression and the generation of nitric oxide (NO), a suppressor of PG synthesis (1-3).Recently, the serine/threonine protein kinase AMP-activated protein kinase (AMPK) was observed to exert antiinflammatory effects in tissues other than cartilage, mediated in part by suppression of NF-B activation (4-10). AMPK is a "super-regulator" of

Objective The etiology of chondrocyte mitochondrial dysfunction in OA is incompletely understood. OA chondrocytes are deficient in active AMPK-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), metabolic biosensors that modulate the mitochondrial biogenesis “master regulator” peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α. Moreover, PGC-1α critically mediates AMPK anti-catabolic activity in chondrocytes. Here, we tested the hypotheses that mitochondrial biogenesis is deficient in human OA chondrocytes, which functionally increases chondrocyte pro-catabolic responses, but is reversed by activation of the AMPK-SIRT1-PGC-1α pathway. Methods We studied human knee chondrocytes, human and mouse knee cartilages. We examined expression and activity (phosphorylation) of AMPKα, and SIRT1 and PGC-1α, and defined and compared mitochondrial content and functions including oxidative phosphorylation (OXPHOS) with expression of mitochondrial biogenesis factors (mitochondrial transcriptional factor A (TFAM), nuclear respiratory factors (NRFs)). Results Human knee OA chondrocytes had decreased mitochondrial biogenesis capacity, linked to reduced AMPKα activity and decreased SIRT1, PGC-1α, TFAM, and NRF1,2 expression. Human knee OA and aged mouse knee cartilages had decreased TFAM and ubiquinol-cytochrome c reductase core protein I (UQCEC1), a subunit of mitochondrial complex III, in situ. Functionally, chondrocyte TFAM knockdown inhibited mitochondrial biogenesis and enhanced pro-catabolic responses to IL-1β. Last, pharmacologic AMPK activation by A-769662 increased PGC-1α via SIRT1, and reversed impairments in mitochondrial biogenesis, OXPHOS, and intracellular ATP in human knee OA chondrocytes. Conclusions Mitochondrial biogenesis is deficient in human OA chondrocytes and this promotes chondrocyte pro-catabolic responses. Activation of the AMPK-SIRT1-PGC-1α pathway reverses these effects, mediated by TFAM, suggesting translational potential to limit OA progression.

Microcrystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate (MSU) deposited in synovium and articular cartilage initiate joint inflammation and cartilage degradation in large part by binding and directly activating resident cells. TLRs trigger innate host defense responses to infectious pathogens, and the expression of certain TLRs by synovial fibroblasts has revealed the potential for innate immune responses to be triggered by mesenchymally derived resident cells in the joint. In this study we tested the hypothesis that chondrocytes also express TLRs and that one or more TLRs centrally mediate chondrocyte responsiveness to CPPD and MSU crystals in vitro. We detected TLR2 expression in normal articular chondrocytes and up-regulation of TLR2 in osteoarthritic cartilage chondrocytes in situ. We demonstrated that transient transfection of TLR2 signaling-negative regulator Toll-interacting protein or treatment with TLR2-blocking Ab suppressed CPPD and MSU crystal-induced chondrocyte release of NO, an inflammatory mediator that promotes cartilage degeneration. Conversely, gain-of-function of TLR2 in normal chondrocytes via transfection was associated with increased CPPD and MSU crystal-induced NO release. Canonical TLR signaling by parallel pathways involving MyD88, IL-1R-associated kinase 1, TNF receptor-associated factor 6, and IκB kinase and Rac1, PI3K, and Akt critically mediated NO release in chondrocytes stimulated by both CPPD and MSU crystals. We conclude that CPPD and MSU crystals critically use TLR2-mediated signaling in chondrocytes to trigger NO generation. Our results indicate the potential for innate immunity at the level of the articular chondrocyte to directly contribute to inflammatory and degenerative tissue reactions associated with both gout and pseudogout.

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