Alpha-7 Nicotinic Receptor Dampens Murine Osteoblastic Response to Inflammation and Age-Related Osteoarthritis.

Introduction: Osteoarthritis (OA) is a whole-joint disease characterized by a low-grade inflammation that is involved in both cartilage degradation and subchondral bone remodeling. Since subchondral bone has a cholinergic innervation and that acetylcholine (Ach) might have an anti-inflammatory effect through the α7 nicotinic Ach receptor (α7nAchR), we aimed (i) to determine the expression of non-neuronal cholinergic system and nicotinic receptor subunits by murine and human osteoblasts, (ii) to address the role of α7nAchR in osteoblastic response to inflammation, and (iii) to study the role of α7nAchR in a spontaneous aging OA model. Methods: Primary cultures of WT and α7nAchR knock-out mice (Chrna7-/-) murine osteoblasts and of subchondral bone human OA osteoblasts were performed. The expressions of the non-neuronal cholinergic system and of the nAchR subunits were assessed by PCR. In vitro, IL1ß-stimulated WT, Chrna7-/-, and human osteoblasts were pretreated with nicotine. At 24 h, expressions of interleukin-6 (IL6) and metalloproteinase-3 and -13 (MMP), RANK-ligand (RANKL), and osteoprotegerin (OPG) were quantified by qPCR and ELISA. Spontaneous aging OA was evaluated and compared between male WT and Chrna7-/- mice of 9 and 12 months. Results: Murine WT osteoblasts express the main components of the cholinergic system and α7 subunit composing α7nAchR. Nicotine partially prevented the IL1ß-induced expression and production of IL6, MMP3, and RANKL in WT osteoblasts. The effect for IL6 and MMP was mediated by α7nAchR since nicotine had no effect on Chrna7-/- osteoblasts while the RANKL decrease persisted. Chrna7-/- mice displayed significantly higher cartilage lesions than their WT counterparts at 9 and 12 months, without difference in subchondral bone remodeling. Human OA osteoblasts also expressed the non-neuronal cholinergic system and α7 subunit as well as CHRFAM7A, the dominant negative duplicate of Chrna7. Nicotine pretreatment did not significantly reduce IL6 and MMP3 production in IL-1ß-stimulated human osteoarthritic osteoblasts (n = 4), possibly due to CHRFAM7A. Conclusion: Cholinergic system counteracts murine osteoblastic response to IL-1ß through α7nAchR. Since α7nAchR deletion may limit cartilage degradation during murine age-related OA, enhancing cholinergic system could be a new therapeutic target in OA but may depend on CHRFAM7A expression.

SIRT1 directly activates autophagy in human chondrocytes.

Osteoarthritis (OA) is the most common form of arthritis worldwide with no effective treatment. Ageing is the primary risk factor for OA. We sought to investigate if there is a distinct and functional convergence of ageing-related mechanisms SIRT1 and autophagy in chondrocytes. Our results show that, levels of SIRT1 are decreased in human normal aged and OA cartilage compared with young cartilage. Moreover, silencing SIRT1 in chondrocytes lead to decreased expression of chondrogenic markers but did not alter the expression of catabolic proteases. In contrast, activation of SIRT1 increased autophagy in chondrocytes by the deacetylation of lysine residues on crucial autophagy proteins (Beclin1, ATG5, ATG7, LC3). This activation was shown to be mTOR/ULK1 independent. Our results indicate that maintenance of autophagy in chondrocytes by SIRT1 is essential for preserving cartilage integrity throughout life and therefore is a target for drug intervention to protect against OA.

Spermidine restores dysregulated autophagy and polyamine synthesis in aged and osteoarthritic chondrocytes via EP300.

Ageing is the primary risk factor for osteoarthritis (OA). A decline in the ageing-associated process of autophagy is suggested as a potential contributor to OA development. Polyamines such as spermidine decrease during ageing, contributing to impaired autophagy and reduced cellular function. However, the role of polyamines and their effect on the regulatory mechanism governing autophagy in aged and arthritic cartilage tissue has not been established. Elucidating if polyamine regulation of autophagy is impaired during ageing and OA in chondrocytes may lead to improved treatment approaches to protect against cartilage degradation. Our results indicate that polyamine synthesis was decreased in aged and OA cartilage, along with reduced autophagy activity, evidenced by decreased autophagy-related gene and protein expression and autophagosome formation. Importantly, spermidine treatment increased the expression of the acetyltransferase EP300, which binds to crucial autophagy proteins, Beclin1 and LC3, and elevates chondrocyte autophagy. Our data indicate spermidine prevents the ageing- and OA-related decrease in autophagy and may protect against OA development.

Nociceptive Sensitizers Are Regulated in Damaged Joint Tissues, Including Articular Cartilage, When Osteoarthritic Mice Display Pain Behavior.

OBJECTIVE: Pain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify pain-sensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OA-related pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation. METHODS: Ten-week-old mice underwent sham surgery, partial meniscectomy, or surgical destabilization of the medial meniscus (DMM). Pain-related behavior as determined by a variety of methods (testing of responses to von Frey filaments, cold plate testing for cold sensitivity, analgesiometry, incapacitance testing, and forced flexion testing) was assessed weekly. Once pain-related behavior was established, RNA was extracted from either whole joints or microdissected tissue samples (articular cartilage, meniscus, and bone). Reverse transcription-polymerase chain reaction analysis was performed to analyze the expression of 54 genes known to regulate pain sensitization. Cartilage injury assays were performed using avulsed immature hips from wild-type or genetically modified mice or by explanting articular cartilage from porcine joints preinjected with pharmacologic inhibitors. Levels of nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbent assay. RESULTS: Mice developed pain-related behavior 8 weeks after undergoing partial meniscectomy or 12 weeks after undergoing DMM. NGF, bradykinin receptors B1 and B2, tachykinin, and tachykinin receptor 1 were significantly regulated in the joints of mice displaying pain-related behavior. Little regulation of inflammatory cytokines, leukocyte activation markers, or chemokines was observed. When tissue samples from articular cartilage, meniscus, and bone were analyzed separately, NGF was consistently regulated in the articular cartilage. The other pain sensitizers were also largely regulated in the articular cartilage, although there were some differences between the 2 models. NGF and tachykinin were strongly regulated by simple mechanical injury of cartilage in vitro in a transforming growth factor ß-activated kinase 1-, fibroblast growth factor 2-, and Src kinase-dependent manner. CONCLUSION: Damaged joint tissues produce proalgesic molecules, including NGF, in murine OA.

Respiratory infections cause the release of extracellular vesicles: implications in exacerbation of asthma/COPD.

BACKGROUND: Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs) in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1ß/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1ß/IL-18, neutrophilia and subsequent disease exacerbations. METHODS: To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis. RESULTS: Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1ß/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia. CONCLUSIONS: This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

Aging mechanisms in arthritic disease.

Arthritic disease is one of the most common age-related pathologies worldwide. The erosion of cartilaginous tissues from articular surfaces within the joint and the failure to efficiently repair and regenerate this region with age lead to debilitating joint destruction, severe pain, and a crippling loss of function. In addition to the accumulative damage brought about by years of mechanical forces acting upon this region of tissue, there are also defects in underlying biological mechanisms which predispose the older population to excessive joint erosion. This occurs as aberrations in normal chondrocyte biology lead to a reduction in crucial matrix proteins and inhibitory molecules, and elevated production of destructive enzymes. The end result is an accelerated loss of articular cartilage and increased erosion of the joint. As a significant global link exists between aging and the onset of arthritis, this review will consider whether factors known to affect lifespan may also play a role in arthritic disease.