Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells

Abstract

BackgroundOsteoarthritis (OA) is a chronic debilitating joint disorder of particularly high prevalence in the elderly population. Intra-articular basic calcium phosphate (BCP) crystals are present in the majority of OA joints and are associated with severe degeneration. They are known to activate macrophages, synovial fibroblasts, and articular chondrocytes, resulting in increased cell proliferation and the production of pro-inflammatory cytokines and matrix metalloproteases (MMPs). This suggests a pathogenic role in OA by causing extracellular matrix degradation and subchondral bone remodelling. There are currently no disease-modifying drugs available for crystal-associated OA; hence, the aim of this study was to explore the inflammatory pathways activated by BCP crystals in order to identify potential therapeutic targets to limit crystal-induced inflammation.MethodsPrimary human macrophages and dendritic cells were stimulated with BCP crystals, and activation of spleen tyrosine kinase (Syk), phosphoinositide-3 kinase (PI3K), and mitogen-activated protein kinases (MAPKs) was detected by immunoblotting. Lipopolysaccharide (LPS)-primed macrophages were pre-treated with inhibitors of Syk, PI3K, and MAPKs prior to BCP stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). Aa an alternative, cells were treated with synovial fluid derived from osteoarthritic knees in the presence or absence of BCP crystals, and gene induction was assessed by real-time polymerase chain reaction (PCR).ResultsWe demonstrate that exposure of primary human macrophages and dendritic cells to BCP crystals leads to activation of the membrane-proximal tyrosine kinases Syk and PI3K. Furthermore, we show that production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1β and phosphorylation of downstream MEK and ERK MAPKs is suppressed following treatment with inhibitors of Syk or PI3K. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule S100A8 and MMP1 in a Syk-dependent manner and that synovial fluid from OA patients together with BCP crystals exacerbates these effects.ConclusionsWe identify Syk and PI3K as key signalling molecules activated by BCP crystals prior to inflammatory cytokine and DAMP expression and therefore propose that Syk and PI3K represent potential targets for the treatment of BCP-related pathologies.

S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation

“…B). We have previously demonstrated that DAMPS, including BCP crystals and cholesterol crystals, drive S100A8 expression in a Syk/PI3K and Erk dependent manner, and while heme (50 μM) was found to induce MAP kinase phosphorylation within 5 min of incubation (Supplementary Material; Supplementary Fig. 1) in human Mϕ s, pretreatment with the Syk inhibitor, piceatannol, or the Erk inhibitor, PD98059, did not affect heme‐induced S100A8 gene expression, indicating that Syk signaling does not mediate the effects of heme on S100A8 expression.…”

“…High concentrations of S100A8 and S100A9 are reported in inflammatory conditions, such as rheumatoid arthritis, myocardial infarction, and Crohn's disease, and these proteins may exert some of their effects by enabling NLRP3 inflammasome formation and IL‐1β processing via TLR4‐ and reactive oxygen species‐dependent mechanisms . Furthermore, major DAMPs, such as basic calcium phosphate (BCP), associated with osteoarthritis, and cholesterol have been shown to induce S100 protein expression in immune cells …”

Pyroptosis Plays a Role in Osteoarthritis

“…Furthermore, humans with obesity have elevated blood LPS levels. LPS activates caspase-4/5/11, which initiates pyroptosis via the noncanonical inflammasome pathway [99] at both the systemic and tissue levels, leading to increased inflammatory chondrocyte death and loss of cartilage.…”

Disease-Associated Particulates and Joint Inflammation; Mechanistic Insights and Potential Therapeutic Targets

“…We recently demonstrated that, like MSU crystals, BCP crystals also activate, Syk, and its downstream interacting partner, PI3K, in primary macrophages, and that pharmacological inhibition of these kinases leads to reduced MMP expression and pro-inflammatory cytokine production ( 53 , 54 ). Whether Syk and/or PI3K activity is heightened in OA joints is yet to be determined, however, targeting membrane-proximal events induced by BCP crystals is a therapeutic option worth exploring.…”