Abstract
Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1β release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1β release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.
Highlights
Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR)
The [Ca2+]ex-induced IL-1β response was found to be significantly diminished in CaSR-deficient THP-1 cells, while their response to adenosine triphosphate (ATP) or monosodium urate (MSU) crystals was unaffected (Fig. 1a)
This CaSR effect was confirmed by experiments with peripheral blood monocytes from mice with a myeloid-specific CaSR ablation (B6.129P2-Lyz2tm1(cre)Ifo7jxCaSRΔflox/Δflox), which were found to respond with significantly decreased IL-1β secretion upon stimulation with [Ca2+]ex in the presence of 5.6 mM [Pi]ex (Fig. 1b), while their ATP response was unaltered (Supplementary Fig. 1a). [Ca2+]ex-induced IL-1β responses were dependent on the NLRP3 inflammasome both in THP-1 cells and in peripheral blood monocytes, as demonstrated by experiments with a NLRP3 knock-down THP-1 cell line and with peripheral blood monocytes treated with the NLRP3-specific inhibitor MCC950 (Supplementary Fig. 1b, c)
Summary
Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1β release in response to CaSR signaling. Homeostasis of [Ca2+]ex is intricately linked with the anion phosphate [Pi], the clinical relevance of which is most apparent in end-stage chronic kidney disease (CKD). In those patients, hyperphosphatemia is associated with excess mortality, systemic inflammation and vascular calcification[15], especially in cases of severe hyperphosphatemia which is defined as serum levels higher than 4.54 mM16. Clinical evidence for an aggravating effect of inflammasome activation in atherosclerosis comes from the CANTOS study, which showed the benefit of IL-1β blockade in patients with previous myocardial infarction[26]
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