ARTICLE https://doi.org/10.1038/s41467-020-17749-6 OPEN Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis Elisabeth Jäger 1,9, Supriya Murthy 1,9, Caroline Schmidt 1, Magdalena Hahn 1, Sarah Strobel1, Anna Peters 2, Claudia Stäubert 2, Pelin Sungur 3, Tom Venus4, Mandy Geisler5, Veselina Radusheva1, Stefanie Raps1, Kathrin Rothe1, Roger Scholz6, Sebastian Jung1, Sylke Wagner1, Matthias Pierer1, Olga Seifert1, Wenhan Chang 7, Irina Estrela-Lopis4, Nora Raulien1, Knut Krohn 8, Norbert Sträter5, ✉ ✉ Stephanie Hoeppener 3, Torsten Schöneberg2, Manuela Rossol 1,10 & Ulf Wagner 1,10 1234567890():,; 2+ 2+ Increased extracellular Ca concentrations ([Ca ]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extra- osseous 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 2+ by increases in [Ca ]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+]inafflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflam- mation 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. 1 Department of Internal Medicine, Division of Rheumatology, Leipzig University, Liebigstraße 19, 04103 Leipzig, Germany. 2 Rudolf Schönheimer Institute of Biochemistry, Faculty of Medicine, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany. 3 Jena Center for Soft Matter (JCSM), Friedrich-Schiller-University Jena, Humboldtstraße 10, 07743 Jena, Germany. 4 Institute for Medical Physics and Biophysics, Leipzig University, Härtelstraße 16-18, 04107 Leipzig, Germany. 5 Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany. 6 Department of Orthopaedic, Trauma and Plastic Surgery, Leipzig University, Liebigstraße 20, Leipzig, Germany. 7 UCSF Department of Veterans Affairs Medical Center, San Francisco, CA, USA. 8 DNA Core Unit Leipzig, Liebigstraße 19, 04103 Leipzig, Germany. 9These authors contributed equally: Elisabeth Jäger, Supriya Murthy. ✉ 10These authors jointly supervised this work: Manuela Rossol, Ulf Wagner. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2020) 11:4243 | https://doi.org/10.1038/s41467-020-17749-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17749-6 – 2+ β he calcium-sensing receptor (CaSR) has been shown to technology (CRISPR Cas9-CaSR). The [Ca ]ex-induced IL-1 trigger NLRP3 inflammasome activation both in vivo and response was found to be significantly diminished in T 1,2 fl fi in vitro and has been described to mediate in ammatory CaSR-de cient THP-1 cells, while their response to adenosine effects in human cryopyrin-associated periodic syndromes triphosphate (ATP) or monosodium urate (MSU) crystals was (CAPS)1 and in the carrageenan-induced foot pad swelling mouse unaffected (Fig. 1a). This CaSR effect was confirmed by experi- 2 2+ model . Increased [Ca ]ex also triggers constitutive macro- ments with peripheral blood monocytes from mice with a pinocytosis in monocytes and macrophages via G protein- myeloid-specific CaSR ablation (B6.129P2-Lyz2tm1(cre)Ifo7jx- mediated CaSR signaling3. The receptor is expressed in numer- CaSRΔflox/Δflox), which were found to respond with significantly 2+ ous cell types, such as monocytes, macrophages, smooth muscle decreased IL-1β secretion upon stimulation with [Ca ]ex in the fl and endothelial cells, and contributes to in ammatory responses presence of 5.6 mM [Pi]ex (Fig. 1b), while their ATP response was 4 5 2+ in allergic asthma and inflammatory lung disease , after myo- unaltered (Supplementary Fig. 1a). [Ca ]ex-induced IL-1β cardial infarction6 and in obesity7. responses were dependent on the NLRP3 inflammasome both 2+ Short-lived increases in [Ca ]ex occur in the interstitial fluid in THP-1 cells and in peripheral blood monocytes, as demon- around activated8–10 or dying cells2. More longstanding increases strated by experiments with a NLRP3 knock-down THP-1 cell 2+ 11–13 of [Ca ]ex have been reported at sites of chronic infections line and with peripheral blood monocytes treated with the 14 2+ fi and dialysis-related peritonitis . Homeostasis of [Ca ]ex is NLRP3-speci c inhibitor MCC950 (Supplementary Fig. 1b, c). 2 2+ intricately linked with the anion phosphate [Pi], the clinical As shown previously , [Ca ]ex-induced NLRP3 inflamma- relevance of which is most apparent in end-stage chronic kidney some activation does not occur when monocytes are primed with disease (CKD). In those patients, hyperphosphatemia is asso- lipopolysaccharide (LPS) and stimulated only briefly with fl 2+ ciated with excess mortality, systemic in ammation and vascular increased [Ca ]ex, which is in contrast to the ATP response. calcification15, especially in cases of severe hyperphosphatemia Maximum IL-1β release occurs when LPS is added simulta- fi 16 2+ which is de ned as serum levels higher than 4.54 mM . neously with increased [Ca ]ex, but requires considerably longer Coronary artery calcifications, which indicate perturbations of stimulation compared to ATP (Supplementary Fig. 1d). Accord- 2+ 17,18 [Ca ]ex and [Pi] homeostasis, occur in CKD , but also in ingly, in all subsequent experiments monocytes were stimulated 2+ coronary artery disease and other vascular degenerative with [Ca ]ex and LPS simultaneously. 19,20 fl 2+ β diseases . Failure to prevent ectopic mineralization due to The in uence of [Pi]ex on [Ca ]ex-induced IL-1 release in 2+ [Ca ]ex and [Pi] overload is common in tissues with localized monocytes was investigated using customized RPMI1640 media fl 21,22 2+ in ammation and cell necrosis , and occurs also in aorta and with [Pi] ranging from 1 to 5.6 mM, whereby [Ca ] was added carotid and coronary arteries in the autoimmune disease rheu- as calcium chloride. Due to protein binding, the final concentra- matoid arthritis (RA)23. Importantly, activation of the NLRP3 tion of ionized calcium in the tissue culture medium is always inflammasome has been linked to vascular damage and excess lower than the added calcium chloride concentration, which is mortality in atherosclerosis24 and myocardial infarction25. Clin- indicated in all figures (detailed analysis in Rossol et al., 20122) fl ≥ ical evidence for an aggravating effect of in ammasome activation unless indicated otherwise. At 3mM of [Pi]ex, increased 2+ β in atherosclerosis comes from the CANTOS study, which showed [Ca ]ex-triggered concentration-dependent IL-1 release (Fig. the benefit of IL-1β blockade in patients with previous myocardial 1c) and ASC (apoptosis-associated speck-like protein containing infarction26. a CARD) speck formation (Supplementary Fig. 1e) was observed. 2+ In RA, increased CaSR expression has been associated with No [Ca ]ex effect was discernible at low [Pi]ex. High [Pi]ex 27 2+ severe coronary artery calcification , but the role of the receptor combined with low [Ca ]ex did also not induce IL-1β release in arthritis has not been investigated. In this disease, the invading (Fig. 1c). 2+ pannus forms an osteo-immunological interphase with the bone In serum-free buffer or medium, an increase of [Ca ] and [Pi] matrix at the sites of erosive lesions. At erosion sites beneath beyond the solubility product immediately leads to precipitation 2+ osteoclasts, extremely high [Ca ]ex values of 40 mM have been of macroscopically visible calcium orthophosphate crystals. In 28 2+ fl measured . Indirect evidence suggests that besides Ca ,Pi, and vivo, calcium phosphate precipitation in body uids is prevented the Ca2+-binding crystallization inhibitor fetuin-A are also lib- by the presence of the serum protein fetuin-A and other Ca2+- erated from the extracellular matrix during osteoclastic bone binding proteins. Fetuin-A is present in high concentrations in resorption29–31. Fetuin-A is known to stabilize calcium phosphate fetal bovine serum (FBS), and addition of FBS completely crystal precursors during bone mineralization as colloids and is prevented the microscopically detectable calcium orthophosphate one of the most abundant non-collagenous proteins in bone32. crystal formation in RPMI1640 cell culture medium upon 2+ 2+ This study investigates the mechanism of [Ca ]ex-induced addition of 2.5 mM Ca . However, using dynamic light 2+ IL-1β release in RA, and the function of [Pi]ex in [Ca ]ex- scattering analysis (DLS), we were able to detect nanometer- mediated inflammasome activation. We find that increased sized particles if Ca2+ was added to RPMI1640/10% FBS. The 2+ [Ca ]ex in the presence of increased [Pi]ex and fetuin-A leads to particles were below the detection limit of light microscopy due to the formation of fetuin-A-based calciprotein particles (CPPs), their size between 60 and 100 nm, but were detectable by DLS and simultaneously induces CaSR signaling, which triggers CPP already seconds after addition of Ca2+ to FBS-supplemented uptake and subsequently NLRP3 inflammasome activation. This culture medium (Fig. 1d, e). The particles developed only at β ≥ 2+ CaSR-mediated process and the resulting IL-1 release are 3mMof[Pi]ex, and not in the absence of [Ca ] (see Fig. 1d). enhanced in RA. Allosteric enhancement of CaSR signaling Their size increased only marginally over 20 h of incubation in vivo leads to aggravation of arthritis, which emphasizes the (Fig. 1e). In parallel to particle formation, [Ca2+] decreased due pivotal role of the receptor in this disease. to the incorporation of Ca2+ in particles.