Background/Purpose: Monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals are potent inducers of inflammation by activation of the inflammasome in neutrophils phagocytosing the crystals and undergoing cell death by extracellular trap formation (NETosis), resulting in flares of gout or pseudogout. However, crystal deposition does not always lead to inflammatory flares. Whether there are homeostatic mechanisms preventing activation of the inflammasome in the presence of crystal deposits is unclear. We have investigated the role of extracellular trap formation of macrophages (MET) stimulated with MSU and CPP crystals.

Methods: MET formation was assessed in THP-1 cells, differentiated with phorbol-12-myristate-13-acetate (PMA), and human monocytes from healthy donors, isolated by magnetic bead separation and differentiated in vitro with GM-CSF or M-CSF respectively, to obtain M1 and M2 macrophages. Macrophages were stimulated with MSU and CPP crystals with or without priming with IFNg/LPS. METs were detected by DNA staining with SytoxGreen and Hoechst and immunostaining of citrullinated Histone 3 and myeloperoxidase and subsequently quantified with an automated imaging system. IL-1b production was measured by ELISA. To assess the dependence of METosis on NLRP3, macrophages were stimulated in the presence or absence of inhibitors of NLRP3, caspase 1 and in THP-1 cells deficient for NLRP3 and gasdermin D (GSDMD). In addition, MET formation was assessed in peritoneal cavity macrophages of C57BL6/c and caspase-1/-11^KO, NRLP3^KO and GSDMD^KO mice.

Results: THP-1 cells were able to release MET after both MSU and CPP crystal stimulation independent of priming with LPS/IFNg. Whereas crystal stimulation of primary human monocytes did not lead to MET formation, in vitro differentiated M1 and M2 macrophages released METs upon stimulation with crystals. In contrast, crystal stimulation alone did not result in IL-1b production but required LPS/IFNg priming in M1 macrophages, whereas M2 did not secrete IL-1b irrespective of priming. Using pharmacological inhibitors of NLRP3 and Caspase 1/4 did not inhibit MET formation after crystal stimulation. Likewise, MET were found in crystal stimulated THP-1 cells deficient for NLRP3 and GSDMD. Furthermore, MET formation could be detected in peritoneal macrophages from wild type C57BL6/c as well as NLRP3, Caspase-1 and GSDMD deficient mice, documenting independence of inflammasome activation in tissue resident macrophages.

Conclusion: We report the novel observation of MSU and CPP crystal induced formation of MET. METosis required differentiation of monocytes to M1 or M2 macrophages, was independent of inflammasome activation and did not induce IL-1b secretion in the absence of priming with LPS/IFNg. These results suggest that MET formation may represent an alternative mechanism by which macrophages may trap crystals without provoking pyroptosis and inflammatory flares.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/macrophage-extracellular-traps-induced-by-monosodium-urate-or-calcium-pyrophosphate-crystals-form-independently-of-nlrp3-inflammasome-activation/