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. 2020 Apr 1;10:5748. doi: 10.1038/s41598-020-62727-z

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Role of CD44 receptor in mediating monosodium urate monohydrate (MSU) crystal phagocytosis and downstream inflammation in bone marrow derived macrophages (BMDMs) from Cd44^+/+ and Cd44^−/− mice and impact of antibody-mediated receptor shedding on MSU phagocytosis by murine macrophages. BMDMs were treated with MSU (100 μg/mL) for 4 hours to evaluate phagocytosis and for 6 hours to evaluate interleukin-1 beta (IL-1β) gene expression and production. BMDMs were stimulated with lipopolysaccharide (LPS; 100 ng/ml) for 72 hours as a positive control. Lactate dehydrogenase (LDH) release from BMDMs was determined at 1 and 4 hours following incubation with MSU crystals and LDH activity level was used to calculate percent cytotoxicity. We utilized the IM7 clone, which recognizes all CD44 isoforms and causes shedding of the extracellular domain. Anti-CD44 and isotype control (IC) antibodies (2 μg/mL for both antibodies) were incubated with J774A.1 murine macrophages for 4 hours. Data from 3–5 independent experiments are presented with mean and S.D. highlighted. *p < 0.001; **p < 0.01; ***p < 0.05; n.s.: non significant. Scale bar = 50 μm. Dashed line represents gene expression in untreated controls. (a) Phagocytosis of FITC-labeled antibody-coated beads was lower in Cd44^−/− BMDMs. (b) A representative image showing MSU crystal internalization in Cd44^+/+ and Cd44^−/− BMDMs following incubation with MSU crystals for 4 hours. Arrows point to cells with internalized MSU crystals. (c) MSU phagocytosis was reduced in Cd44^−/− BMDMs. (d) Percent cytotoxicity was lower in Cd44^−/− BMDMs. (e) MSU-stimulated IL-1β gene expression was lower in Cd44^−/− BMDMs. (f) MSU and LPS-stimulated IL-1β secretion was lower in Cd44^−/− BMDMs. (g) Representative images depicting the impact of anti-CD44 antibody treatment on MSU phagocytosis by murine J774A.1 macrophages. Arrows point to cells with internalized MSU crystals. (h) Anti-CD44 antibody treatment reduced MSU phagocytosis by murine macrophages.