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Supplementary Material for: CD300a Regulates Mouse Macrophage Functionality in Allergic Inflammation

posted on 2023-03-16, 05:30 authored by Puzzovio P.G., Levy B.D., Levi-Schaffer F.
Background: CD300a is an inhibitory receptor (IR) expressed on several leukocytes, including mast cells (MCs) and macrophages (MΦ), important cells in allergic inflammation (AI). We have previously characterized CD300a role on MCs and in vivo in mouse models of allergy, in which the absence of CD300a resulted in increased inflammatory features and delayed resolution. However, the exact mechanism of this delayed resolution is unclear. Our hypothesis is that MΦ, important players in resolution, might be impaired when CD300a is absent. Objectives: The aim of the study was to investigate CD300a-dependent functionality of mouse MΦ. Method: MΦ were purified from the peritoneum of wild-type (WT) and CD300a−/− mice naïve and 48 h and 96 h after challenge with ovalbumin/alum. Phenotype switching was analyzed via specific M1-M2 inducers and markers. MΦ phagocytotic ability was assessed via Staphylococcus aureus pHrodo-conjugated bioparticles. The influence of MCs on MΦ was investigated by incubating WT MΦ with supernatants from non-activated and IgE-activated bone marrow-derived MCs (BMMCs) and analyzing functional responses. Results: Naïve CD300a−/− MΦ presented with increased sensitivity to activation when treated with LPS. Absence of CD300a results in increased Arg1 expression and increased IL-6 release when MΦ are purified from allergic peritonitis-induced mice. Similar results were obtained when CD300a−/− MΦ were purified 96 h after challenge. On the other hand, CD300a absence did not affect phagocytosis. WT MΦ incubated with supernatants of non-activated and IgE-activated BMMCs presented with increased iNOS expression and decreased Arg1 levels. Conclusions: The IR CD300a controls the activation state of MΦ, and its absence could augment the inflammatory state seen in CD300a−/− mice. Moreover, MCs can also influence MΦ phenotype switching. This may partially explain the delayed AI resolution seen in these mice.