Brief report: Granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and NLRP3 inflammasome up-regulation in an in vivo mouse model.

ABSTRACT

OBJECTIVE: To determine the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of inflammatory macrophages in an in vivo model of monosodium urate monohydrate (MSU) crystal-induced inflammation. METHODS: C57BL/6J mice were treated with either clodronate liposomes to deplete peritoneal macrophages or GM-CSF antibody and were then challenged by intraperitoneal injection of MSU crystals. Peritoneal lavage fluid was collected, and cellular infiltration was determined by flow cytometry. Purified resident and MSU crystal-recruited monocyte/macrophages were stimulated ex vivo with MSU crystals. The interleukin-1ß (IL-1ß) levels in lavage fluids and ex vivo assay supernatants were measured. GM-CSF-derived and macrophage colony-stimulating factor (M-CSF)-derived macrophages were generated in vitro from bone marrow cells. Protein expression of IL-1ß, caspase 1, NLRP3, and ASC by in vitro- and in vivo-generated monocyte/macrophages was analyzed by Western blotting. RESULTS: Depletion of resident macrophages lowered MSU crystal-induced IL-1ß and GM-CSF levels in vivo as well as IL-1ß production by MSU crystal-recruited monocytes stimulated ex vivo. GM-CSF neutralization in vivo decreased MSU crystal-induced IL-1ß levels and neutrophil infiltration. MSU crystal-recruited monocyte/macrophages from GM-CSF-neutralized mice expressed lower levels of the macrophage marker CD115 and produced less IL-1ß following ex vivo stimulation. These monocytes exhibited decreased expression of NLRP3, pro/active IL-1ß, and pro/active caspase 1. In vitro-derived GM-CSF-differentiated macrophages expressed higher levels of NLRP3, pro/active IL-1ß, and pro/active caspase 1 compared to M-CSF-differentiated macrophages. CONCLUSION: GM-CSF plays a key role in the differentiation of MSU crystal-recruited monocytes into proinflammatory macrophages. GM-CSF production may therefore contribute to the exacerbation of inflammation in gout.