Tofacitinib inhibits granulocyte-macrophage colony-stimulating factor-induced NLRP3 inflammasome activation in human neutrophils

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine that activates myeloid cells to initiate tissue inflammation. However, the molecular actions of GM-CSF against innate immunity are still poorly characterized. Here, we investigated the in vitro effects of GM-CSF on the activation of human myeloid lineages, neutrophils, and the underlying intracellular signaling mechanism, including inflammasome activation. Methods: Human neutrophils were stimulated with GM-CSF in the presence or absence of tofacitinib. The cellular supernatants were analyzed for interleukin-1 beta (IL-1 beta) and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1 beta mRNA expressions in human neutrophils were analyzed by real-time polymerase chain reaction. Protein phosphorylation of neutrophils was assessed by Western blot using phospho-specific antibodies. Results: Stimulation with GM-CSF alone, but not tumor necrosis factor-alpha, was shown to increase the release of IL-1 beta and cleaved caspase-1 (p20) from human neutrophils. Tofacitinib, which inhibits GM-CSF-induced Janus kinase 2 (Jak2)-mediated signal transduction, completely abrogated GM-CSF-induced IL-1 beta and caspase-1 (p20) secretion from neutrophils. GM-CSF stimulation also induced pro-IL-1 beta mRNA expression in neutrophils and induced NLR family pyrin domain-containing 3 (NLRP3) protein expression. Although tofacitinib pretreatment marginally inhibited GM-CSF-induced pro-IL-1 beta mRNA expression, tofacitinib completely abrogated NLRP3 protein expression in neutrophils. Conclusions: These results indicate that GM-CSF signaling induces NLRP3 expression and subsequent IL-1 beta production by affecting neutrophils, which may cause the activation of innate immunity. Therefore, GM-CSF is a key regulator of the NLRP3 inflammasome and IL-1 beta production by activating innate immune cells. This process can be blocked by tofacitinib, which interferes with JAK/STAT signaling pathways.