The pyroptotic cell death effector gasdermin D (GSDMD) is required for murine models of hereditary inflammasome-driven, IL-1 beta-dependent, autoinflammatory disease, making it an attractive therapeutic target. However, the importance of GSDMD for more common conditions mediated by pathological IL-1 beta activation, such as gout, remain unclear. In this study, we address whether GSDMD and the recently described GSDMD inhibitor necrosulfonamide (NSA) contribute to monosodium urate (MSU) crystal-induced cell death, IL-1 beta release, and autoinflammation. We demonstrate that MSU crystals, the etiological agent of gout, rapidly activate GSDMD in murine macrophages. Despite this, the genetic deletion of GSDMD or the other lytic effector implicated in MSU crystal killing, mixed lineage kinase domain-like (MLKL), did not prevent MSU crystal-induced cell death. Consequently, GSDMD or MLKL loss did not hinder MSU crystal-mediated release of bioactive IL-1 beta. Consistent with in vitro findings, IL-1 beta induction and autoinflammation in MSU crystal-induced peritonitis was not reduced in GSDMD-deficient mice. Moreover, we show that the reported GSDMD inhibitor, NSA, blocks inflammasome priming and caspase-1 activation, thereby preventing pyroptosis independent of GSDMD targeting. The inhibition of cathepsins, widely implicated in particle-induced macrophage killing, also failed to prevent MSU crystal-mediated cell death. These findings 1) demonstrate that not all IL-1 beta-driven autoinflammatory conditions will benefit from the therapeutic targeting of GSDMD, 2) document a unique mechanism of MSU crystal-induced macrophage cell death not rescued by pan-cathepsin inhibition, and 3) show that NSA inhibits inflammasomes upstream of GSDMD to prevent pyroptotic cell death and IL-1 beta release.

Granulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF deficient mice and in anti-G-CSF monoclonal antibody treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF deficient and anti-G-CSF monoclonal antibody treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF driven neutrophil responses in ocular autoimmunity, operatin within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis.

Flow cytometry can be used to study STAT phosphorylation on a per cell basis. Cells are fixed, permeablized, and stained with antibodies that specifically bind to the phosphorylated form of the STAT protein. This allows the tyrosine phosphorylation of a single STAT to be studied within a heterogeneous cell population and/or the phosphorylation of several STATs within the one cell type.