Regulation of inflammasome and NET formation in neutrophils by PI3K signalling

Abstract

Introduction: The PI3K signalling pathway controls many physiological processes including inflammation, chemotaxis, proliferation, phagocytosis and microbicidal activities in immune cells such as macrophages and dendritic cells. However, it’s role in neutrophils especially in the regulation of inflammasome activation and neutrophil extracellular traps (NET) formation is yet to be understood. Here, I investigated the role of p110δ isoform of the PI3K, which is uniquely expressed in leukocytes, in neutrophilic inflammatory responses. Methods: Bone marrow derived neutrophils (BMDN) were isolated from both WT and P110δD910A mice. BMDN from both mice were challenged with LPS and Nigericin, the levels of IL-6 and IL-1β were determined by ELISA. In some experiments, NET formation was assessed using immunofluorescence microscopy and picogreen assay. Chemotaxis towards MIP1α and CXCL1 was assessed using a transwell assay system. BMDN from WT mice were treated with a pharmacological inhibitor of p110δ(CAL101) to validate the above experiments. Western blot was done to assess protein levels in pro-caspase1, ASC, NLRP3 (inflammasome proteins) and pro-IL1β. Also, WT and P110δD910A mice were challenged with LPS and NET production was measured. Results: We observed significant impairment to core neutrophilic functions such as proinflammatory cytokine IL-6 production, NET formation and migration by neutrophils from p110δD910A mice compared to their WT counterparts. However, there was no significant difference in IL-1β or pro-caspase1 production by WT and p110δD910A neutrophils. The same result was observed in CAL101-treated BMDN. Sepsis induced P110δD910A mice were also impaired in their ability to produce NET when compared to their WT counterparts. Conclusion: These findings suggest that signaling via the p110δ isoform of PI3K plays an important role in regulating neutrophil migration and NET formation but is dispensable for inflammasome response.