PP4 modulates macrophage-neutrophil crosstalk to restrict CCL5 -driven NETosis in sepsis.

Abstract

Sepsis is a life-threatening clinical syndrome caused by a dysregulated innate immune response to infection, resulting in excessive systemic inflammation, multi-organ failure, and persistently high mortality rates. In fatal human sepsis, PP4 expression is markedly reduced in myeloid cells, suggesting a protective role against dysregulated innate immunity. However, its role in macrophage-neutrophil crosstalk during sepsis environment remains unclear. To delineate its cell-type specificity, we generated myeloid cell-specific PP4 knockout mice and investigated PP4's function in innate immune regulation during sepsis. PP4-deficient mice exhibited significantly increased susceptibility to sepsis, with severe tissue damage following cecal ligation and puncture (CLP) or endotoxin challenge. Mechanistically, PP4 modulates macrophage-neutrophil crosstalk during the sepsis environment, with its loss leading to dysregulated CCL5/CCR5 signaling, driving excessive neutrophil activation. Elevated macrophage-derived CCL5 enhanced PAD4-dependent NETosis, ROS production, and elastase activity via CCR5, while CCR5 inhibition effectively mitigated neutrophil hyperactivity. At the molecular level, PP4 directly dephosphorylated TBK1, thereby inactivating IRF3 and suppressing macrophage-driven CCL5 production. Furthermore, ERK1/2 phosphorylation upregulated CCR5 expression in PP4-deficient neutrophils post-CLP, amplifying the CCL5/CCR5-mediated NETosis response. Notably, transfection with wild-type PP4-but not a phosphatase-deficient mutant-reduced LPS-mediated CCR5 expression in neutrophils, thereby limiting ROS production and NETs formation. These findings establish PP4 as a critical regulator of CCL5/CCR5-driven NETosis, uncovering a novel therapeutic target for modulating innate immune responses in sepsis.