CYLD and PP4 Coordinate TLR4 Signaling to Suppress NET Formation and Lung Injury in Sepsis 2254452

Abstract

Abstract Introduction Sepsis, a leading cause of death in critical care, involves life-threatening organ dysfunction triggered by an uncontrolled immune response. One of its most severe outcomes is acute lung injury (ALI), driven by excessive inflammation and neutrophil hyperactivation. This study examines how two key regulators–CYLD and PP4–modulate TLR4 signaling, NET formation, and cytokine responses during sepsis-induced ALI. Methods Mechanism of PP4 and CYLD by LPS exposure was determined in cell cultures and CLP mice model. NETs formation were analyzed by Western blotting, flow cytometry and immunofluorescence live-cell staining, and cytokines was measured by Multiplex Cytokine Assay. Results CYLD and PP4 function as coordinated regulators of inflammatory signaling during sepsis-induced acute lung injury (ALI). CYLD binds to somatic nuclear autoantigenic sperm protein (sNASP) to inhibit TRAF6 activation, while PP4 dephosphorylates sNASP to restore the CYLD/sNASP/TRAF6 complex, thereby suppressing proinflammatory cytokine release. In vivo, mice treated with wild-type CYLD showed protection against CLP-induced lung injury, whereas myeloid-specific PP4 knockout mice exhibited heightened susceptibility to sepsis, marked by elevated IL-6, TNF-α, MIP-1α, and CCL5 levels, severe tissue damage, and excessive NET formation. Mechanistically, PP4 dampens macrophage-neutrophil crosstalk by dephosphorylating TBK1 and modulating LAP-dependent NETosis, while CYLD restrains TRAF6-driven cytokine signaling. Loss of PP4 also enhances ERK1/2 activation and CCR5 expression in neutrophils, amplifying CCL5-mediated NET formation and oxidative stress. Conclusion Together, CYLD and PP4 form a complementary network that limits TLR4-triggered inflammation and preserves immune homeostasis during sepsis. Funding Source National Science and Technology Council, Taiwan Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)