Sepsis-induced NET formation requires MYD88 but is independent of GSDMD and PAD4

Abstract

Neutrophils are peripheral blood-circulating leukocytes that play a pivotal role in host defense against bacterial pathogens which upon activation, they release web-like chromatin structures called neutrophil extracellular traps (NETs). Here, we analyzed and compared the importance of myeloid differentiation factor 88 (MYD88), peptidyl arginine deiminase 4 (PAD4), and gasdermin D (GSDMD) for NET formation in vivo following sepsis and neutrophilia challenge. Injection of lipopolysaccharide (LPS)/E. coli or the transgenic expression of granulocyte colony-stimulating factor (G-CSF), each induced NET-mediated lethal vascular occlusions in mice with combined genetic deficiency in Dnase1 and Dnase1l3 (D1/D1l3-/-). In accordance with the signaling of toll-like receptors, Myd88/D1/D1l3-/- animals were protected from the formation of lethal intravascular NETs during septic conditions. However, this protection was not observed during neutrophilia. It was unexpected to find that both Gsdmd/D1/D1l3-/- and Pad4/D1/D1l3-/- mice were fully capable of forming NETs upon LPS/E.coli challenge. Sepsis equally triggered a similar inflammatory response in these mice characterized by formation of DNA-rich thrombi, vessel occlusions, and mortality from pulmonary embolism, compared to D1/D1l3-/- mice. Pharmacologic GSDMD inhibitors did not reduce PMA-stimulated NET formation in ex vivo models either. Similarly, neither Pad4 nor GSDMD deficiency affected intravascular occlusive NET formation upon neutrophilia challenge. The magnitude of NET production, multi-organ damage, and lethality were comparable to those observed in challenged control mice. In conclusion, our data indicate that NET formation during experimental sepsis and neutrophilia is regulated by distinct stimulus-dependent pathways that may be independent of canonical PAD4 and GSDMD. Key points- Sepsis triggers vaso-occlusive NET formation in Dnase1/Dnase1l3-deficient mice in a myeloid differentiation factor 88-dependent manner - Peptidyl arginine deiminase 4 and gasdermin D are dispensable for NET formation in sepsis and neutrophilia models - Myeloid differentiation factor 88, peptidyl arginine deiminase 4 and gasdermin D differ in their importance for NET formation in vivo