Placental-derived HTRA1 cleaves alpha-1-antitrypsin resulting in the generation of neonatal NET-inhibitory peptides.
Neonatal neutrophils from HTRA1-/- mice become NET competent earlier after birth compared to HTRA1+/+ littermate controls.
Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (PMNs) fail to form NETs due to circulating NET-Inhibitory Peptides (NIPs) -cleavage fragments of alpha-1-antitrypsin (A1AT). However, how fetal and neonatal blood NIPs are generated remains unknown. The placenta expresses High-Temperature Requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1-knock out mice (HTRA1-/-). We found that umbilical cord blood plasma has elevated HTRA1 levels compared to adult plasma, and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of similar molecular weight to previously identified NIPs that block NET formation by adult neutrophils. We demonstrated that neonatal mouse pup plasma contains A1AT fragments which inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. LPS-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation following birth. However, plasma from HTRA1-/- pups had no detectable NIPs and PMNs from HTRA1-/- pups became NET competent earlier after birth compared to HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.