Placental HTRA1 cleaves α1-antitrypsin to generate a NET-inhibitory peptide.

Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (polymorphonuclear leukocytes [PMNs]) fail to form NETs due to circulating NET-inhibitory peptides (NIPs), cleavage fragments of α1-antitrypsin (A1AT). How fetal and neonatal blood NIPs are generated remains unknown, however. 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 knockout mice (HTRA1-/-). We found that umbilical cord blood plasma has elevated HTRA1 levels compared with adult plasma and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of molecular weight similar to previously identified NIPs that block NET formation by adult neutrophils. We showed that neonatal mouse pup plasma contains A1AT fragments that inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. Lipopolysaccharide-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation after birth. However, plasma from HTRA1-/- pups had no detectable NIPs, and PMNs from HTRA1-/- pups became NET competent earlier after birth compared with 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.

Campbell RA ，Campbell HD ，Bircher JS ，de Araujo CV ，Denorme F ，Crandell JL ，Rustad JL ，Monts J ，Cody MJ ，Kosaka Y ，Yost CC ... -  《-》

Alpha-1-antitrypsin: a novel human high temperature requirement protease A1 (HTRA1) substrate in human placental tissue.

Frochaux V ，Hildebrand D ，Talke A ，Linscheid MW ，Schlüter H ... -  《PLoS One》

Alpha-1 Antitrypsin-Induced Endoplasmic Reticulum Stress Promotes Invasion by Extravillous Trophoblasts.

Yoshida K ，Kusama K ，Fukushima Y ，Ohmaru-Nakanishi T ，Kato K ，Tamura K ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation.

Yost CC ，Schwertz H ，Cody MJ ，Wallace JA ，Campbell RA ，Vieira-de-Abreu A ，Araujo CV ，Schubert S ，Harris ES ，Rowley JW ，Rondina MT ，Fulcher JM ，Koening CL ，Weyrich AS ，Zimmerman GA ... -  《-》

The serine protease HtrA1 contributes to the formation of an extracellular 25-kDa apolipoprotein E fragment that stimulates neuritogenesis.

Apolipoprotein-E (apoE) is a glycoprotein highly expressed in the brain, where it appears to play a role in lipid transport, β-amyloid clearance, and neuronal signaling. ApoE proteolytic fragments are also present in the brain, but the enzymes responsible for apoE fragmentation are unknown, and the biological activity of specific apoE fragments remains to be determined. Here we utilized SK-N-SH neuroblastoma cells differentiated into neurons with all-trans-retinoic acid (ATRA) to study extracellular apoE proteolysis. ApoE fragments were detectable in culture supernatants after 3 days, and their levels were increased for up to 9 days in the presence of ATRA. The concentration of apoE fragments was positively correlated with levels of the neuronal maturation markers (PSD95 and SMI32). The most abundant apoE fragments were 25- and 28-kDa N-terminal fragments that both contained sialylated glycosylation and bound to heparin. We detected apoE fragments only in the extracellular milieu and not in cell lysates, suggesting that an extracellular protease contributes to apoE fragmentation. Of note, siRNA-mediated knockdown of high-temperature requirement serine peptidase A1 (HtrA1) and a specific HtrA1 inhibitor reduced apoE 25-kDa fragment formation by 41 and 86%, respectively. Recombinant 25-kDa fragment apoE and full-length apoE both stimulated neuritogenesis in vitro, increasing neuroblastoma neurite growth by more than 2-fold over a 6-day period. This study provides a cellular model for assessing apoE proteolysis, indicates that HtrA1 regulates apoE 25-kDa fragment formation under physiological conditions, and reveals a new neurotrophic function for the apoE 25-kDa fragment.

Muñoz SS ，Li H ，Ruberu K ，Chu Q ，Saghatelian A ，Ooi L ，Garner B ... -  《-》