Placental HTRA1 Protease Cleaves Alpha-1-Antitrypsin and Generates Neonatal NET-Inhibitory Factor

BACKGROUND: Neutrophil extracellular traps (NET) are extracellular lattices of decondensed chromatin associated with anti-microbial proteins and degradative enzymes released by polymorphonuclear leukocytes (PMN) to trap and kill invading microbes. Dysregulated NET formation, however, contributes to inflammatory tissue damage. We have identified a novel NET-inhibitory peptide, neonatal NET-Inhibitory Factor (nNIF), present in the fetal circulation. nNIF is formed as a carboxy-terminus cleavage fragment of alpha-1 antitrypsin (AAT), an abundant, circulating protease inhibitor with homologs in human and mouse blood. However, the exact mechanisms by which nNIF is generated in fetal and neonatal blood remains unknown.

OBJECTIVE: High temperature requirement A 1 (HTRA1) is expressed in the placenta during fetal development and inhibits AAT. We hypothesized that placentally expressed HTRA1, a serine protease, regulates the formation of NET-inhibitory peptides, such as nNIF, through cleavage of AAT.

DESIGN/METHODS: Term and preterm placenta were lysed and probed for HTRA1 expression. HTRA1 and AAT plasma expression from term and preterm infants and adults were determined by ELISA. Recombinant, bioactive HTRA1 or placenta-eluted HTRA1 were incubated with AAT and the generation of carboxy-terminus fragments of AAT was assessed using western blotting and mass spectrometry. Fragments of AAT generated by HTRA1 were incubated with LPS-stimulated PMNs and NET formation was examined qualitatively using live cell imaging and quantitatively using a high throughput fluorescence assay. The effect of the HTRA-AAT cleavage fragment on reactive oxygen species generation, neutrophil chemotaxis, phagocytosis, and bacterial killing was measured using flow cytometry, a modified Boyden chamber asssay, neutrophil labeled Escherichia coli uptake assay, and a bacterial killing assay with a pathogenic strain of Escherichia coli, respectively. Finally, NET formation was evaluated qualitatively and quantitatively in murine PMNs isolated from neonatal WT and HTRA1^-/- pups between 1-3, 4-6 and 7-10 days after birth to determine when PMNs become NET-competent.

RESULTS: Term and preterm infant placentas express HTRA1, and we detected significantly (p<0.05) higher levels of HTRA1 in plasma from term (465.1±71.8 µg/mL) and preterm (385.9±71.3 µg/mL) infant cord blood compared to adults (58.6±11.6 µg/mL). Recombinant, bioactive HTRA1 and placenta-derived HTRA1 incubated with AAT generate a 4kD AAT fragment based on western blot and mass spectrometry similar to the nNIF fragment found in cord blood from term and preterm infants. Pre-incubation of this fragment with LPS-stimulated PMNs significantly inhibits NET formation (p<0.05). The cleavage fragment from HTRA1-AAT, however, has no effect on reactive oxygen species generation, chemotaxis, or phagocytosis. However, incubation of this fragment with LPS-stimulated PMNs significantly (p<0.05) reduces NET-associated bacterial killing by 62% compared to a scrambled HTRA-AAT control peptide. In addition, the HTRA1-AAT fragment significantly (p<0.05) reduces nuclear decondensation by 93% compared to LPS-stimulated PMN, suggesting this fragment inhibits PAD4 activation similar to other NIFs previously examined. Neonatal murine plasma contains a 4kD AAT fragment which inhibits NET formation by adult mouse PMNs, indicating that nNIF generation is conserved in mice. Neonatal PMNs stimulated with LPS exhibit delayed NET formation following birth with PMNs becoming NET-competent by day 8 of life. However, neonatal PMNs from pups born from HTRA1^-/- deficient mice generate significantly (p<0.05) more NETs between day 4-6 of life compared to WT controls, suggesting that HTRA1 regulates NET formation through nNIF production.

CONCLUSIONS: Placental HTRA1 interacts with AAT to generate a carboxy-terminus cleavage fragment of AAT with identical NET-inhibitory properties to nNIF. Our data strongly indicate that placental HTRA1 generates nNIF in the fetal circulation. We speculate that nNIF participates in the required tolerance to new microbial antigens encountered during the transition to extrauterine life.