A SerpinB1 Regulatory Mechanism Is Essential for Restricting Neutrophil Extracellular Trap Generation

Abstract

NETosis (NET generation), a programmed death pathway initiated in mature neutrophils by pathogens and inflammatory mediators, can be a protective process that sequesters microbes and prevents spread of infection, but can also be a pathological process that causes inflammation and serious tissue injury. Little is known about the regulatory mechanism. Previously we demonstrated that serpinb1-deficient mice are highly susceptible to pulmonary bacterial and viral infections due to inflammation and tissue injury associated with increased neutrophilic death. Here we used in vitro and in vivo approaches to investigate whether SerpinB1 regulates NETosis. We found that serpinb1-deficient bone marrow and lung neutrophils are hyper-susceptible to NETosis induced by multiple mediators in both NADPH-dependent and independent manner, indicating a deeply rooted regulatory role in NETosis. This role is further supported by increased nuclear expansion (representing chromatin decondensation) of PMA-treated serpinb-1-deficient neutrophils compared to wild-type, by migration of SerpinB1 from the cytoplasm to the nucleus of human neutrophils coincident with, or before, early conversion of lobulated (segmented) nuclei to delobulated (spherical) morphology, and by finding that exogenous rSerpinB1 abrogates NET production. NETosis of serpinb1-deficient neutrophils is also increased in vivo during Pseudomonas aeruginosa lung infection. The findings identify a previously unrecognized regulatory mechanism involving SerpinB1 that restricts the production of NETs.

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

“…These results demonstrate that nNIF, or a larger protein that encompasses it, is an endogenous inhibitor of NET formation in cord blood plasma from preterm and term neonates ( Figure 2B). Commercially available, active, full-length A1AT purified from human plasma and recombinant A1AT did not inhibit NET formation ( Figure 2E), consistent with previous reports (33,34), indicating that intact A1AT does not contribute to NET-inhibitory activity.…”

“…Both inhibited LPS-stimulated NET deployment in multiple experiments ( Figure 2E, Figure 3A, and Supplemental Figure 2). PMA is commonly employed as a potent nonphysiologic agonist to induce NET formation in vitro (1,6,33,41). nNIF and CRISPP, but not CRISPP-SCR, blocked PMA-stimulated NET deployment ( Figure 3B).…”

Pseudogout-Associated Inflammatory Calcium Pyrophosphate Dihydrate Microcrystals Induce Formation of Neutrophil Extracellular Traps

“…The two other reports that studied MSU crystal-stimulated NETs did not address the question of ROS involvement (26,27). Although requirement of NADPH oxidase for NET formation in response to bacteria and PMA is well accepted, recent data show that NETs can also be induced in an NADPH oxidase-independent manner by the calcium ionophore, ionomycin, MIP-2, or platelet-activating factor (39,61,62). This supports that requirement of NADPH oxidase for NETs depends on the stimulus (39,62).…”

Macrophage Clearance of Neutrophil Extracellular Traps Is a Silent Process

“…oxidase (17), and NET formation is largely NADPH oxidase dependent (2,37), although examples do exist of NADPH oxidaseindependent NET induction (38). Of note, the very recent observation that NADPH oxidase (Nox2)-deficient mice display exacerbated lupus-like symptoms implies that NET formation per se does not contribute to SLE (39).…”