Background Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE−/− mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking. Objective We wished to establish if neutrophils from NE−/− mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4−/−) mice, and how this might impact venous thrombosis, a model where NETs are produced and are crucial to thrombus development. Methods We performed in vitro NET assays using neutrophils from wild-type (WT), NE−/−, SerpinB1 (SB1−/−), and NE−/−SB1−/− mice. We compared WT and NE−/− animals in the inferior vena cava stenosis model of deep vein thrombosis (DVT). Results NE-deficiency resulted in a small reduction in ionomycin-induced NET formation in vitro without affecting histone citrullination. However, NET production in response to PMA or PAF was normal in neutrophils from two independent NE-deficient mouse lines, or in NE−/−SB1−/− as compared to SB1−/− neutrophils. NE-deficiency or inhibition did not prevent NETosis in vivo and DVT outcome. Conclusions NE is not required for NET formation in mice. NE−/− mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4−/− mice in in vitro NETosis assays or experimental venous thrombosis. Our study suggests that NET-targeted therapies need to be highly effective to have an impact on DVT.

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.

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.

Because viral clearance was unimpaired, the study highlights the critical role of serpinB1 in mitigating inflammation and restricting pro-inflammatory cytokine production in influenza infection.

Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-κB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-κB at 276th serine residue. These modifications enhance the interaction of NF-κB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication.

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SerpinB1 is an endogenous inhibitor of serine proteases recognized for its anti-inflammatory and host-protective properties. Although loss of serpinB1 in mice does not result in gross immune deregulation, serpinb1a(-/-) mice display increased mortality and inflammation-associated morbidity upon challenge with influenza virus. Here, we show that IL-17A(+) γδ and CD4(+) Th17 cells are already expanded in the lungs of serpinb1a(-/-) mice at steady-state. Both γδ and αβ(+) CD4(+) CCR6(+) T cells isolated from the lungs of naive serpinb1a(-/-) mice displayed a skewed transcriptional profile relative to WT cells, including increased Th17 signature transcripts [Il17a, l17f, and Rorc (RORγt)] and decreased Th1 signature transcripts [Ifng, Cxcr3, and Tbx21 (T-bet)] in γδ T cells. In addition to the lung, IL-17A(+) γδ and CD4(+) Th17 cells were increased in the spleen of naive serpinb1a(-/-) mice, despite normal αβ and γδ T cell development in the thymus. Within the γδ T cell compartment, loss of serpinb1a prompted selective expansion of Vγ4(+) and Vγ6/Vδ1(+) cells, which also displayed elevated expression of the proliferating cell nuclear antigen, Ki-67, and IL-17A. Given that serpinb1a is preferentially expressed in WT IL-17A(+) γδ and CD4(+) Th17 cell subsets vis-à-vis other T cell lineages, our findings reveal a novel function of serpinB1 in limiting untoward expansion of lymphocytes with a Th17 phenotype.

A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced acute lung injury (ALI) has been shown; however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species and has been implicated in endothelial injury in ALI. Pulmonary microvascular endothelial cells (PMVEC) monolayers were grown on Transwell inserts and incubated with wild-type and NADPH oxidase-deficient AM in the presence or absence of cytomix (equimolar TNF-alpha, IL-1beta, and IFN-gamma). Injury to the monolayers was assessed by trans-PMVEC Evans blue (EB)-labeled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers, and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC cocultures, we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NADPH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute to PMVEC injury in our studies. From our findings we have concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability.