Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1

Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer

Chronic cigarette smoke exposure and pneumococcal infection induce oropharyngeal microbiota dysbiosis and contribute to long-lasting lung damage in mice

Environmental factors, such as cigarette smoking or lung infections, may influence chronic obstructive pulmonary disease (COPD) progression by modifying the respiratory tract microbiome. However, whether the disease itself induces or maintains dysbiosis remains undefined. In this longitudinal study, we investigated the oropharyngeal microbiota composition and disease progression of mice (in cages of 5-10 mice per cage) before, during and up to 3 months after chronic cigarette smoke exposure or exposure to room air for 6 months. Cigarette smoke exposure induced pulmonary emphysema measurable at the end of exposure for 6 months, as well as 3 months following smoke exposure cessation. Using both classical culture methods and 16S rRNA sequencing, we observed that cigarette smoke exposure altered the relative composition of the oropharyngeal microbiota and reduced its diversity (P <0.001). More than 60 taxa were substantially reduced after 6 months of smoke exposure (P <0.001) However, oropharyngeal microbiota disordering was reversed 3 months after smoke exposure cessation and no significant difference was observed compared to age-matched control mice. The effects of lung infection with Streptococcus pneumoniae on established smoke-induced emphysema and on the oropharyngeal microbiota were also evaluated. Inoculation with S. pneumoniae induced lung damage and altered the microbiota composition for a longer time compared to control groups infected but not previously exposed to smoke (P=0.01). Our data demonstrate effects of cigarette smoke and pneumococcus infection leading to altered microbiota and emphysema development. The reversal of the disordering of the microbiota composition, but not lung damage, following smoke exposure cessation and after clearance of infection suggest that changes in lung structure are not sufficient to sustain a disordered microbiota in mice. Whether changes in the airway microbiota contribute to inducing emphysema requires further investigation

Cathepsin G inhibition by Serpinb1 and Serpinb6 prevents programmed necrosis in neutrophils and monocytes and reduces GSDMD-driven inflammation

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a(-/-) mice. Furthermore, Sb1a.Sb6a(-/-) mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a(-/-) macrophages showed increased IL-1 beta release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD

The Genetic Background of Mice Influences the Effects of Cigarette Smoke on Onset and Severity of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS

Transgenic Mice Expressing Human Proteinase 3 Exhibit Sustained Neutrophil-Associated Peritonitis.

Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation