The SERPINA1 gene encodes alpha1-antitrypsin (AAT), an acute phase glycoprotein and serine protease inhibitor that is mainly (80–90%) produced in the liver. Point mutations in the SERPINA1 gene can lead to the misfolding, intracellular accumulation, and deficiency of circulating AAT protein, increasing the risk of developing chronic liver diseases or chronic obstructive pulmonary disease. Currently, siRNA technology can knock down the SERPINA1 gene and limit defective AAT production. How this latter affects other liver genes is unknown. Livers were taken from age- and sex-matched C57BL/6 wild-type (WT) and Serpina1 knockout mice (KO) aged from 8 to 14 weeks, all lacking the five serpin A1a-e paralogues. Total RNA was isolated and RNA sequencing, and transcriptome analysis was performed. The knockout of the Serpina1 gene in mice changed inflammatory, lipid metabolism, and cholesterol metabolism-related gene expression in the liver. Independent single-cell sequencing data of WT mice verified the involvement of Serpina1 in cholesterol metabolism. Our results from mice livers suggested that designing therapeutic strategies for the knockout of the SERPINA1 gene in humans must account for potential perturbations of key metabolic pathways and consequent mitigation of side effects.

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease–antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency–related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae . AAT-KO mice were highly susceptible to S. pneumoniae , as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae –infected AAT-KO mice. Treatment of S. pneumoniae –infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae –infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency.

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins (“spike”). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1β mRNA expression and protein release were significantly inhibited (by about 46–50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

C-terminal fragments of alpha1-antitrypsin (AAT) occur in human biological fluids and tissues, including normal and emphysema lungs. We developed novel mouse monoclonal antibody (IgM, C42 mAb) against C-36 peptide resulting from elastase-associated cleavage of AAT. We addressed the usefulness of the C42 mAb in dot and western blots, immunomicroscopy and flow cytometry. According to the dot-blots, C42 mAb detect C-36 peptide at the range of 0.1-0.05 µg and show no cross-reactivity with native, polymeric or oxidized full-length AAT, AAT-elastase complex and shorter C-terminal fragments of AAT. The epitope recognized by the C42 mAb is unknown but it becomes unavailable after peptide denaturation and separation by electrophoresis following blotting. Because many antibodies towards conformational epitopes did not bind their target proteins in the western blot assays, we think that the C42 mAb is conformational structure-dependent. On the other hand, C42 mAb, unconjugated or conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays specifically detected C-36 peptide in human blood cells. The distributions of the C-36 peptide and the full-length AAT protein in human neutrophils and adherent PBMCs we assessed by laser scanning confocal microscopy. The 3D reconstruction of the confocal sections revealed only a partial co-localization between C-36 and AAT signals. The AAT we detected throughout the cell but mostly at the cell periphery, whereas C-36 peptide was predominantly present in perinuclear areas. Hence, C42 mAb may be a useful tool in respiratory research studies associated with health and disease.

Chronic obstructive pulmonary disease (COPD) is associated with chronic neutrophilic inflammation and pathological changes in circulating levels of alpha1-antitrypsin (AAT), an inhibitor of neutrophil proteases. Variants of C-terminal fragments of AAT have also been identified in human body fluids and tissues, including lungs. However, their biological role and clinical relevance remain largely unknown. Originally it has been thought that these fragments are generated during unspecific cleavage of AAT by metalloproteases. The results of our studies led to unexpected finding that human peripheral blood leukocytes express and release short transcripts, corresponding to 36 amino acid C-terminal peptide of AAT (C-36). Here we show that the hydrophobic C-36 peptide of AAT added to human neutrophil cultures stimulates cell migration and adhesion and induces free radical and cytokine release. Moreover, C-36 forms amyloid-like fibrils, in vitro, which can activate neutrophils. We hypothesize that the C-fragment of AAT may serve as a new marker in COPD patients with and without inherited AAT deficiency. In this study we present data on monoclonal antibody that specifically recognizes C-36 peptide but does not react with full-length native or modified forms of AAT such as oxidized, polymeric or complexed with elastase. The applications we used for antibody validation include dot blots, western blots, flow cytometry, and immunohistochemistry. We also provide evidence that cellular expression of C-36 is associated with more severe stages of COPD. This finding encourages to re-evaluate the significance of AAT fragments.