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Pneumonia is a global cause of mortality, and this provides a strong incentive to improve the mechanistic understanding of innate immune responses in the lungs. Here, we characterized the involvement of the cytokine interleukin (IL)-26 in bacterial lung infection. We observed markedly increased concentrations of IL-26 in lower airway samples from patients with bacterial pneumonia and these correlated with blood neutrophil concentrations. Moreover, pathogen-associated molecular patterns (PAMPs) from both Gram-negative and -positive bacteria increased extracellular IL-26 concentrations in conditioned media from human models of alveolar epithelial cells, macrophages, and neutrophils in vitro . Stimulation with IL-26 inhibited the inherent release of neutrophil elastase and myeloperoxidase in unexposed neutrophils. This stimulation also inhibited the expression of activity makers in neutrophils exposed to Klebsiella pneumoniae. In addition, priming of human lung tissue ex vivo with exogenous IL-26 potentiated the endotoxin-induced increase in mRNA for other cytokines involved in the innate immune response, including the master Th17-regulator IL-23 and the archetype inhibitory cytokine IL-10. Finally, neutralization of endogenous IL-26 clearly increased the growth of Klebsiella pneumoniae in the macrophage culture. These findings suggest that IL-26 is involved in bacterial lung infection in a complex manner, by modulating critical aspects of innate immune responses locally and systemically in a seemingly purposeful manner and by contributing to the killing of bacteria in a way that resembles an antimicrobial peptide. Thus, IL-26 displays both diagnostic and therapeutic potential in pneumonia and deserves to be further evaluated in these respects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The editor declared a past collaboration with one of the authors, KC, at the time of review., (Copyright © 2021 Che, Paulsson, Piersiala, Sax, Mboob, Rahman, Rekha, Säfholm, Adner, Bergman, Cardell, Riesbeck and Lindén.)

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and V H 1D1.43 from two large phage-displayed human Fab and V H libraries. After fusion with human IgG1 Fc, both of them (V H -Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with V H -Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both V H -Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. V H -Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

Neutrophil extracellular traps (NETs), built from mitochondrial or nuclear DNA, proteinases, and histones, entrap and eliminate pathogens in the course of bacterial or viral infections. Neutrophils' activation and the formation of NETs have been described as major risk factors for acute lung injury, multi-organ damage, and mortality in COVID-19 disease. NETs-related lung injury involves both epithelial and endothelial cells, as well as the alveolar-capillary barrier. The markers for NETs formation, such as circulating DNA, neutrophil elastase (NE) activity, or myeloperoxidase-DNA complexes, were found in lung specimens of COVID-19 victims, as well as in sera and tracheal aspirates obtained from COVID-19 patients. DNA threads form large conglomerates causing local obstruction of the small bronchi and together with NE are responsible for overproduction of mucin by epithelial cells. Various components of NETs are involved in the pathogenesis of cytokine storm in SARS-CoV-2 pulmonary disease. NETs are responsible for the interplay between inflammation and thrombosis in the affected lungs. The immunothrombosis, stimulated by NETs, has a poor prognostic significance. Better understanding of the role of NETs in the course of COVID-19 can help to develop novel approaches to the therapeutic interventions in this condition.

Objective: To evaluate the role of immune cells and their effector cytokines in the pathogenesis and progression of knee osteoarthritis (OA) in matched OA synovial fluid (SF) and synovial tissue samples., Methods: Cells from matched samples of synovial tissue and SF acquired from individuals undergoing total knee replacement for OA (n = 39) were characterized for immune cell-associated surface markers and intracellular cytokine expression using polychromatic flow cytometry. Additional individuals with radiographic knee OA (Kellgren/Lawrence severity grades ≥1) who had available etarfolatide (inflammatory cell) imaging (n = 26) or baseline and 3-year data on progression of radiographic knee OA (n = 85) were also assessed. SF cytokine concentrations in all cohorts were evaluated for associations with synovial tissue and SF cell phenotypes and severity of radiographic knee OA., Results: Macrophages (predominant in the synovial tissue, 53% of total cells) and neutrophils (predominant in the SF, 26% of total cells) were the major immune cell populations identified in the OA knee joints, exhibiting expression of or association with transforming growth factor β1 (TGFβ1) and elastase, respectively, in the SF. Expression levels of TGFβ1 and elastase were significantly associated with severity of radiographic knee OA. Baseline SF concentrations of TGFβ1 and elastase along with radiographic knee OA severity scores were predictive of knee OA progression, with areas under the receiver operating characteristic curves of 0.810 (for TGFβ1), 0.806 (for elastase), and 0.846 (for both TGFβ1 and elastase combined), with greater stability of prediction when both markers were utilized., Conclusion: Our findings demonstrate the hitherto underappreciated role of neutrophils in the sterile inflammatory process and progression of OA. Two soluble mediators, SF elastase and TGFβ1, are strong predictors of knee OA progression, reflecting a synergistic role of neutrophil and macrophage populations in the pathogenesis and worsening of OA that could potentially be utilized to identify patients who may have a greater risk of more rapid disease progression., (© 2020, American College of Rheumatology.)

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity., (Copyright © 2020 © 2020 Kasperkiewicz et al. Published by Elsevier Inc. All rights reserved.)

Qin Pi ( Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8 E )-4''- O -methylligstroside ( 1 ), (8 E )-4''- O -methyldemethylligstroside ( 2 ), and 3'',4''-di- O -methyl-demethyloleuropein ( 3 ), have been isolated from the stem bark of Fraxinus chinensis , together with 23 known compounds ( 4 - 26 ). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8 E )-4''- O -methylligstroside ( 1 ), (8 E )-4''- O -methyldemethylligstroside ( 2 ), 3'',4''-di- O -methyldemethyloleuropein ( 3 ), oleuropein ( 6 ), aesculetin ( 9 ), isoscopoletin ( 11 ), aesculetin dimethyl ester ( 12 ), fraxetin ( 14 ), tyrosol ( 21 ), 4-hydroxyphenethyl acetate ( 22 ), and (+)-pinoresinol ( 24 ) exhibited inhibition (IC 50 ≤ 7.65 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1 , 9 , 11 , 14 , 21 , and 22 inhibited fMLP/CB-induced elastase release with IC 50 ≤ 3.23 μg/mL. In addition, compounds 2 , 9 , 11 , 14 , and 21 showed potent inhibition with IC 50 values ≤ 27.11 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines , tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1 , 9 , and 14 . Compounds 1 , 9 , and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1 , 9 , and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1 , 9 , and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. The mechanism behind the formation of sialoliths has been elusive. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland, and often results in sialadenitis with the need for surgical intervention. Here, we show by the use of immunohistochemistry, immunofluorescence, computed tomography (CT) scans and reconstructions, special dye techniques, bacterial genotyping, and enzyme activity analyses that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths in humans. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in the dense aggregation of the latter, and the subsequent mineralization creates alternating layers of dense mineral, which are predominantly calcium salt deposits and DNA. The further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis, in which an immune system-mediated response essentially participates in the physicochemical process of concrement formation and growth.

Type 1 diabetes is an autoimmune disease resulted from self-destruction of insulin-producing pancreatic β cells. However, the pathological pathways that trigger the autoimmune destruction remain poorly understood. Clinical studies have demonstrated close associations of neutrophils and neutrophil elastase (NE) with β-cell autoimmunity in patients with Type 1 diabetes. The present study aims to investigate the impact of NE inhibition on development of autoimmune diabetes in NOD mice. NE pharmacological inhibitor (sivelestat) or biological inhibitor (elafin) was supplemented into NOD mice to evaluate their effects on islet inflammation and diabetogenesis. The impact of NE inhibition on innate and adaptive immune cells was measured with flow cytometry and immunohistochemistry. A significant but transient increase in neutrophil infiltration accompanied with elevated NE activity was observed in the neonatal period of NOD mice. Treatment of NOD mice with sivelestat or elafin at the early age led to a marked reduction in spontaneous development of insulitis and autoimmune diabetes. Mechanistically, inhibition of NE significantly attenuated infiltration of macrophages and islet inflammation, thus ameliorating cytotoxic T cell-mediated autoimmune attack of pancreatic β cells. In vitro studies showed that NE directly induced inflammatory responses in both min6 β cells and RAW264.7 macrophages, and promoted macrophage migration. These findings support an important role of NE in triggering the onset and progression of β-cell autoimmunity, and suggest that pharmacological inhibition of NE may represent a promising therapeutic strategy for treatment of autoimmune diabetes., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Sepsis is a life-threatening disease induced by a systemic inflammatory response, which leads to organ dysfunction and mortality. In sepsis, the host immune response is depressed and unable to cope with infection; no drug is currently available to treat this. The lungs are frequently the starting point for sepsis. This study aimed to identify potential genes for diagnostics and therapeutic purposes in sepsis by a comprehensive bioinformatics analysis. Our criteria are to unravel sepsis-associated signature genes from gene expression datasets. Differentially expressed genes (DEGs) were identified from samples of sepsis patients using a meta-analysis and then further subjected to functional enrichment and protein‒protein interaction (PPI) network analysis for examining their potential functions. Finally, the expression of the topmost upregulated genes ( ARG1 , IL1R2 , ELANE , MMP9 ) was quantified by reverse transcriptase-PCR (RT-PCR), and myeloperoxidase ( MPO ) expression was confirmed by immunohistochemistry (IHC) staining in the lungs of a well-established sepsis mouse model. We found that all the four genes were upregulated in semiquantitative RT-PCR studies; however, MMP9 showed a nonsignificant increase in expression. MPO staining showed strong immunoreactivity in sepsis as compared to the control. This study demonstrates the role of significant and widespread immune activation ( IL1R2 , MMP9 ), along with oxidative stress ( ARG1 ) and the recruitment of neutrophils, in sepsis ( ELANE , MPO )., Competing Interests: The authors declare no conflict of interest.

The last few decades of protease research has confirmed that a number of important biological processes are strictly dependent on proteolysis. Neutrophil elastase (NE) is a critical protease in immune response and host defense mechanisms in both physiological and disease-associated conditions. Particularly, NE has been identified as a promising biomarker for early diagnosis of lung inflammation. Recent studies have shown an increasing interest in developing methods for NE activity imaging both in vitro and in vivo. Unlike anatomical imaging modalities, functional molecular imaging, including enzymatic activities, enables disease detection at a very early stage and thus constitutes a much more accurate approach. When combined with advanced imaging technologies, opportunities arise for measuring imbalanced proteolytic activities with unprecedented details. Such technologies consist in building the highest resolved and sensitive instruments as well as the most specific probes based either on peptide substrates or on covalent inhibitors. This review outlines strengths and weaknesses of these technologies and discuss their applications to investigate NE activity as biomarker of pulmonary inflammatory diseases by imaging.

Mast cells (MCs) are critical mediators of inflammation; however, their microbicidal activity against invading pathogens remains largely unknown. Here, we describe a nonpreviously reported antibacterial mechanism used by MCs against Coxiella burnetii , the agent of Q fever. We show that C. burnetii interaction with MCs does not result in bacterial uptake but rather induces the formation of extracellular actin filaments named cytonemes. MC cytonemes express cathelicidin and neutrophil elastase and mediate the capture and destruction of entrapped bacteria. We provide evidence that MC cytoneme formation and microbicidal activity are dependent on the cooperation of the scavenger receptor CD36 and Toll-like receptor 4. Taken together, our results suggest that MCs use an extracellular sophisticated mechanism of defense to eliminate intracellular pathogens, such as C. burnetii , before their entry into host cells. IMPORTANCE Mast cells (MCs) are found in tissues that are in close contact with external environment, such as skin, lungs, or intestinal mucosa but also in the placenta during pregnancy. If their role in mediating allergic conditions is established, several studies now highlight their importance during infection with extracellular pathogens. This study showed a new and effective antimicrobial mechanism of MCs against Coxiella burnetii , an intracellular bacterium whose infection during pregnancy is associated with abortion, preterm labor, and stillbirth. The data reveal that in response to C. burnetii , MCs release extracellular actin filaments that contain antimicrobial agents and are capable to trap and kill bacteria. We show that this mechanism is dependent on the cooperation of two membrane receptors, CD36 and Toll-like receptor 4, and may occur in the placenta during pregnancy by using ex vivo placental MCs. Overall, this study reports an unexpected role for MCs during infection with intracellular bacteria and suggests that MC response to C. burnetii infection is a protective defense mechanism during pregnancy., (Copyright © 2019 Mezouar et al.)

Background: Intravenous immunoglobulin (IVIg) treatment partially replaces antibody defects and modulates innate and adaptive immune cells in patients with primary antibody deficiencies., Materials and Methods: This study was focused on the evaluation of the effects of in vivo IVIg administration on neutrophils from patients with common variable immune disorders (CVID). We examined polymorphonuclear neutrophil (PMN) phagocytosis, PMN oxidative burst, release of neutrophil elastase, serum level of interleukin-8 and PMN expression of CXCR1, CD11c and CD66b., Results: CVID patients on chronic IVIg treatment had reduced elastase release, but normal expression of CXCR1, CD66b and CD11c receptors on PMN, normal phagocytic ability and normal secretion of interleukin-8. We found that IVIg infusions rapidly reduced the serum level of interleukin-8, the expression of its receptor, CXCR1, and the release of neutrophil elastase, suggesting that IVIg exert a dampening effect on neutrophil activity. In contrast, IVIg infusions did not alter neutrophil phagocytosis or the expression of the other receptors analysed., Discussion: These findings add further information regarding the anti-inflammatory role of immunoglobulins and suggest additional benefits in keeping with recent attempts to use new therapies targeting neutrophil inflammation.

Cystic fibrosis (CF) lung disease is characterized by aggressive neutrophil-dominated inflammation mediated in large part by neutrophil elastase (NE), an omnivorous protease released by activated or disintegrating neutrophils and a key therapeutic target. To date, several short-term studies have shown that anti-NE compounds can inhibit NE and have anti-inflammatory effects. However, progression to large-scale or multicenter clinical trials has been hampered by the fact that the current gold standard methodology of evaluating airway NE inhibition, bronchoalveolar lavage (BAL), is invasive, difficult to standardize across sites and excludes those with severe lung disease. Attempts to utilize sputum that is either spontaneously expectorated (SS) or induced (IS) have been hindered by poor reproducibility, often due to the various processing methods employed. In this study, we evaluate TEmperature-controlled Two-step Rapid Isolation of Sputum (TETRIS), a specialized method for the acquisition and processing of SS and IS. Using TETRIS, we show for the first time that NE activity and cytokine levels are comparable in BAL, SS and IS samples taken from the same people with CF (PWCF) on the same day once this protocol is used. We correlate biomarkers in TETRIS-processed IS and clinical outcome measures including FEV 1 , and show stability and reproducible inhibition of NE over time in IS processed by TETRIS. The data offer a tremendous opportunity to evaluate prognosis and therapeutic interventions in CF and to study the full spectrum of people with PWCF, many of whom have been excluded from previous studies due to being unfit for BAL or unable to expectorate sputum., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Purpose: We report normal neutrophil count in a mother, who carries the same ELANE mutation as her daughter with severe congenital neutropenia. We hypothesized that the mother possessed wild- and mutant-type clones and the wild-type clones could generate neutrophils, whereas the mutant clones could not., Methods: We confirmed mutant variant ratio by sequence signals and measured the frequency of the mutant allele by subcloning in various cell types. We established the ELANE-mutated and non-mutated induced pluripotent stem cells (iPSCs) from the mother's T cells and compared granulopoiesis between these iPSCs., Results: In the sequence analysis of isolated peripheral blood (PB), nail and hair, the mutant variant was detected in approximately 40-60% of lymphocytes, monocytes, hematopoietic progenitor cells, and hair as well as in a small percentage of nail, but in none of the neutrophils. In the subcloning analysis of extracted DNA from CD3 + and CD34 + cells, the mutant allele was identified in 37.5% and 38.1%, respectively. We reprogrammed the mother's PB cells and established the ELANE-mutated and non-mutated iPSCs. Granulopoiesis from mutated iPSCs revealed little sensitivity to granulocyte colony-stimulating factor in comparison with non-mutated iPSCs., Conclusions: These observations strongly suggest that mutant-carrying neutrophils did not appear in the mother's PB because mutated clones could not differentiate into neutrophils. The mother's normal hematological phenotype could be explained by the perseverance of normal, non-mutated granulopoiesis.

Objective: The aim of this study was to detect the oxidative stress response in the rat model of obesity, asthma and obese asthma. Meanwhile, we aimed to investigate the inhibitory effect of neutrophil elastase inhibitor (NEI) on cellular oxidative stress in the body and whether it exerted an effect on the oxidative stress response in obese asthma through the Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (Keap1/Nrf2) pathway., Materials and Methods: The obesity and asthma models were established using a total of 70 Sprague-Dawley (SD) rats. All rats were randomly divided into 7 groups. The rats with normal weight were divided into the control (CTR) group (n=10), asthma (ATM) group (n=10) and ATM+NEI group (n=10). Meanwhile, the obese rats were divided into the obesity (OBS) group (n=10), the OBS+NEI group (n=10), the OBS+ATM group (n=10) and the OBS+ATM+NEI group (n=10). After modeling, rats in NEI intervention groups were injected with Sivelestat (5 mg/kg) via the caudal vein twice a day for 1 week. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Lung tissues of rats were collected for hematoxylin-eosin (HE) staining. Meanwhile, the levels of reactive oxygen species (ROS) in heart, lung and kidney tissues were detected via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The activities of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), H2O2 and total superoxide dismutase (T-SOD) in the heart, lung and kidney tissues were detected using the colorimetric method. The mRNA and protein expressions of Keap1 and Nrf2 messenger ribonucleic acid expressions in the heart, lung and kidney tissues were measured via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, respectively., Results: NEI significantly improved the symptoms and lung pathology in rats with asthma. The level of ROS in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly increased. However, NEI markedly inhibited the level of ROS in rats with asthma. The activities of antioxidant stress-related enzymes (reduced GSH, GSH-Px, H2O2 and SOD) in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group were significantly decreased. However, NEI markedly promoted the activities of the related antioxidant enzymes in oxidative stress response in asthma rats. Besides, the Keap1/Nrf2 signaling pathway in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly inhibited, while NEI activated the Keap1/Nrf2 signaling pathway in rats with asthma., Conclusions: NEI promotes the release of a variety of antioxidant factors, enhances the activity of antioxidant enzymes and improves the symptoms of rats with obese asthma. The possible underlying mechanism may be the activation of the Keap1/Nrf2 signaling pathway.

Ostertagia ostertagi (OO) is a widespread parasite that causes chronic infection in cattle and leads to annual losses of billions of dollars in the cattle industry. It remains unclear why cattle are unable to mount an effective immune response despite a large influx of immune cells to the infected abomasal mucosa and draining lymph nodes. Neutrophils, the immune system's first responders, have the capacity to release neutrophil extracellular traps (NETs) to contain various pathogens, including some parasites. In the present study, the mechanisms by which O. ostertagi influences bovine NET formation were investigated. O. ostertagi larval soluble extract (OO extract) was able to induce typical NETs by purified neutrophils in vitro, confirmed by co-localization of extracellular DNA with typical NET-associated proteins histone and neutrophil elastase (NE). Consistent with existing literature, inhibition assays demonstrated that these OO extract-induced NETs were dependent upon the enzymes NADPH oxidase and myeloperoxidase (MPO). Live OO stage 4 larvae (L4) stimulated neutrophils to form NETs similar to those induced by OO extract. Bovine neutrophils also released NETs in response to Caenorhabditis elegans, a free-living soil nematode, suggesting that bovine NET production may be a conserved mechanism against a broad range of nematodes. This is the first report demonstrating O. ostertagi-induced NET formation by bovine neutrophils, a potentially underappreciated mechanism in the early immune response against nematode infections.

Maintaining balanced levels of IL-1β is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1 -/- mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1 -/- mice with S. aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL-1β. Ifnlr1 -/- mice treated with recombinant IL-1β displayed increased bacterial burdens in the airway and lung. IL-1β levels in neutrophils from Ifnlr1 -/- infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase-1 had reduced IL-1β levels 4 h after infection, due to reductions or absence of active caspase-1 respectively, but levels at 24 h were comparable to WT infected mice. Ifnlr1 -/- infected mice had decreases in both active caspase-1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL-1β processing. By inhibiting neutrophil elastase, we were able to decrease IL-1β levels by 39% in Nlrp3 -/- infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL-1β processing, via inflammasome-dependent and -independent mechanisms., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acute lung injury (ALI) is a serious complication among patients with acute kidney injury (AKI) that is a systemic inflammatory disease with high morbidity and mortality. The pathophysiology of AKI-associated ALI is poorly understood. G-CSF regulates the production and function of neutrophils that mediate lung injury via elastase and other mediators. Here, we used a mouse model of adenine-induced AKI to determine the roles of G-CSF and neutrophil elastase in AKI-associated ALI. We confirmed that ALI was associated with high serum G-CSF levels, and elevated neutrophil elastase activity in the lungs and serum of mice with adenine-induced AKI. Systemic administration of G-CSF-specific neutralizing antibody normalized granulopoiesis, pulmonary neutrophil infiltration, and neutrophil elastase activity, conferring improved lung architecture in mice with adenine-induced AKI. Further studies revealed that macrophages secreted G-CSF upon urea stimulation. Consequently, G-CSF could be a target for new anti-lung injury strategy in patients with AKI., (Copyright © 2018 Elsevier B.V. All rights reserved.)

During NET formation, the content of neutrophils granules is released into the intercellular milieu. Consisting of many proteases and ROS species, formed NETs were shown to degrade cytokines (Schauer, Nat Med, 2014); while the content of neutrophil's azurophilic granules proved to contain glycosidases, secreted upon activation (Thaysen-Andersen, JBC, 2015), and formation of autoantibodies to neutrophil beta-glucoronidase was connected with the level of anti-MPO antibodies (Ab) (Martensson, Autoimmunity, 1992). Taking into account these facts, we aimed to investigate the possibility of NET-related changes in glycan composition on circulating IgG molecules and IgG-IgM immune complexes in multiple sclerosis (MS). This autoimmune disorder still has no reliable detection markers or established ways of treatment, besides widely accepted interferon therapy, making it a particularly interesting clinical condition. By applying capture lectin-ELISA, we analysed binding of α2,6 sialyl-specific lectins SNA, PSqL, and core α1,6-fucose specific lectin AAL to circulating IgG and related complexes in five groups of MS patients: untreated (17 persons); undergoing therapy with interferon (IFN) β-1 b (15 persons), corticosteroids (methylprednisolone) (12 persons) and anti-B-cell monoclonal Ab (12 persons: Ocrelizumab, 6 persons and alemtuzumab, 6 persons). A group of 23 healthy donors served as control. Significant increase in neutrophil elastase activity, observed in the group of patients under corticosteroid treatment was also accompanied by sialyl-specific PSqL and SNA lectin binding to captured IgG molecules. Subsequent analysis demonstrated that sialic acid residues were exposed on free IgG and on circulating IgG-IgM immune complexes. Increased lectin binding was not observed for anti-myelin basic protein (one of the major autoAb in MS) Ab compared to total serum Ab. IFN therapy was accompanied by low neutrophil elastase activity and low amount of circulating immune complexes. Incubation of in vitro generated NETs with human serum revealed the digestion of high-molecular weight immune complexes with subsequent exposure of hidden glycoepitops. Obtained data indicate the potential of neutrophil-derived proteases to modify (partially degrade) circulating immune complexes leading to exposure of internal glycoepitops.

Thrombin-derived C-terminal peptides (TCPs) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. Employing a combination of nuclear magnetic resonance spectroscopy (NMR), biophysical, mass spectrometry and cellular studies combined with in silico multiscale modelling, we here determine the bound conformation of HVF18 (HVFRLKKWIQKVIDQFGE), a TCP generated by neutrophil elastase, in complex with bacterial lipopolysaccharide (LPS) and define a previously undisclosed interaction between TCPs and human CD14. Further, we show that TCPs bind to the LPS-binding hydrophobic pocket of CD14 and identify the peptide region crucial for TCP interaction with LPS and CD14. Taken together, our results demonstrate the role of structural transitions in LPS complex formation and CD14 interaction, providing a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.

Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus-derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.-Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Antileukoproteinase or secretory leukocyte peptidase inhibitor is a small protein which protects the mucosal linings against excessive proteolysis, inflammation, and microbial infection. We discovered that gelatinase B or matrix metalloproteinase (MMP)-9, a secreted zinc-dependent endopeptidase typically found at sites of inflammation, destroys antileukoproteinase by cleavages within both of its two functional domains: the anti-microbial N-terminal and the anti-proteolytic C-terminal domains. Cleaved antileukoproteinase possessed a significantly lower ability to bind lipopolysaccharides (LPS) and a reduced capacity to inhibit neutrophil elastase (NE) activity. Whereas intact antileukoproteinase repressed proinflammatory transcript [prostaglandin-endoperoxide synthase 2 ( PTGS2 ) and IL6 ] synthesis and protein secretion [e.g., of MMP-9] in human CD14 + blood monocytes stimulated with LPS, this effect was reduced or lost for cleaved antileukoproteinase. We demonstrated the in vivo presence of antileukoproteinase cleavage fragments in lower airway secretions of non-cystic fibrosis bronchiectasis patients with considerable levels of neutrophils and, hence, elastase and MMP-9 activity. As a comparison, other MMPs (MMP-2, MMP-7, and MMP-8) and serine proteases (NE, cathepsin G, and proteinase 3) were also able to cleave antileukoproteinase with similar or reduced efficiency. In conclusion, in specific mucosal pathologies, such as bronchiectasis, neutrophils, and macrophage subsets control local immune reactions by proteolytic regulation, here described as the balance between MMPs (in particular MMP-9), serine proteases and local tissue inhibitors.

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro , and in vivo ., Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo ., Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding., Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia.

Infection with the helminth parasite Strongyloides stercoralis ( Ss ) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation., (© 2017 John Wiley & Sons Ltd.)

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated K d of 38.7 nm Furthermore, we showed that NRP1 binds to the RR X R motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium., Objective: We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease., Methods: Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF, and hematopoietic cell-specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM-CSF and follistatin-like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase., Results: OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3-5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45 + cells were OSM + , and of the OSM + cells, 56% ± 7% were CD16 + Siglec-8 - , indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45 + events from matched blood samples (n = 5) were OSM + , suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM + neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM-CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P < .001) in peripheral blood neutrophils in vitro. OSM + neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P < .05)., Conclusions: Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Inflammation plays a significant role in the development of obesity-related complications, but the molecular events that initiate and propagate such inflammation remain unclear. Here, we report that mice fed a high-fat diet (HFD) for as little as 1-3 days show increased differentiation of myeloid progenitors into neutrophils and monocytes but reduced B lymphocyte production in the bone marrow. Levels of neutrophil elastase (NE) and the nuclear factors CCAAT/enhancer-binding protein α (C/EBPα) and growth factor-independent 1 (GFI-1) are elevated in hematopoietic stem and progenitor cells from HFD-fed mice, but mice lacking either NE or C/EBPα are resistant to HFD-induced myelopoiesis. NE deletion increases expression of the inhibitory isoform of p30 C/EBPα, impairs the transcriptional activity of p42 C/EBPα, and reduces expression of the C/EBPα target gene GFI-1 in hematopoietic stem and progenitor cells, suggesting a mechanism by which NE regulates myelopoiesis. Furthermore, NE deletion prevents HFD-induced vascular leakage. Thus, HFD feeding rapidly activates bone marrow myelopoiesis through the NE-dependent C/EBPα-GFI-1 pathway preceding vascular damage and systemic inflammation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective and Design: Arthritic gout is caused by joint inflammation triggered by the damaging effects of monosodium uric acid (MSU) crystal accumulation in the synovial space. Neutrophils play a major role in mediating joint inflammation in gout. Along with neutrophils, other immune cells, such as macrophages, are present in inflamed joints and contribute to gout pathogenesis. Neutrophils form neutrophil extracellular traps (NETs) in response to MSU crystals. In the presence of MSU crystals, macrophages release IL-1β, a cytokine crucial to initiate gout pathogenesis and neutrophil recruitment. Our research investigated interactions between human macrophages and neutrophils in an in vitro model system and asked how macrophages affect NET formation stimulated by MSU crystals., Materials or Subjects: Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF., Treatment: Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1β or anakinra prior to stimulation by MSU crystals., Method: Interaction of neutrophils with MSU crystals was evaluated by live imaging using confocal microscopy. The presence of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured by ELISA. NET formation was quantitated by Sytox Orange-based extracellular DNA release assay and NE-DNA ELISA. AggNET formation was assessed by macroscopic evaluation., Results: We found that crystal- and cell-free supernatants of macrophages stimulated with MSU crystals promote MSU crystal-stimulated NET formation in human neutrophils. This observation was confirmed by additional assays measuring the release of MPO, NE, and the enzymatic activity of NE. MSU crystal-induced NET formation remained unchanged when neutrophil supernatants were tested. IL-1β is a crucial cytokine orchestrating the onset of inflammation in gout and is known to be released in large amounts from macrophages following MSU crystal stimulation. We found that recombinant IL-1β strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1β alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1β is mainly responsible for this effect., Conclusions: Macrophage-derived IL-1β enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1β affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra., Competing Interests: The authors have no financial conflicts of interest to report.

Problem: Neutrophil extracellular traps (NETs) were recently described as a mechanism for microbial killing in the amniotic cavity of women with intra-amniotic infection. Such a clinical condition can result in acute chorioamnionitis, a placental lesion characterized by the infiltration of maternal neutrophils in the chorioamniotic membranes. Herein, we investigated whether these infiltrating neutrophils form NETs in the chorioamniotic membranes from women who underwent spontaneous term or preterm labor with acute chorioamnionitis., Method of Study: Chorioamniotic membrane samples were collected from women who underwent spontaneous term or preterm labor with acute chorioamnionitis (n=10 each). Controls included chorioamniotic membrane samples from women who delivered at term or preterm with or without labor in the absence of acute chorioamnionitis (n=10 each). NETs were visualized and semiquantified in the chorioamniotic membranes by using antibodies against neutrophil elastase and histone H3 in combination with DAPI staining., Results: Neutrophil extracellular traps were abundant in the chorioamniotic membranes from women who underwent spontaneous term or preterm labor with acute chorioamnionitis. NETs were rarely found, or not visualized at all, in the chorioamniotic membranes from women who delivered at term or preterm with or without labor in the absence of acute chorioamnionitis., Conclusion: Neutrophil extracellular traps are abundant in the chorioamniotic membranes from women who underwent spontaneous term or preterm labor with acute chorioamnionitis. These findings suggest that chorioamniotic neutrophils can form NETs as a mechanism of host defense against infection or danger signals., (© Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Here we investigated the cutaneous CD32A and CD89 expression in relation to the neutrophil elastase (NE) expression and serum level of anti-desmoglein 1 and 3 (DSG1/DSG3) IgG in pemphigus, anti-BP180/BP230 IgG in bullous pemphigoid (BP), anti-gliadin nonapeptides (npG), tissue (tTG), and epidermal transglutaminases (eTG) IgA in dermatitis herpetiformis (DH). The examined material consisted of skin/mucosal tissues and sera. In total, 87 patients were studied. Immunohistochemistry on paraffin-embedded sections with quantitative digital morphometry was used to measure the intensity of CD32A/CD89/NE expressions. Levels of anti-DSG1/DSG3 IgG, anti-BP180/BP230 IgG, and anti-npG/tTG/eTG IgA were evaluated with ELISAs. CD32A was abundantly expressed in cutaneous lesions in pemphigus and BP. We found no statistically significant correlation between the CD32A/CD89 and NE expression intensities in pemphigus, BP, and DH. There was a significant correlation between CD89 expression and anti-npG IgA in DH. Our results revealed a lack of correlation between CD32A expressions and anti-DSG1/DSG3 IgG levels in pemphigus, anti-BP180/BP230 IgG in BP as well as CD89 expression and anti-tTG/eTG IgA in DH. CD89 seems to be linked with gluten intolerance in DH rather than with proteolytic destruction of dermal-epidermal junction. CD32A appears to play an important role in mediating skin injury in pemphigus and BP, but probably independently from specific autoantibodies.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

Cystic fibrosis (CF) is a chronic pulmonary disease that is associated with persistent microbial infection and chronic neutrophil infiltration, and also with elevated production of the pro-inflammatory cytokine IL-17A (IL-17). In the current study, we examined IL-17 and the inducible IL-17RC receptor subunit in neutrophils from Pseudomonas aeruginosa infected F508del CF patients at the time of pulmonary exacerbation, and again following intravenous antibiotic treatment. Neutrophils expressed Il17a and Il17rc transcripts and protein at the time of pulmonary exacerbation, which were absent following antibiotic treatment. Further, CF sputum induced IL-23 - dependent Il17rc expression in neutrophils from healthy individuals. Similarly, IL-17 producing neutrophils were detected in F508del and Cftr(-/-) mice infected intranasally with P. aeruginosa. In the sputum of CF subjects, the percentage IL-17 producing neutrophils correlated with elastase and MMP9 activity; therefore, this population of neutrophils may be an important contributor to the severity of pulmonary disease in CF patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Alpha-1 antitrypsin deficiency (AATD) is characterized by low levels of circulating alpha-1 antitrypsin and an increased risk for emphysema, liver disease, and panniculitis. The reduced levels of alpha-1 antitrypsin in AATD predispose the lung to unopposed proteolytic activity, predominantly from neutrophil-derived proteases, chiefly neutrophil elastase. This leads to emphysema. The mechanisms subtending the liver disease are less well understood, but are probably due to a "gain-of function" inflammatory process in the liver, stoked by intracellular retention of aberrantly folded alpha-1 antitrypsin. The panniculitis associated with AATD is most likely due to unopposed proteolytic activity in the skin. Although AATD has been traditionally viewed as a condition arising from a protease-antiprotease imbalance in the lung, it is increasingly recognized that AATD is an inflammatory disorder, both in the lung and in the extrapulmonary manifestations associated with the condition. This inflammation is predominantly neutrophil driven, and there are several alpha-1 antitrypsin-related mechanisms involved in potentiating this neutrophilic response. The rationale for AAT augmentation therapy in AATD is classically based on restoring the antiprotease balance in the lung, but its beneficial effects may also be exerted systemically, further exposing the pathogenesis of AATD-related disease and indicating a potential usage for alpha-1 antitrypsin in other inflammatory conditions.

The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a high-affinity T-cell receptor-like murine monoclonal antibody, 8F4, that binds to the PR1/HLA-A2 complex, mediates lysis of AML and inhibits leukemia colony formation. Here, we explored whether 8F4 was active in vivo against chemotherapy-resistant AML, including secondary AML. In a screening model, coincubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG (NOD scid IL-2 receptor γ-chain knockout) mice. In a treatment model of established human AML, administration of 8F4 significantly reduced or eliminated AML xenografts and extended survival compared with isotype antibody-treated mice. Moreover, in secondary transfer experiments, mice inoculated with bone marrow from 8F4-treated mice showed no evidence of AML engraftment, supporting the possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML.

As the immune pathways involved in the pathogenesis of type 1 diabetes (T1D) are not fully understood, biomarkers implicating novel mechanisms of disease are of great interest and call for independent evaluation. Recently, it was reported that individuals with T1D display dramatic elevations in circulating components of neutrophil extracellular traps (NETs), indicating a potential role for NETosis in T1D. Our aim was to evaluate further the potential of NET-associated proteins as novel circulating biomarkers in T1D. We tested serum from subjects with T1D (n = 44) with a median age of 26·5 years and a median duration of 2·2 years, along with 38 age-matched controls. T1D subjects did not show elevations in either neutrophil elastase (NE) or proteinase 3 (PR3), as reported previously. In fact, both NE and PR3 levels were reduced significantly in T1D subjects, particularly in subjects within 3 years of diagnosis, consistent with the known reduction in neutrophil counts in recent-onset T1D. Indeed, levels of both NE and PR3 correlated with absolute neutrophil counts. Therefore, while not ruling out potential local or transient spikes in NETosis activity, the levels of these serum markers do not support a role for systemically elevated NETosis in the T1D population we studied. Rather, a modest reduction in these markers may reflect other important aspects of disease activity associated with reduced neutrophil numbers., (© 2016 British Society for Immunology.)

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema.

Secretory leukocyte protease inhibitor (SLPI), a ∼12kDa nonglycosylated cationic protein, is emerging as an important regulator of innate and adaptive immunity and as a component of tissue regenerative programs. First described as an inhibitor of serine proteases such as neutrophil elastase, this protein is increasingly recognized as a molecule that benefits the host via its anti-proteolytic, anti-microbial and immunomodulatory activities. Here, we discuss the diverse functions of SLPI. Moreover, we review several novel layers of SLPI-mediated control that protect the host from excessive/dysregulated inflammation typical of infectious, allergic and autoinflammatory diseases and that support healing responses through affecting cell proliferation, differentiation and apoptosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Infections in cystic fibrosis (CF), often involving Pseudomonas aeruginosa, result from a dysregulated airway immunity where one hallmark is the accumulation of necrotic and apoptotic immune cells, in particular neutrophils. In addition, neutrophils actively release DNA, forming neutrophil extracellular traps (NETs) that contain antimicrobial proteins. Altogether, free DNA in complex with actin accumulates in the airway lumen, resulting in highly viscous sputum that provides an anionic matrix, binding cationic antimicrobial proteins. In this study, granulocyte chemotactic protein 2 (GCP-2)/CXCL6, a neutrophil-activating chemokine with bactericidal properties, was detected in the airway epithelium of CF patients and was also present in azurophilic and specific granules of neutrophils. Elastase of neutrophils, but not of P. aeruginosa, completely degraded CXCL6 (chemokine (C-X-C motif) ligand 6). In addition, CXCL6 colocalized with extracellular DNA in both CF sputa and in in vitro-formed NETs. In vitro, CXCL6 bound DNA with a KD of 2,500 nM. Interestingly, both the bactericidal and the receptor-activating properties of CXCL6 (against neutrophils) remained largely unaffected in the presence of DNA. However, the chemotactic properties of CXCL6 were reduced by the presence of DNA. Taken together, CXCL6 is expressed in CF, retaining its functional properties even after binding to the anionic scaffold that extracellular DNA provides in CF.

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

The important human gram positive bacterial pathogen Streptococcus pyogenes employs various virulence factors to promote inflammation and to facilitate invasive disease progression. In this study we explored the relation of the secreted streptococcal cysteine proteases IdeS and SpeB, and neutrophil (PMN) proteases. We found that SpeB is resistant to proteolytic attack in an inflammatory environment, emphasizing the importance of SpeB for streptococcal pathogenicity, while PMN enzymes and SpeB itself process the IgG degrading endopeptidase IdeS. Processing occurs as NH2-terminal cleavage of IdeS resulting in reduced immunorecognition of the protease by specific antibodies. While the endopeptidase retains IgG cleaving activity, its ability to suppress the generation of reactive oxygen species is abolished. We suggest that the cleavage of NH2-terminal peptides by SpeB and/or neutrophil proteases is a mechanism evolved to prevent early inactivation of this important streptococcal virulence factor, albeit at the cost of impaired functionality., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

On the model of chronic obstructive pulmonary disease, the effect of therapy with low-molecular-weight peptides on restructuring and functional activity of bronchial epithelium for restoring the immune and barrier function of the lungs and prevention of inflammatory process progression was studied. Chronic obstructive pulmonary disease was modeled in rats by 60-day intermittent exposure to NO2. Administration of tetrapeptide Bronchogen for 1 month eliminates symptoms of remodeling of the bronchial epithelium and lung tissue typical of chronic obstructive pulmonary disease (goblet cell hyperplasia, squamous metaplasia, lymphocytic infiltration and emphysema, and restoration of ciliated cells). Enhanced production of secretory IgA, a local immunity marker, attested to normalization of functional activity of bronchial epithelium, while normalization of cell composition and profile of proinflammatory cytokines in the bronchoalveolar space reflected reduction of neutrophilic inflammation.

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-β-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis., (© Society for Leukocyte Biology.)