Neutrophil Proteases Research Articles

Targeting neutrophil serine proteases in bronchiectasis.

Persistent neutrophilic inflammation is a central feature in both the pathogenesis and progression of bronchiectasis (BE). Neutrophils release neutrophil serine proteases (NSPs), such as neutrophil elastase, cathepsin G and proteinase 3. When chronically high levels of free NSP activity exceed those of protective antiproteases, structural lung destruction, mucosal-related defects, further susceptibility to infection and worsening of clinical outcomes can occur. Despite the defined role of prolonged, high levels of NSPs in BE, no drug that controls neutrophilic inflammation is licensed for the treatment of BE. Previous methods of suppressing neutrophilic inflammation (such as direct inhibition of neutrophil elastase) have not been successful; however, an emerging therapy designed to address neutrophil-mediated pathology, inhibition of the cysteine protease cathepsin C (CatC, also known as dipeptidyl peptidase 1), is a promising approach to ameliorate neutrophilic inflammation, since this may reduce the activity of all NSPs implicated in BE pathogenesis, and not just neutrophil elastase. Current data suggest that CatC inhibition may effectively restore the protease-antiprotease balance in BE and improve disease outcomes as a result. Clinical trials for CatC inhibitors in BE have reported positive Phase III results. In this narrative review, we discuss the role of high NSP activity in BE, and how this feature drives the associated morbidity and mortality seen in BE. This review discusses therapeutic approaches aimed at treating neutrophilic inflammation in the BE lung, summarising clinical trial outcomes, and highlighting the need for more treatment strategies that effectively address chronic neutrophilic inflammation in BE.

A neutrophil elastase-generated mature form of IL-33 is a potent regulator of endothelial cell activation and proliferative retinopathy

Human interleukin-33 (IL-33) is a 270 amino acid protein that belongs to the IL-1 cytokine family and plays an important role in various inflammatory disorders. Neutrophil proteases (Cathepsin G and Elastase) and mast cell proteases (tryptase and chymase) regulate the activity of IL-33 by processing full-length IL-33 into its mature form. There is little evidence on the role of these mature forms of IL-33 in retinal endothelial cell signaling and pathological retinal angiogenesis. Here, we cloned, expressed, and purified the various mature forms of human IL-33 and then evaluated the effects of IL-3395-270, IL-3399-270, IL-33109-270, and IL-33112-270 on angiogenesis in human retinal microvascular endothelial cells (HRMVECs). We observed that IL-3395-270, IL-3399-270, IL-33109-270, and IL-33112-270 significantly induced HRMVEC migration, tube formation and sprouting angiogenesis. However, only IL-3399-270 could induce HRMVEC proliferation. We used a murine model of oxygen-induced retinopathy (OIR) to assess the role of these mature forms of IL-33 in pathological retinal neovascularization. Our 3′-mRNA sequencing and signaling studies indicated that IL-3399-270 and IL-33109-270 were more potent at inducing endothelial cell activation and angiogenesis than the other mature forms. We found that genetic deletion of IL-33 significantly reduced OIR-induced retinal neovascularization in the mouse retina and that intraperitoneal administration of mature forms of IL-33, mainly IL-3399–270 and IL-33109–270, significantly restored ischemia-induced angiogenic sprouting and tuft formation in the hypoxic retinas of IL-33–/– mice. Thus, our study results suggest that blockade or inhibition of IL-33 cleavage by neutrophil proteases could help mitigate pathological angiogenesis in proliferative retinopathies.

Neutrophil extracellular traps - an a-list-actor in a variety of diseases.

Neutrophil extracellular traps (NETs) represent a response mechanism in which activated neutrophils release DNA-based webs, adorned with histones and neutrophil proteases, to capture and eliminate invasive microorganisms. However, when these neutrophils become excessively activated, much more proteases associated with NETs are liberated into surrounding tissues or bloodstreams, thereby altering the cellular milieu and causing tissue damage. Recent research has revealed that NETs may play significant roles in the emergence and progression of various diseases, spanning from infections, inflammation to autoimmune disorders and cancers. In this review, we delve deeply into the intricate and complex mechanisms that underlie the formation of NETs and their profound interplay with various clinical pathologies. We aim to describe the application perspectives of NETs related proteins in specific disease diagnosis and treatment.

Exogenous serpin B1 restricts immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block in vitro granzyme-mediated target cell killing by CD8 T or NK cells.

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.

Neutrophil serine proteases in cystic fibrosis: role in disease pathogenesis and rationale as a therapeutic target.

Chronic airway inflammation is a central feature in the pathogenesis of bronchiectasis (BE), which can be caused by cystic fibrosis (CFBE; hereafter referred to as CF lung disease) and non-CF-related conditions (NCFBE). Inflammation in both CF lung disease and NCFBE is predominantly driven by neutrophils, which release proinflammatory cytokines and granule proteins, including neutrophil serine proteases (NSPs). NSPs include neutrophil elastase, proteinase 3 and cathepsin G. An imbalance between NSPs and their antiproteases has been observed in people with CF lung disease and people with NCFBE. While the role of the protease/antiprotease imbalance is well established in both CF lung disease and NCFBE, effective therapies targeting NSPs are lacking. In recent years, the introduction of CF transmembrane conductance regulator (CFTR) modulator therapy has immensely improved outcomes in many people with CF (pwCF). Despite this, evidence suggests that airway inflammation persists, even in pwCF treated with CFTR modulator therapy. In this review, we summarise current data on neutrophilic inflammation in CF lung disease to assess whether neutrophilic inflammation and high, uncontrolled NSP levels play similar roles in CF lung disease and in NCFBE. We discuss similarities between the neutrophilic inflammatory profiles of people with CF lung disease and NCFBE, potentially supporting a similar therapeutic approach. Additionally, we present evidence suggesting that neutrophilic inflammation persists in pwCF treated with CFTR modulator therapy, at levels similar to those in people with NCFBE. Collectively, these findings highlight the ongoing need for new treatment strategies targeting neutrophilic inflammation in CF lung disease.

A novel in vitro cell model of the proteinase/antiproteinase balance observed in alpha-1 antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting from mutations in the alpha-1 antitrypsin (AAT) protein, a major systemic antiproteinase, resulting in reduced/no release of AAT, disrupting the proteinase/antiproteinase balance. A sustained imbalance can cause structural changes to the lung parenchyma, leading to emphysema. Predicting and assessing human responses to potential therapeutic candidates from preclinical animal studies have been challenging. Our aims were to develop a more physiologically relevant in vitro model of the proteinase/antiproteinase balance and assess whether the data generated could better predict the efficacy of pharmacological candidates to inform decisions on clinical trials, together with expected biomarker responses. Methods: We developed an in vitro model assessing the proteinase/antiproteinase balance by the changes in the fibrinogen cleavage products of neutrophil elastase (NE) and proteinase 3 (PR3). This allowed the assessment of physiological and pharmaceutical neutrophil serine proteinase (NSP) inhibitors to determine the putative threshold at which the maximal effect is achieved. Results: AAT significantly reduced NE and PR3 activity footprints, with the maximal reduction achieved at concentrations above 10μM. The inhibitor MPH966 alone also significantly reduced NE footprint generation in a concentration-dependent manner, leveling out above 100nM but had no effect on the PR3 footprint. At levels of AAT consistent with AATD, MPH966 had an additive effect, reducing the NE activity footprint more than either inhibitor alone. Conclusion: Our results support an inhibitor threshold above which the activity footprint generation appears resistant to increasing dosage. Our model can support the testing of inhibitors, confirming activity biomarkers as indicators of likely pharmaceutical efficacy, the assessment of NSP activity in the pathophysiology of emphysema, and the likely function of biological or pharmacological inhibitors in disease management.

Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding.

Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.

Neutrophil-Derived Proteases in Lung Inflammation: Old Players and New Prospects

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation.

Oropharyngeal Candidiasis, Candida Albicans Infections, and Oral Immune Mediators Associated with Oral Human Immunodeficiency Virus: A Literature Review

Introduction: While Candida albicans are usually commensal and present in low density in the oral cavity of healthy individuals, an immunocompromised immune system can lead to the overgrowth of this fungal species, leading to oral microbiome “dysbiosis” and activation of immune responses. In severe cases, C. albicans overgrowth can lead to an oral infection called oropharyngeal candidiasis (OPC), which is associated with inflammation, lesions, and sores of the oral cavity. While human immunodeficiency virus (HIV) infection has been linked to an increased risk of OPC, few studies have associated OPC and C. albicans infection with subsequent risk for oral HIV acquisition. Evidence on the oral microbiome and how it can alter HIV risk is also lacking. Methods: A literature search was performed on PubMed, Google Scholar, and the University of Alberta Library Database from inception to November 2023. Results and Discussion: The risk of oral HIV transmission is low. OPC and C. albicans fungal infection can increase HIV susceptibility by activating immune responses associated with the clearance of microbial pathogens, inducing inflammation and elevations in cytokines related to HIV risk including IL-17, IL-6 and IL-1ß. Persistent C. albicans infections also promote the recruitment of T-helper 17 cells, which is a common HIV target cell, and neutrophils, which releases neutrophil proteases upon inflammation and mediates the cleavage of tight junction proteins, ultimately disrupting the oral microbiome. Conclusion: Increased immune cell recruitment to the mucosa and increased epithelial breakdown may facilitate the diffusion and access of HIV virions across the epithelium to immune target cells, suggesting that OPC and C.albicans infections has the potential to increase risk for oral HIV acquisition. Limited evidence of the relationship between C. albicans density, OPC, and oral HIV risk, warrants high-quality cross-sectional studies in the future.

Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation.

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.

Cardiovascular disease in Alpha 1 antitrypsin deficiency: an observational study assessing the role of neutrophil proteinase activity and the suitability of validated screening tools

BackgroundAlpha 1 Antitrypsin Deficiency (AATD) is a rare, inherited lung disease which shares features with Chronic Obstructive Pulmonary Disease (COPD) but has a greater burden of proteinase related tissue damage. These proteinases are associated with cardiovascular disease (CVD) in the general population. It is unclear whether patients with AATD have a greater risk of CVD compared to usual COPD, how best to screen for this, and whether neutrophil proteinases are implicated in AATD-associated CVD. This study had three aims. To compare CVD risk in never-augmented AATD patients to non-AATD COPD and healthy controls (HC). To assess relationships between CVD risk and lung physiology. To determine if neutrophil proteinase activity was associated with CVD risk in AATD. Cardiovascular risk was assessed by QRISK2® score and aortic stiffness measurements using carotid-femoral (aortic) pulse wave velocity (aPWV). Medical history, computed tomography scans and post-bronchodilator lung function parameters were reviewed. Systemic proteinase 3 activity was measured. Patients were followed for 4 years, to assess CVD development.Results228 patients with AATD, 50 with non-AATD COPD and 51 healthy controls were recruited. In all COPD and HC participants, QRISK2® and aPWV gave concordant results (with both measures either high or in the normal range). This was not the case in AATD. Once aPWV was adjusted for age and smoking history, aPWV was highest and QRISK2® lowest in AATD patients compared to the COPD or HC participants. Higher aPWV was associated with impairments in lung physiology, the presence of emphysema on CT scan and proteinase 3 activity following adjustment for age, smoking status and traditional CVD risk factors (using QRISK2® scores) in AATD. There were no such relationships with QRISK2® in AATD. AATD patients with confirmed CVD at four-year follow up had a higher aPWV but not QRISK2® at baseline assessment.ConclusionaPWV measured CVD risk is elevated in AATD. This risk is not captured by QRISK2®. There is a relationship between aPWV, lung disease and proteinase-3 activity. Proteinase-driven breakdown of elastin fibres in large arteries and lungs is a putative mechanism and forms a potential therapeutic target for CVD in AATD.

Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase.

Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M-1s-1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.

Elevated first-trimester neutrophil elastase and proteinase 3 increase the risk of gestational diabetes mellitus and adverse fetal outcomes

BackgroundChronic inflammation plays a vital role in the development of gestational diabetes mellitus (GDM). Studies in mouse models show that neutrophil serine proteases (NSPs), neutrophil elastase (NE) and proteinase-3 (PR3) are important drivers of chronic inflammation with consequent metabolic disturbances. This study evaluated the association of NE and PR3 with GDM development and adverse fetal outcomes.Method(s)This was a prospective cohort study. Serum PR3 and NE concentration was measured in all enrolled pregnant women in the first and the second trimester to determine the connection between NSPs and GDM and adverse fetal outcomes. Logistic regression, spline regression and linear regression analyses were applied to investigate the association of NE or PR3 with GDM development and adverse fetal outcomes. The concentration of NE and PR3 in placental biopsies was evaluated by semi-quantitative analysis of immunohistochemistry staining.Result(s)NE or PR3 concentration in the first trimester, rather than the second, increased more significantly in women with GDM than in those without, regardless of pre-pregnancy body mass index and age. There was a stepwise increase in GDM occurrence as well as comprehensive adverse fetal outcomes across tertiles of NE and PR3. NE and PR3 were positively associated with neutrophil count, pre-pregnancy BMI, plasma glucose level and newborn weight. Logistic regression revealed NE or PR3 to be independent risk factors for the development of GDM and comprehensive adverse fetal outcomes. Spline regression showed a significant increased risk of GDM occurrence and comprehensive adverse fetal outcomes when serum NE concentration exceeded 417.60 ng/mL and a similar result for PR3 and GDM occurrence when the latter exceeded 88.52 ng/mL. Immunohistochemistry data confirmed that enriched NE and PR3 content in placental tissue may have contributed to the development of GDM.Conclusion(s)This work demonstrates that excessive first-trimester NE and PR3 increase the risk of GDM development and comprehensive adverse fetal outcomes.

Metabolic Dysfunction-Associated Steatohepatitis Detected by Neutrophilic Crown-Like Structures in Morbidly Obese Patients: A Multicenter and Clinicopathological Study.

Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of metabolic dysfunction-associated steatotic liver disease (MASLD), and closely associated with a high risk of liver-related morbidity and mortality. Although enhanced neutrophil infiltration of the liver is a histological hallmark of MASH, the morphological pattern of hepatic neutrophils and their relevance to the definition of MASH remain unknown. This clinicopathological study aimed to determine the association of neutrophilic crown-like structures (CLSs) in liver biopsies and evaluate their relevance to the histological diagnosis of MASH. A total of 483 morbidly obese adults who underwent bariatric surgery were recruited. Neutrophilic CLSs in liver biopsies were detected by immunohistochemistry for neutrophil elastase and proteinase 3. All participants were classified into 4 histological subgroups: no MASLD (118, 24.4%), MASLD (76, 15.7%), borderline MASH (185, 38.3%), and definite MASH (104, 21.5%). In the discovery cohort (n = 379), the frequency of neutrophilic CLSs increased in line with the severity of liver disease. The number of neutrophilic CLSs was positively correlated with established histological characteristics of MASH. At a cutoff value of <0.3 per 20× microscopic field, the number of neutrophilic CLSs yielded a robust diagnostic accuracy to discriminate no MASLD and MASLD from borderline MASH and definite MASH; a cutoff at >1.3 per 20× microscopic field exhibited a statistically significant accuracy to distinguish definite MASH from other groups (no MASLD, MASLD, and borderline MASH). The significance of neutrophilic CLSs in identifying borderline MASH and definite MASH was confirmed in an external validation cohort (n = 104). The frequency of neutrophilic CLSs was significantly higher than that of macrophagic CLSs. In conclusion, neutrophilic CLSs in the liver represent a typical histological characteristic of MASH and may serve as a promising indicator to improve the diagnostic accuracy of MASH during histological assessment of liver biopsies.

Dipeptidyl peptidase 1 inhibition as a potential therapeutic approach in neutrophil-mediated inflammatory disease.

Neutrophils have a critical role in the innate immune response to infection and the control of inflammation. A key component of this process is the release of neutrophil serine proteases (NSPs), primarily neutrophil elastase, proteinase 3, cathepsin G, and NSP4, which have essential functions in immune modulation and tissue repair following injury. Normally, NSP activity is controlled and modulated by endogenous antiproteases. However, disruption of this homeostatic relationship can cause diseases in which neutrophilic inflammation is central to the pathology, such as chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency, bronchiectasis, and cystic fibrosis, as well as many non-pulmonary pathologies. Although the pathobiology of these diseases varies, evidence indicates that excessive NSP activity is common and a principal mediator of tissue damage and clinical decline. NSPs are synthesized as inactive zymogens and activated primarily by the ubiquitous enzyme dipeptidyl peptidase 1, also known as cathepsin C. Preclinical data confirm that inactivation of this protease reduces activation of NSPs. Thus, pharmacological inhibition of dipeptidyl peptidase 1 potentially reduces the contribution of aberrant NSP activity to the severity and/or progression of multiple inflammatory diseases. Initial clinical data support this view. Ongoing research continues to explore the role of NSP activation by dipeptidyl peptidase 1 in different disease states and the potential clinical benefits of dipeptidyl peptidase 1 inhibition.

The proteolytic airway environment associated with pneumonia acts as a barrier for treatment with anti-infective antibodies

Like pneumonia, coronavirus disease 2019 (COVID-19) is characterized by a massive infiltration of innate immune cells (such as polymorphonuclear leukocytes) into the airways and alveolar spaces. These cells release proteases that may degrade therapeutic antibodies and thus limit their effectiveness. Here, we investigated the in vitro and ex vivo impact on anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) IgG1s and other IgG subclasses (IgG2 and IgG4) of the neutrophil elastase, proteinase 3 and cathepsin G (the three main neutrophil serine proteases) found in endotracheal aspirates from patients with severe COVID-19. Although the IgGs were sensitive to neutrophil serine proteases, IgG2 was most resistant to proteolytic degradation. The two anti-SARS CoV2 antibodies (casirivimab and imdevimab) were sensitive to the lung’s proteolytic environment, although neutrophil serine protease inhibitors only partly limited the degradation. Overall, our results show that the pneumonia-associated imbalance between proteases and their inhibitors in the airways contributes to degradation of antiviral antibodies.

IL-27 Promotes Neutrophil Extracellular Trap Generation in Sickle Cell Disease

Introduction: Neutrophil Extracellular Traps (NETs) are neutrophil-derived damage-associated molecular patterns (DAMPs) composed of decondensed chromatin decorated with citrullinated histones and neutrophil proteases. Recently, we have shown that caspase-4-dependent activation (cleavage) of the pore-forming protein, Gasdermin-D (GSDMD) in neutrophils promotes NETs generation leading to lung injury in SCD. IL-27, an anti-inflammatory cytokine with some pro-inflammatory properties is significantly elevated in the plasma of SCD patients, however, it remains debatable whether IL-27 signaling in neutrophils serves to promote or inhibit NETs generation in SCD. Methods: Citrated blood samples from SCD or race matched control humans were incubated with saline, hemin (20 mM), or hemin (20 mM) + recombinant human IL-27 (200 ng/mL) for 15 minutes at room temperature. Platelet-poor-plasma from the three treatment groups were incubated with sytox green and fluorescent Abs against neutrophil elastase (NE) and histones for in situ staining of extracellular DNA, NE and histones, respectively. Imaging flow cytometry was used to detect circulating NETs (cNETs) as 1-2 mm fragments triple-positive for extracellular DNA, NE, and histones. Results: Incubation with hemin led to significant increase in cNETs concentration in SCD but not control human blood, which was further increased following incubation of SCD human blood with hemin in presence of IL-27. Conclusion: Our current findings suggest that IL-27 signaling promotes NETs generation by neutrophils in SCD patient blood. Currently, studies are underway to identify the molecular mechanism underlying IL-27-dependent NETs generation in SCD.

Mature IL-36γ Induces Stratum Corneum Exfoliation in Generalized Pustular Psoriasis by Suppressing Corneodesmosin

Loss-of-function sequence variations in the IL36RN gene encoding IL-36 receptor antagonist cause familial generalized pustular psoriasis, which begins shortly after birth and is difficult to treat, and its effects on the epidermis are unclear. This study investigated the involvement of IL-36 receptor agonists in the epidermal formation of generalized pustular psoriasis. We found that the IL-36 receptor agonists, especially mature IL-36γ, stimulated IL-8 and pro-IL-36γ production in the epidermis while downregulating the genes encoding epidermal cornified envelope-related proteins, for example, corneodesmosin. IL-36 receptor antagonist and monoclonal anti-IL-36γ antibodies counteracted the effect of mature IL-36γ on corneodesmosin in keratinocytes in a dose-dependent manner. In the epidermis of patients with generalized pustular psoriasis with IL36RN loss-of-function sequence variations, pro-IL-36γ was overproduced in the epidermis, and corneodesmosin protein expression was markedly decreased in the region of giant subcorneal pustules (Kogoj's spongiform pustules), with high neutrophil infiltration. IL-8 induced by mature IL-36γ stimulated the infiltration of several neutrophils in the epidermis. The newly produced pro-IL-36γ is cleaved to the mature form by neutrophil proteases. This newly produced mature IL-36γ was predicted to further suppress the gene expression of corneodesmosin, leading to significant stratum corneum exfoliation and formation of the pustules. Overall, our results elucidate the mechanism underlying the formation of Kogoj's spongiform pustules in generalized pustular psoriasis.

Citrulline is not a major determinant of autoantibody reactivity to neutrophil extracellular traps.

One of the main strategies of neutrophils in responding to microbial infections is the formation of neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin associated with antimicrobial proteins. Citrullination plays an important role during NET formation and a substantial fraction of NET-associated proteins appeared to be citrullinated. The release of citrullinated intracellular proteins from netting neutrophils led to the hypothesis that the production of anti-citrullinated protein autoantibodies by autoimmune patients, in particular patients with rheumatoid arthritis, might be initiated when citrullinated NET components are not properly cleared and are exposed to the immune system. Here, we discuss the processes that lead to NET formation, including the role of peptidylarginine deiminase activation and our current knowledge on citrullinated NET-associated proteins. Citrulline-dependent epitopes do not appear to play a major role in the recognition of NETs by autoantibodies from rheumatoid arthritis and systemic lupus erythematosus patients, even though anti-NET autoantibodies are frequently observed in sera from these patients. The neutrophil proteases associated with NETs have a major impact on the integrity of NET-associated proteins when NET formation is induced by activating isolated human neutrophils. Cleavage/degradation of these proteins also resulted in a strong reduction of the reactivity with autoantibodies. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.