Neutrophil-Derived Peptidyl Arginine Deiminase Activity Contributes to Pulmonary Emphysema by Enhancing Elastin Degradation.

In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and in vitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Monocyte NLRP3 inflammasome and interleukin-1ß activation modulated by alpha-1 antitrypsin therapy in deficient individuals.

INTRODUCTION: Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1ß secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS: Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1ß secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS: C3d acting via CR3 induces NLRP3 and pro-IL-1ß production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1ß secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1ß (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION: These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

Effect of elexacaftor/tezacaftor/ivacaftor on airway and systemic inflammation in cystic fibrosis.

Treatment with elexacaftor/tezacaftor/ivacaftor (ETI) has been shown to improve lung function in people with cystic fibrosis (PWCF). However, its biological effects remain incompletely understood. Here we describe alterations in pulmonary and systemic inflammation in PWCF following initiation of ETI. To address this, we collected spontaneously expectorated sputum and matching plasma from PWCF (n=30) immediately prior to ETI therapy, then again at 3 and 12 months. Within 3 months, PWCF demonstrated reduced activity of neutrophil elastase, proteinase three and cathepsin G, and decreased concentrations of interleukin (IL)-1ß and IL-8 in sputum, accompanied by decreased Pseudomonas burden and restoration of secretory leukoprotease inhibitor levels. Once treated with ETI, all airway inflammatory markers studied in PWCF had reduced to levels found in matched non-CF bronchiectasis controls. In PWCF with advanced disease, ETI resulted in decreased plasma concentrations of IL-6, C-reactive protein and soluble TNF receptor one as well as normalisation of levels of the acute phase protein, alpha-1 antitrypsin. These data clarify the immunomodulatory effects of ETI and underscore its role as a disease modifier.

Trikafta Rescues CFTR and Lowers Monocyte P2X7R-induced Inflammasome Activation in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) is caused by mutations in the CFTR (CF transmembrane conductance regulator) gene and is characterized by sustained inflammation. ATP triggers IL-1ß secretion via P2X7R (P2X7 receptor) and activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome. Objectives: To explore the effect of the CFTR modulator elexacaftor/tezacaftor/ivacaftor (Trikafta) on CFTR expression and the ATP/P2X7R signaling axis in monocytes and on circulating proinflammatory markers. Methods: Inflammatory mediators were detected in blood from 42 patients with CF before and after 3 months of Trikafta therapy. Markers of inflammasome activation and IL-1ß secretion were measured in monocytes before and after stimulation with ATP and LPS, in the presence or absence of the P2X7R inhibitor A438079. Measurements and Main Results: P2X7R is overexpressed in CF monocytes, and receptor inhibition decreased NLRP3 expression, caspase-1 activation, and IL-1ß secretion. In vitro and in vivo, P2X7R expression is regulated by CFTR function and intracellular chloride (Cl-) levels. Trikafta therapy restored CFTR expression yet decreased P2X7R in CF monocytes, resulting in normalized Cl- and potassium efflux, and reduced intracellular calcium levels. CFTR modulator therapy decreased circulating levels of ATP and LPS and reduced inflammasome activation and IL-1ß secretion. Conclusions: P2X7R expression is regulated by intracellular Cl- levels and in CF monocytes promotes inflammasome activation. Trikafta therapy significantly increased CFTR protein expression and reduced ATP/P2X7R-induced inflammasome activation. P2X7R may therefore be a promising target for reducing inflammation in patients with CF who are noneligible for Trikafta or other CFTR modulator therapy.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, µ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent µ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 µg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.

Targeting of Glycosaminoglycans in Genetic and Inflammatory Airway Disease.

In the lung, glycosaminoglycans (GAGs) are dispersed in the extracellular matrix (ECM) occupying the interstitial space between the capillary endothelium and the alveolar epithelium, in the sub-epithelial tissue and in airway secretions. In addition to playing key structural roles, GAGs contribute to a number of physiologic processes ranging from cell differentiation, cell adhesion and wound healing. Cytokine and chemokine-GAG interactions are also involved in presentation of inflammatory molecules to respective receptors leading to immune cell migration and airway infiltration. More recently, pathophysiological roles of GAGs have been described. This review aims to discuss the biological roles and molecular interactions of GAGs, and their impact in the pathology of chronic airway diseases, such as cystic fibrosis and chronic obstructive pulmonary disease. Moreover, the role of GAGs in respiratory disease has been heightened by the current COVID-19 pandemic. This review underlines the essential need for continued research aimed at exploring the contribution of GAGs in the development of inflammation, to provide a better understanding of their biological impact, as well as leads in the development of new therapeutic agents.

A Review of Alpha-1 Antitrypsin Binding Partners for Immune Regulation and Potential Therapeutic Application.

Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.

Circulating Truncated Alpha-1 Antitrypsin Glycoprotein in Patient Plasma Retains Anti-Inflammatory Capacity.

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele (Q0bolton ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0bolton-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0bolton-AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0bolton-AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0bolton-AAT bound IL-8 and leukotriene B4, comparable to healthy control M-AAT, and significantly decreased leukotriene B4-induced neutrophil adhesion (p = 0.04). Through a mechanism involving increased mRNA stability (p = 0.007), ataluren treatment of HEK-293 significantly increased Q0bolton-AAT mRNA expression (p = 0.03) and Q0bolton-AAT truncated protein secretion (p = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0bolton-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype.

Alpha-1 Antitrypsin Augmentation Inhibits Proteolysis of Neutrophil Membrane Voltage-Gated Proton Channel-1 in Alpha-1 Deficient Individuals.

Background and Objectives: Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of immunomodulatory functions. Individuals with the genetic condition of alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. As peripheral neutrophil hyper-responsiveness in AATD leads to excessive degranulation and increased migration to the airways, we examined the expression of the membrane voltage-gated proton channel-1 (HVCN1), which is integrally linked to neutrophil function. The objectives of this study were to evaluate altered HVCN1 in AATD neutrophils, serine protease-dependent degradation of HVCN1, and to investigate the ability of serum AAT to control HVCN1 expression. Materials and Methods: Circulating neutrophils were purified from AATD patients (n = 20), AATD patients receiving AAT augmentation therapy (n = 3) and healthy controls (n = 20). HVCN1 neutrophil expression was assessed by flow cytometry and Western blot analysis. Neutrophil membrane bound elastase was measured by fluorescence resonance energy transfer. Results: In this study we demonstrated that HVCN1 protein is under-expressed in AATD neutrophils (p = 0.02), suggesting a link between reduced HVCN1 expression and AAT deficiency. We have demonstrated that HVCN1 undergoes significant proteolytic degradation in activated neutrophils (p < 0.0001), primarily due to neutrophil elastase activity (p = 0.0004). In addition, the treatment of AATD individuals with AAT augmentation therapy increased neutrophil plasma membrane HVCN1 expression (p = 0.01). Conclusions: Our results demonstrate reduced levels of HVCN1 in peripheral blood neutrophils that may influence the neutrophil-dominated immune response in the AATD airways and highlights the role of antiprotease treatment and specifically AAT augmentation therapy in protecting neutrophil membrane expression of HVCN1.

Activation of complement component 3 is associated with airways disease and pulmonary emphysema in alpha-1 antitrypsin deficiency.

INTRODUCTION: Alpha-1 antitrypsin (AAT) deficiency (AATD) is associated with early onset emphysema. The aim of this study was to investigate whether AAT binding to plasma constituents could regulate their activation, and in AATD, exploit this binding event to better understand the condition and uncover novel biomarkers of therapeutic efficacy. METHODS: To isolate AAT linker proteins, plasma samples were separated by size exclusion chromatography, followed by co-immunoprecipitation. AAT binding proteins were identified by mass spectrometry. Complement turnover and activation was determined by ELISA measurement of C3, C3a and C3d levels in plasma of healthy controls (n=15), AATD (n=51), non-AATD patients with obstructive airway disease (n=10) and AATD patients post AAT augmentation therapy (n=5). RESULTS: Direct binding of complement C3 to AAT was identified in vivo and in vitro. Compared with healthy controls, a breakdown product of C3, C3d, was increased in AATD (0.04 µg/mL vs 1.96 µg/mL, p=0.0002), with a significant correlation between radiographic pulmonary emphysema and plasma levels of C3d (R2=0.37, p=0.001). In vivo, AAT augmentation therapy significantly reduced plasma levels of C3d in comparison to patients not receiving AAT therapy (0.15 µg/mL vs 2.18 µg/mL, respectively, p=0.001). DISCUSSION: Results highlight the immune-modulatory impact of AAT on the complement system, involving an important potential role for complement activation in disease pathogenesis in AATD. The association between plasma C3d levels and pulmonary disease severity, that decrease in response to AAT augmentation therapy, supports the exploration of C3d as a candidate biomarker of therapeutic efficacy in AATD.

α1 Antitrypsin therapy modulates the neutrophil membrane proteome and secretome.

Obstructive pulmonary disease in patients with α1 antitrypsin (AAT) deficiency (AATD) occurs earlier in life compared with patients without AATD. To understand this further, the aim of this study was to investigate whether AATD presents with altered neutrophil characteristics, due to the specific lack of plasma AAT, compared with non-AATD COPD.This study focussed on the neutrophil plasma membrane and, by use of label-free tandem mass spectrometry, the proteome of the neutrophil membrane was compared in forced expiratory volume in 1 s (FEV1)-matched AATD, non-AATD COPD and in AATD patients receiving weekly AAT augmentation therapy (n=6 patients per cohort). Altered protein expression in AATD was confirmed by Western blot, ELISA and fluorescence resonance energy transfer analysis.The neutrophil membrane proteome in AATD differed significantly from that of COPD as demonstrated by increased abundance and activity of primary granule proteins including neutrophil elastase on the cell surface in AATD. The signalling mechanism underlying increased degranulation involved Rac2 activation, subsequently resulting in proteinase-activated receptor 2 activation by serine proteinases and enhanced reactive oxygen species production. In vitro and ex vivo, AAT reduced primary granule release and the described plasma membrane variance was resolved post-AAT augmentation therapy in vivo, the effects of which significantly altered the AATD neutrophil membrane proteome to that of a non-AATD COPD cell.These results provide strong insight into the mechanism of neutrophil driven airways disease associated with AATD. Therapeutic AAT augmentation modified the membrane proteome to that of a typical COPD cell, with implications for clinical practice.

Specific Inhibition of the NLRP3 Inflammasome as an Antiinflammatory Strategy in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) pulmonary disease is characterized by chronic infection with Pseudomonas aeruginosa and sustained neutrophil-dominant inflammation. The lack of effective antiinflammatory therapies for people with CF (PWCF) represents a significant challenge.Objectives: To identify altered immunometabolism in the CF neutrophil and investigate the feasibility of specific inhibition of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome as a CF antiinflammatory strategy in vivo.Methods: Key markers of increased aerobic glycolysis, known as a Warburg effect, including cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, succinate, HIF-1α (hypoxia-inducible factor-1α), lactate, and the IL-1ß precursor pro-IL-1ß, as well as caspase-1 activity and processing of pro-IL-1ß to IL-1ß by the NLRP3 inflammasome, were measured in neutrophils from blood and airway secretions from healthy control subjects (n = 12), PWCF (n = 16), and PWCF after double-lung transplantation (n = 6). The effects of specific inhibition of NLRP3 on airway inflammation and bacterial clearance in a murine CF model were subsequently assessed in vivo.Measurements and Main Results: CF neutrophils display increased aerobic glycolysis in the systemic circulation. This effect is driven by low-level endotoxemia, unaffected by CFTR (cystic fibrosis transmembrane conductance regulator) modulation, and resolves after transplant. The increased pro-IL-1ß produced is processed to its mature active form in the LPS-rich CF lung by the NLRP3 inflammasome via caspase-1. Specific NLRP3 inhibition in vivo with MCC950 inhibited IL-1ß in the lungs of CF mice (P < 0.0001), resulting in significantly reduced airway inflammation and improved Pseudomonas clearance (P < 0.0001).Conclusions: CF neutrophil immunometabolism is altered in response to inflammation. NLRP3 inflammasome inhibition may have an antiinflammatory and anti-infective role in CF.

Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α.

Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.

The Human Cathelicidin Antimicrobial Peptide LL-37 Promotes the Growth of the Pulmonary Pathogen Aspergillus fumigatus.

The pulmonary mucus of cystic fibrosis (CF) patients displays elevated levels of the cathelicidin antimicrobial peptide LL-37, and the aim of this work was to assess the effect of LL-37 on the growth of Aspergillus fumigatus, a common pathogen of CF patients. Exposure of A. fumigatus to LL-37 and its derived fragment RK-31 (1.95 µg/ml) for 24 h had a positive effect on growth (199.94% ± 6.172% [P < 0.05] and 218.20% ± 4.63% [P < 0.05], respectively), whereas scrambled LL-37 peptide did not (85.12% ± 2.92%). Exposure of mycelium (preformed for 24 h) to 5 µg/ml intact LL-37 for 48 h increased hyphal wet weight (4.37 ± 0.23 g, P < 0.001) compared to the control (2.67 ± 0.05 g) and scrambled LL-37 (2.23 ± 0.09 g) treatments. Gliotoxin secretion from LL-37 exposed hyphae (169.1 ± 6.36 ng/mg hyphae, P < 0.05) was increased at 24 h compared to the results seen with the control treatment (102 ± 18.81 ng/mg hyphae) and the scrambled LL-37 treatment (96.09 ± 15.15 ng/mg hyphae). Shotgun proteomic analysis of 24-h LL-37-treated hyphae revealed an increase in the abundance of proteins associated with growth (eukaryotic translation initiation factor 5A [eIF-5A] [16.3-fold increased]), tissue degradation (aspartic endopeptidase [4.7-fold increased]), and allergic reactions (Asp F13 [10-fold increased]). By 48 h, there was an increase in protein levels indicative of cellular stress (glutathione peroxidase [9-fold increased]), growth (eIF-5A [6-fold increased]), and virulence (RNase mitogillin [3.7-fold increased]). These results indicate that LL-37 stimulates A. fumigatus growth and that this stimulation can result in increased fungal growth and secretion of toxins in the lungs of CF patients.

Hermansky-Pudlak syndrome with a novel genetic variant in HPS1 and subsequent accelerated pulmonary fibrosis: significance for phenocopy diseases.

The Hermansky-Pudlak syndrome (HPS) is a collection of autosomal-recessive disorders characterised by tyrosinase-positive oculocutaneous albinism (OCA), bleeding diatheses and, in selected individuals, early-onset accelerated pulmonary fibrosis, neutropaenia and granulomatous colitis. We describe a young man who presented following a self-directed literature review prompted by severe bleeding complications following minor surgical and dental procedures in the context of OCA. HPS was clinically suspected, with subsequent genetic testing confirming biallelic mutations in the HPS1 gene. Of interest, this is the only described HPS type 1 patient with two different (compound heterozygote) splice site variants in HPS1 In addition to detailing a novel genetic result and outlining the progressive clinical course of disease in this case, we discuss the management of HPS, the prognostic value of subtype analysis and the technical difficulties relating to transplantation in the case of HPS-associated advanced pulmonary fibrosis. This case also illustrates the concept of lung phenocopy relationships and the potential for elucidating the pathogenesis of more common pulmonary disorders by studying genetic diseases that result in similar phenotypes. Furthermore, it re-emphasises the importance of the patient voice, particularly with regard to complex diagnoses and rare diseases.

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated.

The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

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.

A neutrophil intrinsic impairment affecting Rab27a and degranulation in cystic fibrosis is corrected by CFTR potentiator therapy.

Studies have endeavored to reconcile whether dysfunction of neutrophils in people with cystic fibrosis (CF) is a result of the genetic defect or is secondary due to infection and inflammation. In this study, we illustrate that disrupted function of the CF transmembrane conductance regulator (CFTR), such as that which occurs in patients with ∆F508 and/or G551D mutations, correlates with impaired degranulation of antimicrobial proteins. We demonstrate that CF blood neutrophils release less secondary and tertiary granule components compared with control cells and that activation of the low-molecular-mass GTP-binding protein Rab27a, involved in the regulation of granule trafficking, is defective. The mechanism leading to impaired degranulation involves altered ion homeostasis caused by defective CFTR function with increased cytosolic levels of chloride and sodium, yet decreased magnesium measured in CF neutrophils. Decreased magnesium concentration in vivo and in vitro resulted in significantly decreased levels of GTP-bound Rab27a. Treatment of G551D patients with the ion channel potentiator ivacaftor resulted in normalized neutrophil cytosolic ion levels and activation of Rab27a, thereby leading to increased degranulation and bacterial killing. Our results confirm that intrinsic alterations of circulating neutrophils from patients with CF are corrected by ivacaftor, thus illustrating additional clinical benefits for CFTR modulator therapy.