AAT Deficiency Research Articles

Indirect effect of alpha-1-antitrypsin on endotoxin-induced IL-1β secretion from human PBMCs.

Human alpha-1-antitrypsin (AAT) encoded by the SERPINA1 gene, is an acute phase glycoprotein that regulates inflammatory responses via both protease inhibitory and non-inhibitory activities. We previously reported that AAT controls ATP-induced IL-1β release from human mononuclear cells by stimulating the release of small bioactive molecules. In the current study, we aimed to elucidate the identity of these putative effectors released from human PBMCs in response to AAT, which may inhibit the LPS-induced release of IL-1β. We pre-incubated human PBMCs alone or with different preparations of AAT (4 mg/ml) for 30 min at 37°C, 5% CO2, and collected cell supernatants filtered through centrifugal filters (cutoff 3 kDa) to eliminate AAT and other high molecular weight substances. Supernatants passed through the filters were used to culture PBMCs isolated from the autologous or a heterologous donors with or without adding LPS (1 μg/ml) for 6 h. Unexpectedly, supernatants from PBMCs pre-incubated with AAT (Zemaira®), but not with other AAT preparations tested or with oxidized AAT (Zemaira®), lowered the LPS-induced release of IL-1β by about 25%–60% without affecting IL1B mRNA. The reversed-phase liquid chromatography coupled with mass spectrometry did not confirm the hypothesis that small pro-resolving lipid mediators released from PBMCs after exposure to AAT (Zemaira®) are responsible for lowering the LPS-induced IL-1β release. Distinctively from other AAT preparations, AAT (Zemaira®) and supernatants from PBMCs pre-treated with this protein contained high levels of total thiols. In line, mass spectrometry analysis revealed that AAT (Zemaira®) protein contains freer Cys232 than AAT (Prolastin®). Our data show that a free Cys232 in AAT is required for controlling LPS-induced IL-1β release from human PBMCs. Further studies characterizing AAT preparations used to treat patients with inherited AAT deficiency remains of clinical importance.

Improving the Laboratory Diagnosis of M-like Variants Related to Alpha1-Antitrypsin Deficiency.

Alpha1-antitrypsin (AAT) is a serine protease inhibitor that is encoded by the highly polymorphic SERPINA1 gene. Mutations in this gene can lead to AAT deficiency (AATD), which is associated with an increased risk of lung and/or liver disease. On the basis of electrophoretic migration, AAT variants are named with capital letters; M (medium) signifies the normal protein. Among pathological variants, the M-like ones represent a heterogeneous group of rare allelic variants that exhibit the same electrophoretic pattern as the M wild-type protein, which makes them difficult to detect with routine methods. In order to avoid their misdiagnosis, the present study defines and validates effective methods for the detection of two pathogenic M-like variants, Mwurzburg and Mwhitstable. Comparison of protein phenotypes using isoelectric focusing of samples that presented the Mwurzburg variant, as revealed by exons 5 sequencing, identified a particular electrophoretic pattern amenable to the Mwurzburg protein. The specific phenotyping pattern was retrospectively validated, thus enabling the detection of 16 patients with Mwurzburg variant among the subjects already tested but not sequenced according to our diagnostic algorithm. The Mwhitstable allele was detected by intron 4 sequencing of SERPINA1 gene. Mwurzburg and Mwhitstable are often misdiagnosed and the introduction of diagnostic improvements can help the clinical management, especially in patients with established lung disease without any other reported risk factors.

Alu RNA induces NLRP3 expression through TLR7 activation in α-1-antitrypsin-deficient macrophages.

α-1 antitrypsin (AAT) is a serine protease inhibitor that plays a pivotal role in maintaining lung homeostasis. The most common AAT allele associated with AAT deficiency (AATD) is PiZ. Z-AAT accumulates in cells due to misfolding, causing severe AATD. The major function of AAT is to neutralize neutrophil elastase in the lung. It is generally accepted that loss of antiprotease function is a major cause of COPD in individuals with AATD. However, it is now being recognized that the toxic gain-of-function effect of Z-AAT in macrophage likely contributes to lung disease. In the present study, we determined that TLR7 signaling is activated in Z-MDMs, and the expression level of NLRP3, one of the targets of TLR7 signaling, is significantly higher in Z- compared with M-MDMs. We also determined that the level of endosomal Alu RNA is significantly higher in Z-compared with M-MDMs. Alu RNA is a known endogenous ligand that activates TLR7 signaling. Z-AAT likely induces the expression of Alu elements in MDMs and accelerates monocyte death, leading to the higher level of endosomal Alu RNA in Z-MDMs. Taken together,this study identifies a mechanism responsible for the toxic gain of function of Z-AAT macrophages.

Liver gene therapy: The magic bullet for the sick lung

Alpha1-antitrypsin (AAT) deficiency is one of the most common genetic diseases and is due to pathogenic variants in the SERPINA1 gene that encodes for AAT, an acute-phase protein with anti-inflammatory and immunomodulatory properties. Although more than 150 pathogenic variants have been described, 95% of individuals with severe AAT deficiency are homozygotes for the Z allele, a substitution of glutamic acid for lysine at residue 342 (p.Glu342Lys) in SERPINA1. This single amino acid change makes Z-AAT prone to aggregation mostly in hepatocytes, the cells that abundantly synthetize and secrete AAT into the circulation.

Α1-ANTITRYPSIN DEFICIENCY IN GREEKS: FOCUS ON RARE VARIANTS

TYPE: Abstract TOPIC: Genetic and Developmental Disorders PURPOSE: This study aimed to investigate genotype and phenotype characteristics of a Greek cohort of A1AT deficiency patients. METHODS: A1AT deficiency patients followed-up in Greece and analyzed in A1AT Laboratory, Universitätsklinikum Gießen und Marburg GmbH, Germany were enrolled in the study. Patients underwent A1AT measurement for early emphysema RESULTS: Included are 24 adults, 54.2% males, 28.6% never-smokers, diagnosed at a median age (IQR) of 49 (43.0-59.5) years with A1AT levels of 0.255 (0.185-0.380) g/L. Patients presented with a median (IQR) FEV1 (% predicted), FEV1/FVC% and DLCO (% predicted) of 40.9 (29.9, 59.8), 50.5 (39.3, 63.0) and 52.8 (35.8, 68.0) respectively. Ten patients (41.7%) presented PI*ZZ genotype, 4 patients (16.7%) PI*Q0Q0 genotypes and 3 patients (12.5%) PI*ZQ0 genotypes. Q0 alleles corresponded to PI*Q0-Bellingham (1/7pts), PI*Q0-Amersfoort (3/7 pts) and PI*Q0-Granitefalls 3/7pts), ultra-rare null variants. Four patients (16.7%) were carriers of at least one of the following rare variants MHeerlen, MProcida and MMalton. In 3 patients (12.5%) sequencing indicated other ultra-rare mutations. Null variants were significantly associated with lowest A1AT levels and lower FVC values, compared to other variants [p=0.023 and p=0.034 respectively]. A1AT protein levels significantly correlated to FEF25-75% (% predicted) [p=0.013, r=0.559] CONCLUSIONS: The present study provides new evidence about an increased incidence of rare and ultra-rare A1AT variants in Greece of clear pathogenic significance, confirming an established North to South European geographical trend in A1AT deficiency. CLINICAL IMPLICATIONS: The interpretation of novel SERPINA1 variants highlighted the importance of unravelling new mechanisms of A1AT deficiency pathophysiology in a personalized medicine era DISCLOSURE: Nothing to declare. KEYWORD: A1 ANTITRYPSIN DEFIECIENCY

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

LMAN1-MCFD2 complex is a cargo receptor for the ER-Golgi transport of α1-antitrypsin.

α1-antitrypsin (AAT) is a serine protease inhibitor synthesized in hepatocytes and protects the lung from damage by neutrophil elastase. AAT gene mutations result in AAT deficiency (AATD), which leads to lung and liver diseases. The AAT Z variant forms polymer within the endoplasmic reticulum (ER) of hepatocytes and results in reduction in AAT secretion and severe disease. Previous studies demonstrated a secretion defect of AAT in LMAN1 deficient cells, and mild decreases in AAT levels in male LMAN1 and MCFD2 deficient mice. LMAN1 is a transmembrane lectin that forms a complex with a small soluble protein MCFD2. The LMAN1–MCFD2 protein complex cycles between the ER and the Golgi. Here, we report that LMAN1 and MCFD2 knockout (KO) HepG2 and HEK293T cells display reduced AAT secretion and elevated intracellular AAT levels due to a delayed ER-to-Golgi transport of AAT. Secretion defects in KO cells were rescued by wild-type LMAN1 or MCFD2, but not by mutant proteins. Elimination of the second glycosylation site of AAT abolished LMAN1 dependent secretion. Co-immunoprecipitation experiment in MCFD2 KO cells suggested that AAT interaction with LMAN1 is independent of MCFD2. Furthermore, our results suggest that secretion of the Z variant, both monomers and polymers, is also LMAN1-dependent. Results provide direct evidence supporting that the LMAN1–MCFD2 complex is a cargo receptor for the ER-to-Golgi transport of AAT and that interactions of LMAN1 with an N-glycan of AAT is critical for this process. These results have implications in production of recombinant AAT and in developing treatments for AATD patients.

Genetic testing of allelic variants of PIZ (GLU342Lys, RS28929474) and PIS (GLU264Val, RS17580) of SERPINA1 gene in children with bronchial asthma

According to world publications, mutations in the SERPINA1 gene may be a genetic risk factor for severe chronic obstructive pulmonary disease and, consequently, rapid progression of respiratory dysfunction. This disease leads to a decrease in the level of alpha-1-antitrypsin protein. It is inherited by autosomal recessive type, but there are registered cases of codominance. In the absence of treatment, diseases of the respiratory system become chronic and lead to disability in adulthood.
 Early diagnosis of AAT deficiency is important to prevent complications and reduce mortality among people with this pathology. Due to these factors, genetic testing of SERPINA1 gene mutations in children with chronic lung diseases is appropriate to detect and prevent severe complications, associated with AATD.
 The aim of this work is to improve the effectiveness of early diagnosis of AAT deficiency in children with bronchial asthma and recurrent obstructive bronchitis by identifying different genotypes and phenotypes of A1AT deficiency, studying their relationship with the clinical course of respiratory diseases in children

Leave no one behind: inclusion of alpha-1 antitrypsin deficiency patients in COVID-19 vaccine trials.

The coronavirus disease of 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, continues to present an unprecedented challenge worldwide. Emerging evidence suggests that α-1 antitrypsin (A1AT), a circulating protein with protective effects on the lung and other vital organs, plays a critical role in preventing SARS-CoV-2 infection and may be a promising therapeutic option for patients with COVID-19. A1AT deficiency (AATD) is characterized by dysfunctional or insufficient levels of A1AT. Recently, we have proposed that AATD patients are a vulnerable population for COVID-19. Patients with AATD may derive limited benefit from the current COVID-19 vaccines and continue to rely on conventional medical therapy and behavioral adaptations to mitigate the risk of infection. Unfortunately, this population has not been included in the COVID-19 vaccine clinical trials and studies have yet to characterize the safety, immunogenicity, and ultimately, the efficacy of COVID-19 vaccines for AATD patients. Re-evaluation of the COVID-19 vaccine safety and immunogenicity will further promote informed decision-making for vaccination in AATD individuals and contribute to reduce morbidity and mortality from COVID-19 infection.

Frequency of alpha-1 antitrypsin deficiency and unexpected results in COPD patients in Turkey; rare variants are common.

Alpha-1 antitrypsin (α1-AT) is a protease inhibitor that is largely released from liver cells. It inhibits neutrophil elastase and its deficiency increases the risk of developing chronic obstructive pulmonary disease (COPD). The frequency of α1-AT deficiency has been reported with different prevalence rates in different parts of the world. The most common α1-AT variant causing α1-AT deficiency is the Pi*Z allele. In this study, we aimed to determine the frequency of the α1-AT genotypic variant in COPD patients in our country. In this study, 196 consecutive COPD patients admitted to our clinic were included. In addition to recording the demographic data of the volunteers, a dry drop of blood sample was taken from the fingertip for the SERPINA1 genotype study. One hundred and fifty-eight (80.6%) of the patients were male and the mean age was 56.92 ± 9.84 years. A variant in the SERPINA1 gene was detected in a total of 14 (7.1%) COPD patients. Pi*ZZ homozygous variant was detected in only 1 (0.51%) patient, while Pi*MZ was detected in 3 (1.53%) patients. The Pi*S variant was never detected. Various rare heterozygous variants were detected in 9 (4.6%) patients and a single point mutation was found in one (0.51%) patient. Serum α1-AT levels were significantly lower in patients with variants compared to the Pi*MM group (p < 0.001). In this study, which investigated the genotypic α1-AT variant frequency in COPD patients for the first time in our country, we found that the percentage of homozygous Pi*ZZ patients was 0.51%, but when heterozygous α1-AT gene variant and single point mutation were included, the frequency was 7.1%. At the same time, while the Pi*S variant was never detected, rare variants were found more frequently than expected.

A Novel Detection Method to Identify Individuals with Alpha-1 Antitrypsin Deficiency: Linking Prescription of COPD Medications with the Patient-Facing Electronic Medical Record.

Alpha-1 antitrypsin deficiency (AATD) is under-recognized, prompting the need for enhanced detection strategies. The primary aim of this study is to determine the feasibility of using the electronic medical record (EMR) and linked electronic patient messages (EPM) to encourage AATD testing by patients with chronic obstructive pulmonary disease (COPD). Study participants were eligible, untested adult patients who were prescribed an inhaled medication which is exclusively Food and Drug Administration-approved for treating COPD. Eligible patients received a message with basic information about AATD and availability of free, home-based AATD testing. Through a collaboration with the Alpha-1 Foundation's Alpha-1 Coded Testing (ACT) study, patients referred to home-based testing through EPM were flagged. The effectiveness of the electronic message was evaluated by the proportion of patients who underwent testing, and the rate of detecting individuals with severe deficiency of AAT among those tested. A total of 12,369 patients on eligible inhalers were screened; 5430 patients met all criteria and received an EPM. During the study, 396 patients (7.3%) fully requested an ACT kit. Of these, 209 patients (52.8%) returned the test sample and received genotyping results; 65.5%, had a normal AAT genotype (PI*MM), 31.6% were heterozygotes for a deficient allele (PI*MS, PI*MZ and PI*M/Null rare), and 2.9% had severe deficiency of alpha-1 antitrypsin (PI*SZ, PI*ZZ, PI*S/Null rare). While the response rate and test return rate were low, the rate of detecting individuals with AATD using this detection strategy exceeds that of many prior strategies. As such, while requiring independent validation in other populations, this detection strategy holds promise.

Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease.

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αβ-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.

Un déficit sévère en alpha1-antitrypsine révélé par un ictère néonatal

AbstractAlpha 1-antitrypsin is a glycoprotein mainly produced by hepatocytes; the main role is the protection of pulmonary parenchyma by inhibiting pro-inflammatory proteases like elastase released by neutrophils during inflammation. One of the most common genetic diseases is severe AAT deficiency. The clinical manifestation are pulmonary involvement with emphysema, liver disease, which may progress to cirrhosis and more rarely panniculitis; its biological translation is a decrease of AAT serum level. We report here the case of a severe deficit discovered in a newborn with neonatal jaundice.

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.

Therapeutic benefits of recombinant alpha1-antitrypsin IgG1 Fc-fusion protein in experimental emphysema

BackgroundAlpha-1 antitrypsin (AAT) is a major serine protease inhibitor. AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In well-selected AATD patients, therapy with plasma-derived AAT (pAAT), “augmentation therapy”, provides modest clinical improvement but is perceived as cumbersome with weekly intravenous infusions. Using mouse models of emphysema, we compared the effects of a recombinant AAT-IgG1 Fc-fusion protein (AAT-Fc), which is expected to have a longer half-life following infusion, to those of pAAT.MethodsIn an elastase model of emphysema, mice received a single intratracheal instillation of porcine pancreatic elastase (PPE) or human leucocyte elastase (hLE). AAT-Fc, pAAT, or vehicle was administered intraperitoneally 1 day prior to or 3 weeks following elastase instillation. Lung function and histology assessments were performed at 7 and 32 days after elastase instillation. In a cigarette smoke (CS) model of emphysema, mice were exposed to CS daily, 5 days a week, for 6 months and AAT-Fc, pAAT, or vehicle were administered every 10 days during the last 3 months of CS exposure. Assessments were performed 3 days after the last CS exposure. Immune responses to lung elastin peptide (EP) and the effects of AAT-Fc or pAAT treatment on dendritic cell (DC) function were determined ex vivo.ResultsBoth elastase instillation and CS exposure triggered emphysema-like alveolar enlargement, increased lung compliance, and increased markers of inflammation compared to controls. Administration of AAT-Fc either prior to or following elastase instillation or during CS exposure provided greater protection than pAAT against alveolar enlargement, lung dysfunction, and airway inflammation. When challenged ex vivo with EP, spleen mononuclear cells from elastase-exposed mice exhibited dose-dependent production of IFNγ and IL-17, suggesting immune reactivity. In co-culture experiments with splenic CD4+ T cells isolated from elastase-exposed mice, AAT-Fc treatment prior to EP-priming of bone marrow-derived dendritic cells inhibited the production of IFNγ and IL-17.ConclusionsCompared to pAAT, AAT-Fc more effectively prevented or attenuated elastase- and CS-induced models of emphysema. These effects were associated with immunomodulatory effects on DC activity. AAT-Fc may provide a therapeutic option to individuals with AATD- and CS-induced emphysema.

Hypoimmune induced pluripotent stem cell–derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

COVID-19 infection in severe Alpha 1-antitrypsin deficiency: Looking for a rationale

The clinical manifestations of COVID-19 are heterogeneous: 46.4% of patients admitted into hospital reported to have at least one comorbidity. Comorbidities such as COPD, diabetes, hypertension and malignancy predispose patients with Covid-19 to adverse clinical outcomes. Alpha 1-antitrypsin deficiency (AATD) is a genetic disorder caused by pathological mutation(s) in the SERPINA1 gene resulting in an imbalance in proteinase activity which may lead to premature emphysema and COPD.Our aim was to investigate whether people with severe AAT deficiency (AATD) have an increased risk of (severe) COVID-19 infection.We collected data on COVID-19 symptoms, laboratory-confirmed infection, hospitalization and treatment by means of a telephone survey, directly administered to Italian severe AATD subjects in May 2020. We then compared our findings with data collected by the Istituto Superiore di Sanità on the total population in Italy during the same period.We found an higher frequency of SARS-CoV-2 infection in our cohort (3.8%) compared to national data regarding infection, thus giving severe AATD a relative risk of 8. 8 (95%CI 5.1-20,0; p<0.0001) for symptomatic SARS-CoV-2 infection. Moreover, the relative risk (RR) was higher in AATD patients with pre-existing lung diseases (RR 13.9; 95%CI 8.0-33.6; p<0.001), but with a similar death rate (1 in 8, 12.5%) compared to the general population (13.9%; RR 0.9).These preliminary findings highlight the importance of close surveillance in the spread of COVID-19 in patients with severe AATD and underlines the need for further studies into the role of the antiprotease shield in preventing SARS-Cov-2 infection.

Reinforcement of the Unfolded Protein Response Mitigates Cytotoxicity Induced by Human Z‐Type α1‐Antitrypsin

Deficient human α1‐antitrypsin (AAT) variants are involved in pulmonary emphysema and liver cirrhosis. Especially, the Z‐type AAT (Z AAT) variant folds very slowly, and thus accumulates protein folding intermediates prone to aggregation in the endoplasmic reticulum (ER) of hepatocytes. Misfolded proteins accumulated in the ER lead to persistent ER stress and subsequent cell death. In this study, the contribution of unfolded protein response (UPR) in Z AAT‐induced cytotoxicity was investigated. Deletions of each UPR element severely hampered growth of Z AAT‐overexpressing yeast cells. Overexpression of UPR elements, except kar2, alleviated the slow growth phenotype of Z AAT‐overexpressing cells, possibly through augmentation of folding capacity. Furthermore, reinforcement of UPR elements promoted extracellular secretion of Z AAT, which may have mitigated the plasma deficiency of AAT. Our results therefore provide further information on therapeutic strategies to address protein folding diseases.

Hepatocyte proteomes reveal the role of protein disulfide isomerase 4 in alpha 1-antitrypsin deficiency

Background & AimsA single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention.MethodsOur functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease.ResultsWe report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease.ConclusionsPDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD.Lay summaryProtein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.

Impact of serine protease inhibitor alpha1-antitrypsin on expression of endoplasmic reticulum stress-induced proinflammatory factors in adipocytes

Obesity-induced endoplasmic reticulum (ER) stress contributes to low-grade chronic inflammation in adipose tissue and may cause metabolic disorders such as diabetes mellitus and dyslipidemia. Identification of high serpina A1 (alpha-1 antitrypsin, A1AT) expression in mouse adipose tissue and adipocytes prompted us to explore the role of A1AT in the inflammatory response of adipocytes under ER stress. We aimed to determine the role of A1AT expression in adipocytes with ER stress during regulation of adipocyte homeostasis and inflammation. To this end, we chemically induced ER stress in A1AT small interfering RNA-transfected differentiating adipocytes using thapsigargin. Induction of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker, by thapsigargin was lower in A1AT-deficient SW872 adipocytes. Thapsigargin or the proinflammatory cytokine tumor necrosis factor (TNF)α increased basal expression of cytokines such as interleukin (IL)-1β and IL-8 in both SW872 and primary omental adipocytes. This thapsigargin- or TNFα-induced expression of proinflammatory genes was increased by A1AT deficiency. These findings indicate that adipose A1AT may suppress the ER stress response to block excessive expression of proinflammatory factors, which suggests that A1AT protects against adipose tissue dysfunction associated with ER stress activation.