In α-1 antitrypsin (AAT) deficiency (AATD), emphysema is classically linked to protease-antiprotease imbalance caused by decreased antiprotease AAT due levels and function. This decrease is secondary to the impaired release of Z-AAT polymers from hepatocytes carrying Pi*Z, E342K mutation in SERPENA1 gene. Whether the accumulation of Z-AAT polymers in distal lungs contributes directly to emphysema pathogenesis has remained unexplored due to the lack of suitable model systems. We characterized lung injury and airspace enlargement in a Z-AAT-overexpressing murine model. We generated Z-AAT Serpina1Null mice overexpressing human (E342K) SERPENA1 in Serpina1Null mice and analyzed pulmonary phenotypes in young and aged animals, complemented by translational studies using primary cells, bronchoalveolar lavage fluid (BALf), and lung tissue from individuals who have never smoked and individuals with AATD. Young Z-AAT Serpina1Null mice accumulated Z-AAT polymers in hepatocytes, plasma, and BALf, exhibited spontaneous neutrophilic lung inflammation, increased alveolo-capillary permeability, and premature airspace enlargement, which was worse in older Z-AAT Serpina1Null mice. Moreover, Z-AAT polymers accumulated in alveolar type-2 epithelial (AT2) cells and lung macrophages, associated with endoplasmic reticulum (ER) stress, mitochondria dysfunction, and incomplete autophago-lysosomal fusion, which we recapitulated in lung samples from individuals with AATD. These findings support the pathogenic role of Z-AAT polymer accumulation in distal lung epithelium as a driver of epithelial, endothelial, and macrophage dysfunction linked to AATD emphysema.

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder caused by mutations in SERPINA1, leading to chronic obstructive pulmonary disease (COPD) and liver disease. Neutrophils are key regulators of inflammatory signaling networks; however, their dysregulation in AATD and the underlying molecular mechanisms remain poorly understood. Here, we employed a multi-omics approach integrating RNA sequencing (RNA-seq) and metabolomics to comprehensively characterize neutrophil dysfunction in AATD. RNA-seq analysis of blood neutrophils from AATD individuals revealed transcriptional dysregulation in genes involved in intracellular signaling, immune response regulation, and metabolic adaptation. Isolation and characterization of neutrophil-derived extracellular vesicles (EV) demonstrated an increased plasma burden of neutrophil elastase (NE)-rich EV with elevated surface-bound NE. Metabolomic profiling revealed that these EVs are enriched with pro-inflammatory metabolites linked to dysregulated signaling pathways. Integrated transcriptomic and metabolomic network analysis showed that altered neutrophil gene expression and signaling pathways reshape EV metabolic cargo, linking metabolic reprogramming to inflammatory signal transduction in AATD. Furthermore, differentially expressed EV metabolites may modulate gene expression in recipient cells, sustaining chronic inflammation in AATD. The observed upregulation of interferon, pattern recognition receptors, and cytokine-mediated signaling pathways in neutrophils suggests a potential feedback loop amplifying inflammation in AATD and COPD.

α-1 Antitrypsin deficiency (AATD) is a genetic disorder characterized by accumulation of misfolded Z α-1 antitrypsin (ZAAT) in hepatocytes, leading to liver injury and metabolic dysfunction. There is no therapy to reduce ZAAT accumulation and restore proteostasis. Pioglitazone activates AMP-activated protein kinase (AMPK), enhance autophagy, and modulate endoplasmic reticulum stress responses, suggesting a potential effect on ZAAT clearance. Our objective is to examine whether pioglitazone can protect against AATD-mediated liver disease. Huh7.5 cells expressing ZAAT (HuhZ) and Pi*Z transgenic mice were used to investigate pioglitazone treatment on hepatic ZAAT accumulation, autophagy activation, and AMPK signaling. Histological, molecular, and metabolic analyses were conducted to assess changes in ZAAT content, autophagy markers, AMPK phosphorylation, and proteostasis. Pioglitazone significantly reduced intracellular ZAAT and decreased lipid droplet accumulation in HuhZ cells. Pioglitazone markedly lowered hepatic ZAAT content in Pi*Z mice, suggesting enhanced degradation. This reduction was mediated through the AMPK pathway, indicated by increased phosphorylation of AMPK and ULK1. Pioglitazone induced autophagy, shown by decreased p62 and increased ATG5 and LC3B-II. This is indicative of enhanced autophagy. Although total hepatic AAT levels were reduced, periodic acid-Schiff with diastase-positive ZAAT aggregates exhibited only a downward trend, suggesting these may be more resistant to clearance. These findings demonstrate pioglitazone reduces hepatic ZAAT accumulation by activating AMPK and inducing autophagy in AATD-associated liver disease, supporting its potential for therapeutic repurposing. As pioglitazone is FDA-approved with benefits for metabolic liver health, further studies are warranted to evaluate efficacy in restoring proteostasis and reducing hepatic ZAAT.NEW & NOTEWORTHY α-1 Antitrypsin deficiency (AATD)-mediated liver disease lacks therapies that reduce hepatic ZAAT accumulation and liver manifestations. We demonstrate that pioglitazone activates AMPK and induces autophagy, leading to decreased ZAAT and improved proteostasis in Pi*Z mouse livers and human hepatocyte models. As an FDA-approved drug with metabolic benefits, pioglitazone holds promise for repurposing in AATD-related liver disease. These findings offer a mechanistic rationale for targeting autophagy to alleviate hepatic injury in protein misfolding disorders.

Alpha-1 antitrypsin, the most abundant protease inhibitor within the plasma, plays a crucial role in regulating neutrophils' function during inflammation. Alpha-1 antitrypsin deficiency is associated with excessive neutrophilic inflammation, yet the mechanisms underlying alpha-1 antitrypsin's role in neutrophil trafficking remain poorly understood. Here, we demonstrate alpha-1 antitrypsin is essential for maintaining neutrophil polarity, directional migration, and tissue infiltration during inflammation. Using alpha-1 antitrypsin-knockout mice, we found these mice present increased numbers of neutrophils in the bone marrow, impaired mobilization, and reduced liver neutrophil infiltration following lipopolysaccharide-induced systemic inflammation. Flow cytometry and immunohistochemistry revealed alpha-1 antitrypsin-knockout neutrophils had lower CD44 expression and defective F-actin polarization leading to impaired chemotaxis. Importantly, low expression of CD44 prevented efficient adhesion and transmigration of alpha-1 antitrypsin-knockout neutrophils across liver sinusoidal endothelial cells. Furthermore, chemotaxis assays showed alpha-1 antitrypsin-knockout neutrophils in alpha-1 antitrypsin-deficient media displayed random motility and loss of directional migration toward fMLP (N-Formyl-Met-Leu-Phe), suggesting a critical role for alpha-1 antitrypsin in neutrophil trafficking. Additionally, plasma alpha-1 antitrypsin deficiency delayed neutrophils' rate of phagocytosis. Mechanistically, alpha-1 antitrypsin deficiency resulted in excessive ERK1/2 (Extracellular Signal-Regulated Kinase 1/2) activation in alpha-1 antitrypsin-knockout neutrophils, driving an interleukin-10-enriched environment while suppressing expression of CXCL1 (C-X-C Motif Chemokine Ligand 1) and CXCL10 (C-X-C Motif Chemokine Ligand 10), chemokines essential for neutrophil recruitment. Notably, exposure to wild-type plasma with sufficient alpha-1 antitrypsin restored ERK1/2 activation, CD44 expression, and chemokine levels in alpha-1 antitrypsin-knockout neutrophils, confirming the role of circulating alpha-1 antitrypsin in maintaining neutrophil function. These findings highlight alpha-1 antitrypsin as a key regulator of neutrophil trafficking, adhesion, and immune signaling, with implications for alpha-1 antitrypsin deficiency-related inflammatory disorders.

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by accumulation of misfolded α-1 antitrypsin within hepatocytes. AATD patients are prone to develop liver disease that remains undiagnosed until the late stages of the disease. Due to challenges in manipulating the α-1 antitrypsin genes in mice, determining a true loss of function of α-1 antitrypsin in previous AATD mouse models has been challenging. Here, we report generation and liver characterization of a new humanized transgenic mouse model for AATD with a background of a CRISPR-Cas9 generated SERPINA1-null mouse. Male and female transgenic mice for normal (Pi*M) and mutant (Pi*Z) variants of human α-1 antitrypsin at 4-6 months of age were subjected to this study. The accumulation of human α-1 antitrypsin in the hepatocytes and fibrotic features of the liver were monitored by performing an in vivo study. We demonstrate a strong liver phenotype satisfying clinically relevant manifestations of liver pathology associated with AATD, including hepatic accumulation of human α-1 antitrypsin globules, liver deposition of extracellular matrix proteins, hepatic ER stress, and liver fibrosis in Pi*Z mice, in addition to mild systemic inflammation. In addition to major phenotypic criteria of AATD-associated liver fibrosis, accompanying single-nucleus RNA-seq data demonstrate activation of pathways associated with liver metabolic changes, inflammation, and regeneration. Data from this study suggest our humanized transgenic AATD mouse model could provide a suitable model to study α-1 antitrypsin loss of function, replicate the pathophysiology of AATD associated liver disease, and evaluate therapeutic reagents against this disease. ### Competing Interest Statement The authors have declared no competing interest.

Background and Aims: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder associated with liver disease, ranging from fibrosis to hepatocellular carcinoma. The disease remains asymptomatic until its final stages when liver transplantation is the only available therapy. Biomarkers offer an advantage for disease evaluation. The presence of microRNAs (miRNAs) in plasma extracellular vesicles (EVs) presents a noninvasive approach to assess the molecular signatures of the disease. In this study, we aimed to identify miRNA biomarkers to distinguish molecular signatures of the liver disease associated with AATD in AATD individuals. Methods: Using small RNA sequencing and qPCR, we examined plasma EV miRNAs in healthy controls (n = 20) and AATD patients (n = 17). We compared the EV miRNAs of AATD individuals with and without liver disease, developing an approach for detecting liver disease. A set of miRNAs identified in the AATD testing cohort was validated in a separate cohort of AATD patients (n = 45). Results: We identified differential expression of 178 EV miRNAs in the plasma of the AATD testing cohort compared to controls. We categorized AATD individuals into those with and without liver disease, identifying 39 differentially expressed miRNAs. Six miRNAs were selected to test their ability to discriminate liver disease in AATD. These were validated for their specificity and sensitivity in an independent cohort of 45 AATD individuals. Our logistic model established composite scores with threeand four-miRNA combinations, achieving areas under the curve of 0.737 and 0.751, respectively, for predicting AATD liver disease. Conclusions: We introduce plasma EV-derived miRNAs as potential biomarkers for evaluating AATD liver disease. Plasma EV-associated miRNAs may represent a mo lecular signature of AATD liver disease and could serve as valuable tools for its detection and monitoring.

Liver fibrosis associated with increased mortality is caused by activation of hepatic stellate cells and excessive production and accumulation of extracellular matrix in response to fibrotic insults. It has been shown that in addition to liver inflammation, systemic inflammation also contributes to liver fibrogenesis. A deeper understanding of mechanisms that control liver fibrotic response to intra- and extra-hepatic inflammation is essential to develop novel clinical strategies against this disease. Extracellular vesicles (EV) have been recognized as immune mediators that facilitate activation of hepatic stellate cells. In inflammatory diseases, activated neutrophils release neutrophil elastase (NE) bound to EV, which has been identified as a significant contributor to inflammation by promoting immune cell activation. Here, we aimed to explore the role of inflammation derived plasma EV-associated NE in liver fibrogenesis and its potential mechanisms. We show EV-associated NE induces activation, proliferation and migration of hepatic stellate cells by promoting activation of the ERK1/2 signaling pathway. This effect did not occur through EV without surface NE, and Sivelestat, a NE inhibitor, inhibited activation of the ERK1/2 signaling pathway mediated by EV-associated NE. Moreover, we found plasma EV-associated NE increases deposition of collagen1 and α-smooth muscle actin in the liver of a mouse model of liver fibrosis (Mdr2-/-). Notably, this effect does not occur in control mice without preexisting liver disease. These data suggest that EV-associated NE is a pro-fibrogenic factor for hepatic stellate cell activation via the ERK1/2 signaling pathway in pre-existing liver injuries. Inhibition of the plasma EV-associated NE in inflammatory conditions may be a therapeutic target for liver fibrosis in patients with inflammatory diseases.

Background and Aims:Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterized by the misfolding and accumulation of the mutant variant of alpha-1 antitrypsin (AAT) within hepatocytes, which limits its access to the circulation and exposes the lungs to protease-mediated tissue damage. This results in progressive liver disease secondary to AAT polymerization and accumulation, and chronic obstructive pulmonary disease (COPD) due to deficient levels of AAT within the lungs. Our goal was to characterize the unique effects of COPD secondary to AATD on liver disease and gene expression. Methods:A subcohort of AATD individuals with COPD (n = 33) and AATD individuals without COPD (n = 14) were evaluated in this study from our previously reported cross-sectional cohort. We used immunohistochemistry to assess the AATD liver phenotype, and RNA sequencing to explore liver transcriptomics. We observed a distinct transcriptomic profile in liver tissues from AATD individuals with COPD compared to those without. Results:A total of 339 genes were differentially expressed. Canonical pathways related to fibrosis, extracellular matrix remodeling, collagen deposition, hepatocellular damage, and inflammation were significantly upregulated in the livers of AATD individuals with COPD. Histopathological analysis also revealed higher levels of fibrosis and hepatocellular damage in these individuals. Conclusions:Our data supports a relationship between the development of COPD and liver disease in AATD and introduces genes and pathways that may play a role in AATD liver disease when COPD is present. We believe addressing lung impairment and airway inflammation may be an approach to managing AATD-related liver disease.

BACKGROUND:Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by misfolding and accumulation of mutant alpha-1 antitrypsin (ZAAT) in the endoplasmic reticulum of hepatocytes. Hepatic ZAAT aggregates acquire a toxic gain-of-function that impacts the endoplasmic reticulum which is theorized to cause liver disease in individuals with AATD who present asymptomatic until late-stage cirrhosis. Currently, there is no treatment for AATD-mediated liver disease except liver transplantation. In our study of mitochondrial RNA, we identified that Sirtuin3 (SIRT3) plays a role in the hepatic phenotype of AATD. METHODS:Utilizing RNA and protein analysis in an in vitro AATD model, we investigated the role of SIRT3 in the pathophysiology of AATD-mediated liver disease while also characterizing our novel, transgenic AATD mouse model. RESULTS:We show lower expression of SIRT3 in ZAAT-expressing hepatocytes. In contrast, the overexpression of SIRT3 increases hepatic ZAAT degradation. ZAAT degradation mediated by SIRT3 appeared independent of proteasomal degradation and regular autophagy pathways. We observed that ZAAT-expressing hepatocytes have aberrant accumulation of lipid droplets, with ZAAT polymers localizing on the lipid droplet surface in a direct interaction with Perilipin2, which coats intracellular lipid droplets. SIRT3 overexpression also induced the degradation of lipid droplets in ZAAT-expressing hepatocytes. We observed that SIRT3 overexpression induces lipophagy by enhancing the interaction of Perilipin2 with HSC70. ZAAT polymers then degrade as a consequence of the mobilization of lipids through this process. CONCLUSIONS:In this context, SIRT3 activation may eliminate the hepatic toxic gain-of-function associated with the polymerization of ZAAT, providing a rationale for a potential novel therapeutic approach to the treatment of AATD-mediated liver disease.

Abstract Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder associated with a 5–tenfold decrease in lung levels of alpha-1-antitrypsin (AAT) and an increased risk for obstructive lung disease. α-defensins are cationic broad-spectrum cytotoxic and pro-inflammatory peptides found in the azurophilic granules of neutrophils. The concentration of α-defensins is less than 30 nM in the bronchoalveolar lavage fluid of healthy controls but is up to 6 μM in AATD individuals with significant lung function impairment. Alveolar macrophages are generally classified into pro-inflammatory (M1) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. Therefore, monocyte-macrophage differentiation should be tightly controlled to maintain lung integrity. In this study, we determined the effect of α-defensins on monocyte-macrophage differentiation and identified the molecular mechanism of this effect. The results of this study demonstrate that 2.5 μM of α-defensins inhibit the phosphorylation of ERK1/2 and STAT3 and suppress the expression of M2 macrophage markers, CD163 and CD206. In addition, a scratch assay shows that the high concentration of α-defensins inhibits cell movement by ~ 50%, and the phagocytosis assay using flow cytometry shows that α-defensins significantly reduce the bacterial phagocytosis rate of monocyte-derived macrophages (MDMs). To examine whether exogenous AAT is able to alleviate the inhibitory effect of α-defensins on macrophage function, we incubated MDMs with AAT prior to α-defensin treatment and demonstrate that AAT improves the migratory ability and phagocytic ability of MDMs compared with MDMs incubated only with α-defensins. Taken together, this study suggests that a high concentration of α-defensins inhibits the activation of ERK/STAT3 signaling, negatively regulates the expression of M2 macrophage markers, and impairs innate immune function of macrophages.

ABSTRACTAlpha-1 antitrypsin deficiency (AATD) is a rare genetic disease characterized by reduced circulating levels of alpha-1 antitrypsin (AAT) due to the retention of misfolded AAT in the hepatocytes. The toxic AAT aggregates in hepatocytes cause liver fibrosis, cirrhosis, and hepatocellular carcinoma. Most patients remain asymptomatic until the final stage in which liver transplantation becomes the only treatment. Timely diagnosis of AATD liver disease plays a critical role in intervention and finding curative solutions. Assessing the prevalence and severity of AATD liver disease remains challenging due to limitations in current methods. Liver biopsy, the gold standard for evaluating the hepatic AAT accumulation, the initiating stage for AATD liver disease, is hindered by invasiveness and sampling errors. To address these limitations, we conducted a study using unique and precious clinical samples. We analyzed plasma extracellular vesicle (EV) derived miRNAs and liver transcriptomes from AATD individuals to develop a sensitive and noninvasive diagnostic approach for AATD liver disease. In the testing stage of our study, we enrolled 17 AATD individuals with different stages of liver disease, as determined by liver biopsy, and 20 controls. We identified differential expression of 178 miRNAs within the AATD group compared to controls by miRNA sequencing. Among those miRNAs, we selected miR-223-3p, miR-23a-3p, miR-15b-5p, let-7a-5p, let-7f-5p, and miR-374a-5p for further validation in an independent cohort of 45 AATD individuals. Using a logistic model that combined three miRNAs, we achieved an AUC of 0.737 for detecting AATD liver disease. Adding a fourth miRNA into this model increased the AUC to 0.751. The changes in EV miRNAs are correlated to dysregulated expression of liver mRNAs in AATD individuals with different stages of liver disease. We propose that plasma-circulating EV exhibit distinct miRNAs in AATD individuals and could serve as clinically significant biomarkers for the early detection of AATD liver disease.

The SERPINA1 gene encodes the serine protease inhibitor alpha -1 antitrypsin (AAT) and is located on chromosome 14q31- 32.3 in a cluster of homologous genes likely formed by exon duplication. AAT has a variety of anti-inflammatory properties. Its clinical relevance is best illustrated by the genetic disease alpha -1 antitrypsin deficiency (AATD) which is associated with an increased risk for chronic obstructive pulmonary disease (COPD) and cirrhosis. While 2 single nucleotide polymorphisms (SNPs), S and Z, are responsible for more than 95% of all individuals with AATD, there are a number of rare variants associated with deficiency and dysfunction, as well as those associated with normal levels and function. Our laboratory has identified a number of novel AAT alleles that we report in this manuscript. We screened more than 500,000 individuals for AATD alleles through our testing program over the past 20 years. The characterization of these alleles was accomplished by DNA sequencing, measurement of AAT plasma levels and isoelectric focusing at pH 4-5. We report 22 novel AAT alleles discovered through our screening programs, such as Zlittle rock and QOchillicothe, and review the current literature of known AAT genetic variants.

Background Severe acute respiratory syndrome caused by a novel coronavirus 2 (SARS-CoV-2) has infected more than 18 million people worldwide. The activation of endothelial cells is a hallmark of signs of SARS-CoV-2 infection that includes altered integrity of vessel barrier and endothelial inflammation. Objectives Pulmonary endothelial activation is suggested to be related to the profound neutrophil elastase (NE) activity, which is necessary for sterilization of phagocytosed bacterial pathogens. However, unopposed activity of NE increases alveolocapillary permeability and extracellular matrix degradation. The uncontrolled protease activity of NE during the inflammatory phase of lung diseases might be due to the resistance of exosome associated NE to inhibition by alpha-1 antitrypsin. Method 31 subjects with a diagnosis of SARS-CoV2 infection were recruited in the disease group and samples from 30 voluntaries matched for age and sex were also collected for control. Results We measured the plasma levels of exosome-associated NE in SARS-CoV-2 patients which, were positively correlated with sign of endothelial damage in those patients as determined by plasma levels of LDH. Notably, we also found strong correlation with plasma levels of alpha-1 antitrypsin and exosome-associated NE in SARS-CoV-2 patients. Using macrovascular endothelial cells, we also observed that purified NE activity is inhibited by purified alpha-1 antitrypsin while, NE associated with exosomes are resistant to inhibition and show less sensitivity to alpha-1 antitrypsin inhibitory activity, in vitro. Conclusions Our results point out the role of exosome-associated NE in exacerbation of endothelial injury in SARS-CoV-2 infection. We have demonstrated that exosome-associated NE could be served as a new potential therapeutic target of severe systemic manifestations of SARS-CoV-2 infection.

Alpha-1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full-length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R-like ER kinase and inositol-requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro-apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.