Fazirsiran for Adults With Alpha-1 Antitrypsin Deficiency Liver Disease: A Phase 2 Placebo Controlled Trial (SEQUOIA).

BACKGROUND & AIMS: Homozygous ZZ alpha-1 antitrypsin (AAT) deficiency produces mutant AAT (Z-AAT) proteins in hepatocytes, leading to progressive liver fibrosis. We evaluated the safety and efficacy of an investigational RNA interference therapeutic, fazirsiran, that degrades Z-AAT messenger RNA, reducing deleterious protein synthesis. METHODS: This ongoing, phase 2 study randomized 40 patients to subcutaneous placebo or fazirsiran 25, 100, or 200 mg. The primary endpoint was percent change in serum Z-AAT concentration from baseline to week 16. Patients with fibrosis on baseline liver biopsy received treatment on day 1, at week 4, and then every 12 weeks and had a second liver biopsy at or after weeks 48, 72, or 96. Patients without fibrosis received 2 doses on day 1 and at week 4. RESULTS: At week 16, least-squares mean percent declines in serum Z-AAT concentration were -61%, -83%, and -94% with fazirsiran 25, 100, and 200 mg, respectively, vs placebo (all P < .0001). Efficacy was sustained through week 52. At postdose liver biopsy, fazirsiran reduced median liver Z-AAT concentration by 93% compared with an increase of 26% with placebo. All fazirsiran-treated patients had histologic reduction from baseline in hepatic globule burden. Portal inflammation improved in 5 of 12 and 0 of 8 patients with a baseline score of >0 in the fazirsiran and placebo groups, respectively. Histologic meta-analysis of histologic data in viral hepatitis score improved by >1 point in 7 of 14 and 3 of 8 patients with fibrosis of >F0 at baseline in the fazirsiran and placebo groups, respectively. No adverse events led to discontinuation, and pulmonary function tests remained stable. CONCLUSIONS: Fazirsiran reduced serum and liver concentrations of Z-AAT in a dose-dependent manner and reduced hepatic globule burden. (ClinicalTrials.gov, Number NCT03945292).

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.

Lung Inflammation in alpha-1-antitrypsin deficient individuals with normal lung function.

BACKGROUND: Alpha-1-antitrypsin deficient (AATD) individuals are prone to develop early age of onset chronic obstructive pulmonary disease (COPD) more severe than non-genetic COPD. Here, we investigated the characteristics of lower respiratory tract of AATD individuals prior to the onset of clinically significant COPD. METHODS: Bronchoalveolar lavage was performed on 22 AATD with normal lung function and 14 healthy individuals. Cell counts and concentrations of proteases, alpha-1-antitrypsin and proinflammatory mediators were determined in the bronchoalveolar lavage fluid from study subjects. In order to determine the airway inflammation, we also analyzed immune cell components of the large airways from bronchial biopsies using immunohistochemistry in both study subjects. Finally, we made comparisons between airway inflammation and lung function rate of decline using four repeated lung function tests over one year in AATD individuals. RESULTS: AATD individuals with normal lung function had 3 folds higher neutrophil counts, 2 folds increase in the proteases levels, and 2-4 folds higher levels of IL-8, IL-6, IL-1ß, and leukotriene B4 in their epithelial lining fluid compared to controls. Neutrophil elastase levels showed a positive correlation with the levels of IL-8 and neutrophils in AATD epithelial lining fluid. AATD individuals also showed a negative correlation of baseline FEV1 with neutrophil count, neutrophil elastase, and cytokine levels in epithelial lining fluid (p < 0.05). In addition, we observed twofold increase in the number of lymphocytes, macrophages, neutrophils, and mast cells of AATD epithelial lining fluid as compared to controls. CONCLUSION: Mild inflammation is present in the lower respiratory tract and airways of AATD individuals despite having normal lung function. A declining trend was also noticed in the lung function of AATD individuals which was correlated with pro-inflammatory phenotype of their lower respiratory tract. This results suggest the presence of proinflammatory phenotype in AATD lungs. Therefore, early anti-inflammatory therapies may be a potential strategy to prevent progression of lung disease in AATD individuals.

Alpha-defensins inhibit ERK/STAT3 signaling during monocyte-macrophage differentiation and impede macrophage function.

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.

Characterization of hepatic inflammatory changes in a C57BL/6J mouse model of alpha1-antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by a hepatic accumulation of mutant alpha-1 antitrypsin (ZAAT). Individuals with AATD are prone to develop a chronic liver disease that remains undiagnosed until late stage of the disease. Here, we sought to characterize the liver pathophysiology of a human transgenic mouse model for AATD with a manifestation of liver disease compared with normal transgenic mice model. Male and female transgenic mice for normal (Pi*M) and mutant variant (Pi*Z) human alpha-1 antitrypsin at 3 and 6 mo of age were subjected to this study. The progression of hepatic ZAAT accumulation, hepatocyte injury, steatosis, liver inflammation, and fibrotic features were monitored by performing an in vivo study. We have also performed a Next-Gene transcriptomic analysis of the transgenic mice liver tissue 16 h after lipopolysaccharide (LPS) administration to delineate liver inflammatory response in Pi*Z mice as compared with Pi*M. Our results show hepatic ZAAT accumulation, followed by hepatocyte ballooning and liver steatosis developed at 3 mo in Pi*Z mice compared with the mice carrying normal variant of human alpha-1 antitrypsin. We observed higher levels of hepatic immune cell infiltrations in both 3- and 6-mo-old Pi*Z mice compared with Pi*M as an indication of liver inflammation. Liver fibrosis was observed as accumulation of collagen in 6-mo-old Pi*Z liver tissues compared with Pi*M control mice. Furthermore, the transcriptomic analysis revealed a dysregulated liver immune response to LPS in Pi*Z mice compared with Pi*M. Of particular interest for translational work, this study aims to establish a mouse model of AATD with a strong manifestation of liver disease that will be a valuable in vivo tool to study the pathophysiology of AATD-mediated liver disease. Our data suggest that the human transgenic mouse model of AATD could provide a suitable model for the evaluation of therapeutic approaches and preventive reagents against AATD-mediated liver disease.NEW & NOTEWORTHY We have characterized a mouse model of human alpha-1 antitrypsin deficiency with a strong manifestation of liver disease that can be used as an in vivo tool to test preventive and therapeutic reagents. Our data explores the altered immunophenotype of alpha-1 antitrypsin-deficient liver macrophages and suggests a relationship between acute inflammation, immune response, and fibrosis.

Cigarette smoke exposed airway epithelial cell-derived EVs promote pro-inflammatory macrophage activation in alpha-1 antitrypsin deficiency.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder most commonly secondary to a single mutation in the SERPINA1 gene (PI*Z) that causes misfolding and accumulation of alpha-1 antitrypsin (AAT) in hepatocytes and mononuclear phagocytes which reduces plasma AAT and creates a toxic gain of function. This toxic gain of function promotes a pro-inflammatory phenotype in macrophages that contributes to lung inflammation and early-onset COPD, especially in individuals who smoke cigarettes. The aim of this study is to determine the role of cigarette exposed AATD macrophages and bronchial epithelial cells in AATD-mediated lung inflammation. METHODS: Peripheral blood mononuclear cells from AATD and healthy individuals were differentiated into alveolar-like macrophages and exposed to air or cigarette smoke while in culture. Macrophage endoplasmic reticulum stress was quantified and secreted cytokines were measured using qPCR and cytokine ELISAs. To determine whether there is "cross talk" between epithelial cells and macrophages, macrophages were exposed to extracellular vesicles released by airway epithelial cells exposed to cigarette smoke and their inflammatory response was determined. RESULTS: AATD macrophages spontaneously produce several-fold more pro-inflammatory cytokines as compared to normal macrophages. AATD macrophages have an enhanced inflammatory response when exposed to cigarette smoke-induced extracellular vesicles (EVs) released from airway epithelial cells. Cigarette smoke-induced EVs induce expression of GM-CSF and IL-8 in AATD macrophages but have no effect on normal macrophages. Release of AAT polymers, potent neutrophil chemo attractants, were also increased from AATD macrophages after exposure to cigarette smoke-induced EVs. CONCLUSIONS: The expression of mutated AAT confers an inflammatory phenotype in AATD macrophages which disposes them to an exaggerated inflammatory response to cigarette smoke-induced EVs, and thus could contribute to progressive lung inflammation and damage in AATD individuals.

Relationship between alpha-1 antitrypsin deficiency and obstructive sleep apnea.

PURPOSE: This study aimed to identify if individuals with mild to severe alpha-1 antitrypsin deficiency (AATD) are at higher risk for developing obstructive sleep apnea (OSA) than the general population. METHODS: A seven-question sleep apnea risk assessment questionnaire, STOP-BAG, was applied to 2338 participant responses from the Alpha-1 Coded Testing Study (ACT) and 4638 participant responses from the Kentucky Behavioral Risk Factor Survey (KyBRFS). Propensity scores were generated from a logistic regression model using continuous variables of age and body mass index (BMI). STOP-BAG scores were analyzed using chi-square analysis on this matched cohort to assess OSA risk in AATD. RESULTS: Self-reported OSA was higher in the KyBRFS cohort (14.5%) than in individuals with mild or severe AATD (11.2%) (p = 0.012). However, a higher percentage of the AATD cohort met clinically meaningful thresholds for STOP-BAG scores ≥ 5 (22.7%) than the KyBRFS cohort (13.0%) (p = 0.001). These differences persisted despite 1:1 propensity score matching on age and BMI to account for differences in baseline characteristics. No statistically significant difference in OSA risk between AATD genotypes was found. CONCLUSION: AATD appears to have higher risk for OSA than the general population. The 11.2% prevalence of diagnosed OSA in the AATD population is much lower than symptom scores would predict. Further studies are needed to validate the possibility that elastin loss is involved in OSA pathogenesis.

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease.

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.

Modulation of calreticulin expression reveals a novel exosome-mediated mechanism of Z variant α1-antitrypsin disposal.

α1-Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT-ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT-ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.

Alpha-1 Antitrypsin Deficiency Liver Disease, Mutational Homogeneity Modulated by Epigenetic Heterogeneity With Links to Obesity.

Alpha-1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5-methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early-stage AATD and the ZZ genotype, genome-wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver-impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome-wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well-known sex-specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk.

Alpha-1 antitrypsin deficient individuals have circulating extracellular vesicles with profibrogenic cargo.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD)-mediated liver disease is a toxic "gain-of-function" inflammation in the liver associated with intracellular retention of mutant alpha-1 antitrypsin. The clinical presentation of the disease includes fibrosis, cirrhosis and liver failure. However, the pathogenic mechanism of AATD-mediated liver disease is not well understood. Here, we investigated the role of plasma extracellular vesicles (EVs) in progression of AATD-mediated liver disease. METHODS: EVs were isolated from plasma of AATD individuals with liver disease and healthy controls. Their cytokines and miRNA content were examined by multiplex assay and small RNA sequencing. The bioactivity of EVs was assessed by qPCR, western blot analysis and immunofluorescent experiments using human hepatic stellate cells (HSCs) treated with EVs isolated from control or AATD plasma samples. RESULTS: We have found that AATD individuals have a distinct population of EVs with pathological cytokine and miRNA contents. When HSCs were cultured with AATD plasma derived-EVs, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with healthy control plasma EVs. CONCLUSION: AATD individuals have a distinct population of EVs with abnormal cytokine and miRNA contents and the capacity to activate HSCs and mediate fibrosis. Better understanding of the components which cause liver inflammation and fibrogenesis, leading to further liver injury, has the potential to lead to the development of new treatments or preventive strategies to prevent AATD-mediated liver disease. Video abstract.

Chemokine signaling axis between endothelial and myeloid cells regulates development of pulmonary hypertension associated with pulmonary fibrosis and hypoxia.

Pulmonary hypertension complicates the care of many patients with chronic lung diseases (defined as Group 3 pulmonary hypertension), yet the mechanisms that mediate the development of pulmonary vascular disease are not clearly defined. Despite being the most prevalent form of pulmonary hypertension, to date there is no approved treatment for patients with disease. Myeloid-derived suppressor cells (MDSCs) and endothelial cells in the lung express the chemokine receptor CXCR2, implicated in the evolution of both neoplastic and pulmonary vascular remodeling. However, precise cellular contribution to lung disease is unknown. Therefore, we used mice with tissue-specific deletion of CXCR2 to investigate the role of this receptor in Group 3 pulmonary hypertension. Deletion of CXCR2 in myeloid cells attenuated the recruitment of polymorphonuclear MDSCs to the lungs, inhibited vascular remodeling, and protected against pulmonary hypertension. Conversely, loss of CXCR2 in endothelial cells resulted in worsened vascular remodeling, associated with increased MDSC migratory capacity attributable to increased ligand availability, consistent with analyzed patient sample data. Taken together, these data suggest that CXCR2 regulates MDSC activation, informing potential therapeutic application of MDSC-targeted treatments.

Analysis of the MILES cohort reveals determinants of disease progression and treatment response in lymphangioleiomyomatosis.

INTRODUCTION: The Multicenter International Lymphangioleiomyomatosis (LAM) Efficacy of Sirolimus (MILES) trial revealed that sirolimus stabilised lung function in patients with moderately severe LAM. The purpose of this study was to further examine the MILES cohort for the effects of racial, demographic, clinical and physiological patient characteristics on disease progression and treatment response in LAM. METHODS: MILES subjects were stratified on the basis of menopausal status (pre-menopausal/post-menopausal), race (Asian/Caucasian), bronchodilator responsiveness (present/absent), initial forced expiratory volume in 1 s (FEV1; 51-70% versus ≤50% predicted) and tuberous sclerosis complex (TSC) association (yes/no). A linear mixed effects model was used to compare slope differences, and nonparametric tests were used to compare medians and proportions between treatment groups in each stratum. RESULTS: In the MILES placebo group, pre-menopausal patients declined 5-fold faster than post-menopausal patients (mean±se FEV1 slope -17±3 versus -3±3 mL·month-1; p=0.003). Upon treatment with sirolimus, both the pre-menopausal (-17±3 versus -1±2 mL·month-1; p<0.0001) and post-menopausal patients (-3±3 versus 6±3 mL·month-1; p=0.04) exhibited a beneficial response in mean±se FEV1 slope compared with the placebo group. Race, LAM subtype, bronchodilator responsiveness or baseline FEV1 did not impact the rate of disease progression in the placebo group or treatment response in the sirolimus group. Menopausal status and race had differential effects on the adverse event profile of sirolimus. Baseline serum vascular endothelial growth factor (VEGF)-D >600 pg·mL-1 identified subgroups of patients who were more likely to decline on placebo and respond to treatment with sirolimus. CONCLUSIONS: In LAM patients, treatment with sirolimus is beneficial regardless of menopausal status, race, bronchodilator responsiveness, baseline FEV1 or TSC association. Serum VEGF-D and menopausal status can help inform therapeutic decisions.

Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival.

Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

Myeloid-derived Suppressor Cells Are Necessary for Development of Pulmonary Hypertension.

Pulmonary hypertension (PH) complicates the care of patients with chronic lung disease, such as idiopathic pulmonary fibrosis (IPF), resulting in a significant increase in morbidity and mortality. Disease pathogenesis is orchestrated by unidentified myeloid-derived cells. We used murine models of PH and pulmonary fibrosis to study the role of circulating myeloid cells in disease pathogenesis and prevention. We administered clodronate liposomes to bleomycin-treated wild-type mice to induce pulmonary fibrosis and PH with a resulting increase in circulating bone marrow-derived cells. We discovered that a population of C-X-C motif chemokine receptor (CXCR) 2+ myeloid-derived suppressor cells (MDSCs), granulocytic subset (G-MDSC), is associated with severe PH in mice. Pulmonary pressures worsened despite improvement in bleomycin-induced pulmonary fibrosis. PH was attenuated by CXCR2 inhibition, with antagonist SB 225002, through decreasing G-MDSC recruitment to the lung. Molecular and cellular analysis of clinical patient samples confirmed a role for elevated MDSCs in IPF and IPF with PH. These data show that MDSCs play a key role in PH pathogenesis and that G-MDSC trafficking to the lung, through chemokine receptor CXCR2, increases development of PH in multiple murine models. Furthermore, we demonstrate pathology similar to the preclinical models in IPF with lung and blood samples from patients with PH, suggesting a potential role for CXCR2 inhibitor use in this patient population. These findings are significant, as there are currently no approved disease-specific therapies for patients with PH complicating IPF.

Whole-Genome Sequencing in Severe Chronic Obstructive Pulmonary Disease.

Genome-wide association studies have identified common variants associated with chronic obstructive pulmonary disease (COPD). Whole-genome sequencing (WGS) offers comprehensive coverage of the entire genome, as compared with genotyping arrays or exome sequencing. We hypothesized that WGS in subjects with severe COPD and smoking control subjects with normal pulmonary function would allow us to identify novel genetic determinants of COPD. We sequenced 821 patients with severe COPD and 973 control subjects from the COPDGene and Boston Early-Onset COPD studies, including both non-Hispanic white and African American individuals. We performed single-variant and grouped-variant analyses, and in addition, we assessed the overlap of variants between sequencing- and array-based imputation. Our most significantly associated variant was in a known region near HHIP (combined P = 1.6 × 10-9); additional variants approaching genome-wide significance included previously described regions in CHRNA5, TNS1, and SERPINA6/SERPINA1 (the latter in African American individuals). None of our associations were clearly driven by rare variants, and we found minimal evidence of replication of genes identified by previously reported smaller sequencing studies. With WGS, we identified more than 20 million new variants, not seen with imputation, including more than 10,000 of potential importance in previously identified COPD genome-wide association study regions. WGS in severe COPD identifies a large number of potentially important functional variants, with the strongest associations being in known COPD risk loci, including HHIP and SERPINA1. Larger sample sizes will be needed to identify associated variants in novel regions of the genome.

Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.

BACKGROUND & AIMS: Alpha-1 antitrypsin deficiency (AATD) is an uncommonly recognized cause of liver disease in adults, with descriptions of its natural history limited to case series and patient-reported data from disease registries. Liver pathology is limited to selected patients or unavailable. Therefore, we aimed to determine the prevalence and severity of liver fibrosis in an adult AATD population who were not known to have cirrhosis, while defining risk factors for fibrosis and testing non-invasive markers of disease. METHODS: A total of 94 adults with classic genotype 'PI*ZZ' AATD were recruited from North America and prospectively enrolled in the study. Liver aminotransferases and markers of synthetic function, transient elastography, and liver biopsy were performed. RESULTS: The prevalence of clinically significant liver fibrosis (F ≥ 2) was 35.1%. Alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase values were higher in the F ≥ 2 group. Metabolic syndrome was associated with the presence of clinically significant fibrosis (OR 14.2; 95% CI 3.7-55; p <0.001). Additionally, the presence of accumulated abnormal AAT in hepatocytes, portal inflammation, and hepatocellular degeneration were associated with clinically significant fibrosis. The accuracy of transient elastography to detect F ≥ 2 fibrosis was fair, with an AUC of 0.70 (95% CI 0.58-0.82). CONCLUSIONS: Over one-third of asymptomatic and lung affected adults with 'PI*ZZ' AATD have significant underlying liver fibrosis. Liver biopsies demonstrated variable amounts of accumulated Z AAT. The risk of liver fibrosis increases in the presence of metabolic syndrome, accumulation of AAT in hepatocytes, and portal inflammation on baseline biopsy. The results support the hypothesis that liver disease in this genetic condition may be related to a "toxic gain of function" from accumulation of AAT in hepatocytes. LAY SUMMARY: Individuals diagnosed with classic alpha-1 antitrypsin deficiency (ZZ) are at risk of liver injury and scarring, because of the accumulation of abnormal alpha-1 antitrypsin in the liver. A liver biopsy in ZZ individuals can demonstrate the accumulation of alpha-1 antitrypsin within the liver and identify if any associated liver scarring is present. Indviduals with large amounts of alpha-1 antitrypsin on biopsy may be at risk of liver injury and fibrosis. Additional common medical conditions of diabetes, obesity, high cholesterol, and hypertension (known as metabolic syndrome) are associated with a greater degree of liver injury. CLINICAL TRIAL NUMBER: clinicaltrials.gov NCT01810458.