Pulmonology
Abstract
Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell–mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease–inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.
Authors
Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal
Abstract
Background: The complement system plays a key role in host defense but is activated by ischemia-reperfusion injury (IRI). Primary graft dysfunction (PGD) is a form of acute lung injury occurring predominantly due to IRI, which worsens survival after lung transplantation (LTx). Local complement activation is associated with acute lung injury, but whether it is more reflective of allograft injury compared to systemic activation remains unclear. We proposed that local complement activation would help identify those who develop PGD post-LTx. We also aimed to identify which complement activation pathways are associated with PGD. Methods: We performed a multicenter cohort study at the University of Pennsylvania and Washington University. Bronchoalveolar lavage (BAL) and plasma specimens were obtained from recipients within 24 h post-LTx. PGD was scored based on the consensus definition. Complement activation products and components of each arm of the complement cascade were measured using ELISA. Results: In both cohorts, sC4d and sC5b-9 levels were increased in BAL of subjects with PGD compared to those without PGD. Subjects with PGD also had higher C1q, C2, C4, and C4b, compared to subjects without PGD, suggesting classical and lectin pathway involvement. Ba levels were higher in subjects with PGD, suggesting alternative pathway activation. Among lectin pathway-specific components, MBL and FCN-3 had a moderate-to-strong correlation with the terminal complement complex in the BAL but not in the plasma. Conclusion: Complement activation fragments are detected in the BAL within 24 h post-LTx. Components of all three pathways are locally increased in subjects with PGD. Our findings create a precedent for investigating complement-targeted therapeutics to mitigate PGD. Funding: This research was supported by the National Institutes of Health (NIH), American Lung Association, Children’s Discovery Institute, the Robert Wood Johnson Foundation, the Cystic Fibrosis Foundation, the Barnes-Jewish Hospital Foundation, The Danish Hearth Foundation], The Danish Research Foundation of Independent Research, The Svend Andersen Research Foundation and the Novo Nordisk Research Foundation.
Authors
Hrishikesh S. Kulkarni, Kristy Ramphal, Lina Ma, Melanie Brown, Michelle L. Oyster, Kaitlyn Speckhart, Tsuyoshi Takahashi, Derek E. Byers, Mary K. Porteous, Laurel Kalman, Ramsey R. Hachem, Melanie Rushefski, Ja'Nia McPhatter, Marlene Cano, Daniel Kreisel, Masina Scavuzzo, Brigitte Mittler, Edward Cantu, Katrine Pilely, Peter Garred, Jason D. Christie, John Atkinson, Andrew E. Gelman, Joshua M. Diamond
Abstract
Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient’s risk of severe COPD. We determined 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in IGSF3 (immunoglobulin superfamily, member 3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs of which, rs1414272 and rs12066192 were directly- and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide- and sphingosine-1 phosphate-, but low glycosphingolipids- and gangliosides levels; were less apoptotic and more adherent; with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and a weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.
Authors
Kelly S. Schweitzer, Natini Jinawath, Raluca Yonescu, Kevin Ni, Natalia Rush, Varodom Charoensawan, Irina Bronova, Evgeny Berdyshev, Sonia M. Leach, Lucas A. Gillenwater, Russell P. Bowler, David B. Pearse, Constance A. Griffin, Irina Petrache
Abstract
The acute respiratory distress syndrome (ARDS) results from overwhelming pulmonary inflammation. Prior bulk RNA sequencing provided limited insights into ARDS pathogenesis. We used single cell RNA sequencing to probe ARDS at a higher resolution. Peripheral blood mononuclear cells of patients with pneumonia and sepsis with early ARDS were compared to that of sepsis patients who did not develop ARDS. Monocyte clusters from ARDS patients revealed multiple distinguishing characteristics in comparison to monocytes from patients without ARDS including down-regulation of SOCS3 expression accompanied by a pro-inflammatory signature with up-regulation of multiple type I IFN-induced genes, especially in CD16+ cells. To generate an ARDS risk score, we identified up-regulation of 29 genes in the monocytes of these patients, and 17 showed a similar profile in cells of patients in independent cohorts. Monocytes had increased expression of RAB11A, known to inhibit neutrophil efferocytosis, ATP2B1, a calcium pump that exports Ca2+ implicated in endothelial barrier disruption, and SPARC, associated with processing of pro-collagen to collagen. These data show that monocytes of ARDS patients up-regulate expression of genes not just restricted to those associated with inflammation. Together, our findings identify molecules that are likely involved in ARDS pathogenesis that may inform biomarker and therapeutic development.
Authors
Yale Jiang, Brian R. Rosborough, Jie Chen, Sudipta Das, Georgios D. Kitsios, Bryan J. McVerry, Rama K. Mallampalli, Janet S. Lee, Anuradha Ray, Wei Chen, Prabir Ray
Abstract
Background Currently recommended traditional spirometry outputs do not reflect their relative contributions to airflow, and we hypothesized that machine learning algorithms can be trained on spirometry data to identify these structural phenotypes. Methods Participants enrolled in a large multicenter study (COPDGene) were included. The data points from expiratory flow-volume curves were trained using a deep learning model to predict structural phenotypes of COPD on computed tomography (CT), and results were compared with traditional spirometry metrics and an optimized random forest classifier. Area under the receiver operating characteristic curve (AUC) and weighted F-score were used to measure the discriminative accuracy of a fully convolutional neural network, Random Forest, and traditional spirometry metrics to phenotype CT as normal, emphysema-predominant (>5% emphysema), airway-predominant (Pi10>median), and mixed phenotypes. Similar comparisons were made for the detection of functional small airway disease phenotype (fSAD>20% on parametric response mapping). Results Among 8,980 individuals, neural network was more accurate in discriminating predominant emphysema/airway phenotypes (AUC 0.80, 95%CI 0.79-0.81) than traditional measures of spirometry, FEV1/FVC (AUC 0.71, 95%CI 0.69-0.71) and FEV1 %predicted (AUC 0.70, 95%CI 0.68-0.71) ), and random forest classifier (AUC 0.78, 95%CI 0.77-0.79). The neural network was also more accurate in discriminating predominant emphysema/small airway phenotypes (AUC 0.91, 95%CI 0.90-0.92) than FEV1/FVC (AUC 0.80, 95%CI 0.78-0.82), FEV1 %predicted (AUC 0.83, 95%CI 0.80-0.84), and with comparable accuracy with random forest classifier (AUC 0.90, 95%CI 0.88-0.91). Conclusions Structural phenotypes of COPD can be identified from spirometry using deep learning and machine learning approaches, demonstrating their potential to identify individuals for targeted therapies.
Authors
Sandeep Bodduluri, Arie Nakhmani, Joseph M. Reinhardt, Carla G. Wilson, Merry-Lynn N. McDonald, Ramaraju Rudraraju, Byron C Jaeger, Nirav R. Bhakta, Peter J. Castaldi, Frank C. Sciurba, Chengcui Zhang, Purushotham V. Bangalore, Surya P. Bhatt
Abstract
BACKGROUND. The numbers of fatal cases of Coronavirus Disease 2019 (COVID-19) continue to increase rapidly around the world. We aim to retrospectively investigate potential roles of factors, mainly immunologic parameters, in early predicting outcomes of patients with COVID-19. METHODS. A total of 1,018 patients confirmed COVID-19 were enrolled in our retrospective study from two centers. The data of clinical features, laboratory tests, immunological tests, radiological findings, and outcomes were collected. Univariate and multivariable logistic regression analysis were performed to evaluate factors associated with in-hospital mortality. Receiver operator characteristic (ROC) curves and survival curves were plotted to evaluate the clinical usefulness. RESULTS. Compared to the survival patients, the counts of all T lymphocytes subsets were markedly lower in non-survivors(P < 0.001), especially in CD8+ T cells (96.89 vs 203.98 cells/μl, P < 0.001) . Among all tested cytokines, IL-6 elevated most significantly with an upward trend of more than ten times (56.16 vs 5.36 pg/mL, P < 0.001). By a multivariable logistic regression analysis, two immunological indicators were found to be associated with in-hospital mortality, including IL-6 > 20 pg/mL (OR = 9.781; 95%CI, 6.304–15.174; P < 0.001) and CD8+ T cell count < 165 cells/μl (OR = 5.930; 95%CI, 3.677–9.562; P < 0.001), after adjusting confounding factors (age, gender, and underlying diseases). All the patients were divided into four groups according to levels of IL-6 and CD8+ T cells. The group with IL-6 > 20 pg/mL and CD8+ T cell count < 165 cells/μl had more old and male patients, as well as more proportion of patients with comorbidities, ventilation, ICU admission, shock, and death than those of any other group (P < 0.001). Furthermore, the ROC curve of the model combining IL-6 (>20 pg/mL) and CD8+ T cell count(<165 cells/μl) displayed more favorable discrimination than that of CURB-65 score (area under curve (AUC) = 0.907 vs 0.843, P < 0.001). Hosmer-Lemeshow test showed a good fitting of the model with no statistical significance (P = 0.581). CONCLUSIONS. We firstly identify two reliable prognostic indicators, IL-6 (>20 pg/mL) and CD8+ T cell count (<165 cells/μl), which can accurately stratify patients into risk categories and predict mortality of patients with COVID-19. Those two indicators combined may guide clinicians to evaluate patient prognosis and make appropriate decisions.
Authors
Miao Luo, Jing Liu, Weiling Jiang, Shuang Yue, Huiguo Liu, Shuang Wei
Abstract
Patients with hereditary or acquired hemolytic anemias have a high risk of developing in-situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice for the first time to assess the pathogenesis of pulmonary thrombosis following deionized-water induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravenous ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet-P2Y12 purinergic-receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation leading to thrombosis in the pre-capillary pulmonary arterioles and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.
Authors
Tomasz Brzoska, Ravi Vats, Margaret F. Bennewitz, Egemen Tutuncuoglu, Simon C. Watkins, Margaret V. Ragni, Matthew D. Neal, Mark T. Gladwin, Prithu Sundd
Abstract
BACKGROUND. Dysregulation of L-arginine metabolism has been proposed to occur in severe asthma patients. The effects of L-arginine supplementation on L-arginine metabolite profiles in these patients is unknown. We hypothesized that severe asthmatics with low fractional exhaled nitric oxide (FeNO) would have fewer asthma exacerbations with the addition of L-arginine to their standard asthma medications compared to placebo and would demonstrate the greatest changes in metabolite profiles. METHODS. Participants were enrolled in a single-center, cross-over, double-blinded, L-arginine intervention trial at the University of California-Davis (NCT01841281). Subjects received placebo or L-arginine, dosed orally at 0.05mg/kg (ideal body weight) twice daily. The primary endpoint was moderate asthma exacerbations. Longitudinal plasma metabolite levels were measured using mass spectrometry. A linear mixed-effect model with subject-specific intercepts was used for testing treatment effects. RESULTS. A cohort of 50 subjects was included in the final analysis. L-arginine did not significantly decrease asthma exacerbations in the overall cohort. Higher citrulline levels and a lower arginine availability index (AAI) were associated with higher FeNO (P-value = 0.005 and 2.51 x 10–9 respectively). Higher AAI was associated with lower exacerbation events. The eicosanoid prostaglandin H2 (PGH2) and Nα-Acetyl-L-arginine were found to be good predictors for differentiating clinical responders and non-responders. CONCLUSIONS. There was no statistically significant decrease in asthma exacerbations in the overall cohort with L-arginine intervention. PGH2, Nα-Acetyl-L-arginine and the AAI could serve as predictive biomarkers in future clinical trials that intervene in the arginine metabolome.
Authors
Shu-Yi Liao, Megan R. Showalter, Angela L. Linderholm, Lisa M. Franzi, Celeste Kivler, Yao Li, Michael R. Sa, Zachary A. Kons, Oliver Fiehn, Lihong Qi, Amir A. Zeki, Nicholas J. Kenyon
Abstract
BACKGROUND. Severe acute respiratory coronavirus 2 (SARS-CoV-2) caused coronavirus disease 2019 (COVID-19) has become a pandemic. This study addressed the clinical and immunopathological characteristics of severe COVID-19. METHODS. Sixty-nine COVID-19 patients were classified into as severe and non-severe groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from two deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations. RESULTS. Circulating cytokines, including IL8, IL6, TNFα, IP10, MCP1, and RANTES, were significantly elevated in severe COVID-19 patients. Dynamic IL6 and IL8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II, type I pneumocytes and endothelial cells, leading to severe lung damage through cell pyroptosis and apoptosis. In severe cases, lymphopenia, neutrophilia, depletion of CD4+ and CD8+ T lymphocytes, and massive macrophage and neutrophil infiltrates were observed in both blood and lung tissues. CONCLUSIONS. A panel of circulating cytokines could be used to predict disease deterioration and inform clinical interventions. Severe pulmonary damage was predominantly attributed to both SARS-CoV-2 caused cytopathy and immunopathologic damage. Strategies that encourage pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of severe COVID-19 patients.
Authors
Shaohua Li, Lina Jiang, Xi Li, Fang Lin, Yijin Wang, Boan Li, Tianjun Jiang, Weimin An, Shuhong Liu, Hongyang Liu, Pengfei Xu, Lihua Zhao, Lixin Zhang, Jinsong Mu, Hongwei Wang, Jiarui Kang, Yan Li, Lei Huang, Caizhong Zhu, Shousong Zhao, Jiangyang Lu, Junsheng Ji, Jingmin Zhao
Abstract
Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol-25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds that of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the anti-inflammatory nuclear receptor, Liver X Receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural (oxy)sterols, 25HC is uniquely induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGFb. CH25H/25HC/LXR is thus an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.
Authors
Jennifer H. Madenspacher, Eric D. Morrell, Kymberly M. Gowdy, Jeffrey G. McDonald, Bonne M. Thompson, Ginger W. Muse, Jennifer Martinez, Seddon Y. Thomas, Carmen Mikacenic, Jerry A. Nick, Edward Abraham, Stavros Garantziotis, Renee D. Stapleton, Julie M. Meacham, Mary Jane Thomassen, William J. Janssen, Donald N. Cook, Mark M. Wurfel, Michael B. Fessler
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