BACKGROUND Persistent cough and dyspnea are prominent features of post-acute sequelae of SARS-CoV-2 (also termed ’Long COVID’); however, physiologic measures and clinical features associated with these pulmonary symptoms remain poorly defined. Using longitudinal pulmonary function testing (PFTs) and CT imaging, this study aimed to identify the characteristics and determinants of pulmonary Long COVID. METHODS This single-center retrospective study included 1,097 patients with clinically defined Long COVID characterized by persistent pulmonary symptoms (dyspnea, cough, and chest discomfort) lasting for ≥1 month after resolution of primary COVID infection. RESULTS After exclusion, a total of 929 patients with post-COVID pulmonary symptoms and PFTs were stratified diffusion impairment and restriction as measured by percent predicted diffusion capacity for carbon monoxide (DLCO) and total lung capacity (TLC). Dyspnea was the predominant symptom in the cohort (78%) and had similar prevalence regardless of degree of diffusion impairment or restriction. Longitudinal evaluation revealed diffusion impairment (DLCO ≤80%) and pulmonary restriction (TLC ≤80%) in 51% of the cohort overall (n=479). In multivariable logistic regression analysis (adjusted odds ratio; aOR, 95% confidence interval [CI]), invasive mechanical ventilation during primary infection conferred the greatest increased odds of developing pulmonary Long COVID with diffusion impairment and restriction (aOR=10.9 [4.09-28.6]). Finally, a sub-analysis of CT imaging identified radiographic evidence of fibrosis in this patient population. CONCLUSIONS Longitudinal PFT measurements in patients with prolonged pulmonary symptoms after SARS-CoV-2 infection revealed persistent diffusion impaired restriction as a key feature of pulmonary Long COVID. These results emphasize the importance of incorporating PFTs into routine clinical practice for evaluation of patients with prolonged pulmonary symptoms after resolution of SARS-CoV-2. Subsequent clinical trials should leverage combined symptomatic and quantitative PFT measurements for more targeted enrollment of pulmonary Long COVID patients. FUNDING This work was supported by the National Institute of Allergy and Infectious Diseases (AI156898, K08AI129705), the National Heart, Lung, and Blood Institute (HL153113, OTA21-015E, HL149944), and the COVID-19 Urgent Research Response Fund established by the Hugh Kaul Precision Medicine Network at the University of Alabama at Birmingham.
Michael John Patton, Donald Benson, Sarah W. Robison, Dhaval Raval, Morgan L. Locy, Kinner Patel, Scott Grumley, Emily B. Levitan, Peter Morris, Matthew Might, Amit Gaggar, Nathaniel Erdmann
Mechanisms underlying maintenance of pathological vascular hypermuscularization are poorly delineated. Herein, we investigated retention of smooth muscle cells (SMCs) coating normally unmuscularized distal pulmonary arterioles in pulmonary hypertension (PH) mediated by chronic hypoxia ± Sugen 5416, and reversal of this pathology. With hypoxia in mice or culture, lung endothelial cells (ECs) upregulated hypoxia-inducible factor (Hif)-1a and 2a which induce platelet-derived growth factor-B (PDGF-B), and these factors reduced to normoxic levels with re-normoxia. Re-normoxia reversed hypoxia-induced pulmonary vascular remodeling, but with EC HIFα over-expression during re-normoxia, pathological changes persisted. Conversely, after establishment of distal muscularization and PH, EC-specific deletion of Hif1a, Hif2a, or Pdgfb induced reversal. In human idiopathic pulmonary artery hypertension, HIF1A, HIF2A, PDGFB and autophagy-mediating gene products, including Beclin1, were upregulated in pulmonary artery SMCs and/or lung lysates. Furthermore, in mice, hypoxia-induced EC-derived PDGF-B upregulated Beclin1 in distal arteriole SMCs, and after distal muscularization was established, re-normoxia, EC Pdgfb deletion or treatment with STI571 (which inhibits PDGF receptors) downregulated SMC Beclin1 and other autophagy products. Finally, SMC-specific Becn1 deletion induced apoptosis, reversing distal muscularization and PH mediated by hypoxia ± Sugen 5416. Thus, chronic hypoxia induction of HIFα-to-PDGF-B axis in ECs is required for non-cell autonomous Beclin1-mediated survival of pathological distal arteriole SMCs.
Fatima Z. Saddouk, Andrew P. Kuzemczak, Junichi Saito, Daniel M. Greif
NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodelling in vivo. NKX2-5 is up-regulated in scleroderma (SSc) patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells, halted vascular smooth muscle proliferation and migration, enhanced contractility and blocked the expression of the extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodelling and halted the progression to hypertension in the mouse chronic hypoxia mouse model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGFβ and further enhanced by hypoxia. The effect of TGFβ was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodelling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.
Ioannis Papaioannou, Athina Dritsoula, Ping Kang, Reshma S. Baliga, Sarah L. Trinder, Emma Cook, Shiwen Xu, Adrian Hobbs, Christopher P. Denton, David J. Abraham, Markella Ponticos
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.
Idil Apak Evans, Xingshen Sun, Bo Liang, Amber R. Vegter, Lydia Guo, Thomas J. Lynch, Yan Zhang, Yulong Zhang, Yaling Yi, Yu Yang, Zehua Feng, Soo Yeun Park, Amanita Shonka, Hannah McCumber, Lisi Qi, Peipei Wu, Guangming Liu, Allison Lacina, Kai Wang, Katherine N. Gibson-Corley, David K. Meyerholz, Dominique H. Limoli, Bradley H. Rosen, Ziying Yan, Douglas J. Bartels, John F. Engelhardt
Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modelling and network correlation analyses identified 118 differentially expressed proteins, significant through three complementary analytical pipelines. Candidate biomarkers were subsequently analysed in two validation cohorts of differing ethnicity using antibody-based proximity extension assays. TB-specific host biomarkers were confirmed. A six-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14 and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts. This biomarker panel exceeds the World Health Organisation Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.
Hannah F. Schiff, Naomi F. Walker, Cesar Ugarte-Gil, Marc Tebruegge, Antigoni Manousopoulou, Spiros D. Garbis, Salah Mansour, Pak Ho Wong, Gabrielle Rockett, Paolo Piazza, Mahesan Niranjan, Andres F. Vallejo, Christopher H. Woelk, Robert J. Wilkinson, Liku B. Tezera, Diana Garay-Baquero, Paul Elkington
IL-33 is a cytokine central to type 2 immune pathology in chronic airway disease. This cytokine is abundantly expressed in the respiratory epithelium and increased in disease, but how expression is regulated is undefined. Here we show that increased IL33 expression occurs from multiple noncanonical promoters in human chronic obstructive pulmonary disease (COPD), and it facilitates production of alternatively spliced isoforms in airway cells. We found that phorbol 12-myristate 13-acetate (PMA) can activate IL33 promoters through protein kinase C in primary airway cells and lines. Transcription factor (TF) binding arrays combined with RNA interference identified activator protein (AP) TFs as regulators of baseline and induced IL33 promoter activity. ATAC-Seq and ChIP-PCR identified chromatin accessibility and differential TF binding as additional control points for transcription from noncanonical promoters. In support of a role for these TFs in COPD pathogenesis, we found that AP-2 (TFAP2A, TFAP2C) and AP-1 (FOS and JUN) family members are upregulated in human COPD specimens. This study implicates integrative and pioneer TFs in regulating IL33 promoters and alternative splicing in human airway basal cells. Our work reveals a potentially novel approach for targeting IL-33 in development of therapeutics for COPD.
Heather E. Raphael, Ghandi F. Hassan, Omar A. Osorio, Lucy S. Cohen, Morgan D. Payne, Ella Katz-Kiriakos, Ishana Tata, Jamie Hicks, Derek E. Byers, Bo Zhang, Jen Alexander-Brett
Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3), a mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila, is expressed in mouse and human macrophages. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung disease.
S. Marina Casalino-Matsuda, Fei Chen, Francisco J. Gonzalez-Gonzalez, Hiroaki Matsuda, Aisha Nair, Hiam Abdala-Valencia, G.R. Scott Budinger, Jin-Tang Dong, Greg J. Beitel, Peter H.S. Sporn
There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non–AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1–/– mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix–stabilizing therapy in emphysema.
Mohit Ojha, Noah J. Smith, Andrew J. Devine, Rashika Joshi, Emily M. Goodman, Qiang Fan, Richard Schuman, Aleksey Porollo, J. Michael Wells, Ekta Tiwary, Matthew R. Batie, Jerilyn Gray, Hitesh Deshmukh, Michael T. Borchers, Samuel A. Ammerman, Brian M. Varisco
BACKGROUND. Information about the size, airway location, and longitudinal behavior of mucus plugs in asthma is needed to understand their role in mechanisms of airflow obstruction and to rationally design muco-active treatments. METHODS. Computed tomography (CT) lung scans from 57 asthma patients were analyzed to quantify mucus plug size and airway location, and paired CT scans obtained 3 years apart were analyzed to determine plug behavior over time. Radiologist annotations of mucus plugs were incorporated in an image-processing pipeline to generate size and location information that was related to measures of airflow. RESULTS. The length distribution of 778 annotated mucus plugs was multimodal and a 12 mm length defined short (“stubby”, ≤12 mm) and long (“stringy”, >12 mm) plug phenotypes. High mucus plug burden was disproportionately attributable to stringy mucus plugs. Mucus plugs localized predominantly to airway generations 6 to 9, and 47% of plugs in baseline scans, persisted in the same airway for three years, and fluctuated in length and volume. Mucus plugs in larger proximal generations had greater effects on spirometry measures than plugs in smaller distal generations, and a model of airflow that estimates the increased airway resistance attributable to plugs predicted higher impact for proximal and more numerous mucus plugs. CONCLUSIONS. Persistent mucus plugs in proximal airway generations occur in asthma and demonstrate a stochastic process of formation and resolution over time. Proximal airway mucus plugs are consequential for airflow and are in locations amenable to treatment by inhaled muco-active drugs or bronchoscopy. TRIAL REGISTRATION. Clinicaltrials.gov NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01759186, NCT01716494, and NCT01760915 FUNDING. NIH Grants: R01 HL080414, UG1 HL139106, P01 HL107202, U01 HL146002, U10 HL109172, U10 HL109168, U10 HL109152, U10 HL109257, U10 HL109146, U10 HL109250, U10 HL109164, U10 109086, and T32 HL007185, F32 HL162422. The following companies provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up: AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi–Genzyme– Regeneron, and TEVA. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.
Brendan K. Huang, Brett M. Elicker, Travis S. Henry, Kimberly G. Kallianos, Lewis D. Hahn, Monica Tang, Franklin Heng, Charles E. McCulloch, Nirav R. Bhakta, Sharmila Majumdar, Jiwoong Choi, Loren C. Denlinger, Sean B. Fain, Annette T. Hastie, Eric A. Hoffman, Elliot Israel, Nizar N. Jarjour, Bruce D. Levy, David T. Mauger, Kaharu Sumino, Sally E. Wenzel, Mario Castro, Prescott G. Woodruff, John V. Fahy
Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation and airways hyperresponsiveness which is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase (DCLK)-1+ tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57Bl/6 mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ positive tuft cells in the large airways. Compared to wild-type mice, RV-infected Pou2f3–/– mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early life viral infections could potentiate type 2 inflammatory responses to future infections.
Yiran Li, Mingyuan Han, Shilpi Singh, Haley A. Breckenridge, Jordan E. Kreger, Claudia C. Stroupe, Daniel A. Sawicky, Shiuhyang Kuo, Adam M. Goldsmith, Fang Ke, Anukul T. Shenoy, J. Kelley Bentley, Ichiro Matsumoto, Marc B. Hershenson
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