Defining Small Airways Disease as a Therapeutic Target in Post-Acute Sequela of COVID-19 (PASC)

FY21 PRMRP Focus Area: This project investigates novel mechanisms of exercise limitation and dyspnea after SARS-CoV-2 infection. This project is directly responsive to the research Topic Area of Respiratory Health. The project will address the Focus Area: "Research focused on acute and chronic lung injury/disorders due to viral infections, such as SARS-CoV-2." Background: Persistent dyspnea after SARS-CoV-2 infection is a major health crisis. As of August 2021, over 200 million people worldwide and 36 million people in the United States have been infected with SARS-CoV-2, the virus that causes COVID-19. While treatments for the acute illness have improved, multiple observational studies have reported persistent debilitating symptoms among those who have recovered from COVID-19, now referred to as Post-Acute Sequelae of SARS-CoV-2 infection (PASC). Estimates of the prevalence and duration of PASC vary, ranging from as few as 13.3% to as many as 76% of patients with symptoms at 6 months. Given the overwhelming number of COVID-19 cases, PASC is expected to represent a considerable healthcare burden. Despite this high prevalence, the etiology of PASC is largely unclear. Persistent dyspnea, or shortness of breath, is a common complaint in patients with PASC, which may arise from dysfunction in the lung and/or other organ systems. However, routine diagnostic testing, such as pulmonary function testing (PFT) and chest high-resolution computed tomography (HRCT), have failed to identify a precise cause of dyspnea in PASC. In fact, in a large proportion of PASC patients with significant dyspnea, almost all standard diagnostics may be normal. Thus, dyspnea in PASC is poorly characterized with no effective treatment. Precisely defining the pathophysiologic abnormalities is necessary to identify effective, personalized therapy to treat dyspnea in PASC. Rationale: As part of our established Coronavirus Recovery (CORE) Clinic at Massachusetts General Hospital (MGH), we have identified a subset of dyspneic PASC patients with mosaicism and associated air-trapping on expiratory images obtained by HRCT, findings that have also been reported by other groups. Mosaicism with air-trapping is a sign of small airway inflammation and/or fibrosis, which has previously been reported after other respiratory viral infections. We therefore propose that dyspnea in at least a subset of PASC patients stems from microscopic airway pathology. The therapeutic implications of this hypothesis, however, are limited by the heterogeneous nature of PASC, the suboptimal sensitivity of HRCT for small airway disease, and the fact that microscopic airway pathology may be due to ongoing inflammation and/or fibrosis. Heterogeneous, poorly understood syndromes such as PASC are difficult to treat because therapies that are appropriate for some etiologies may be ineffective in others (e.g., corticosteroids used to treat airway inflammation may be less effective than anti-fibrotic therapies for fibrotic airways). The limited spatial resolution of HRCT also means that even dyspneic PASC patients without HRCT findings may have small airway disease. Objective: We propose a study to investigate the precise etiology of dyspnea in patients with PASC and identify targeted treatments using an innovative combination of advanced cardiopulmonary exercise testing (CPET), endobronchial optical coherence tomography (EB-OCT), and single-cell RNA sequencing (scRNAseq) and proteomics of bronchoscopic samples. CPET enables the comprehensive characterization and phenotyping of multisystem contributions to dyspnea. EB-OCT is a bronchoscope-compatible, minimally invasive imaging modality, with microscopic resolution (< 10 microns) greatly exceeding that of HRCT. EB-OCT detects microscopic pathology, and can distinguish ongoing inflammation from fibrosis, with high sensitivity and specificity. scRNAseq and proteomics of bronchoscopic samples provides molecular characterization of the lung epithelium and immune landscape, and thus, can facilitate identification of novel, targetable biologic pathways. Hypothesis: We hypothesize that a subset of PASC patients presenting with significant dyspnea and lack of extrapulmonary causes of dyspnea on CPET will have evidence of small airway inflammation and/or fibrosis on EB-OCT. We also hypothesize that cellular and molecular analysis of bronchoscopic samples will yield distinct molecular and cellular signatures, which may lead to additional, novel therapeutic strategies. Aim 1: To determine whether microscopic small airway pathology is the primary etiology of dyspnea in a subset of PASC patients with normal cardiopulmonary assessment. Aim 2: To profile distinct cellular and molecular signatures associated with small airway pathology in PASC. Study Design: To accomplish the research proposed here, we will leverage the unique resources of MGH. These include our large and growing cohort of PASC patients, our nationally recognized CPET laboratory, and our unique experience in EB-OCT and research bronchoscopy with cellular transcriptional profiling. We will recruit dyspneic PASC subjects to undergo pulmonary function testing, HRCT, and CPET. Aim 1: We will perform EB-OCT in PASC subjects (n=60) with dyspnea despite normal peak oxygen consumption on CPET, both with (n=30) and without (n=30) mosaicism and air-trapping on expiratory HRCT, and age/sex-matched healthy control subjects (n=20). On EB-OCT imaging, we will evaluate for microscopic airway pathology, including inflammation and fibrosis. We will use our previously published methods to quantify the following parameters: airway fibrosis, airway inflammation, interstitial fibrosis, obliterative fibrosis, organizing pneumonia, bronchiectasis, and mucus volume, and viscosity. We will compare quantified EB-OCT results across the groups and correlate the results with clinical and physiologic data. Aim 2: During the same procedure, we will also collect bronchial alveolar lavage and endobronchial brushings, guided by EB-OCT, for cellular and molecular analysis. We will compare differences in cellular and gene expression profiles of airway epithelial and immune cells across the groups and correlate these data with the EB-OCT, clinical and physiologic data from Aim 1. All investigators will be blinded to group designation during data collection and analysis. This proposal brings together two Principal Investigators and one Co-Investigator, each with complementary primary expertise. Together, the proposed team possesses a unique combination of skills and a track record of success using CPET, EB-OCT, and molecular techniques for minimally invasive characterization of microscopic lung pathology. Short- and Long-Term Impacts: The data generated by this proposal will transform our understanding of the pathophysiology of dyspnea in PASC and directly enable personalized treatment strategies. Our robust PASC cohort, comprehensive CPET laboratory, and track record of success using EB-OCT and molecular techniques for minimally invasive characterization of microscopic lung pathology make us uniquely poised to succeed. Upon completion of this proposal, we will have conducted the first-ever assessment of microscopic lung disease pathophysiology along with detailed phenotyping and profiling of molecular and cellular alterations in the airways of dyspneic patients with PASC and compiled comprehensive exercise performance data in a large set of PASC patients. We expect that we will confirm our hypothesis and demonstrate that a subset of dyspneic patients with PASC have normal CPET and microscopic airway inflammation and/or fibrosis on EB-OCT as the primary pathologic abnormality. We expect that PASC patients with evidence of mosaicism on HRCT will have higher amounts of airway pathology as compared to PASC patients with normal HRCT, but that both groups will have evidence of airway disease as compared to healthy controls. We further expect that we will see phenotypic and transcriptional changes in both epithelial and immune cell populations isolated from the subjects. Specifically, we expect to detect evidence of airway epithelial stress and upregulation of pro-fibrotic mediators in PASC patients with microscopic evidence of fibrosis. In these patients, we also expect to find enrichment of monocyte-derived populations that persist after the resolution of acute lung injury, and myeloid-epithelial interactions that may propagate ongoing fibrotic changes. We expect a pro-inflammatory signature in these monocyte-derived cells and/or absence of pro-repair gene networks that promote tissue homeostasis and injury resolution. Comparison of scRNAseq data from diseased airways compared to normal appearing airways (as determined by EB-OCT) will help determine if changes are localized only to disease regions or are present more globally in the lung. scRNAseq will also likely identify unique changes in the profiled cells that identify novel pathogenic mechanisms of PASC associated disease and could identify diagnostic disease biomarkers. Additionally, it could help determine whether the small airway pathologies seen in PASC patients are similar to other fibrotic or airway diseases, or whether PASC represents a novel syndrome. This would be the first step in identifying potential therapeutic targets. If pro-fibrotic pathways are upregulated, this would support the use of established anti-fibrotic therapies targeted to fibrogenic mediators such as TGF-ß or fibroblast growth factor receptors, or the development of novel therapeutics targeting newly identified remodeling pathways. If airway inflammation is present in patients with PASC, this would support the use of steroids or other immune modulators that may be targeted towards specific inflammatory cytokines and chemokines and their receptors. Even if our hypothesis is incorrect, and no abnormalities are identified by EB-OCT or scRNAseq of airway cells, these studies will still result in a wealth of information on the physiology and pathophysiology of PASC patients. 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