Chen Xi Yang

Background: Approximately 10% of individuals who recover from COVID-19 experience residual respiratory symptoms impacting their quality of life, but the mechanisms behind pulmonary long COVID (PLC) are largely unknown. Objectives: We characterized airway and circulating immune cells in patients with and without PLC. Methods: Participants were recruited and allocated into two groups: 1) PLC, defined by a St. George's Respiratory Questionnaire (SGRQ) total score of >10 at least three months following an acute SARS-CoV-2 infection with self-reported new or worsening symptoms, and 2) controls, defined by SGRQ ≤10 with or without a prior history of COVID. We performed research bronchoscopy and obtained bronchoalveolar lavage (BAL) in seven PLC patients and seven age- and sex-matched control subjects. Single-cell RNA sequencing (scRNAseq) was performed on the BAL cells. Peripheral blood mononuclear cells (PBMCs) were cryopreserved in 30 participants (17 PLC, 13 controls) for proteomic analysis. Serum was submitted for microarray detection of auto-IgG antibodies. Results: We annotated 105 836 cells using scRNAseq and found that CD4 ⁺ T cells were credibly increased in participants with PLC. scRNAseq revealed up-regulation of anti-viral pathways including those related to interferon signaling in T cells as well as antigen presenting cells. In PBMCs, T cells expressing both CD4 and CD8 were elevated in PLC participants. Autoantibodies targeting histone 2B and histone 3 were significantly increased in PLC participants (adj.p<0.05). Conclusions: PLC is associated with dysregulation of T cell mediated immunity, which may be related to autoimmunity. These cells represent potential novel therapeutic targets in patients suffering from PLC.

Asthma is characterized by persistent airway epithelial dysfunction and remodelling of the reticular basement membrane (RBM). In healthy airways, the RBM is primarily composed of the extracellular matrix (ECM) proteins laminin and collagen-IV, but in remodelled asthmatic airways, the RBM has increased deposition of collagen-I, -III and fibronectin. Here, we systematically compared the effects of collagen-I, -III, -IV, fibronectin, laminin, and bovine serum albumin (BSA) control on bronchial epithelial cells (BECs) from six healthy controls and seven individuals with asthma. Epithelial attachment, spreading and barrier function were assessed in real time over 72 h using electrical cell–substrate impedance sensing. Cell culture supernatants were analyzed for release of epithelial cytokines, thymic stromal lymphopoietin (TSLP), interleukin (IL)-6, IL-8, and IL-11 using ELISA. BECs from both control and asthma donors had faster cell attachment, spreading, and barrier formation on collagen-I, -III, -IV, and fibronectin compared to laminin and BSA. BECs from both control and asthma donors cultured on collagen -I and -III produced more TSLP, but had no effect on IL-6, IL-8, and IL-11 expression. In summary, remodelling of the RBM in asthma may promote epithelial barrier formation whilst simultaneously enhancing epithelial-derived Th2 inflammation through increased TSLP release.

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by global immune dysregulation. The most abundant immune cells in the distal airways and alveolar compartments are macrophages and lymphocytes. These cells have been poorly characterized in human airways due to their plasticity and numerous subtypes. There is a marked scarcity of information in COPD airway, limiting our understanding of the inflammatory process that drives COPD progression. Objectives: The primary aim of this study was to characterize the differences in composition of the immune cells in the alveolar space of patients with COPD versus healthy controls and the relation with severity of airflow limitation at a single cell resolution. Methods: Individuals with and without COPD were recruited. All participants underwent bronchoscopy, where a single cell suspension was created from the retrieved bronchoalveolar lavage and then sequenced using single cell RNA sequencing technology. Full pulmonary function tests, thoracic computed tomography (CT) and xenon-129 hyperpolarized gas magnetic resonance imaging (MRI) were performed on participants for phenotyping. Results: From 17 participants, a total of 97,254 cells were recovered with 52,840 cells belonging to the COPD group (N = 10). The COPD immune landscape was perturbed and characterized by an expansion of non-typable macrophages, increased neutrophils and classical monocytes, and significant changes in regulatory and double-negative T lymphocytes compared to controls (FDR < 0.05). The percentage of these cells were further altered with increasing GOLD (Global initiative for chronic Obstructive Lung Disease) severity stages. The percentage of double negative T cells in BAL was significantly related to airway luminal area on CT; whereas that of regulatory T cells was inversely related to MRI gas transfer and DLCO of COPD patients. Conclusions: The immune cell transcriptomic profile of COPD BAL reveals global immune dysregulation, with increased abundance of proinflammatory and profibrotic macrophages, monocytes and neutrophils, potentially driving airway remodeling. Regulatory T cells may play a significant role in gas exchange perturbations of COPD.

BACKGROUND: Mucus plugs form in acute asthma and persist in chronic disease. Although eosinophils are implicated in mechanisms of mucus pathology, many mechanistic details about mucus plug formation and persistence in asthma are unknown. METHODS: Using histology and spatial, single-cell proteomics, we characterized mucus-plugged airways from nontransplantable donor lungs of 14 patients with asthma (9 with fatal asthma and 5 with nonfatal asthma) and individuals acting as controls (10 with chronic obstructive pulmonary disease and 14 free of lung disease). Additionally, we used an airway epithelial cell–eosinophil (AEC-eosinophil) coculture model to explore how AEC mucus affects eosinophil degranulation. RESULTS: Asthma mucus plugs were tethered to airways showing infiltration with innate lymphoid type 2 cells and hyperplasia of smooth muscle cells and MUC5AC-expressing goblet cells. Asthma mucus plugs were infiltrated with immune cells that were mostly dual positive for eosinophil peroxidase (EPX) and neutrophil elastase, suggesting that neutrophils internalize EPX from degranulating eosinophils. Indeed, eosinophils exposed to mucus from IL-13–activated AECs underwent CD11b- and glycan-dependent cytolytic degranulation. Dual-positive granulocytes varied in frequency in mucus plugs. Whereas paucigranulocytic plugs were MUC5AC rich, granulocytic plugs had a mix of MUC5AC, MUC5B, and extracellular DNA traps. Paucigranulocytic plugs occurred more frequently in (acute) fatal asthma and granulocytic plugs predominated in (chronic) nonfatal asthma. CONCLUSION: Together, our data suggest that mucin-rich mucus plugs in fatal asthma form because of acute goblet cell degranulation in remodeled airways and that granulocytic mucus plugs in chronic asthma persist because of a sustaining niche characterized by epithelial cell–mucin-granulocyte cross-talk. FUNDING: NIH grants HL080414, HL107202, and AI077439.

Background: The role of epigenetic ageing in the environmental pathogenesis and prognosis of fibrotic interstitial lung disease (fILD) is unclear. We evaluated whether ambient particulate matter with diameter ≤2.5 μm (PM_2.5) and neighbourhood disadvantage exposures are associated with accelerated epigenetic ageing, and whether epigenetic age is associated with adverse clinical outcomes in patients with fILD.Methods: This multicentre, international, cohort study included patients with fILD from the University of Pittsburgh (UPitt, n=306) and University of British Columbia (UBC, n=170). 5-year PM_2.5exposures were estimated using satellite-derived hybrid models. Neighbourhood disadvantage was calculated using US and Canadian census-based metrics. Epigenetic age difference (EAD=epigenetic age−chronological age) was calculated using GrimAge analysis of blood DNA methylation data. Linear models assessed associations of exposures with EAD. Cox models assessed associations of EAD with transplant-free survival. Causal mediation analysis evaluated EAD mediation of exposure–survival relationships.Results: Median epigenetic age was 11.7 years older than chronological age in patients with fILD. In combined cohort analysis, each interquartile range (IQR) increase in PM_2.5was associated with 2.88 years (95% CI 1.39–4.38; p2.5–survival relationship and 59% of the neighbourhood disadvantage–survival relationship. Epigenetic age was also more strongly associated with transplant-free survival than chronological age.Conclusions: Epigenetic age acceleration is associated with worse survival and mediates adverse exposure impacts in fILD.

While spatial transcriptomics offer valuable insights into gene expression patterns within the spatial context of tissue, many technologies do not have a single-cell resolution. Here, we present SMART, a marker gene-assisted deconvolution method that simultaneously infers the cell type-specific gene expression profile and the cellular composition at each spot. Using multiple datasets, we show that SMART outperforms the existing methods in realistic settings. It also provides a two-stage approach to enhance its performance on cell subtypes. The covariate model of SMART enables the identification of cell type-specific differentially expressed genes across conditions, elucidating biological changes at a single-cell-type resolution.

Aim: To elucidate the important cellular and molecular drivers of pulmonary long COVID, we generated a single-cell transcriptomic map of the airway mucosa using bronchial brushings from patients with long COVID who reported persistent pulmonary symptoms.Method: Adults with and without long COVID were recruited from the general community in Greater Vancouver, Canada. The cohort was divided into those with pulmonary long COVID, which was defined as persons with new or worsening respiratory symptoms following ≥12 weeks from their initial acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (n=9); and control subjects defined as SARS-CoV-2 infected persons whose acute respiratory symptoms had fully resolved or individuals who had no history of acute coronavirus disease 2019 (COVID-19) (n=9). These participants underwent bronchoscopy from which a single cell suspension was created from bronchial brush samples and then sequenced.Results: A total of 56 906 cells were recovered for the downstream analysis, with 34 840 cells belonging to the pulmonary long COVID group, which strikingly showed a unique cluster of neutrophils in the pulmonary long COVID group (p<0.05). Ingenuity Pathway Analysis revealed that the neutrophil degranulation pathway was enriched across epithelial cell clusters. Differential gene expression analysis between the pulmonary long COVID and control groups demonstrated upregulation of inflammatory chemokines and epithelial barrier dysfunction across epithelial cell clusters, as well as over-expression of mucin genes across secretory cell clusters.Conclusion: A single-cell transcriptomic landscape of the small airways suggest that neutrophils may play a significant role in mediating the chronic small airway inflammation driving pulmonary symptoms of long COVID.

Background: DNA methylation may be a link between HIV, aging, and the increased risk of lung comorbidities. We investigated whether bronchoalveolar lavage (BAL) cells of people living with HIV (PLWH) demonstrate epigenetic disruptions and advanced epigenetic aging. Methods: BAL cell DNA methylation from 25 PLWH and 16 HIV-uninfected individuals were tested for differential methylation of Alu and LINE-1 sites, markers of aging. We used a weighted gene correlation network analysis to identify HIV- and age-associated co-methylation networks. We tested the effect of HIV on DNA methylation using a robust linear model (false discovery rate < 0.10). Results: The BAL cells of PLWH were marked by global hypomethylation in both Alu and LINE-1 elements. Six co-methylated CpG networks were identified that were significantly associated with age; of these, the red module was significantly differentially methylated in PLWH and enriched pathways (e.g., Ras signaling and T-cell receptors). We identified 6428 CpG sites associated with HIV. Conclusions: We have shown here for the first time that alterations in the DNA methylation of BAL cells in the lung with HIV show a pattern of advanced aging. This study strongly supports that HIV may contribute to an increased the risk of lung comorbidities through the epigenetics of aging.

Shareable abstract Current cannabis smoking is significantly associated with faster peripheral blood epigenetic age acceleration; interestingly, cannabis smoking cessation is shown to normalise this age acceleration signal.https://bit.ly/3x7s2CU