Hong Dang

Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with pre-existing lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model ( Scnn1b -Tg), Scnn1b -Tg and WT mice were inoculated with a mouse adapted SARS-CoV-2 virus (SARS-CoV-2MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry (IHC), single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b -Tg mice inoculated with SARS-CoV-2MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b -Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b -Tg mice. However, Scnn1b -Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were administered a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia while enteral prednisone suppressed IL33 expression and disease. The eosinophilic pneumonia in Scnn1b -Tg mice after SARS-CoV-2MA10 infection mimics reports of eosinophilic pneumonia in humans post-SARS-CoV-2, suggesting targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.

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.

Heat wave burden is rapidly rising relative to pre-industrial levels and exacerbating human respiratory illnesses. Yet, how to protect human airways, short of deep and sustained reductions in greenhouse gas emissions, remains unclear. Here, we find by climate modeling, continuum mechanics analysis, in vitro cell culture models, and human clinical data, that heat waves increase risks of cough hypersensitivity, which can be reduced by alkaline aerosols of endogenous salt ions. We predict that the mouth breathing of indoor and outdoor air during heat waves, by exposing human airways to aridity, causes mucosal collapse onto cilia and inflammatory compression of airway epithelial cells. Using airway lining interface cultures of human bronchial epithelial cells, we find that mucosal collapse alters the expression of genes associated with airway hypersensitivity, including TRPV4, PIEZO1, SCNN1A, SCNN1B, ANO1, and CFTR. Our theoretical and experimental models indicate that mucosal collapse can be reversed by inhalation of alkaline (pH > 8) hypertonic divalent salts for 4–6 hours by globular protein translocation toward the airway epithelium. We find, on analysis of cough recordings in a recently published clinical trial, that inhaling alkaline hypertonic divalent salt aerosols with pH > 8 every 4–6 hours significantly reduced placebo-adjusted cough bouts (34%) (n = 8) (p = 0.01) among refractory chronic cough patients. Inhaled aerosols of the same composition with pH < 8 had no effect on cough bouts (-12%) (n = 4). Aerosols with endogenous ions may help reduce global warming threats to human respiratory health.

The long-term pulmonary sequelae of SARS-CoV-2 respiratory infections reflect infection severity, innate and adaptive immunity, and respiratory epithelial repair. This study investigated the acute and reparative responses as a function of age and sex in primary human bronchial epithelial (HBE) cultures utilizing a 14-day SARS-CoV-2 infection protocol. SARS-CoV-2 infection peaked at 3 days post-infection (dpi) with an ∼ 2 log titer suppression at 14 dpi. SARS-CoV-2 infection induced interferon, interferon-induced gene, and cell damage responses. No age- or sex-dependent effects on SARS-CoV-2 infection were detected. Airway epithelia repaired to an abnormal mucus metaplastic/inflammatory state that reflected potentially beneficial and adverse consequences at 14 dpi. Repair processes were infection severity-dependent, not sex-dependent, and were more robust in young donor cultures. Analyses of long-COVID subjects with persistent pulmonary fibrosis or persistent bronchitic airway diseases exhibited expression of HBE 14 dpi failed repair gene signatures, including ISG gene signatures. Human airway epithelial repair post-SARS-CoV-2 is prolonged and incomplete in vitro over 14 days, and persistently abnormal repair may contribute to phenotypes of people with long-COVID pulmonary syndrome.

In vitro models play a major role in studying airway physiology and disease. However, the native lung’s complex tissue architecture and nonepithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue have not been established. Here, we describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology; characteristic epithelial, endothelial, stromal, and immune cell populations; and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate viable explants with recovery of function 14 days postthaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. Our study examined ICAM-1, an adhesion molecule that is expressed on most cell types and plays important roles in leukocyte adhesion, migration, and motility. The data demonstrate that ICAM-1 is necessary for the clearance of S. pneumoniae and for the resolution of pneumonia but is not required for the recruitment of neutrophils or inflammatory macrophages into the pneumonic lung parenchyma or the alveoli/airways.

Introduction: Chronic primary pain conditions (CPPCs) are linked to catecholamine activation of peripheral adrenergic receptors. Yet, catecholamine-dependent epigenetic mechanisms, such as microRNA (miRNA) regulation of mRNA transcripts, remain largely unknown. Objectives: We sought to identify RNA species correlated with case status in 3 pain cohorts, to validate RNAs found to be dysregulated in a mouse model of CPPC onset, and to directly test the role of adrenergic receptors in miRNA regulation. Furthermore, we tested antinociceptive effects of miR-374 overexpression. Methods: We used RNA-seq and quantitative reverse transcription polymerase chain reaction to measure RNA expression in 3 pain cohorts. Next, we validated identified RNAs with quantitative reverse transcription polymerase chain reaction in a mouse model of CPPC onset, measuring expression in plasma, peripheral (adipose, muscle, dorsal root ganglia [DRG]), and central (spinal cord) tissues. Then, we stimulated adrenergic receptors in primary adipocyte and DRG cultures to directly test regulation of microRNAs by adrenergic signaling. Furthermore, we used in vitro calcium imaging to measure the antinociceptive effects of miR-374 overexpression. Results: We found that one miRNA family, miR-374, was downregulated in the plasma of individuals with temporomandibular disorder, fibromyalgia syndrome, or widespread pain following a motor vehicle collision. miR-374 was also downregulated in plasma, white adipose tissue, and spinal cord from mice with multisite mechanical sensitivity. miR-374 downregulation in plasma and spinal cord was female specific. Norepinephrine stimulation of primary adipocytes, but not DRG, led to decreased miR-374 expression. Furthermore, we identified tissue-specific and sex-specific changes in the expression of predicted miR-374 mRNA targets, including known (HIF1A, NUMB, TGFBR2) and new (ATXN7, CRK-II) pain targets. Finally, we demonstrated that miR-374 overexpression in DRG neurons reduced capsaicin-induced nociceptor activity. Conclusions: Downregulation of miR-374 occurs between adrenergic receptor activation and mechanical hypersensitivity, and its adipocyte source implicates adipose signaling in nociception. Further study of miR-374 may inform therapeutic strategies for the millions worldwide who experience CPPCs.

Enhancers are genomic sequences that function as regulatory elements capable of increasing the transcription of a given gene often located at a considerable distance. The broadly accepted model of enhancer activation involves bringing an enhancer-bound activator protein complex into close spatial proximity to its target promoter through chromatin looping. Equally relevant to the work described herein, roles for guanine (G) rich sequences in transcriptional regulation are now widely accepted. Non-coding G-rich sequences are commonly found in gene promoters and enhancers, and various studies have described specific instances where G-rich sequences regulate gene expression via their capacity to form G-quadruplex (G4) structures under physiological conditions. In light of this, our group previously performed a search for long human genomic stretches significantly enriched for minimal G4 motifs (referred to as LG4s herein) leading to the identification of 301 LG4 loci with a density of at least 80 GGG repeats / 1,000 basepairs (bp) and averaging 1,843 bp in length. Further, in agreement with previous reports indicating that minimal G4s are highly enriched in promoters and enhancers, we found 217/301 LG4 sequences overlap a GeneHancer annotated enhancer, and the gene promoters regulated by these LG4 enhancers were found to be similarly, markedly enriched with G4-capable sequences. Importantly, while the generally accepted model for enhancer:promoter specificity maintains that interactions are dictated by enhancer- and promoter-bound transcriptional activator proteins, the current study was designed to test an alternative hypothesis: that LG4 enhancers physically interact with their cognate promoters via a direct G4:G4 DNA-based mechanism. As such, this work employs a combination of informatic mining and locus-specific immunoprecipitation strategies to establish the spatial proximity of enhancer:promoter pairs within the nucleus then biochemically confirms the ability of individual LG4 ssDNAs to directly and specifically interact with DNA sequences found in their target promoters. In addition, we also identify four single nucleotide polymorphisms (SNPs), occurring within a LG4 enhancer on human chromosome 5, significantly associated with Cystic Fibrosis (CF) lung disease severity (avg. p value = 2.83E-9), presumably due to their effects on the expressions of CF-relevant genes directly regulated by this LG4 enhancer (e.g., EXOC3 and CEP72). Graphical AbstractIn brief: LG4 enhancers physically interact with gene promoters by forming composite G4 structures where both the LG4 and cognate promoter contribute half of the necessary sequence for G4 formation.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O ₂ , airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na ⁺ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na ⁺ channel) subunit expression. The combination of increased Na ⁺ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA–in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Rationale: Lung disease is the major cause of morbidity and mortality in persons with cystic fibrosis (pwCF). Variability in CF lung disease has substantial non-CFTR (CF transmembrane conductance regulator) genetic influence. Identification of genetic modifiers has prognostic and therapeutic importance. Objectives: Identify genetic modifier loci and genes/pathways associated with pulmonary disease severity. Methods: Whole-genome sequencing data on 4,248 unique pwCF with pancreatic insufficiency and lung function measures were combined with imputed genotypes from an additional 3,592 patients with pancreatic insufficiency from the United States, Canada, and France. This report describes association of approximately 15.9 million SNPs using the quantitative Kulich normal residual mortality-adjusted (KNoRMA) lung disease phenotype in 7,840 pwCF using premodulator lung function data. Measurements and Main Results: Testing included common and rare SNPs, transcriptome-wide association, gene-level, and pathway analyses. Pathway analyses identified novel associations with genes that have key roles in organ development, and we hypothesize that these genes may relate to dysanapsis and/or variability in lung repair. Results confirmed and extended previous genome-wide association study findings. These whole-genome sequencing data provide finely mapped genetic information to support mechanistic studies. No novel primary associations with common single variants or rare variants were found. Multilocus effects at chr5p13 (SLC9A3/CEP72) and chr11p13 (EHF/APIP) were identified. Variant effect size estimates at associated loci were consistently ordered across the cohorts, indicating possible age or birth cohort effects. Conclusions: This premodulator genomic, transcriptomic, and pathway association study of 7,840 pwCF will facilitate mechanistic and postmodulator genetic studies and the development of novel therapeutics for CF lung disease.