Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

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 O2, 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 ß 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.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection.

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Increased colonic expression of ACE2 associates with poor prognosis in Crohn's disease.

The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. We examined the expression of colonic ACE2 in 67 adult CD and 14 NIBD control patients using RNA-seq and quantitative (q) RT-PCR. We validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. Colonic ACE2 expression was significantly higher in a subset of adult CD patients which was defined as the ACE2-high CD subset. IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of CD diagnosis, and a Cox regression analysis found that high ACE2 levels is an independent risk factor for surgery (OR 2.17; 95% CI, 1.10-4.26; p = 0.025). Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that can impact CD disease-related outcomes.

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo.

The spike aspartic acid-614 to glycine (D614G) substitution is prevalent in global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains, but its effects on viral pathogenesis and transmissibility remain unclear. We engineered a SARS-CoV-2 variant containing this substitution. The variant exhibits more efficient infection, replication, and competitive fitness in primary human airway epithelial cells but maintains similar morphology and in vitro neutralization properties, compared with the ancestral wild-type virus. Infection of human angiotensin-converting enzyme 2 (ACE2) transgenic mice and Syrian hamsters with both viruses resulted in similar viral titers in respiratory tissues and pulmonary disease. However, the D614G variant transmits significantly faster and displayed increased competitive fitness than the wild-type virus in hamsters. These data show that the D614G substitution enhances SARS-CoV-2 infectivity, competitive fitness, and transmission in primary human cells and animal models.

SARS-CoV-2 D614G Variant Exhibits Enhanced Replication ex vivo and Earlier Transmission in vivo.

The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.