Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

Rationale: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been testedin vivo,given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure.Objectives: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis.Methods: Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes.Measurements and main results: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy.Conclusions: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.

In the era of combined antiretroviral therapy (cART), lung diseases such as chronic bronchitis (CB) and COPD are common among persons living with HIV (PLWH), particularly smokers. Although smoking is highly prevalent among PLWH, HIV may be an independent risk factor for lung diseases; however, the role of HIV and cigarette smoke (CS) and their potential interaction in the development of chronic lung diseases among PLWH has not been delineated. To investigate this interaction, cynomolgus macaques were exposed to CS and/or simian-adapted human immunodeficiency virus (SHIV) and treated with cART. The development of CB and the lung functions were evaluated following CS±SHIV treatment. The results showed that in the lung, SHIV was a strong independent risk factor for goblet cell metaplasia/hyperplasia and mucus formation, MUC5AC synthesis, loss of tight junction proteins, and increased expression of Th2 cytokines/transcription factors. In addition, SHIV and CS synergistically reduced the lung function and increased the extrathoracic tracheal ring thickness. Interestingly, SHIV-infection generated significant numbers of HIV-gp120⁺ epithelial cells (HGECs) in small airways and alveoli and their numbers doubled in CS+SHIV-infected lungs. We conclude that even with cART, SHIV independently induces CB and pro-COPD changes in the lung and the effects are exacerbated by CS.

Cigarette smoke (CS) exposure is one of the primary risk factors associated with the chronic mucous hypersecretion (CMH). The antiapoptotic protein, Bcl-2 sustains hyperplastic mucous cells, and the airway epithelium of ex-smokers with CMH as well as mice exposed to chronic CS showed increased Bcl-2 expression. Therefore, we investigated whether Bcl-2 plays a role in CS-induced mucous expression. Primary airway epithelial cells (AECs) of murine and human origin were treated with CS extract (CSE), and there was a concentration- and time-dependent increase in secretory mucin (MUC5AC), mucous regulator (SPDEF) and Bcl-2 expression. Using differentiated human AECs cultured on air-liquid interface, EGFR and ERK1/2 pathways were interrogated. Bcl-2 activity was blocked using a small molecule BH3 mimetic ABT-263 that disrupts the Bcl-2 interaction with pro-apoptotic proteins. The ABT-263 treatment resulted in the downregulation of CSE-induced mucus expression and disrupted the EGFR-signaling while inducing the apoptosis and the pro-apoptotic protein, Bik expression. This strategy significantly suppressed the mainstream CS-induced mucous phenotype in a 3-D human airway epithelium model. Therefore, the present study suggests that CS induces Bcl-2 expression to help promote mucous cell survival; and small molecule BH3 mimetics targeting Bcl-2 could be useful in suppressing the CS-induced mucous response.

The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve the barrier function of the airway epithelium and reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury; thus, it was further studied using biochemical, genetic, and biophysical methods to ascertain the mechanism of action via the improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression, and motion were required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that the improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.

We developed a novel imaging-based analysis to detect coordination of ciliary motion and optimal coordination, a process called metachrony. We found that metachrony is key to the optimization of ciliary-mediated mucus transport in both ferret and human tracheal tissue. This process appears to be regulated through calcium-dependent mechanisms. This study demonstrates the capacity to measure a key feature of ciliary coordination that may be important in genetic and acquired disorders of ciliary function.