To the best of our knowledge, this is the first study to demonstrate the expression of COVID-19 receptor ACE2 and protease TMPRSS2 in patients with IPF through immunohistochemical methods. We have shown that ACE2 and TMPRSS2 are co-expressed in FSP-1 positive fibroblasts in human lung fibrotic tissues and in an animal lung fibrosis model. Importantly, PM upregulated ACE2 and TMPRSS2 protein levels, which were suppressed by KC deletion or treatment with reparixin, an inhibitor of chemokine receptors. These results provide insights into the worsening and risk factors of COVID-19 in patients with IPF.
COVID-19 is a viral disease with respiratory failure caused by SARS-CoV-2 entering cells through host ACE2 and TMPRSS2. ACE2, a functional receptor for SARS-CoV, is present in the lung alveolar epithelial cells . We also found that alveolar lung cells were positive for ACE2 and TMPRSS2. Given that SARS-CoV-2 cell entry depends on host ACE2 and TMPRSS2 , the expression levels of ACE2 and TMPRSS2 may be assumed to affect transmissibility and susceptibility. Although the factors regulating ACE2 and TMPRSS2 are still unknown, both are co-expressed in lung type II pneumocytes as revealed by single-cell RNA-sequencing datasets .
These data indicated that alveolar lung cells in normal human lungs might be possible entry sites for SARS-CoV-2.
In light of the clinical manifestation of other comorbidities, ACE2 expression is elevated in the lungs of patients with morbidities correlated to severe COVID-19 . Smoking was found to upregulate ACE2 expression . Moreover, TMPRSS2 expression was found to be significantly elevated in smokers .The incremental expression of ACE2 and TMPRSS2 may explain the increased infection rate in smokers compared with nonsmokers . However, the levels of ACE2 and TMPRSS2 mRNA are controversial in IPF patients. The mRNA level of ACE2 was increased in patients with IPF and interstitial lung disease (ILD) compared to controls, but that of TMPRSS2 did not show a difference in those with IPF . There was no difference in gene expression of ACE2 and TMPRSS2 between IPF and healthy individuals, regardless of early and advanced IPF . Collectively, previous studies showed stable levels of ACE2 and TMPRSS2 in patients with IPF.However, in our results, ACE2 and TMPRSS2 were expressed in lung tissue and co-localized with FSP-1 positive fibroblasts, and exhibited enhanced expression after the development of lung fibrosis. Although there are no reports on the incidence of SARS-CoV-2 infection in patients with IPF, such patients are extremely susceptible to COVID-19, when ACE2 and TMPRSS2 are co-expressed.
Air pollution, a major problem for the general population, is caused by a complex mixture of solid and liquid particles suspended in the air and various gases such as ozone (O3), NO2, and carbon monoxide (CO). Air pollutants can change host immunity to respiratory viral infections related to individual preexisting pulmonary conditions such as IPF, asthma, and chronic obstructive pulmonary disease (COPD) . Johannsson et al. demonstrated an important relationship between the risk of acute exacerbation of IPF and O3 and NO2 . IPF mortality was significantly associated with cumulative air pollution exposure to PM2.5 with a hazard ratio (HR) of 7.93 (95% confidence interval (CI) 2.93 to 21.33) and particulate matter 10 (PM10) with a HR of 2.01 (95% CI 1.07–3.77) . The mechanisms of exposure to PM include EMT formation and fibronectin expression, and activation of transcription factors ETS-1 and NF-κB . In addition, based on our findings, PM induced deterioration of pulmonary fibrosis in a bleomycin animal model and increased the expression of ACE2 and TMPRSS2. ACE2 expression in the mice lung increased at 2 and 5 days after PM instillation, and PM2.5-induced acute lung injury in ACE2 KO mice was more severe than that in the wild-type . This could possibly be due to ACE2 having a protective role against acute lung injury . Furthermore, TMPRSS2 enhances viral spread and pathogenesis by antibody-mediated neutralization and activation of virus-cell fusion . Moreover, the formation of PM and viral microdroplets may also affect the severity of COVID-19 infection . Based on these findings, we speculate that a combination of air pollution and pulmonary fibrosis will accelerate the spread and deterioration of COVID-19.
Although acute exacerbation of idiopathic pulmonary fibrosis (IPF-AE) is clinically important, the role of cytokines require further understanding. High interlukin-8 levels in the cytokine profile reflect lung inflammatory processes in IPF-AE patients . The human IL-8 analog, KC, can aggravate inflammation and increase the levels of ACE2 and TMPRSS2. Moreover, as IPF progresses, fibrosis spreads widely in the alveolar walls, compromising alveoli function and gas exchange. However, the molecular mechanisms underlying fibrotic development have not been clarified. IL-8 mediates IPF mesenchymal progenitor cell fibrogenicity and prompts fibrotic progression by recruiting macrophages . We further showed that the fibrosis status was reversed in KC-deficient mice compared with the wild-type when bleomycin and PM were received simultaneously. In the model of bleomycin-induced pulmonary fibrosis, treatment with reparixin improved the lung pathology and reduced collagen deposition. Reparixin, an inhibitor of IL-8 receptor such as CXCR1/2, inhibits neutrophil influx, fibrogenic cytokine, and decreases pulmonary fibrosis by blocking CXCR2 . These data suggest that IL-8 and its downstream signaling induce lung function damage and upregulate ACE2 and TMPRSS2 expression. Furthermore, in IPF patients, Pirfenidone, a commercial antifibrotic drug with poor unknown mechanism, reduced disease progression, and increased lung function, exercise tolerance, and progression-free survival compared to placebo . It has been suggested, there is no rationality to discontinue conventional antifibrotic therapy in patients with IPF who are infected with COVID-19 .
COVID-19 is an emerging pathogen, and modifying exposure of patients with IPF to air pollution is impossible. Our study is therefore subject to possible limitations, but human tissue and animal models were used in the study. Our results are mainly based on histological methods. Further studies are warranted to validate these results. Moreover, we failed to demonstrate the origins of FSP-1 positive fibroblasts co-expressing ACE2 and TMPRSS2 in human and murine IPF tissues and more susceptible to SARS-CoV-2 infection. Since previous studies demonstrated FSP-1 or a-smooth muscle actin (a-SMA) positive fibroblasts as derivatives from the lung residential alveolar epithetical cells, bone marrow mesenchymal cells , or endothelium , we cannot exclude any of these origins as the sources of ACE2 and TMPRSS2 expression in IPF lung tissues.