PM2.5 Exposure Facilitates SARS-CoV-2 Infection through ACE2/TMPRSS2 Regulation and Suppression of Anti-Viral Response

BackgroundEpidemiological studies suggest an interaction between air pollution including particulate matter <2.5 {micro}m (PM2.5) and coronavirus disease 2019 (COVID-19) mortality and morbidity; however, the underlying mechanisms are not clear. The aim of our study was to investigate effects of PM2.5 on viability, epithelial integrity, and cellular entry of SARS-CoV-2 into airway epithelial cells, and the mechanisms involved. MethodsWe exposed Calu-3 airway epithelial cell cultures to PM2.5 (10, 50, and100 {micro}g/ml) and SARS-CoV-2 (MOI 0.01) for 24 h. The viability of Calu-3 cells and epithelial barrier integrity were determined using MTT assay and immunofluorescence staining for Zonula Occludens-1, respectively. mRNA expression for viral entry-related genes such as angiotensin converting enzyme (ACE)2 and transmembrane protease, serine (TMPRSS)2, and inflammatory and inflammasomal genes, including interleukin (IL)-8,IL-6, nuclear factor (NF)-{kappa}B p65 (RELA), JNK, c-JUN, Caspase-1, IL-1{beta}, NLRP3, was analyzed by qRT-PCR. Intracellular viral spike protein intensity and RNA-dependent RNA polymerase (RdRP) expression were determined using immunofluorescence staining and qRT-PCR, respectively. ELISA was used to analyze the release of inflammatory cytokines (IL-8, IL-6, and GM-CSF). ResultsHigher concentrations of 100{micro}g/ml PM2.5 decreased Calu-3 cell viability (p=0.02) and deteriorated epithelial barrier integrity, while 50 {micro}g/ml of PM2.5 (p<0.01) induced mRNA expression for ACE2 and TMPRSS2. Although PM2.5 alone decreased c-JUN, it did not alter the expression of mRNA for JNK and RELA. In contrast, a combination of SARS-CoV-2 and PM2.5 led to a significant increase in mRNA for both JNK and RELA (p < 0.05 and p < 0.01, respectively) and attenuated c-JUN expression. Moreover, our results indicated an increase in the expression of IL-1{beta}, IL-6, and GM-CSF following exposure to PM2.5 and PM2.5 + SARS-CoV-2, whereas IL-8 was induced only by SARS-CoV-2 exposure. Co-incubation of Calu-3 cells with PM2.5 and SARS-CoV-2 leads to a decrease in IL-8, IL-1{beta}, Caspase-1 (CASP-1), and Interferon gamma (IFNG) expression. Finally, the viral load (RdRP) also increased in the presence of both PM2.5 and the SARS-CoV-2 group. ConclusionOur findings have demonstrated that PM2.5 impaired epithelial integrity and cell viability, whereas it increased the mRNA expression for ACE2 and TMPRSS2, and induced inflammatory changes in Calu-3 cells incubated with SARS-CoV-2. These findings suggest that PM2.5 can facilitate the entry of SARS-CoV-2 into airway epithelial cells, and that both PM2.5 and SARS-CoV-2 can decrease the inflammatory and antiviral responses of the host cell.