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

Background Epidemiological studies suggest an interaction between air pollution including particulate matter <2.5 µm (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.

We exposed Calu-3 airway epithelial cell cultures to PM2.5 (10, 50, and100 µg/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)-κB p65 (RELA), JNK, c-JUN, Caspase-1, IL-1β, 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).

Higher concentrations of 100µg/ml PM2.5 decreased Calu-3 cell viability (p=0.02) and deteriorated epithelial barrier integrity, while 50 µg/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-1β, 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-1β, 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.

Our 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. Competing Interest Statement The authors have declared no competing interest. Footnotes We recently detected some missing data in our manuscript. We fixed all the problems in the current version. -Abstract: add information about IL-1beta and IFNG, and also RdRP gene expression. -Introduction: add two more references in lines 68 and 79. -Method: deleted a repeated section named 2.4. SARS-CoV-2 treatment of Calu-3 cells -Add results part about IFNG: section 3.10 and also figure 9 for IFNG