PM 2.5 Exposure Facilitates SARS-CoV-2 Infection through ACE2/TMPRSS2 Regulation and Suppression of Anti-Viral Response
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Background
Epidemiological studies suggest an interaction between air pollution including particulate matter <2.5 µm (PM 2.5 ) 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 PM 2.5 on viability, epithelial integrity, and cellular entry of SARS-CoV-2 into airway epithelial cells, and the mechanisms involved.
Methods
We exposed Calu-3 airway epithelial cell cultures to PM 2.5 (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-1 β, 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).
Results
Higher concentrations of 100µg/ml PM 2.5 decreased Calu-3 cell viability (p=0.02) and deteriorated epithelial barrier integrity, while 50 µg/ml of PM 2.5 (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 PM 2.5 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 PM 2.5 and PM 2.5 + SARS-CoV-2, whereas IL-8 was induced only by SARS-CoV-2 exposure. Co-incubation of Calu-3 cells with PM 2.5 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 PM 2.5 and the SARS-CoV-2 group.
Conclusion
Our findings have demonstrated that PM 2.5 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 PM 2.5 can facilitate the entry of SARS-CoV-2 into airway epithelial cells, and that both PM 2.5 and SARS-CoV-2 can decrease the inflammatory and antiviral responses of the host cell.
