C57-01 The Effect of PM2.5 and SARS-CoV-2 on ACE2 and TMPSS2 Expression and Anti-Viral Response in Calu-3 Airway Epithelial Cells

Rationale Although studies have shown that air pollutants affect SARS-CoV-2 infection severity, the underlying mechanisms remain unclear. Our aim was to investigate the impacts of particulate matter less than 2.5 um (PM2.5) and SARS-CoV-2 on inflammatory changes and inflammasomal responses of Calu-3 cells. Methods Calu-3 airway epithelial cells were exposed to PM2.5 at concentrations of 10, 50, and 100 ug/mL, and to SARS-CoV-2 at an MOI of 0.01 for 24 hours. Cell viability was evaluated by the MTT assay, and epithelial barrier integrity was assessed through immunofluorescence staining of Zonula Occludens-1. The mRNA expression of viral entry genes—angiotensin-converting enzyme (ACE)2 and transmembrane protease serine (TMPRSS)2—as well as inflammatory and inflammasome-related genes including interleukin (IL)-8, IL-6, nuclear factor (NF)-kB p65 (RELA), JNK, c-JUN, Caspase-1 (CASP1), IL-1B, NLRP3, and interferon gamma (IFNg) was analyzed using qRT-PCR. Intracellular viral spike protein levels and RNA-dependent RNA polymerase (RdRP) expression were examined by immunofluorescence staining and qRT-PCR, respectively, while the release inflammatory cytokines were analyzed by ELISA. Results Higher concentrations of 100ug/mL PM2.5 reduced Calu-3 cell viability (p = 0.02) and damaged epithelial barrier integrity. PM2.5 (50ug/ml) increased mRNA expression for ACE2 and TMPRSS2 (p < 0.01). While PM2.5 alone decreased c-JUN expression, it did not change mRNA levels of JNK and RELA. In contrast, a co-incubation of SARS-CoV-2 and PM2.5 significantly increased mRNA levels for JNK and RELA (p < 0.05 and p < 0.01, respectively) and decreased c-JUN expression (p < 0.01). Additionally, exposure to PM2.5 and lower concentrations of PM2.5 + SARS-CoV-2 led to increased expression of IL-1B, IL-6, and GM-CSF, whereas IL-8 was induced only by SARS-CoV-2. Co-incubation with higher concentrations of PM2.5 (50 and 100 ug/mL) and SARS-CoV-2 decreased the expression of IL-8, IL-1B, CASP-1, and IFNg compared to their respective control groups. Lastly, both 50 ug/mL PM2.5 and SARS-CoV-2 led to an increase in the viral load (RdRP) (p < 0.05). Conclusions Our results show that PM2.5 impair epithelial integrity and cell viability, raises mRNA levels of ACE2 and TMPRSS2, and causes inflammation in Calu-3 cells exposed to SARS-CoV-2. These results suggest that PM2.5 may facilitate SARS-CoV-2 entry into airway epithelial cells, and that both PM2.5 and SARS-CoV-2 can deteriorate the host cell’s inflammatory and antiviral defenses. This abstract is funded by: Turkish Thoracic Society