(485) Chronic sequelae of SARS-CoV-2 in the Scnn1b-Tg mouse model of CF lung disease.

Background: Respiratory viral infections are a common cause of pulmonary exacerbation in people with CF (pwCF) and are known to have prolonged impacts on lung function. SARS-CoV-2 (SARS2) continues to be transmitted in the community and, while effective vaccinations and new therapeutics have reduced acute fatality rates, there is emerging evidence of long-term pulmonary consequences of SARS2 infection including those without pre-existing lung disease. Observational studies of pwCF infected with SARS-CoV-2 have not revealed worse acute disease. However, little is known about the potential chronic consequences of SARS2 in pwCF. Accordingly, we set out to study both the acute and chronic sequelae of SARS 2 infection using a mouse model of CF lung disease.

Methods: Scnn1b transgenic mice (Scnn1b-Tg) and WT littermates were inoculated intranasally with a mouse-adapted SARS2 virus (SARS-CoV-2 MA10). Lung tissue was harvested at 2, 15, 30 and 60 days post inoculation (dpi). Left lobes were prepared as histological sections for analysis by light microscopy (H&E, ABPAS), immunohistochemistry (IHC), and RNA in situ hybridization (ISH). Quantification was performed with Visiopharm software. We also performed single cell RNA sequencing of lung single cell suspensions from Scnn1b-Tg mice and WT littermate controls.

Results:
Scnn1b-Tg mice inoculated with SARS2 exhibited less weight loss than WT mice (95% vs 89% of baseline weight at 2 dpi, p< 0.05). SARS2 nucleocapsid protein and SARS2 spike mRNA, as measured by IHC and RNA-ISH, respectively, were less abundant in Scnn1b-Tg mice than in WT mice. There was evidence of acute lung injury in WT and Scnn1b-Tg mice at 2 and 4 dpi, which resolved by 30 dpi in WT mice. Conversely, histological evaluation of Scnn1b-Tg mice at 15, 30 and 60 dpi demonstrated heterogenous lobar areas of chronic inflammation. Immune phenotyping was performed in WT and Scnn1b-Tg mice over the time course of infection using immunostaining for multiple immune cell markers. Following infection with SARS2, there was a proportional increase in lymphoid cells in WT and Scnn1b-Tgmice at 2 and 4 dpi. In contrast, Scnn1b-Tg mice exhibited a dramatic accumulation of M2 macrophages from 4 through 30dpi, that was not present in WT mice. To investigate the mechanism that may account for the hyperinflammatory phenotype in Scnn1b-Tg mice, scRNA sequencing was performed on Scnn1b-Tg mice and WT mice. There was no differential expression of Ace2 or Tmprss2, SARS2 cell entry factors. Pathway analysis of alveolar macrophages (AM) revealed enrichment for both M1 and M2 macrophage signatures in Scnn1b-Tg mice. Alveolar type 2 (AT2) cells showed upregulation of the alarmin Il33 in Scnn1b-Tg mice. RNA-ISH confirmed Il33 expression, and there was marked upregulation of Il33 in the lobar areas of inflammation seen in Scnn1b-Tg mice from 15 dpi onward.





Conclusions: Scnn1b-Tg mice appear to have less severe acute disease related to SARS2 infection as compared to WT mice, but subsequently they develop persistent alveolar inflammatory lesions dominated by M2 macrophages. We provide evidence, using scRNA sequencing and RNA-ISH, that Il33 expression is increased in AT2 cells from Scnn1b-Tg mice and that AM from Scnn1b-Tg mice are enriched for markers of M2 activation in the naïve state. These findings suggest that abnormal Il33 signalling may contribute to development of chronic post viral lung abnormalities associated with muco-obstructed lung disease.

Acknowledgements: Supported by CFF; HAWKIN21F0

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