Bacterial-induced or passively administered interferon gamma conditions the lung for early control of SARS-CoV-2

Type-1 and type-3 interferons (IFNs) are important for control of viral replication; however, less is known about the role of Type-2 IFN (IFNγ) in anti-viral immunity. We previously observed that lung infection with Mycobacterium bovis BCG achieved though intravenous (iv) administration provides strong protection against SARS-CoV-2 in mice yet drives low levels of type-1 IFNs but robust IFNγ. Here we examine the role of ongoing IFNγ responses to pre-established bacterial infection on SARS-CoV-2 disease outcomes in two murine models. We report that IFNγ is required for iv BCG induced reduction in pulmonary viral loads, an outcome dependent on IFNγ receptor expression by non-hematopoietic cells. Importantly, we show that BCG infection prompts pulmonary epithelial cells to upregulate IFN-stimulated genes with reported anti-viral activity in an IFNγ-dependent manner, suggesting a possible mechanism for the observed protection. Finally, we confirm the anti-viral properties of IFNγ by demonstrating that the recombinant cytokine itself provides strong protection against SARS-CoV-2 challenge when administered intranasally. Together, our data show that a pre-established IFNγ response within the lung is protective against SARS-CoV-2 infection, suggesting that concurrent or recent infections that drive IFNγ may limit the pathogenesis of SARS-CoV-2 and supporting possible prophylactic uses of IFNγ in COVID-19 management.


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Single cell RNA sequencing was performed in one experiment, capturing a total of ~17,000 cells.All other experiments were repeated at least twice.Data provided in the manuscript are pooled from all independent repeats.Information on experimental replication can be found in the figure legends.
Animals were randomly assigned to groups of 3-8 age-and sex-matched mice for each experiment.Additional statement for Optibuild antibodies: "This antibody was developed for use in flow cytometry.The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity.However, verification testing has not been performed on all conjugate lots.Please refer to www.bdbiosciences.com/us/s/resourcesfor technical protocols." [ThermoFisher] , v9.0) was used during acquisation of of flow cytometry data.xPONENT (Luminex, v4.3) was used during acquisition of of cytokine multiplex data.
Single-cell RNA sequencing data generated in this study has been deposited to the NCBI GEO database and is available under the Accession ID GSE236601: https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE236601 Source data are provided with this paper as a Source Data file.No statistical methods were used to pre-determine sample size.Sample size was based on prior experience with animal models of SARS-CoV-2 infection and the typical variability(Hilligan & Namasivayam et al.J Exp Med, 2022: ref33; Oyesola & Hilligan et al.Sci Immunol 2023; Baker et  al.Front Immunol, 2023: ref 39)as well as the availablity of the appropriate genotypes.Groups of 3-8 mice were used for each experiment.Additional information on sample size can be found in the figure legends.
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