Murine Parainfluenza Virus Persists in Lung Innate Immune Cells Sustaining Chronic Lung Pathology

Abstract

Respiratory viruses including the human parainfluenza viruses (hPIVs) are a constant burden to human health, with morbidity and mortality frequently increased after the acute phase of the infection. Although is proven that respiratory viruses can persist in vitro, the mechanisms of virus or viral products persistence, their sources, and their impact on chronic respiratory diseases in vivo are unknown. Here, we used Sendai virus (SeV) to model hPIV infection in mice and test whether virus persistence associates with the development of chronic lung disease. Following SeV infection, virus products were detected in lung macrophages, type 2 innate lymphoid cells (ILC2s) and dendritic cells for several weeks after the infectious virus was cleared. Cells containing viral protein showed strong upregulation of antiviral and type 2 inflammation-related genes that associate with the development of chronic post-viral lung diseases, including asthma. Lineage tracing of infected cells or cells derived from infected cells suggests that distinct functional groups of cells contribute to the chronic pathology. Importantly, targeted ablation of infected cells or those derived from infected cells significantly ameliorated chronic lung disease. Overall, we identified persistent infection of innate immune cells as a critical factor in the progression from acute to chronic post viral respiratory disease.

Figure 1.. Viral antigens and RNA persist in mouse lungs after SeV-driven acute illness.

A. Timeline of the study design. Mice were inoculated with either phosphate-buffered saline (mock) or 5×10⁴ tissue culture infectious dose (TCID₅₀) of SeV 52 per animal. Lungs were analyzed on days 3 and 49 post-infection. B. Disease progression was monitored by measuring weight loss through the experiment. Data are representative of 4 independent experiments (mean ±SD). For SeV-infected and mock groups the area under the curve (AUC) was calculated and t-tests were performed for statistical significance analysis. ****P<0.0001. C. Whole lung homogenates were harvested at days 3 and 49 post-infection and both SeV NP RNA expression and infectious virus titers were quantified by qPCR and infectivity assays respectively. Relative RNA quantitation by qPCR was normalized to mouse GAPDH and b-Actin. Two-way analysis of variance with Holm-Sídák post-test was used to estimate statistical significance between groups. N = 5 animals per group. *P<0.05; **P<0.01; ****P<0.0001. D. SeV-infected lungs were stained for SeV NP (white staining, upper panels) using immunofluorescence and for SeV NP RNA using RNAscope (white staining, lower panels). Nuclear staining (Hoechst for immunofluorescence and DAPI for RNAscope) in blue. Representative images of 3 independent experiments. Scale bars: 100 μm. E. SeV-infected lungs were stained for basal stem cells (Krt5⁺, green staining) and SeV NP (magenta staining) to localize SeV NP⁺ cells in relation to areas displaying chronic lesions (dashed areas, subpanels E1-E3) and unaffected areas (subpanel E4). Arrowheads indicate SeV NP⁺ cells, more detailed in the correspondent zoomed inset panels. Images were taken using a widefield microscope. Left panel, tiling image, 5x magnification. Right subpanels, 20x magnification. Right insets: digital zooms from the correspondent 20x magnification images. Scale bars: Left panel. 500 μm; Subpanels. 100 μm. Images are representative of 3 independent experiments, 5 mice per group.

Figure 2.. Diverse lung immune cells express SeV NP during chronic infection.

A. Characterization of immune cells expressing SeV NP from cryopreserved mouse lungs after 49 dpi by immunofluorescence. Tissue sections were stained for SeV NP (magenta) in combination with the surface markers CD3 (T lymphocytes), CD11c, CD11b (dendritic cell subsets), F4/80 (macrophages), and Thy1.2 (Innate Lymphoid Cells and some T lymphocyte subsets) in green and red. Nuclear staining is displayed in blue. White arrows indicate individual SeV NP⁺ cells. Images were taken in a widefield fluorescence microscope using a 20x magnification scope. Scale bars: 25 μm. Representative images from three independent experiments, 5 mice per condition. Right panels: insets from the dashed areas. B-E. Lungs from SeV-infected mice were harvested at 3 and 49 dpi, enzymatically digested, and analyzed by multiplex spectral flow cytometry with a panel of 16 antibodies to quantify (B and C) and characterize (D and E) SeV⁺ cells. B. Representative dot plots of SeV NP⁺ cells (% of live) comparing acute (3 dpi) with long-term (49 dpi) SeV infection. SeV NP⁺ gates were drawn based on the isotype control and the mock-infected samples. C. Frequency of SeV NP⁺ cells gated on total live cells from SeV 3 days-, SeV 49 days-, and mock-infected lungs. D. Representative histograms of SeV NP⁺ fluorescence intensity from 9 individual cell subsets, B cells, ILC2s, T CD4⁺ lymphocytes, T CD8⁺ lymphocytes, NK cells, Polymorphonuclear cells (PMNs), Alveolar macrophages (AMs), Tissue macrophages (TMs), and Dendritic cells (DCs) at 49 dpi. Histograms from SeV-infected animals are displayed in red while histograms from mock-infected animals are displayed in gray. E. Frequency and mean fluorescence intensity (MFI) of SeV NP⁺ cells within lymphoid- and myeloid-origin cell subsets. All multiple comparisons were done with one-way ANOVA and Holm-Sídák post-test. *P<0.05; **P<0.01; ***P<0.0005; ****P<0.0001. Data representative of two independent experiments, 4–5 animals per condition, total 1 million events acquired per animal.

Figure 3.. Type 2 innate lymphoid cells and macrophages are persistently activated in a type 2 inflammation manner during SeV chronic lung disease.

Lung type 2 innate lymphoid cells (ILC2s) (A-C), and macrophages (D-F) were isolated either from SeV- or mock-infected mouse lungs after 49 dpi and subjected to bulk RNA-seq. A and D. Volcano plots indicating differentially expressed genes detected in ILC2s and macrophages, respectively, from SeV-infected lungs over mock. P<0.05, LogFC>2. B-C. Scattered dot plots showing expression of ILC2 hallmark genes (B) and virus-related ISGs (C). Each dot corresponds to an individual pool of cells (n = 6 animals pooled in pairs per condition). E-F. Scattered dot plots indicating expression of Th2 polarization (E) and phagocytic activity (F) genes from macrophages. Each dot corresponds to cells obtained from an individual animal (minimum n = 3 animals per condition). Data are displayed as mean ± SD. Two-way analysis of variance (ANOVA) with Bonferroni post-test was used to estimate statistical significance between multiple comparisons. *P<0.05; **P<0.01; ****P<0.0001. CPM, copies per million. G. Bubble chart showing gene set enrichment analysis (GSEA) of upregulated genes in ILC2s and macrophages sorted from SeV 49 dpi lungs. Bubble size indicates gene set size per GSEA pathway, while bubble color gradient indicates Normalized Enrichment Scores (NES) values.

Figure 4.. Paramyxovirus infection clearance is not complete and leaves long-term survivor cells in the lower respiratory tract expressing persistent viral RNA and viral proteins.

A. B6.Cg-Gt(ROSA)^tdTom (tdTom) mice were inoculated intranasally with either PBS (mock) or 5×10⁵ TCID₅₀ rSeV-C^eGFP-Cre. Lungs were harvested at 3, and 49 dpi for flow cytometry (FC) analysis, and cell sorting at 49 dpi. Weight loss was recorded up to 21 dpi to monitor disease progression. Data (mean±SD) are representative of 2 individual experiments (minimum 3 mice per group). B. Representative dot plots comparing percentage of tdTom⁺ cells in mouse lungs during acute (3 dpi) and chronic (49 dpi) rSeV- C^eGFP-Cre infection. C. Frequency of tdTom⁺ cells gated on total live cells from rSeV-C^eGFP-Cre 3 days-, rSeV- C^eGFP-Cre 49 days-, and mock-infected lungs. Data are shown as mean±SD. Statistical significance was estimated with one-way ANOVA using Bonferroni post-test. ***P<0.005. D-E. Characterization of immune (CD45⁺) and non-immune (CD45⁻) cell proportions within tdTom⁺ cells. Representative dot plots (D) and quantification (E) of CD45 staining in tdTom⁺ cells during acute (3 dpi) and chronic (49 dpi) rSeV-C ^eGFP-Cre infection. Statistical significance was estimated with two-way ANOVA and Holm-Sídák post-test. *P<0.05. F. Combination of tdTom and SeV NP detection enables sorting of two distinct subsets of VID cells, SeV-infected cells persistently expressing viral antigens (tdTom⁺NP⁺) and SeV-infected/survivor cells only (tdTom⁺NP⁻). Representative dot plots indicating the gating strategy used for sorting 3 cell subpopulations from rSeV-C^eGFP-Cre-infected lungs tdTom⁺NP⁻, tdTom⁺NP⁺, and negative. Negative cells from mock-infected lungs were also sorted. Data representative of 2 individual experiments, 6 mice per condition. G. Sorted cells were pooled (2 mice) and subjected to RNAseq. Normalized viral reads per 10⁸ total reads are displayed per individual pool of tdTom⁺NP⁻ and tdTom⁺NP⁺ cells. H. Coverage analysis indicating normalized viral reads per genome position in each individual cell pool. Viral specific gene regions and reporter genes are indicated in light gray.

Figure 5.. Viral clearance and persistence entails opposing transcriptional programs in long-term SeV-infected lungs.

Viral infected and cells derived from them sorted from tdTom mice infected with rSeV-C^eGFPCre at 49 dpi were subjected to bulk RNAseq and host transcriptome analysis. A. Volcano plots indicating differentially expressed genes (DEGs) in tdTom⁺NP⁻, tdTom⁺NP⁺, and Negative cells against Mock negative cells. B. Venn diagram showing overlapping DEGs from tdTom⁺NP⁻ and tdTom⁺NP⁺, as well as exclusive DEGs from each cell subset. C. Bar graphs showing gene ontology (GO) enrichment analysis of each of the VID cell subsets (tdTom⁺NP⁻ and tdTom⁺NP⁺) exclusive DEGs. D. Heatmaps of selected gene collections from the GO pathways in C. Shown are fold change (FC) values of tdTom⁺NP⁻ and tdTom⁺NP⁻ are displayed. Columns groups are color-coded following the same patterns on C. and B. E. Gene set enrichment assay (GSEA) bubble chart indicating the most significant enriched pathways of tdTom⁺NP⁺ transcriptome signatures in comparison with tdTom⁺NP⁻ cells. Bubble color gradient indicates Normalized enrichment score (NES), and bubble sizes correspond to gene set size on each pathway.

Figure 6.. Chronic lung pathology induced by paramyxovirus infection is dependent on long-term surviving and persistently infected cells.

A. Diphtheria toxin regime treatment and timepoints for tissue harvesting and analysis. B. Disease progression was assessed by monitoring animal weight loss until 26 dpi. Graphs are representative of 2 independent experiments and depict mean weight loss values ±SD, 4 mice per condition. C. Mouse lungs were harvested at 49 dpi and tissue sections were stained with Hematoxylin and Eosin to compare pathological changes between the analysis groups. Top panels indicate representative images of whole lung sections (Brightfield, 5x magnification tiled images, Scale bars: 1 mm) and bottom panels indicate zoomed-in images from the indicated areas (Brightfield, 5x magnification tiled images, scale bars: 100 μm). 4 animals per condition. D. Lung sections from SeV +DT and SeV -DT groups were blindly scored for histopathological changes. Total area affected, percentage of airway structures affected, and intensity of alveolitis, peribroncholitis, and bronchus-associated lymphoid tissue (BALT) expansion were determined for every individual lung sample. Individual weighted scores values ±SD are indicated. Data representative of 2 individual experiments, 4–7 mice per condition. E. Lung sections were stained for the tissue remodeling and chronic lung lesion markers Krt5 (green) and Krt8 (magenta) with immunofluorescence to check for chronic lung lesion progression. Nuclear staining is displayed in blue. The dashed area indicates chronic lung lesions and areas of intense tissue remodeling. Images were taken with a widefield microscope. Upper panels, tiling images, 20x magnification, scale bars: 500 μm. Lower panels: 20x magnification, scale bars: 100 μm. F. Quantification of chronic lung lesion area (%) over total lung section area. Mean values ±SD are shown. Data are representative of two individual experiments, 4–7 mice per condition. Statistical significance was estimated using one-way ANOVA and Bonferroni post-test. *P<0.05.