Journal: bioRxiv

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

doi: 10.1101/2023.11.07.566103

Figure Lengend Snippet: A. Timeline of the study design. Mice were inoculated with either phosphate-buffered saline (mock) or 5×10 4 tissue culture infectious dose (TCID 50 ) 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 β-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.

Article Snippet: Slides were mounted with Fluormount-G (Invitrogen) and images were acquired using a Zeiss Axio observer Widefield fluorescence microscope, using 5x and 20x objectives.

Techniques: Saline, Infection, RNA Expression, Virus, Quantitation Assay, Staining, Immunofluorescence, Microscopy

Journal: bioRxiv

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

doi: 10.1101/2023.11.07.566103

Figure Lengend Snippet: 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.

Article Snippet: Slides were mounted with Fluormount-G (Invitrogen) and images were acquired using a Zeiss Axio observer Widefield fluorescence microscope, using 5x and 20x objectives.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Microscopy, Infection, Multiplex Assay, Flow Cytometry

Journal: bioRxiv

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

doi: 10.1101/2023.11.07.566103

Figure Lengend Snippet: A. Genome schematics showing the insertion of eGFP and the recombinase Cre genes as independent reading frames in the SeV Cantell genome, between the virus genes N and P, to generate a Cre- expressing SeV recombinant virus (rSeV-C eGFPCre ). B. Schematic design showing Cre recombination of the LoxP-STOP-tdTomato reporter gene cassette leading to constitutive expression of the tdTomato fluorescent protein. C. Murine embryonic fibroblasts (MEFs) from either WT C57BL/6 mice or tdTomato reporter mice were infected in vitro at a multiplicity of infection (MOI) of 0.01 TCID 50 /cell to test the robustness of the reporter system. Representative images from two independent experiments were taken after 24 hpi using a widefield fluorescence microscope. eGFP signal (green) and tdTomato signal (red) were overlaid on brightfield images of WT and tdTomato-infected MEFs. 20x magnification. Scale bars: 50 µm.

Article Snippet: Slides were mounted with Fluormount-G (Invitrogen) and images were acquired using a Zeiss Axio observer Widefield fluorescence microscope, using 5x and 20x objectives.

Techniques: Virus, Expressing, Recombinant, Infection, In Vitro, Fluorescence, Microscopy

Journal: bioRxiv

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

doi: 10.1101/2023.11.07.566103

Figure Lengend Snippet: 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.

Article Snippet: Slides were mounted with Fluormount-G (Invitrogen) and images were acquired using a Zeiss Axio observer Widefield fluorescence microscope, using 5x and 20x objectives.

Techniques: Staining, Immunofluorescence, Microscopy