Increased Susceptibility of Plscr1-/- Mice to Influenza Virus Infection

Wt and Plscr1-/- mice were exposed to sublethal (300 pfu, B, C and F-H) or lethal (900 pfu, D and E) IAV (WSN) infection. (A) Scheme of experiment. (B) Whole lungs of Wt mice were analyzed for Plscr1 RNA by qRT-PCR. (C and D) Mean relative weight of mice post sublethal or lethal infection. (E) Survival rate of mice post lethal IAV infection. (F) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. (G) Infectious viral titer in the lungs was assessed by plaque assays. (H) Representative staining for H1N1 in lungs. The scale bars represent 1 mm. Quantification was performed using ImageJ. Data are expressed as mean ± SEM of n = 30 mice/group for weight loss post sublethal infection and n=8 mice/group for weight loss and survival rate post lethal infection. For the rest analysis, n= 5-10 mice/group. All data were pooled from three independent experiments. Logrank (Mantel-Cox) test was used to compare survival rates. Ordinary two-way ANOVA tests were used to compare weight losses. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001. dpi, days post infection. CTCF, Corrected Total Cell Fluorescence.

Increased Lung Inflammation in Plscr1-/- Mice in Influenza Virus Infection

Wt and Plscr1-/- mice were exposed to sublethal (300 pfu) IAV (WSN) infection. (A) Total BAL leukocyte numbers. (B) Differential cell counts in BAL. (C) Representative lung sections stained with H&E. Scale bars represent 3 mm (main) and 200 μm (inlays). (D) Whole lungs were analyzed for Ifn-α, Ifn-β, Ifn-γ and Ifn-λ RNA by qRT-PCR. (E) Tnf-α and Ifn-λ concentrations in BAL by ELISA. Data are expressed as mean ± SEM of n = 3-14 mice/group. All data were pooled from three independent experiments. *p < 0.05, **p < 0.01. dpi, days post infection.

Transcriptional Regulation of IFN-λR1 by PLSCR1 and IFN-λ in IAV Infection

(A-E) Wt and Plscr1-/- mice were exposed to sublethal (300 pfu) IAV (WSN) infection. (A) Heatmap of interferons and their receptors in whole lungs by RNA-seq. (B) Whole lungs were analyzed for Ifn-λr1 by qRT-PCR. (C) Heatmap of differential expressions of all ISGs in whole lungs by RNA-seq. Gene expressions were compared between groups within each row and color-labeled from row minimum (blue) to row maximum (red). (D) Localization of Ifn-λr1+ cells in the lungs of IAV-infected Wt mice at 7 dpi. Sections stained for Ifn-λr1 (red), Foxj1, uteroglobin or Sftpc (green) and DAPI (blue) are shown. (E) Representative staining for Ifn-λr1 in airways or alveoli of IAV-infected Wt and Plscr1-/- mice at 7dpi. Quantifications were performed using ImageJ. (F-G) Calu-3 cells were analyzed for PLSCR1 (F) and IFN-λR1 (G) RNA by qRT-PCR after recombinant IFN-λ and/or α-IFN-λR1 antibody treatment. Data are presented as fold change compared to non-treated group. (H-I) Chromatin-Immunoprecipitation of PLSCR1 and IFN-λR1 promoter in Calu-3 cells followed by standard PCR (H) and real-time quantitative PCR (I). Data are expressed as mean ± SEM of n = 4-12 mice or wells/group. For transcriptomic analysis, 9 mice from each PBS-treated group and 4 mice from each IAV-infected group were pooled together. All data were pooled from three independent experiments. *p < 0.05, **p < 0.01, ***p<0.001. dpi, days post infection. CTCF, Corrected Total Cell Fluorescence. Scale bars represent 50 μm.

Protein Interaction between IFN-λR1 and PLSCR1 in IAV Infection

(A) Co-Immunoprecipitation of Plscr1 and Ifn-λr1 in whole mouse lungs followed by western blot. (B) Proximity ligation assay of Ifn-λr1 and Plscr1 in the lungs of Wt mice infected or uninfected with IAV. Scale bars represent 50 μm. Quantifications were performed using ImageJ. (C) Colocalization of IFN-λR1 (green) and PLSCR1 (red) on Calu-3 cell membranes infected or uninfected with IAV in a nonpermeabilized staining. Scale bars represent 10 μm. Data are expressed as mean ± SEM of n = 6-7 lungs/group. All data were pooled from three independent experiments. *p < 0.05, **p < 0.01. PLA, Proximity Ligation Assay. CTCF, Corrected Total Cell Fluorescence.

Requirement of Both Nuclear and Surface PLSCR1 but Not the Enzymatic Activity in IFN-λR1-Mediated Anti-Influenza Activities

PLSCR1-/- A549 cells were transduced with mutated PLSCR1 plasmids using lentivirus and infected with IAV (PR8) for 24 hours at 1 MOI (A-E) or 10 MOI (F). (A) IFN-λR1 RNA by qRT-PCR. (B) IFN-λR1 proteins by western blotting. (C) Proximity ligation assay of IFN-λR1 and PLSCR1. Scale bars represent 20 μm. Quantifications were performed using ImageJ. (D) Viral RNA load was assessed by quantifying M gene by qRT-PCR. (E) Infectious viral titer was assessed by plaque assays. (F) Cells were stained with crystal violet. Cell viability was quantified using ImageJ. Data are expressed as mean ± SEM of n = 4-13 wells/group. All data were pooled from three independent experiments. ns, not significant, *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001, *****p<0.00001. CTCF, Corrected Total Cell Fluorescence.

Cell-Specific Roles of Plscr1 in Influenza Virus Infection in Mice

Wt mice were exposed to 2500 EID50 IAV (PR8) infection. Lungs were used for single-cell RNA sequencing analysis at 0, 1, 3, 6 and 21 dpi. (A) Two-dimensional UMAP representation of individual cells obtained from different timepoints. (B) Violin plot of aggregated Plscr1 expressions in all epithelial cell clusters. Red dots represent mean expression levels. (C) Violin plot of time-dependent Plscr1 expressions in both ciliated epithelial cell clusters. (D) GO analysis for upregulated pathways in Ciliated Epithelial Cells-1. (E) Heatmap of the most differentially expressed genes of Ciliated Epithelial Cells-1 at different timepoints. (F) Violin plot of aggregated Plscr1 expressions in all immune cell clusters. Red dots represent mean expression levels. (G) Violin plot of time-dependent Plscr1 expressions in alveolar macrophage cluster. (H) Violin plot of time-dependent Plscr1 expressions in neutrophil cluster.

Reduced Susceptibility of Plscr1floxStopFoxj1-Cre+ Mice to Influenza Virus Infection

Plscr1floxStop and Plscr1floxStopFoxj1-Cre+ mice were exposed to sublethal (300 pfu) IAV (WSN) infection and sacrificed at 3 dpi. (A) Schematic representation of the experimental design of of ciliated epithelial cell conditional Plscr1 KI mice. (B) Validation of Plscr1 overexpression in lungs of Plscr1floxStopFoxj1-Cre+ mice by qRT-PCR. (C) Representative immunofluorescent staining for Plscr1, Ifn-λr1 and Foxj1 in lungs. (D) Mean relative weight of mice. (E) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. (F) Infectious viral titer in the lungs was assessed by plaque assays. (G) Total BAL leukocyte numbers. (H) Neutrophil percentages in BAL. (I) Whole lungs were analyzed for Ifn-λr1 RNA by qRT-PCR and Ifn-λr1 protein by western blot. (J) Whole lungs were analyzed for Ifn-α, Ifn-β, Ifn-γ, Ifn-λ RNA by qRT-PCR. (K) Model depicting proposed mechanism of PLSCR1-regulated IFN-λ signaling. Data are expressed as mean ± SEM of n= 3-10 mice/group. ns, not significant, *p < 0.05, **p < 0.01, ***p<0.001. dpi, days post infection.

Model depicting proposed mechanism of PLSCR1-regulated IFN-λ signaling.

Influenza infection is usually detected by intracellular pattern recognition receptors (PRRs) such TLR 3 and 7, RIG-I and MDA5. These PRRs activate the expression of IFN-λ in early infection stage through IRF-3 and NK-κB-controlled transcriptions. IFN-λ secreted by the infected cells interacts with IL-10R2 and IFN-λR1 on neighboring cell surface, which results in activation of expression of various IFN-stimulated genes, including PLSCR1. In ciliated airway epithelial cells, PLSCR1 can further enhance the transcription of IFN-λR1 by directly binding to its promoter region as a transcriptional factor, or interact with IFN-λR1 on the cell membrane.

PCR Primer List

scRNA-Seq Cluster Annotations