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

Wt and Plscr1-/- mice were exposed to sublethal (300 pfu, B, C, G and F) or lethal (900 pfu, D and E) IAV (WSN) infection.

(A) Scheme of experiment.

(B) Whole lungs were analyzed for Plscr1 RNA by qRT-PCR.

(C and D) Mean relative weight of mice post sublethal or lethal infection.

(E) Probability of survival of mice post lethal IAV infection.

(F) Representative staining for H1N1 in lungs. The scale bars represent 1 mm. Quantification was performed using ImageJ.

(G) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR.

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. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001. dpi, days post infection.

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 concentration in BAL by ELISA.

Data are expressed as mean ± SEM of n = 5-10 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) Heatmap of downregulated ISGs in whole lungs by RNA-seq.

(C) Whole lungs were analyzed for Ifn-λr1 by qRT-PCR.

(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 = 4-8 mice or wells/group. 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-D) or 10 MOI (E).

(A) PLSCR1 RNA by qRT-PCR. Data are also presented as fold change compared to uninfected groups.

(B) IFN-λR1 RNA by qRT-PCR.

(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) 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) Aggregated Plscr1 expressions in all epithelial cell clusters.

(C) 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) Aggregated Plscr1 expressions in all immune cell clusters.

(G) Plscr1 expressions in alveolar macrophage cluster.

(H) 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) Generation 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) Total BAL leukocyte numbers.

(G) Neutrophil percentages in BAL.

(H) Whole lungs were analyzed for Ifn-λr1 RNA by qRT-PCR.

(I) Whole lungs were analyzed for Ifn-α, Ifn-β, Ifn-γ, Ifn-λ RNA by qRT-PCR.

(J) 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.

PCR primer list.

ScRNA-seq Cluster Annotations.

Requirement of Plscr1 in IFN-λ Signaling Independent of Viral Titer

Wt and Plscr1-/- mice were intranasally given 2.5 μg/g of body weight of poly(I:C) (HMW) constitutively for 6 days and sacrificed on day 7.

(A) Scheme of experiment.

(B) Total BAL leukocyte numbers.

(C) Differential cell counts in BAL

(D, F-G) Whole lungs were analyzed for Ifn-α, Ifn-β, Ifn-γ, Ifn-λ (D); Plscr1 (F); and Ifn-λr1 (G) RNA by qRT-PCR.

(E) Representative lung sections stained with H&E. Scale bars represent 3 mm (main) and 200 μm (inlays).

Data are expressed as mean ± SEM of n = 5-12 mice/group. All data were pooled from three independent experiments. ns, not significant, *p < 0.05, ***p<0.001.

PLSCR1 Transduction Efficiency and Distribution

PLSCR1 plasmids on PLV-EF1a-IRES-Hygro backbone were packaged into GFP-expressing lentivirus. PLSCR1-/- A549 cells were transduced using lentivirus. After a 10-day hygromycin selection, cells were analyzed using flow cytometry.

(A) Gating strategy for live, GFP+ and PLSCR1+ A549 cells.

(B) Surface, cytoplasm and nuclear expression of PLSCR1. Data are presented as fold change compared to WT-PLSCR1-/- A549 cells.

(C) Lentiviral transduction efficiency.

Unaffected Susceptibility of Plscr1floxStopLysM-Cre+ Mice to Influenza Virus Infection

Plscr1floxStop and Plscr1floxStopLysM-Cre+ mice were exposed to sublethal (300 pfu) IAV (WSN) infection.

(A) Validation of Plscr1 overexpression in lungs of Plscr1floxStopLysM-Cre+ mice by qRT-PCR.

(B) Mean relative weight of mice.

(C) Total BAL leukocyte numbers.

(D) Differential cell counts in BAL.

(E) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR.

(F) Representative lung sections stained with H&E.

(G-I) Whole lungs were analyzed for Ifn-α, Ifn-β, Ifn-γ, Ifn-λ (G); Ifn-λr1 (H); and Plscr1 (I) RNA by qRT-PCR.

Data are expressed as mean ± SEM of n = 15-16 mice/group for weight loss. For the rest analysis, n= 3-7 mice/group. All data were pooled from three independent experiments. ns, not significant, *p < 0.05, **p < 0.01. dpi, days post infection.