Fig 1.
PWD alleles on Chr16 provide complete protection from lethal IAV challenge.
(A–C) Male B6 and PWD mice were challenged with 2 LD50 or 5 LD50 doses of PR8 IAV (LD50 dose was pre-determined on B6 mice). Survival (A) and weight loss (B) are shown, with P values in each panel indicating overall significance of differences between the groups, as determined by Mantel-Cox test and 2-way ANOVA, respectively. (C) On day 22 post-infection, serum was collected from PWD mice and uninfected naïve PWD mice, and anti-IAV antibody titers were determined by ELISA (as described in the Materials and Methods). P values indicate the significance of difference between the indicated groups, as determined by student’s T test. (D, E) Male and female B6 and B6.Chr16PWD (Chr16) mice were challenged with ~LD50 of PR8 IAV. Survival (D) and weight loss (E) are shown, representing pooled data from males and females with P values in each panel indicating significance of differences between the strains, as determined by Mantel-Cox test and 2-way ANOVA, respectively. The number of animals per condition is indicated in each panel.
Fig 2.
Sex differences and normalization of IAV severity in male and female B6 mice.
(A) B6 male and female mice were challenged with equal doses of PR8 IAV, and survival was assessed. (B and C) B6 male and female mice were challenged with sex-adjusted estimated LD50 of PR8 IAV (33% lower dose for females). Significance of differences in survival between females and males was determined using the Mantel-Cox test, significance of differences in weight loss was determined by two-way ANOVA (overall effect of sex). P values are indicated below each panel title. The number of animals per condition is indicated in each panel.
Fig 3.
IAV challenge of B6.ChrPWD consomic mice reveals a wide range of susceptibility phenotypes.
B6 control and B6.ChrPWD male and female mice were infected with sex-adjusted doses of PR8 as in Fig 2B. Significance of difference in survival between B6 and each consomic strain (male and female data pooled by strain) was assessed by Mantel-Cox test. (A) A bubble volcano plot showing significance of survival (-Log10(P)) compared with B6 plotted vs. % survival, across all consomic strains, with strains of interest labeled. The size of the circle indicates the total number of animals studied per strain (N), as indicated. (B) Survival analysis of B6 and consomic mice, with strains of interest labeled (male and female data pooled by strain). Complete data on survival analysis for each strain are presented in Table 1. (C) Comparison of survival between B6 and B6.Chr17PWD consomics, Chr17S (short) and Chr17F (full), (male and female data pooled by strain). ** signifies a P value of <0.01, as determined by Mantel-Cox test.
Table 1.
Survival analysis of IAV-challenged consomic strains using pooled sex data.
The indicated number (N) of each strain were challenged with IAV as described in Fig 3. Male and female data were pooled. Significance of differences in survival between B6 and each consomic strain were assessed using the Mantel-Cox test and the P value are reported, with P<0.05 shown in bold font. Percent of mice surviving to the endpoint on day 20 is shown. Numbers of male and female mice studied are also shown. Data from for B6.Chr17PWD represent pooled data from Chr17S and Chr17F strains. The low N for some strains (Chr13, Chr7, etc.) was a result of low availability of animals due to poor breeding performance.
Fig 4.
Genotype-dependent sex differences in IAV susceptibility of B6.ChrPWD consomic mice.
Male (M) and female (F) B6 control and B6.Chr5PWD (A-D) and B6.ChrX.3PWD (E-H) mice were infected with sex-adjusted doses of PR8 as in Fig 2B. The following comparisons were made: (A and E) male consomic vs. male B6 control, (B and F) female consomic vs. female B6 control, and (C, D, G and H) male vs. female of each consomic. Significance of difference in survival for each comparison assessed by Mantel-Cox test, while significance of difference in weight loss was assessed by two-way ANOVA (overall effect of sex). The P value for each comparison is indicated in each corresponding graph, in red font if below P<0.05. Complete data on survival analysis for all consomic strains are presented in Tables 1 and 2.
Table 2.
Survival analysis of IAV-challenged consomic strains using sex-stratified data.
Male and female consomic mice were challenged with IAV as described in Fig 3. Significance of differences in survival assessed using the Mantel-Cox test and the P value are reported, with P<0.05 shown in bold font. The following three comparisons were made: male vs. females of each consomic strain (Male vs. Female, column 1), consomic males vs. B6 males (Consomic M vs. B6 M, column 2), and consomic females vs. B6 females (Consomic F vs. B6 F, column 3).
Fig 5.
Differences in IAV viral load between B6, ChrX.3PWD, Chr5PWD and male and female mice.
Male and female B6 and ChrX.3 mice (A) or B6 and Chr5 mice (B) were infected with PR8 IAV as in Fig 3. On day 6 post-infection, whole lung RNA was extracted as described in the Materials and Methods. Relative expression of IAV NS1 gene RNA was assessed using qRT-PCR and normalized to host housekeeping gene B2m. Data were analyzed by two-way ANOVA with Sidak’s post-hoc comparisons, with P values representing significance of differences as indicated for each comparison.
Fig 6.
Sex differences in genetically regulated host lung responses in B6 and ChrX.3PWD mice.
Male and female B6 and B6.ChrX.3PWD (ChrX.3) mice were infected with PR8 IAV as in Fig 3. On day 6 post-infection, whole lung RNA was processed for RNA sequencing and analysis as described in Materials and Methods. (A) Principal component analysis was performed in DEseq2, demonstrating clustering of individual samples by sex and genotype, as annotated. (B, C, and D) DEseq2 was used to determine differentially expressed genes between X.3 and B6 mice within males or females, as annotated, using a cutoff of |Log2 (Fold Change)|>0.6 and Padjusted (Padj) <0.05. (B) A row-normalized gene expression heatmap showing DEGs in males, females, or both sexes, as indicated. Volcano plots (C) and Venn diagrams (D) illustrating differential gene expression between ChrX.3 and B6. (E) Biological pathway enrichment analysis on DEGs upregulated in ChrX.3 in males was performed using Gene Ontology as described in Materials and Methods. Top 20 enriched pathways are shown. (F) ImmGen MyGeneSet analysis was performed on DEGs upregulated in ChrX.3 in males as described in the Materials and Methods. Heatmap indicates relative expression of DEGs in cell populations of interest, with each column representing a cell type or cells state within the broader cell categories labeled across the top. (G) A Venn diagram indicating overlap between genes differentially expressed between males vs. females in ChrX.3 and B6 mice.
Table 3.
Positional candidate genotype-dependent DEGs that mapped to ChrX.3 interval.
Genes differentially expressed between ChrX.3 males and B6 males (male), ChrX.3 females and B6 females (female), or in both sexes (both), which mapped to Chr X.3 interval (Chr = X; position = 88,400,000 bp or greater) from the RNAseq data described in Fig 6. Direction of change, up indicates increased expression in ChrX.3 relative to B6, down indicates decreased expression. A cutoff filter of |log2(FoldChange)| = 0.6, Padj = <0.05 was used to identify DEGs.
Table 4.
Sex-dependent DEGs mapping to the sex chromosomes.
Genes differentially expressed between males and females in: B6 mice only (B6; none in this category), ChrX.3 mice only (ChrX.3), or in both strains (both) which mapped to ChrX or ChrY, from the RNAseq data described in Fig 6. Direction of change: up indicates increased expression in males relative to females, down indicates increased expression in females relative to males. A cutoff filter of Padj = <0.05 was used to identify DEGs, without a fold change cutoff.