![Lung-localized immune responses differ between primary and secondary infections. Naïve and memory mice (previously infected with X31 [H3N2]) were challenged with PR8 (H1N1) influenza virus to assess primary and secondary immune responses, respectively. (A and B) Survival curve (A) and weight loss morbidity (B) of mice undergoing primary (red) and secondary (blue) infection, compiled from two independent experiments (n = 22–25 mice/group). Statistical significance was determined by the Mantel–Cox test (survival) and two-way ANOVA (weight loss). (C–G) RNA-seq was performed on whole lung samples of naïve and memory mice at baseline (uninfected, “0 dpi”) and 1–3 dpi for four to five mice per group, compiled from two independent experiments, representative of two independent experiments. (C) Principal component analysis (PCA) plot depicting the stratification of samples based on the top 500 differentially expressed genes. (D) Heatmap showing row-normalized significantly differentially expressed genes (Padj ≤ 0.05, fold change [FC] ≥2) comparing each infected time point with either naïve or memory uninfected controls (0 dpi) and grouped by k-means clustering, delineating 5 clusters (C1–C5). Each column represents the average of four to five biological replicates for each condition and time point. Data are representative of two independent experiments. Statistical significance was calculated by the Wald test. (E) Graphs show top five enriched GO pathways from C1 to C5. Gene ratio, which represents the portion of enriched genes in an ontology, is indicated on the x axis. The dot size depicts the number of genes from each cluster mapping to a specific ontology. Select genes from each cluster are labeled on the right. Statistical significance was calculated by the Wilcoxon rank-sum test. (F) Fold-change expression (mean ± SD) of genes induced within each cluster in the primary (red) and secondary (blue) response relative to baseline. Statistical significance was determined by two-way ANOVA. (G) Quantification of influenza-specific genes encoding HA, NP, and polymerase (PB1 and PB2) proteins in primary (red) and secondary (blue) infections shown as normalized counts extracted from lung RNA-seq data (see Materials and methods). Line graphs are presented as the mean ± SD. Statistical significance was calculated by the Wald test. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05. HA, hemagglutinin; NP, nucleoprotein. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jem/223/1/10.1084_jem.20242307/1/jem_20242307_fig1.png?Expires=1777800547&Signature=y9LA~DVyDdqUSr4muJFcIotYHdj6oC-lo129qOMNzwYxHD-7r98O0UCJDZGKogG2QcCEnzTtQ0N4MfjXovfymIjqrlGUTEByRBH-M6SwOtiRwV3vUedOyDlrqqbJCv0XSUEanP0lma40F2-y7M3cDmETdjNji3ikRvNgiv0dS8kmOTFaDrOrDRqn7ED1A1-R5XK2N-R-KUaKEUq8TCT0~CuWqAu-GDXJbBO5Qi-m7OVoLD~C6fpuRiTnkunUpG0b~iFSFHP1WalxI4BGGhdVBeDdAz7FaaBy8-s~r~asq4QpB~001fbEYFdnoqCZNQO0FO5viwOKC7VjFP0Hm0Ei1w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Lung-localized immune responses differ between primary and secondary infections. Naïve and memory mice (previously infected with X31 [H3N2]) were challenged with PR8 (H1N1) influenza virus to assess primary and secondary immune responses, respectively. (A and B) Survival curve (A) and weight loss morbidity (B) of mice undergoing primary (red) and secondary (blue) infection, compiled from two independent experiments (n = 22–25 mice/group). Statistical significance was determined by the Mantel–Cox test (survival) and two-way ANOVA (weight loss). (C–G) RNA-seq was performed on whole lung samples of naïve and memory mice at baseline (uninfected, “0 dpi”) and 1–3 dpi for four to five mice per group, compiled from two independent experiments, representative of two independent experiments. (C) Principal component analysis (PCA) plot depicting the stratification of samples based on the top 500 differentially expressed genes. (D) Heatmap showing row-normalized significantly differentially expressed genes (Padj ≤ 0.05, fold change [FC] ≥2) comparing each infected time point with either naïve or memory uninfected controls (0 dpi) and grouped by k-means clustering, delineating 5 clusters (C1–C5). Each column represents the average of four to five biological replicates for each condition and time point. Data are representative of two independent experiments. Statistical significance was calculated by the Wald test. (E) Graphs show top five enriched GO pathways from C1 to C5. Gene ratio, which represents the portion of enriched genes in an ontology, is indicated on the x axis. The dot size depicts the number of genes from each cluster mapping to a specific ontology. Select genes from each cluster are labeled on the right. Statistical significance was calculated by the Wilcoxon rank-sum test. (F) Fold-change expression (mean ± SD) of genes induced within each cluster in the primary (red) and secondary (blue) response relative to baseline. Statistical significance was determined by two-way ANOVA. (G) Quantification of influenza-specific genes encoding HA, NP, and polymerase (PB1 and PB2) proteins in primary (red) and secondary (blue) infections shown as normalized counts extracted from lung RNA-seq data (see Materials and methods). Line graphs are presented as the mean ± SD. Statistical significance was calculated by the Wald test. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05. HA, hemagglutinin; NP, nucleoprotein.