Biological sex differences after high and low doses of influenza A virus infection during obesity

Figures

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  Figure 1. Progression of obesity and metabolic states in male and female mice. Male and female mice were fed with a low-fat diet (LFD) or a high-fat diet (HFD) for 14 weeks. Comparisons of (A) body mass after the 14th week, (B) body mass index (BMI) on the day of infection, and (C) gonadal adipose tissue (GAT), (D) inguinal adipose tissue (IAT), and (E) perirenal adipose tissue (PAT) on day of euthanization (i.e., 3 dpi) are compared between male and female mice, with or without obesity. A glucose tolerance test (GTT) was performed on the 14th week of diet treatment. Comparisons of (F) fed glucose, (G) GTT over time, and (H) GTT area under the curve (AUC) are shown. Comparisons of (I) leptin, (J) adiponectin, and (K) total cholesterol in plasma samples from blood collected at the 14th week are shown. Data is presented as mean ± standard error of the mean (SEM) (n=7-10/group). Statistical comparisons were done using one-way ANOVA followed by Tukey’s post-hoc comparisons or Kruskal-Wallis test followed by Dunn’s post-hoc comparisons. Repeated measurements in 1G were compared by repeated measures ANOVA (mixed model) and Tukey’s multiple comparisons. An asterisk (*) indicates a statistically…

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  Figure 2. Morbidity, virus replication, lung histopathology, and cytokine and chemokine responses following a high-dose IAV infection. Male and female mice, with or without obesity, were intranasally inoculated with a high dose (103 TCID50) of the 2009 pandemic H1N1 IAV or vehicle (i.e., medium) only and monitored for body mass and body temperature changes. (A) Percentage change in body mass and (B) body temperature, and (C) survival percentages are shown (n=4-5/group). Subsets of mice (n=3-6/group) were euthanized at 3 days post-infection (dpi); (D) lung viral titers were measured by TCID50 (dashed line, limit of detection), and (E–G) histopathology analysis was performed in the lungs by H & E staining. (E) Total inflammation scores and (F) parenchymal inflammation between the four virus-infected groups are presented. (G) Representative histopathology images of non-obese males (NO-M), males with obesity (O-M), non-obese females (NO-F), and females with obesity (O–F) are shown. Likewise, (H–L) fold changes in cytokines and chemokines IL-1β, IL-23, IL-17A, MCP-1, and MIP-1α in virus-infected mice compared to their medium-inoculated controls were compared. Data is presented as mean ±…

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  Figure 3. Morbidity and virus replication following a low-dose IAV infection. Male and female mice, with or without obesity, were intranasally inoculated with a low dose (i.e., 101.5 TCID50) of the 2009 pandemic H1N1 IAV and monitored for 21 days post-infection (dpi) for changes in body mass and temperature. (A) Absolute (gm) and (B) relative (%) changes in body mass and (C) relative (%) change in body temperature, and (D) survival curves are compared (n=5-8/group). Virus titers in the lungs were measured by TCID50 assay at (E) 3 dpi (n=7-9/group) and (F) 10 dpi (n=3-4/group). Data is shown as mean ± standard error of the mean (SEM). Statistical comparisons were done using one-way ANOVA followed by Tukey’s post-hoc comparisons or Kruskal-Wallis test followed by Dunn’s post-hoc comparisons. Repeated measurements in A-C were compared by repeated measures ANOVA (mixed model) and Tukey’s multiple comparisons. An asterisk (*) indicates a statistically significant difference (p<0.05) while a hash (#) indicates a trend (0.05 ≤ p ≤ 0.1). NO-M, non-obese males; NO-F, non-obese females; O-M, males with obesity; and O-F, females with obesity.

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  Figure 4. Progression of lung inflammation following low-dose IAV infection. Male and female mice, with or without obesity, were intranasally inoculated with a low dose (i.e., 101.5 TCID50) of the 2009 pandemic H1N1 IAV. Subsets of mice were euthanized at 3, 10, and 21 dpi, and histopathology analysis by hematoxylin and eosin (H&E) staining was performed. Total inflammation scores at 3, 10, and 21 dpi together with their representative images for the (A) non-obese males (NO-M), (B) non-obese females (NO-F), (C) males with obesity (O–M), and (D) females with obesity (O–F) are shown (n=2-6/group). Data is shown as mean ± standard error of the mean (SEM). Statistical comparisons were performed using the one-way ANOVA or Kruskal-Wallis test followed by Tukey’s or Dunn’s multiple comparisons, respectively. An asterisk (*) indicates a statistically significant difference (p<0.05) while a hash (#) indicates a trend (0.05 ≤ p ≤ 0.1). V, vessel; PA, pulmonary artery; MSB, main-stem bronchi; Br, bronchiole.

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  Figure 5. Cytokine/chemokine responses in the lungs following low-dose IAV infection. After infection with a low dose (i.e., 101.5 TCID50) of the 2009 pandemic H1N1 IAV, subsets of mice were euthanized at 3-, 10-, and 21-days post-infection (dpi), lungs were collected, and various cytokines and chemokines were measured. Fold changes in cytokines and chemokines in virus-infected mice at different days were determined by comparing to their respective medium-inoculated controls euthanized at 3 dpi. Absolute concentrations and fold changes for (A) TNF-α, (B) IL-1α, (C) IL-12p70, (D) IFN-γ, (E) IL-13, (F) IL-10, (G) IL-27, (H) IL-7, (I) MIP-1α, (J) RANTES, (K) GRO-α, and (L) IP-10, for 3-, 10-, and 21-dpi are shown. Statistical comparisons were made using one-way ANOVA or Kruskal-Wallis test, and p-values were adjusted for multiple comparisons using the Benjamini-Hochberg method. An asterisk (*) indicates a statistically significant difference (p<0.05) while a hash (#) indicates a trend (0.05 ≤ p ≤ 0.1). NO-M, non-obese males; NO-F, non-obese females; O-M, males with obesity; and O-F, females with obesity.

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  Figure 6. Characterization of myeloid and lymphoid cells in the lungs following a low-dose IAV infection. After infection with a low dose (i.e., 101.5 TCID50) of the 2009 pandemic H1N1 IAV, subsets of mice were euthanized at 3 days post-infection (dpi) to determine the frequencies of various myeloid and lymphoid cells in the lungs. In myeloid panel, the frequencies of (A) CD45+ cells, (B) neutrophils, (C) eosinophils, (D) alveolar macrophages, (E) M1 macrophages, (F) M2 macrophages, (G) M1 and (H) M2 type alveolar macrophages, (I) Ly6c+ and (J) Ly6c- monocytes, (K) interstitial macrophages, (L) M1 and (M) M2 type interstitial macrophages, (N) CD11b dendritic cells, and (O) monocyte-derived dendritic cells are compared. Likewise, frequencies of (P) CD3+ T cells, (Q) CD3+CD4+ T helper cells, (R) CD3+CD8+ T cells, (S) gamma delta T cells, and (T) NK cells are compared. Data is shown as mean ± standard error of the mean (SEM) (n=3-5/group). Statistical comparisons were performed using the one-way ANOVA or Kruskal-Wallis test, followed by Tukey’s or Dunn’s multiple comparisons, respectively. An asterisk (*) indicates a statistically significant difference (p<0.05) while a hash (#) indicates a…

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  Figure 7. Comparison of morbidity, virus replication, and pulmonary cytokine and chemokine responses between HFD-treated responder and non-responder females. Female mice were on HFD for 13 to 14 weeks. About 30% of them were non-responders. (A) Body mass, (B) body mass index (BMI), and (C) glucose tolerance test (GTT) area under the curve (AUC) between females with obesity (i.e., the responders) and non-responder females are compared (n=16-23/group). Likewise, (D) survival rates after high-dose IAV infection and (E) low-dose IAV infection are shown (n=4-8/group). Similarly, (F) virus titers and (G) log10-transformed cytokine and chemokine concentrations in the lungs at 3 dpi with a low-dose virus infection are compared (n=5-8/group). Data is shown as mean ± standard error of the mean (SEM). Statistical comparisons were made using the unpaired t-test or Mann-Whitney test, and an asterisk (*) indicates a statistically significant difference (p<0.05) while a hash (#) indicates a trend (0.05 ≤ p ≤ 0.1).