Evaluation of Nutritional Gel Supplementation in C57BL/6J Mice Infected with Mouse-Adapted Influenza A/PR/8/34 Virus

Abstract

Mice are a common animal model for the study of influenza virus A (IAV). IAV infection causes weight loss due to anorexia and dehydration, which can result in early removal of mice from a study when they reach a humane endpoint. To reduce the number of mice prematurely removed from an experiment, we assessed nutritional gel (NG) supplementation as a support strategy for mice infected with mouse-adapted Influenza A/Puerto Rico/8/34 (A/PR/8/34; H1N1) virus. We hypothesized that, compared with the standard of care (SOC), supplementation with NG would reduce weight loss and increase survival in mice infected with IAV without impacting the initial immune response to infection. To assess the effects of NG, male and female C57BL/6J mice were infected with IAV at low, intermediate, or high doses. When compared with SOC, mice given NG showed a significant decrease in the maximal percent weight loss at all viral doses in males and at the intermediate dose for females. Mice supplemented with NG had no deaths for either sex at the intermediate dose and a significant increase in survival in males at the high viral dose. Supplementation with NG did not alter the viral titer or the pulmonary recruitment of immune cells as measured by cell counts and flow cytometry of cells recovered in bronchoalveolar lavage (BAL) fluid in either sex. However, mice given NG had a significant reduction in IL6 and TNFα in BAL fluid and no significant differences in CCL2, IL4, IL10, CXCL1, CXCL2, and VEGF. The results of this study show that as compared with infected SOC mice, infected mice supplemented with NG have reduced weight loss and increased survival, with males showing a greater benefit. These results suggest that NG should be considered as a support strategy and indicate that sex is an important biologic variable in mice infected with IAV.

Figure 1.

NG supplementation in female (A, C, E) and male (B, D, F) mice infected with low influenza dose. (A, B) % of initial body weight following exposure to IAV over 14 dpi. Arrows represent the day SOC (red arrow) and NG (blue arrow) supplemented mice reached their peak weight loss. (C, D) Maximum percent weight loss for individual female (C) and male (D) mice on the study. Analysis was performed using an unpaired, 2-tailed, t tests. (E, F) Kaplan–Meijer survivor graph. Statistics performed using log-ranked (Mantel–Cox) test. Values are the mean ± SEM * indicates a significant finding (P £ 0.05). Females (SOC n = 9; NG n = 9); Males (SOC n = 13; NG n = 14).

Figure 2.

NG supplementation in female (A, C, E) and male (B, D, F) mice infected with intermediate influenza dose. (A, B) % of initial body weight following exposure to IAV over 14 dpi. Arrows represent the day SOC (red arrow) and NG (blue arrow) supplemented mice reached their peak weight loss. (C, D) Maximum percent weight loss for individual female (C) and male (D) mice on the study. Analysis was performed using an unpaired, 2-tailed, t tests. (E, F) Kaplan–Meijer survivor graph. Statistics performed using log-ranked (Mantel–Cox) test. Values are the mean ± SEM. For each data set significance is shown by *, P £ 0.05 and †, P £ 0.01. Females (SOC n = 12; NG n = 13); Males (SOC n = 18; NG n = 14).

Figure 3.

NG supplementation in female (A, C, E) and male (B, D, F) mice infected with high influenza dose. (A, B) % of initial body weight following exposure to IAV over 14 dpi. Arrows represent the day SOC (red arrow) and NG (blue arrow) supplemented mice reached their peak weight loss. (C, D) Maximum percent weight loss for individual female (C) and male (D) mice on the study. Analysis was performed using an unpaired, 2-tailed, t tests. (E, F) Kaplan–Meijer survivor graph. Statistics performed using log-ranked (Mantel–Cox) test. Values are the mean ± SEM. For each data set significance is shown by *, P £ 0.05 and †, P £ 0.01. Females (SOC n = 13; NG n = 12); Males (SOC n = 10; NG n = 10).

Figure 4.

Viral titers were performed at 5 dpi in A) female and B) male mice infected with the intermediate dose of IAV. Values are the mean ± SEM. Analysis performed by unpaired t test. SOC n = 5/sex; NG n = 5/sex.

Figure 5.

Total number of cells and total protein concentration in BAL fluid collected 7 dpi from female (A, C) and male (B, D) mice infected with PBS or the intermediate dose IAV given SOC or NG. (A, B) Total number of cells in BAL fluid in mice. (C, D) Total protein concentration levels from BAL fluid supernatant. Values are the mean ± SEM. Statistics were performed using a one-way ANOVA with Dunnet multiple comparisons test. For each data set significance is shown by *, P £ 0.05, †, P £ 0.01, ‡, P £ 0.001, and §, P £ 0.0001. For females and males, PBS n = 6/sex; SOC n = 10/sex; NG n = 10/sex.

Figure 6.

Total number of CD45+ staining inflammatory cells from BAL fluid collected 7 dpi from female mice infected with PBS or the intermediate dose IAV given SOC or NG. Populations of inflammatory cells identified through flow cytometry. An inflammatory response secondary to IAV infection was confirmed due to the increased levels of inflammatory cells noted between PBS and influenza infected mice. Total no. of A) alveolar macrophages (AM), B) interstitial macrophages (IM), C) neutrophils, D) T-cells, E) B-cells, F) eosinophils. Values are the mean ± SEM. G) Flow cytometry gating strategy used for identification of individual cell populations. Statistics were performed using a one-way ANOVA with Dunnet multiple comparisons test. For each data set significance is shown by *, P £ 0.05, †, P £ 0.01, ‡, P £ 0.001, and §, P £ 0.0001. PBS n = 6; SOC n = 10; NG n = 10.

Figure 7.

Total number of CD45+ staining inflammatory cells from BAL fluid collected 7 dpi from male mice infected with PBS or the intermediate dose IAV given SOC or NG. Populations of inflammatory cells identified through flow cytometry. Total no. of A) AM, B) IM, C) neutrophils, D) T-cells, E) B-cells, F) eosinophils. Values are the mean ± SEM. Statistics were performed using a one-way ANOVA with Dunnet multiple comparisons test. For each data set significance is shown by *, P £ 0.05, †, P £ 0.01, and ‡, P £ 0.001. PBS n = 6; SOC n = 10; NG n = 10.

Figure 8.

Concentration of chemokines/ cytokines from BAL fluid collected 7 dpi from female (Athrough H) and male (I through P) mice infected with the intermediate dose IAV given SOC or NG. Protein concentration of: A and I) IL6, B and J) CCL2, Cand K) TNFa, Dand L) IL4, E/M) IL10, Fand N) CXCL1, G and O) CXCL2, and Hand P) VEGF. Values are mean ± SEM. Statistics were performed using a one-way ANOVA with Dunnet multiple comparisons test. When cytokines/chemokines were nondetectable in mice given PBS, statistical analysis was performed using an unpaired t test. Note that y-axis values are different between graphs. For each data set significance is shown by *, P £ 0.05, †, P £ 0.01, ‡, P £ 0.001, and §, P £ 0.0001. No significance is indicated by ‘ns’ (P ³ 0.05). PBS n = 6/sex; SOC n = 10/sex; NG n = 10/sex.