Protein energy malnutrition decreases immunity and increases susceptibility to influenza infection in mice

Abstract

Background: Protein energy malnutrition (PEM), a common cause of secondary immune deficiency in children, is associated with an increased risk of infections. Very few studies have addressed the relevance of PEM as a risk factor for influenza.

Methods: We investigated the influence of PEM on susceptibility to, and immune responses following, influenza virus infection using isocaloric diets providing either adequate protein (AP; 18%) or very low protein (VLP; 2%) in a mouse model.

Results: We found that mice maintained on the VLP diet, when compared to mice fed with the AP diet, exhibited more severe disease following influenza infection based on virus persistence, trafficking of inflammatory cell types to the lung tissue, and virus-induced mortality. Furthermore, groups of mice maintained on the VLP diet showed significantly lower virus-specific antibody response and a reduction in influenza nuclear protein-specific CD8(+) T cells compared with mice fed on the AP diet. Importantly, switching diets for the group maintained on the VLP diet to the AP diet improved virus clearance, as well as protective immunity to viral challenge.

Conclusions: Our results highlight the impact of protein energy on immunity to influenza infection and suggest that balanced protein energy replenishment may be one strategy to boost immunity against influenza viral infections.

Figure 1.

A, B, Very low protein (VLP) diet leads to reduced growth and a decrease in serum leptin concentration. Mice maintained on the adequate protein (AP) and VLP diets were assessed daily for percent change in original body weight (A) and serum leptin concentration (B) at 5 weeks after beginning the feeding regimen. A, Results are shown from 1 of 2 independent experiments and consists of 5 mice per group. B, Serum leptin concentration is shown from 6 mice in each group. Error bars represent mean ± SEM. The differences in mean body weights between the VLP and AP groups were statistically significant as follows: P < .001 for day 9 and P < .0001 for days 10–38. C, Mice on the VLP diet show increased susceptibility to influenza infection. Mice maintained on the AP or VLP diets were infected with either A/PR8 or A/Mex influenza and assessed for virus-induced mortality (percent survival). Data represent results from 3 independent experiments.

Figure 2.

Influenza A virus–infected mice maintained on a very low protein (VLP) diet show a defect in viral clearance in the lungs. Lung tissues harvested from mice on the adequate protein (AP) or VLP diets, on days 3, 6, 9, and 12 postinfection (A/PR8, 50 MID₅₀; A/Mex, 100 MID₅₀), were homogenized and assayed for virus titer as described in Materials and Methods. Data represent values from 2 independent experiments with each experiment consisting of n = 6 lung tissues per group at each time point. Values represent mean ± SEM.

Figure 3.

Lung tissues from mice fed the very low protein (VLP) diet show increased inflammation following influenza infection. A, Lung tissues from adequate protein (AP) and VLP diet fed mice were harvested at 6 days postinfection (A/PR8, 50 MID₅₀; A/Mex, 100 MID₅₀) and analyzed using histo-chemical staining (hematoxylin and eosin) as described in Materials and Methods. Representative images (magnification, 10 × ) from an experiment consisting of 3 mice per group are shown. B, Lung tissues from mice fed the AP and VLP diets, harvested at days 3 and 6 postinfection, were analyzed for percent neutrophils (CD11b⁺Ly6G⁺) (left panel) and NK cells (CD3⁻NK1.1⁺) (right panel) as described in Materials and Methods. Values in panel B represent mean ± SEM, n = 6/group, from 2 independent experiments.

Figure 4.

Mice maintained on the very low protein (VLP) diet show a decrease in influenza-specific hemagglutination inhibition (HI) antibody titer and NP-specific CD8⁺ T cells. A, Serum samples harvested from mice fed the adequate protein (AP) and VLP diets, at 30 days postinfection (A/PR8, 50 MID₅₀; A/Mex, 100 MID₅₀), were analyzed for HI titer by HI assay as described in Materials and Methods. B, Splenocytes harvested at days 8, 15, 30 days postinfection (A/PR8, 50 MID₅₀) were analyzed for influenza NP-specific CD8 T cells using flow cytometry as described in Materials and Methods. B, Shown is the percent of NP-specific CD8⁺ T cells. Values in panel A are combined from 2 independent experiments. Values in panel B represent mean ± SEM, n ≥ 3 per group.

Figure 5.

A, Schematic representation of diets used, virus infection, and assessments for markers of infection and immunity in the supplemental protein (SP) group of mice. Groups of mice were fed either adequate protein (AP) or very low protein (VLP) diets. Three weeks later, a subgroup of mice fed the VLP diet was switched to the AP diet and is referred to as the SP group. Three weeks later, all 3 groups of mice were infected with influenza virus (A/PR8) and assessed for markers of susceptibility to infection and immunity. B–D, Supplementing the mice fed the VLP diet with the diet containing higher protein energy modifies immune deficits and decreases susceptibility to A/PR8 infection. Mice fed with the AP, VLP, or SP diets were examined for change in body weight prior to switching diet (B) and after infection with A/PR8 (25 MID₅₀) (C). Dotted line indicates the time point when the VLP diet was switched to the AP diet for the SP group of mice. Mice fed with the AP, VLP, or SP diets were examined for mortality following infection with A/PR8 (25 MID₅₀) (D). Data represent results from 2 to 3 independent experiments. Values in panels A represent mean ± SEM. The differences in mean body weights between the VLP and SP groups for preinfection phase (A, left panel) were statistically significant as follows: P < .0001 for days 27, 30, 33, 36, 39, and 42. The differences in mean body weights between the VLP and SP groups for postinfection phase (A, right panel) were statistically significant as follows: day 12, P = .03; day 13, P = .03; day 14, P = .004; day 15, P = .001; day 16, P = .0004; day 17, P = .0001; and day 18, P < .0001.

Figure 6.

Switching from the very low protein (VLP) diet to the adequate protein (AP) diet enhances antiviral innate immune response and promotes virus clearance. Groups of mice fed with the AP, VLP, or supplemental protein (SP) diet regimen were either infected with influenza virus (A/PR8) or administered with phosphate-buffered saline (PBS), as described in Materials and Methods. Lung homogenates were assayed for virus titer on days 6 and 9 postinfection (A) and interferon γ (IFN-γ) on day 6 postinfection (B), as described in Materials and Methods. Data in panels A and B represent results from 2 independent experiments and consists of lung tissue harvested from 6 mice at each time point. Values represent mean ± SEM.

Figure 7.

Switching from very low protein (VLP) diet to adequate protein (AP) diet modulates virus-specific adaptive immunity. Groups of mice fed with the AP, VLP, or supplemental protein (SP) diet were either infected with influenza virus (A/PR8) or administered with phosphate-buffered saline (PBS), as described in Materials and Methods. Percent NP-specific CD8⁺ T cells, percent interferon γ (IFN-γ)+CD4⁺ T cells, and percent IFN-γ+CD8⁺ T cells on days 8, 15, and 30 postinfection in the spelenocytes (A) and hemagglutination inhibition (HI) antibody titer on day 30 postinfection in the serum (B) were analyzed as described in Materials and Methods. A, Data represent results from 2 independent experiments with 3–6 mice per group. B, Each symbol represents an individual mouse from 2 independent experiments consisting of 8 mice per group, and the horizontal line indicates the mean value for the group.