Mice chronically fed high-fat diet have increased mortality and disturbed immune response in sepsis

Abstract

Background: Sepsis is a potentially deadly disease that often is caused by gram-positive bacteria, in particular Staphylococcus aureus (S. aureus). As there are few effective therapies for sepsis, increased basic knowledge about factors predisposing is needed.

Methodology/principal findings: The purpose of this study was to study the effect of Western diet on mortality induced by intravenous S. aureus inoculation and the immune functions before and after bacterial inoculation. Here we show that C57Bl/6 mice on high-fat diet (HFD) for 8 weeks, like genetically obese Ob/Ob mice on low-fat diet (LFD), have increased mortality during S. aureus-induced sepsis compared with LFD-fed C57Bl/6 controls. Bacterial load in the kidneys 5-7 days after inoculation was increased 10-fold in HFD-fed compared with LFD-fed mice. At that time, HFD-fed mice had increased serum levels and fat mRNA expression of the immune suppressing cytokines interleukin-1 receptor antagonist (IL-1Ra) and IL-10 compared with LFD-fed mice. In addition, HFD-fed mice had increased serum levels of the pro-inflammatory IL-1beta. Also, HFD-fed mice with and without infection had increased levels of macrophages in fat. The proportion and function of phagocytosing granulocytes, and the production of reactive oxygen species (ROS) by peritoneal lavage cells were decreased in HFD-fed compared with LFD-fed mice.

Conclusions: Our findings imply that chronic HFD disturb several innate immune functions in mice, and impairs the ability to clear S. aureus and survive sepsis.

Figure 1. Survival and bacterial load after intravenous S. aureus inoculation.

(A) Survival in mice fed: LFD before and after bacterial inoculation (LFD→LFD, n = 21), LFD before and HFD after bacterial inoculation (LFD→HFD, n = 10), HFD before and after bacterial inoculation (HFD→HFD, n = 18), HFD before and LFD after bacterial inoculation (HFD→LFD, n = 12). The HFD→HFD group had increased mortality, compared with LFD→LFD mice (P = 0.02). There was no difference between the LFD→HFD and LFD→LFD group (P = 0.9), or between the HFD→LFD group (P = 1). Log rank test. Data were collected from 2 different experiments. (B) Bacterial load in kidneys 5–7 days after bacterial inoculation in LFD- (n = 7) and HFD-fed (n = 6) mice. Mann-Whitney. Data are depicted as actual values or lowest/highest measurable value if not determinable. Detection limit is <0.01 CFU/2 kidneys (×10⁶), shown by the dotted line. Points represent individual mice; group median are denoted by a line throughout. (C) Survival in LFD-fed C57BL/6 (n = 15) and Ob/Ob (n = 18) mice after S. aureus inoculation. Ob/Ob mice had increased mortality compared with LFD-fed C57Bl/6 mice (P = 0.005). Log rank test.

Figure 2. Cytokine levels in serum, 5–7 days after bacterial inoculation.

(A) Serum levels of IL-1Ra in LFD- (n = 26) and HFD-fed (n = 23) mice. Detection limit is <31 pg/ml. Student's t-test on log data. (B) Serum levels of IL-10 in LFD- (n = 25) and HFD-fed (n = 21) mice. Detection limit is <13 pg/ml. Mann-Whitney. (C) Serum levels of IL-6 in LFD- (n = 25) and HFD-fed (n = 21) mice. Detection limit is <1.3 pg/ml. ANCOVA on log data with experiment as covariate. (D) Serum levels of IL-1β in LFD- (n = 25) and HFD-fed (n = 21) mice. Detection limit is <2 pg/ml. Student's t-test on log data. (E) Serum levels of TNF-α in LFD- (n = 25) and HFD-fed (n = 21) mice. Detection limit is <5.2 pg/ml. ANCOVA on log data with experiment as covariate. Data were collected from 3 different experiments. Data are depicted as actual values or lowest measurable value if not determinable; detection limit is denoted by the dotted line. In the scatter graphs, points represent individual mice; group means/medians are denoted by a line throughout.

Figure 3. Cytokine mRNA expression in gonadal adipose tissue.

(A–E) mRNA expression was measured in uninfected LFD- (n = 8) and HFD-fed (n = 8) mice, and in LFD- (n = 13) and HFD-fed (n = 9) mice 5–7 days after bacterial inoculation. mRNA from the following cytokine genes was measured: (A) Il1rn, (B) Il10, (C) Il6, (D) Il1b, and (E) Tnf,. Welch's test. In the scatter graphs, points represent individual mice (fold change as compared with uninfected LFD-fed mice); group geometrical means are denoted by a line throughout. Statistical comparisons are made between LFD- and HFD-fed mice in both infected and uninfected animals.

Figure 4. Monocyte proportion and function.

(A) Percentage of monocytes (Emr4⁺ cells) in blood, as determined by FACS, in uninfected LFD- (n = 5) and HFD-fed (n = 5) mice (Student's t-test), and 5–7 days after bacterial inoculation in LFD- (n = 7) and HFD-fed (n = 7) mice (Welch's t-test). (B) Emr1 mRNA expression in gonadal adipose tissue in uninfected LFD- (n = 8) and HFD-fed (n = 8) mice, and in LFD- (n = 12) and HFD-fed (n = 9) mice 5–7 days after bacterial inoculation. Welch's t-test. (C) Percentage of phagocytosing monocytes in uninfected LFD- (n = 5) and HFD-fed mice (n = 5). Welch's t-test. (D) Ingested bacteria per monocyte in uninfected LFD- (n = 5) and HFD-fed (n = 5) mice. Student's t-test. In the scatter graphs, points represent individual mice (fold change as compared with uninfected LFD-fed mice); group geometrical means are denoted by a line throughout. Statistical comparisons are made between LFD- and HFD-fed mice in both infected and uninfected animals.

Figure 5. Granulocyte proportion and function.

(A) Percentage of granulocytic neutrophils as determined by differential counts, in uninfected LFD- (n = 26) and HFD-fed (n = 29) mice (Student's t-test, data are collected from 2 different experiments), and 5–7 days after bacterial inoculation in LFD- (n = 7) and HFD-fed (n = 7) mice (Welch's t-test). (B) Percentage of phagocytosing granulocytes in uninfected LFD- (n = 5) and HFD-fed mice (n = 5) (Student's t-test), and 5–7 days after inoculation in LFD- (n = 14) and HFD-fed (n = 13) mice (Student's t-test on log data that were collected from 2 different experiments). (C) Ingested bacteria per granulocyte in uninfected LFD- (n = 5) and HFD-fed mice (n = 5) (Student's t-test), and 5–7 days after bacterial inoculation in LFD- (n = 14) and HFD-fed (n = 13) mice (Student's t-test on log data that were collected from 2 different experiments).

Figure 6. ROS production in lavage cells.

ROS production 5–7 days after bacterial inoculation in LFD- (n = 7) and HFD-fed (n = 6) mice. Peritoneal lavage cells were left unstimulated or stimulated with or PMA. ANCOVA on log data with experimental day as covariate.

Figure 7. Spleen weight and leukocyte fractions.

(A) Spleen weight in uninfected LFD- (n = 13) and HFD-fed (n = 13) mice, and 5–7 days after inoculation in LFD- (n = 16) and HFD-fed (n = 16) mice. Student's t-test on log data. Data were collected from 2 different experiments. (B) Spleenic leukocyte fractions in uninfected LFD- (n = 7) and HFD-fed (n = 8) mice. Emr1 (Welch's t-test), CD4 (Student's t-test), CD8 (Student's t-test), and CD19 (Student's t-test) were used as markers for monocytes/macrophages, T-helper, T cytotoxic, and B cells respectively.