Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF-1α axis plays a key role in host resistance to Plasmodium infection
- Translational Medicine Platform Oswaldo Cruz Foundation, Ribeirão Preto Medical School, University of Sao Paulo, Brazil
- Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of Sao Paulo, Brazil
- Technology Center for Vaccines, Federal University of Minas Gerais, Brazil
- Department of Pathology, University of Michigan Medical School, United States
- Laboratory of Immunopathology, Instituto René Rachou, Fundação Oswaldo Cruz - Minas, Brazil
- Department of Medicine, Division of Infectious Diseases and Immunology, University of Massachusetts Chan Medical School, United States
- Department of Pharmacology, Ribeirão Preto Medical School, University of Sao Paulo, Brazil
Figures
Figure 1
P. chabaudi-infected mice display enhanced TNF production associated with increased disease parameters.
C57BL/6 mice were infected or not i.p. with Pc (105 iRBCs). (A) Parasitemia was determined at 0, 3, 5, 7, 8, 10, and 12 days post-infection (dpi). (B) Blood glucose levels were measured with a glucometer at 0, 3, 5, 7, 8, 10, and 12 dpi. Means of blood glucose levels were compared between infected (red) and uninfected (blue) mice at the same time points. (C) Blood TNF levels were measured by ELISA at 8 dpi. Means of blood TNF levels were compared between infected (red) and uninfected (blue) mice at the same time points. (D) Tnf mRNA from mice liver was determined at 8 dpi. (E) Parasitemia was determined at 8 dpi in C57BL/6 and Tnfrsf1a/b-/- mice. (F) Rectal temperature was determined at 8 dpi in C57BL/6 and Tnfrsf1a/b-/- mice. (G–J) MCP-1, TNF, IFN, and IL-10 were measured by CBA Kit at 8 dpi. (K) Blood glucose levels were measured with a glucometer at 8 dpi. (A, B, and F) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group; Statistical analysis: One-way ANOVA with Tukey post-hoc test. (C and D) Graph shows mean ± SEM of combined data from three independent experiments with four to six mice per group; Statistical analysis: One-way ANOVA with Bonferroni post-hoc test. (E, G, H, I, J, K) Graphs show mean ± SEM of combined data from three independent experiments with four to six mice per group; Statistical analysis: One-way ANOVA with Tukey post-hoc test. Asterisks above the bars indicate comparisons between mice of the same strain before infection and at 8 dpi. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, **** p≤0.0001, ns = non-significant.
Figure 2
The detrimental effects of P. chabaudi infection on physical activity, food intake, and energy metabolism are partially dependent on TNF receptor signaling.
Mouse energy balance from C57BL/6 and Tnfrsf1a/b-/- was assessed using metabolic cages (TSE 959 Systems, Chesterfield, MO) at 3 days prior to infection (uninfected) (C57BL/6: blue, Tnfrsf1a/b-/-: gray) and at 8 days postinfection (dpi) with Pc (105 infected RBCs) (C57BL/6: red, Tnfrsf1a/b-/-: orange). Scores for (A and E) physical activity, (B and F) food intake, (C and G) energy expenditure, (D and H) respiratory exchange, and (I and J) percentage of fat mass and lean mass are depicted. Hourly measurements of such parameters are shown in top row panels for mice 3 days prior to infection (uninfected) and in middle row panels for infected mice. The average values for light and dark cycles in all groups are depicted in bottom row panels. (K and L) Glucose uptake was measured 2-[14 C] deoxyglucose. Experiments were performed at the National Mouse Metabolic Phenotyping Center (MMPC) at UMASS Medical School. Basal glucose uptake in individual organs of infected (red) and uninfected (blue) mice was measured using an intravenous injection of 2-[14 C] deoxyglucose. After 1 h, mice were anesthetized, and tissue samples were taken for organ-specific levels of 2-[14 C] deoxyglucose-6-phosphate. (A - L) Graph shows mean ± SEM of combined data from two independent experiments; Statistical analysis: One-way ANOVA with Tukey post-hoc test. Asterisks above the bars indicate comparisons between mice of the same strain before infection and at 8 dpi. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
Figure 3 with 2 supplements
P. chabaudi infection stimulates increased GLUT1 expression in hepatic non-parenchymal and splenic CD11b+ cells associated with an altered host metabolic profile.
C57BL/6 mice were infected or not i.p. with Pc (105 iRBCs). (A) RNA-seq was performed in livers from infected and uninfected mice. (B) GLUT1 expression in livers of infected (8 dpi) or uninfected mice was evaluated by western blot. (C and D) GLUT1 expression in (C) hepatocytes or (D) hepatic non-parenchymal cells isolated from livers from C57BL/6-infected (8 dpi) or uninfected mice, quantified by western blot (expression of β-actin was used as control). (E) Hepatocytes or (F) hepatic non-parenchymal cells were purified from livers harvested from infected (red - 8 dpi) and uninfected (blue) mice and cultured ex vivo for quantification of extracellular acidification rate (ECAR) by Seahorse XFe96. (G, I, and J) T cells, B cells, and myeloid cells (CD11b+) were purified from splenocytes of infected and uninfected mice using magnetic beads and (G) GLUT1 expression was evaluated by western blot. (I) T and B cells or (J) CD11b+purified from spleens were cultured ex vivo for quantification ECAR. (H, K, L, and M) Graphs showing GLUT1 expression evaluated by flow cytometry in splenic cells from infected (red – 8 dpi) and uninfected (blue) mice. A. Heatmap represents three biological replicates performed in RNA-seq. (B, C, D, and G) Western blot images representative of at least three independent experiments. (E, F, I, and J) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group; Statistical analysis: Two-way ANOVA followed by Sidak’s post-hoc test employing mixed effect analysis provided by Agilent Seahorse XFe96 analyzer software. (H, K, L, and M) Representative dot plots and graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group; Statistical analysis: Student’s t test. Asterisks above the bars indicate comparisons between mice of the same strain before infection and at 8 dpi. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
-
Figure 3—source data 1
PDF file containing original western blots for Figure 3B, C, D and G, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/97759/elife-97759-fig3-data1-v1.zip
-
Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3B, C, D and G.
- https://cdn.elifesciences.org/articles/97759/elife-97759-fig3-data2-v1.zip
Figure 3—figure supplement 1
Gating strategies for flow cytometry analysis from spleens.
(A) Lymphoid cells: B lymphocytes (Live/ Lymphocytes/ CD45+/ CD19+), CD4+T lymphocytes (Live/ Lymphocytes/ CD45+/ CD3+/ CD4+), and CD8+T lymphocytes (Live/ Lymphocytes/ CD45+/ CD3+/ CD8+). (B) Myeloid monocytic cells: (Live/ CD45+/ F4/80+/ CD11b+CD11c+/ Ly6G-). (C) Representative histogram of GLUT1 expression that was evaluated by flow cytometry in splenic cells from infected (red – 8 dpi) and uninfected (blue) mice.
Figure 3—figure supplement 2
Densitometric analysis of GLUT expression in liver and splenic cell populations.
(A) Representative immunoblot and densitometric quantification of GLUT1 protein levels in whole liver extracts from control and infected C57BL/6 mice, normalized to β-actin. (B) Immunoblot analysis and corresponding densitometry of GLUT1 expression in total liver homogenates and isolated hepatocytes from control and infected C57BL/6 mice. (C) GLUT1 protein levels in non-parenchymal liver cells from control and infected C57BL/6 mice, quantified by densitometric analysis and normalized to β-actin. (D) Immunoblot detection and densitometric quantification of GLUT1 expression in splenic T cells, B cells, CD11b+ cells, and total spleen lysates from control and infected C57BL/6 mice. Bars represent control and infected conditions for each genotype, and statistical comparisons are indicated as shown. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
Figure 4 with 1 supplement
P. chabaudi infection triggers increased glycolysis in monocytic cells in a TNF-receptor-dependent way.
C57BL/6 or Tnfrsf1a/b-/- mice were infected or not i.p. with P. chabaudi (105 iRBCs). All experimental groups survived Pc infection throughout the observation period. (A) Glycolytic and gluconeogenesis pathways. Relative expression of mRNA glycolytic (B–E) or gluconeogenesis (F–J) enzymes in livers of control or infected (8 dpi) mice. (K) GLUT1, GLUT2, and NAK-ATPase expression in livers of infected (8 dpi) or uninfected mice evaluated by western blot. (L) Representative histogram and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/Ly6G- cells from spleens of infected (8 dpi) or uninfected C57BL/6 or Tnfrsf1a/b-/- mice. (M) Representative histogram and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/MHCII + cells from spleens of uninfected C57BL/6 or Tnfrsf1a/b-/- mice stimulated or not with TNF (100 ng/ml) for 18 hr. (N and O) CD11b+ cells were purified from spleens harvested from infected (8 dpi) C57BL/6 (red) and Tnfrsf1a/b-/- (orange) mice or uninfected C57BL/6 (blue) and Tnfrsf1a/b-/- (gray) mice and cultured ex vivo for evaluation of ECAR by Seahorse XFe96 Analyzers. (P) Parasitemia was determined at 8 dpi from TNFR1 cKO and WT (Tnfrsf1afl/fl) mice. (Q) Rectal temperature was determined at 8 dpi in WT and TNFR1 cKO at 8 dpi. (R) Blood glucose levels were measured with a glucometer at 8 dpi. (B, C, D, E, F, G, H, I, J, and M) Graphs show mean ± SEM of combined data from two independent experiments with three to five mice per group; Statistical analysis: One-way ANOVA followed by Tukey post-test. (K) Representative dot plot of one representative experiment representative of three independent ones; (L, P, Q, R) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group, Statistical analysis: Student’s t test. (N) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group; (O) Violin plots of combined data from three independent experiments with three to five mice per group – individual dots represent the average of triplicates in each experiment – the difference refers to the number of cells isolated from naïve vs infected animals, which varied. (N and O) Statistical analysis: Two-way ANOVA followed by Sidak’s post-test employing mixed effect analysis provided by Agilent Seahorse XFe96 analyzer software. Asterisks above the bars indicate comparisons between mice of the same strain before infection and at 8 dpi. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
-
Figure 4—source data 1
PDF file containing original western blots for Figure 4K, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/97759/elife-97759-fig4-data1-v1.zip
-
Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4K.
- https://cdn.elifesciences.org/articles/97759/elife-97759-fig4-data2-v1.zip
Figure 4—figure supplement 1
Representative immunoblots and densitometric quantification of GLUT1 and GLUT2 expression in liver samples from control and infected C57BL/6 and Tnfrsf1a/b-/- mice, normalized to Na+/K+-ATPase.
Bars represent control and infected conditions for each genotype, and statistical comparisons are indicated as shown. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
Figure 5 with 2 supplements
HIF-1α contributes to glycolysis induction during Pc infection.
HIF-1α expression in the nuclear extract of cells from spleen (A) and liver (B) of infected (8 dpi) or uninfected C57BL/6 and Tnfrsf1a/b-/- mice evaluated by western blot. (Expression of nucleophosmin [NPM] was used as control). (C) Representative histogram and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/MHCII + cells from spleens of infected (8 dpi) or uninfected HIF-1a cKO and WT (Hif1afl/fl) mice. (D) Representative histogram and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/MHCII + cells from spleens of uninfected HIF-1a cKO and WT mice stimulated or not with TNF (100 ng/ml) for 18 hr. (E and F) CD11b+ cells were purified from spleens harvested from infected (8 dpi) WT (red) and HIF-1a cKO (pink) mice or uninfected WT (Hif1afl/fl) (blue) and HIF-1a cKO (purple) mice and cultured ex vivo for evaluation of ECAR by Seahorse XFe96 Analyzers. (G) Parasitemia was determined at 8 dpi from HIF-1a cKO and WT (Hif1afl/fl) mice. (H) Blood TNF levels were measured by ELISA at 8 dpi. (I) TNF levels in supernatants from splenic cells harvested from infected (8 dpi) or uninfected HIF-1a cKO and WT (Hif1afl/fl) mice and stimulated ex vivo with LPS [1 µg/mL] or not during 24 hr. (A and B) Representative dot plot of one representative experiment representative of three independent ones. (C) Graphs show mean ± SEM of one representative experiment of three independent ones with three to five mice per group; Statistical analysis: Student’s t test. (D) Graphs show mean ± SEM of one representative experiment of two independent ones with three to five mice per group; Statistical analysis: Student’s t test. (E) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group. (F) Violin plots of combined data from three independent experiments with three to five mice per group – individual dots represent the average of triplicates in each experiment – the difference refers to the number of cells isolated from naive vs infected animals, which varied. (E and F) Statistical analysis: Two-way ANOVA followed by Sidak’s post-hoc test employing mixed effect analysis provided by Agilent Seahorse XFe96 analyzer software. (G and H) Graphs show mean ± SEM of combined data from three independent experiments with three to five mice per group; Statistical analysis: Student’s t test. (I) Graphs show mean ± SEM of one representative experiment of three independent ones with three to five mice per group; Statistical analysis: One-way ANOVA followed by Tukey post-hoc test. Asterisks above the bars indicate comparisons between mice of the same strain before infection and at 8 dpi. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
-
Figure 5—source data 1
PDF file containing original western blots for Figure 5A and B indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/97759/elife-97759-fig5-data1-v1.zip
-
Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5A and B.
- https://cdn.elifesciences.org/articles/97759/elife-97759-fig5-data2-v1.zip
Figure 5—figure supplement 1
HIF1-α deficiency results in higher parasitemia following P. chabaudi infection.
Parasitemia from wild type (pink) and HIF-1a cKO (purple) infected mice was determined at 0, 3, 5, 8, 10, 12, 14, 16, 18, 21, and 26 days-post-infection (dpi). ***p≤0.001 (t-test).
Figure 5—figure supplement 2
TNF signaling is required for HIF-1α activation in the liver during P. chabaudi infection.
(A) Immunoblot and densitometric analysis of HIF-1α protein levels in spleen lysates from control and infected C57BL/6 mice, normalized to nucleophosmin (NPM). (B) Representative immunoblot of HIF-1α protein levels in liver extracts from control and infected C57BL/6 and Tnfrsf1a/b-/- mice, with NPM used as a loading control. The accompanying bar graph shows densitometric quantification of HIF-1α normalized to NPM. Bars represent control and infected conditions for each genotype.
Figure 6
Malaria disease parameters in iNOS-deficient mice mirror those observed in TNF receptor-deficient animals.
(A) Nitrite levels in supernatants from CD11b+splenic cells harvested from infected (8 dpi) C57BL/6 or Tnfrsf1a/b-/- mice and stimulated ex vivo with LPS [1 µg/mL] or not during 48 H. (B) Parasitemia was determined at 8 dpi from C57BL/6 and Nos2-/- mice. (C) Rectal temperature was determined at 8 dpi. (D) Blood glucose levels were measured with a glucometer at 8 dpi. (E and F) CD11b+ cells were purified from spleens harvested from infected (8 dpi) C57BL/6 (red) and Nos2 -/- (green) mice or uninfected C57BL/6 (blue) and Nos2-/- (beige) mice and cultured ex vivo for evaluation of ECAR by Seahorse XFe96 Analyzers. (G–J) Relative expression of mRNA glycolytic enzymes in livers of naive or infected (8 dpi) mice. (K) Representative histogram and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/MHCII + cells from spleens of infected (8 dpi) or uninfected C57BL/6 or Nos2-/- mice. (L) Representative histogram and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/MHCII + cells from spleens of uninfected C57BL/6 or Nos2-/- mice stimulated or not with TNF (100 ng/ml) for 18 hr. (M) CD11b+splenic cells were purified from infected C57BL/6 (red - 8 dpi), Nos2-/- (green - 8 dpi), and uninfected mice and cultured ex vivo for quantification of proton efflux rate (PER) using glycolytic rate assay by Seahorse XFe96. (N) Parasitemia was determined during 20 dpi from WT-infected mice treated with 2-[14 C]-deoxyglucose (250 mg/kg daily from 4 to 15 dpi) or PBS. (O) Lactate was measured in the plasma of mice. (A) Graphs show mean ± SEM of one representative experiment of three independent ones with four to five mice per group; Statistical analysis: One-way ANOVA followed by Tukey post-hoc test. (B, C, and D) Graphs show mean ± SEM of one representative experiment of three independent ones with three to five mice per group (B and C) or of combined data from two independent experiments with three to five mice per group (D); Statistical analysis: Student’s t test. (E) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group. (F) Violin plots of combined data from three independent experiments with three to five mice per group – individual dots represent the average of triplicates in each experiment – the difference refers to the number of cells isolated from naïve vs infected animals, which varied. (E and F) Statistical analysis: Two-way ANOVA followed by Sidak’s post-hoc test employing mixed effect analysis provided by Agilent Seahorse XFe96 analyzer software. (G, H, I, J, N, O) Graphs show mean ± SEM of combined data from two independent experiments with three to five mice per group; Statistical analysis: Student’s t test: One-way ANOVA followed by Tukey post-hoc test. (K) Graphs show mean ± SEM of one representative experiment of three independent ones with three to five mice per group; Statistical analysis: Student’s t test. (L) Graphs show mean ± SEM of one representative experiment of two independent ones with three to five mice per group; Statistical analysis: Student’s t test. (M) Graphs show mean ± SEM of one representative experiment of at least three independent ones performed with four to five mice per group; Statistical analysis: Two-way ANOVA followed by Sidak’s post-hoc test employing mixed effect analysis provided by Agilent Seahorse XFe96 analyzer software. Asterisks above the bars indicate comparisons between mice of the same strain before infection and at 8 dpi. Asterisks connected by horizontal lines indicate comparisons between infected groups. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = non-significant.
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF-1α axis plays a key role in host resistance to Plasmodium infection
eLife 13:RP97759.
https://doi.org/10.7554/eLife.97759.4
