P. chabaudi-infected mice display enhanced TNF production associated with increased disease parameters.

C57BL/6 mice were infected or not i.p. with P. chabaudi (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 m-RNA from mice liver was determined at 8 days post- infection (dpi). (E) Parasitemia was determined at 8 days-post-infection (dpi) in C57BL/6 and TNFR deficient mice. (F) Rectal temperature was determined at 8 dpi in C57BL/6 and TNFR deficient mice. (G-J) MCP-1, TNF, IFN and IL-10 was measured by CBA Kit at 8 dpi. (K) Blood glucose levels were measured with a glucometer at 8dpi. A, B and F: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 mice per group; Statistical analysis: One-way ANOVA with Tukey post-hoc test. C and D: Graph shows mean± SEM of combined data from 3 independent experiments with 4 - 6 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 3 independent experiments with 4-6 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.

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 TNFR-/- was assessed using metabolic cages (TSE 959 Systems, Chesterfield, MO) at 3 days prior to infection (uninfected) (C57BL/6: blue, TNFR-/-: gray) and at 8 days postinfection (dpi) with P. chabaudi (105 infected RBCs) (C57BL/6: red, TNFR-/-: 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) percentual 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-[14C] 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-[14C] deoxyglucose. After 1h, mice were anesthetized, and tissue samples were taken for organ-specific levels of 2-[14C] deoxyglucose-6-phosphate. A - L: Graph shows mean ± SEM of combined data from 2 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.

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 P. chabaudi (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 on by western blot. (C and D) GLUT1 expression in (C) hepatocytes or (D) hepatic non-parenchymal cells isolated from livers from C57BL/6 infected (8dpi) 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 3 biological replicates performed in RNA-seq. B, C, D and G: Western blot images representative of at least 3 independent experiments. E, F, I and J: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 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 1 representative experiment of at least 3 independent ones performed with 4-5 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.

P. chabaudi infection triggers increased glycolysis in monocytic cells in a TNF-receptor dependent way.

C57BL/6 or TNFR-/- 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 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 TNFR-/- 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 TNFR-/- mice stimulated or not with TNF (100 ng/ml) for 18h. (N and O) CD11b+ cells were purified from spleens harvested from infected (8 dpi) C57BL/6 (red) and TNFR-/- (orange) mice or uninfected C57BL/6 (blue) and TNFR-/- (gray) mice and cultured ex vivo for evaluation of ECAR by Seahorse XFe96 Analyzers. (P) Parasitemia was determined at 8 days-post-infection (dpi) from TNF-R1aΔLyz2 and Wild-type mice. (Q) Rectal temperature was determined at 8 dpi in WT and TNF- R1aΔLyz2 at 8 dpi. (R) Blood glucose levels were measured with a glucometer at 8dpi. B, C, D, E, F, G, H, I, J and M: Graphs show mean ± SEM of combined data from 2 independent experiments with 3-5 mice per group; Statistical analysis: One-way ANOVA followed by Tukey post-test. K: representative dot plot of 1 representative experiment representative of 3 independent ones; L, P, Q, R: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 mice per group, Statistical analysis: Student’s t test. N: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 mice per group; O: Violin plots of combined data from 3 independent experiments with 3-5 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.

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 TNFR-/- mice evaluated by western blot. (expression of nucleofosmin -NFM- was used as control). (C) Representative histogram and 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-1aΔLyz2 and Wild-type mice. (D) Representative histogram and and graph showing GLUT1 expression evaluated by flow cytometry in CD11b+/F4/80+/CD11c+/MHCII+ cells from spleens of uninfected HIF-1aΔLyz2 and wild-type mice stimulated or not with TNF (100 ng/ml) for 18h. (E and F) CD11b+ cells were purified from spleens harvested from infected (8 dpi) WT (red) and HIF-1aΔLyz2 (pink) mice or uninfected WT (blue) and HIF-1aΔLyz2 (purple) mice and cultured ex vivo for evaluation of ECAR by Seahorse XFe96 Analyzers. (G) Parasitemia was determined at 8 days-post- infection (dpi) from HIF-1aΔLyz2 and Wildtype 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-1aΔLyz2 and Wild-type mice mice and stimulated ex vivo with LPS [1ug/mL] or not during 24H. A and B: representative dot plot of 1 representative experiment representative of 3 independent ones. C: Graphs show mean ± SEM of 1 representative experiment of 3 independent ones with 3-5 mice per group; Statistical analysis: Student’s t test. D: Graphs show mean ± SEM of 1 representative experiment of 2 independent ones with 3-5 mice per group; Statistical analysis: Student’s t test. E: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 mice per group. F: Violin plots of combined data from 3 independent experiments with 3-5 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 and H: Graphs show mean ± SEM of combined data from 3 independent experiments with 3-5 mice per group; Statistical analysis: Student’s t test. I: Graphs show mean ± SEM of 1 representative experiment of 3 independent ones with 3-5 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.

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 TNF-R1-/-mice and stimulated ex vivo with LPS [1ug/mL] or not during 48H. (B) Parasitemia was determined at 8 days-post- infection (dpi) from C57BL/6 and iNOS knockout mice. (C) Rectal temperature was determined at 8 dpi. (D) Blood glucose levels were measured with a glucometer at 8dpi. (E and F) CD11b+ cells were purified from spleens harvested from infected (8 dpi) C57BL/6 (red) and iNOS knockout (green) mice or uninfected C57BL/6 (blue) and iNOS knockout (beige) mice and cultured ex vivo for evaluation of ECAR by Seahorse XFe96 Analyzers. (G-J) Relative expression of glycolytic enzymes in livers of naïve 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 iNOS-/- 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 iNOS-/- mice stimulated or not with TNF (100 ng/ml) for 18h. (M) CD11b+ splenic cells were purified from infected C57BL/6 (red - 8 dpi), iNOS-/- (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 days-post-infection (dpi) from wild-type infected mice treated with 2-[14C]-deoxyglucose (250mg/kg daily from 4 to 15 dpi) or PBS. (O) Lactate was measured in the plasma of mice. A: Graphs show mean ± SEM of 1 representative experiment of 3 independent ones with 4-5 mice per group; Statistical analysis: One-way ANOVA followed by Tukey post-hoc test. B, C and D: Graphs show mean ± SEM of 1 representative experiment of 3 independent ones with 3-5 mice per group (B and C) or of combined data from 2 independent experiments with 3-5 mice per group (D); Statistical Analysis: Student’s t test. E: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 mice per group. F: Violin plots of combined data from 3 independent experiments with 3-5 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 2 independent experiments with 3-5 mice per group; Statistical Analysis: Student’s t test: One-way ANOVA followed by Tukey post-hoc test. K: Graphs show mean ± SEM of 1 representative experiment of 3 independent ones with 3-5 mice per group; Statistical analysis: Student’s t test. L: Graphs show mean ± SEM of 1 representative experiment of 2 independent ones with 3-5 mice per group; Statistical analysis: Student’s t test. M: Graphs show mean ± SEM of 1 representative experiment of at least 3 independent ones performed with 4-5 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.