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. ****P<0, 0001 - t- test. (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. ***P<0, 001 – onewayANOVA. (D) TNF m-RNA 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.*P≤0,1, **P≤0.01, ***P≤0.001, **** P≤0.0001, ns = non-significant onewayANOVA).

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 post-infection (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. ι P≤0.05, ιι P≤0.01, ιιιι P≤0.0001, ns = non-966 significant (comparisons within the same group at 3 days prior to infection vs 8 dpi), *P≤0.05, ** P≤0.01, *** P≤0.001 (comparisons between C57BL/6 and TNF-/- mice) (t-test). (K) 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. (L) Blood glucose levels were measured with a glucometer at 8dpi. ****P<0, 0001 – oneway-ANOVA.

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 the liver 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). Data are representative of a total of 4 experiments with similar profile. (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. ***P≤0.001 and ****P≤0.0001 (t-test. **P≤0.01, ****P≤0.0001, ns = non-significant (two- way ANOVA).

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

C57BL/6 or TNFR-/- mice were infected or not i.p. with P. chabaudi (105 iRBCs). (A) Glycolytic and gluconeogenesis pathways. (B-E) PCR of glycolytic enzymes (F-J) PCR from liver of gluconeogenesis enzymes at 8 dpi. .*P≤0,1, **P≤0.01, ns = non-significant onewayANOVA. (K and L) GLUT1 expression in (K) livers of infected (8 dpi) or uninfected mice was evaluated by western blot. (L) Representative histogram and 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. ****P≤0.0001, ***P≤0.001 and *P≤0.05) (t-test). (M and N) 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≤0.0001 (two-way ANOVA). (O) Parasitemia was determined at 8 days-post-infection (dpi) from TNF-R1aΔLyz2 and Wild-type mice. (P) Rectal temperature was determined at 8 dpi in WT and TNF- R1aΔLyz2 at 8 dpi. (Q) Blood glucose levels were measured with a glucometer at 8dpi. Data are representative of a total of 3-5 experiments with similar profile.

HIF-1α contributes to glycolysis induction during Pc infection. (A) HIF- 1a expression in spleen and (B) liver of infected (8 dpi) or uninfected C57BL/6 and TNFR-/- mice was 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+/Ly6G- cells from spleens of infected (8 dpi) or uninfected HIF-1aΔLyz2 and Wild-type mice. (D and E) 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. ****P≤0.0001 (two-way ANOVA). (F) Parasitemia was determined at 8 days-post- infection (dpi) from HIF-1aΔLyz2 and Wild-type mice. (G) Blood TNF Levels were measured by ELISA at 8 dpi. (H) 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.*P≤0,1, **P≤0.01, ***P≤0.001, **** P≤0.0001, ns = non-significant onewayANOVA). Data are representative of a total of 3-4 experiments with similar profile.

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) or uninfected C57BL/6 mice and stimulated ex vivo with LPS [1ug/mL] or not during 48H.**** P≤0.0001, ns = non- significant onewayANOVA). (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. ****P≤0.0001 (two-way ANOVA). (G-J) PCR from liver of glycolytic enzymes at 8 dpi. *P≤0,1, **P≤0.01, ns = non-significant onewayANOVA. (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) 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.