Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance

Abstract

Host defenses against pathogens are energetically expensive, leading ecological immunologists to postulate that they might participate in energetic trade-offs with other maintenance programs. However, the metabolic costs of immunity and the nature of physiologic trade-offs it engages are largely unknown. We report here that activation of immunity causes an energetic trade-off with the homeothermy (the stable maintenance of core temperature), resulting in hypometabolism and hypothermia. This immunity-induced physiologic trade-off was independent of sickness behaviors but required hematopoietic sensing of lipopolysaccharide (LPS) via the toll-like receptor 4 (TLR4). Metabolomics and genome-wide expression profiling revealed that distinct metabolic programs supported entry and recovery from the energy-conserving hypometabolic state. During bacterial infections, hypometabolic states, which could be elicited by competition for energy between maintenance programs or energy restriction, promoted disease tolerance. Together, our findings suggest that energy-conserving hypometabolic states, such as dormancy, might have evolved as a mechanism of tissue tolerance.

Keywords: caloric restriction; dormancy; hibernation; innate immunity; ketones; metabolism; resistance; thermoneutrality; torpor; triglycerides.

Figure 1.. Activation of immunity by LPS promotes energy conservation.

(A) Summary of the effects of TLR ligands on energy conservation and sickness behaviors. (B) Rate of oxygen consumption (VO₂) in mice housed at 22°C and treated with various doses of LPS (n=3 mice per dose). (C, D) Rate of oxygen consumption (VO₂) in mice housed at 22°C (C) or 30°C (D) and treated with vehicle or LPS (n=4–8 mice per treatment and temperature). Three stages (entry, maintenance, and exit) of the hypometabolic state are indicated (D). (E, F) Total activity in mice housed at 30°C (E) or 22°C (F) and treated with vehicle or LPS (n=4–8 mice per treatment and temperature). (G) Core temperature of mice housed at 22°C or 30°C that were treated with vehicle or LPS (n=6 mice per treatment and temperature). (H, I) Infrared measurements of dorsal surface temperature of mice housed at 30°C or 22°C that were treated with vehicle or LPS (n=5 mice per treatment and temperature); dorsal surface temperature (H) and representative images (I). (J, K) Cumulative food intake in mice housed at 30°C (J) or 22°C (K) that were with vehicle or LPS (n=4–8 mice per treatment and temperature). (L) Blood glucose levels in mice treated with LPS that were housed at 22°C or 30°C (n=12–13 mice per temperature, pooled from multiple experiments). (M) Plasma levels of leptin at various time points after administration of LPS to mice housed at 22°C or 30°C (n=3–4 mice per time point and temperature). Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001. See also Figure S1 and S2.

Figure 2.. Hematopoietic sensing of LPS via TLR4/MyD88 triggers energy conservation independent of sickness behaviors.

(A) Rate of oxygen consumption (VO₂) in C57BL/6J (n=5) and Tlr4^−/− (n=6) mice treated with LPS at 22°C. (B) Core temperature of C57BL/6J (n=6) and Tlr4^−/− (n=5) mice treated with LPS at 22°C. (C) Rate of oxygen consumption (VO₂) in C57BL/6J (n=5) and Myd88^−/− (n=5) mice treated with LPS at 22°C. (D) Core temperature of C57BL/6J (n=5) and Myd88^−/− (n=6) mice treated with LPS at 22°C. (E, F) Rate of oxygen consumption (VO₂) in Tlr4^f/f (n=3) and Tlr4^f/fLyz2^Cre (n=9), or Myd88^f/f (n=6) and Myd88^f/fLyz2^Cre (n=7) mice treated with LPS at 22°C (data pooled from multiple experiments). (G, H) Rate of oxygen consumption (VO₂) in Tlr4^f/f (n=5) and Tlr4^f/fVav1^Cre (n= 4) male mice, or Myd88^f/f (n=4) and Myd88^f/fVav1^Cre (n=8) female mice treated with LPS at 22°C (data pooled from multiple experiments). (I, J) Total activity (I) and cumulative food intake (J) in Tlr4^f/f (n=5) and Tlr4^f/fVav1^Cre (n=4) male mice treated with LPS at 22°C (data pooled from multiple experiments). (K, L) Total activity (K) and cumulative food intake (L) in Myd88^f/f (n=4) and Myd88^f/fVav1^Cre (n=8) female mice treated with LPS at 22°C (data pooled from multiple experiments). Data are presented as mean ± SEM. See also Figure S2 and S3.

Figure 3.. Shift in core set point and Q₁₀ effect suppresses metabolic rate to initiate physiologic trade-off with homeothermy.

(A-C) Temporal analysis of RSC assembly and activity in liver, BAT, and heart of mice treated with LPS. BN-PAGE coupled with in-gel activity assays were used to detect CI- and CIV-containing RSCs, and CII. CIII and CV were detected by immunoblotting. (D) Model for suppression of metabolic rate during LPS-induced hypothermia. RMR: resting metabolic rate at 22°C; BMR: basal metabolic rate at 30°C; HMR: hypothermic metabolic rate. (E, F) Quantification of absolute (E) and relative (F) energetic costs of maintenance programs in mice housed at 22°C or 30°C. (G) Quantification of absolute energy savings in mice treated with LPS at 22°C or 30°C. (H) Quantification of plasma cytokines at various times after treatment of mice with LPS housed at 22°C or 30°C (n=5–10 mice per time point and temperature). SEM values are presented as a heat map. (I) Quantification of relative energy savings in mice treated with LPS at 22°C or 30°C. See also Figure S4.

Figure 4.. Activation of immunity reprograms nutrient metabolism in the liver.

(A) Quantification of β-hydroxybutyrate in plasma of mice treated with LPS (n= 5–14 mice per time point and temperature, pooled from multiple experiments). (B) Quantification of total body, lean, and fat mass in mice administered LPS (n=7–9 mice per temperature, data compiled from multiple experiments). (C) Pathways or processes (Gene Ontologies and IPA) significantly enriched in downregulated genes at the 12-hour time point. (D) Heat maps of genes involved in fatty acid oxidation in livers of mice treated with LPS at 22°C and 30°C (n=4–5 mice per temperature and treatment). (E, F) LC-MS analysis of acylcarnitines in plasma (E) and liver (F) of mice treated with LPS for 12 hours at 30°C (n=5 mice per treatment). Data plotted as log2 fold change. (G) GC-MS analysis of fatty acids in plasma of mice treated with LPS (n=4–5 mice per temperature and time point). (H) Schematic of biochemical pathways involved in ketogenesis in liver; metabolites (black), enzymes (blue), and amino acids (purple). (I) Heat map of genes involved in catabolism of amino acids in livers of mice treated with LPS at 22°C and 30°C (n=4–5 mice per temperature and treatment). (J) GC-MS analysis of amino acids in plasma of mice treated with LPS (n=4–5 mice per temperature and time point). (K) GC-MS analysis of urea in livers of mice treated with LPS (n=4–5 mice per temperature and time point). Mean values are presented in heat maps for plasma metabolites and gene expression, and other data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S5 and Tables S1–3.

Figure 5.. BAT thermogenesis generates heat for exit from hypothermic state.

(A) LC-MS analysis for lipids in plasma of mice treated with LPS (n=4–5 mice per temperature and time point). (B) Photograph of plasma collected at 24 hours after administration of LPS to mice. (C) Heat map of lipogenesis genes in livers of mice treated with LPS (n=4–5 mice per time point and temperature). (D) Quantification of ANGPTL4 by ELISA in plasma of mice treated with LPS (n=5–10 mice per time point and temperature). (E) Quantification of plasma triglycerides 24 hours after administration of LPS to WT and Angptl4^−/− mice that were housed at 30°C (n=3–8 mice per time point and genotype, pooled from multiple experiments). (F) Core temperature of C57BL/6J and Ucp1^−/− mice 24 hours after treatment with LPS (n=3–5 mice per genotype and temperature). (G) Rate of oxygen consumption (VO₂) in C57BL/6J (n=10) and Ucp1^−/− (n=7) mice treated with LPS at 22°C. (H-J) Quantification of tissue damage biomarkers in plasma of C57BL/6J (n=4–9) and Ucp1^−/− (n=4–7) mice 24 hours after treatment with LPS at 22°C; Creatinine (H), Cardiac troponin I (I), Creatine kinase (J). (K) Quantification of plasma cytokines 4 hours after treatment of C57BL/6J and Ucp1^−/− mice with LPS at 22°C (n=8–10 mice per time point and temperature). Mean values are presented as a heat map. (L) Kaplan-Meier survival curves of C57BL/6J (n=5) and Ucp1^−/− (n=5) mice treated with LPS at 22°C and 30°C. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S6.

Figure 6.. Competition for energy promotes plasticity in host defense strategies.

(A, B) Food consumption (A) and change in body weight (B) after injection of 1×10⁴ CFUs of wild type L. monocytogenes into mice housed at 22°C and 30°C (n=13–14 mice per temperature; data combined from 2 separate experiments). (C) Flow cytometric quantification of CD45⁺ cells in liver and spleen 4 days post-infection with L. monocytogenes (n=6–7 mice per temperature). (D) Liver CFUs 4-days post-infection with L. monocytogenes (1×10⁴ CFU, n=12–14 mice per temperature, data pooled from 2 experiments). (E) Reaction norm plots of host health (weight loss) and pathogen burden (L. monocytogenes CFU in liver) 4-days post-infection (n=12–14 mice per temperature, data pooled from 2 experiments). (F) Body mass of mice infected with E. coli (1×10⁷ CFU, n=12 mice per temperature). (G, H) Bacterial CFUs from spleen (G) and liver (H) 1-day post-infection with E. coli (1×10⁷ CFU, n=9 mice per temperature). (I, J) Plasma triglycerides (I, n=9 mice per temperature) and cytokine concentration (J, n=14 mice per temperature) 1-day post-infection with E. coli (1×10⁷ CFU). Mean values of cytokines are plotted as a heat map. MCP-1 is higher in mice housed at 22°C (p<0.05). (K) Body mass of mice infected with E. coli (1×10⁸ CFU, n=19 mice per temperature). (L) Kaplan-Meier survival curves of C57BL/6J mice infected with E. coli (1×10⁸ CFU, n=28–29 mice per temperature; data combined from 3 separate experiments). (M, N) Bacterial CFUs from spleen (M) and liver (N) of C57BL/6J and Ucp1^−/− mice housed at 22°C and infected with E. coli (1×10⁷ CFU, n=3–8 mice per genotype and timepoint). (O) Kaplan-Meier survival curves of C57BL/6J and Ucp1^−/− mice housed at 22°C and infected with E. coli (1×10⁸ CFU, n=10–14 mice per genotype). (P) Core temperature of C57BL/6J and Ucp1^−/− mice housed at 22°C and infected with E. coli (1×10⁸ CFU, n=10–14 mice per genotype). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p <0.0001. See also Figure S7.

Figure 7.. Energy conserving hypometabolic state promotes tolerance during bacterial infection.

(A) Body mass of ad libitum (Ad Lib) fed and every other day fasted (EODF) mice housed at 30°C (n=9 mice per condition). (B-D) Rate of oxygen consumption (VO₂) in C57BL/6J mice fed ad libitum or subjected to EODF at 30°C (n=6 mice per condition). Total VO₂ consumption during fasting (C) and fed (D) days. (E) Core temperature of C57BL/6J mice housed at 30°C that were fed ad libitum (n=3) or subjected to EODF (n=5). (F-H) Total activity in C57BL/6J mice fed ad libitum or subjected to EODF at 30°C (n=6 mice per condition). Total activity during fasting (G) and fed (H) days. (I, J) Body mass (I) and core temperature (J) of C57BL/6J mice infected with E. coli (1×10⁷ CFU) that were fed ad libitum (n=9) or previously subjected to 5 cycles of EODF (n=8) at 30°C. (K) Kaplan-Meier survival curves of C57BL/6J mice infected with E. coli (1×10⁷ CFU) that were fed ad libitum (n=9) or previously subjected to 5 cycles of EODF (n=8) at 30°C. (L) Kaplan-Meier survival curves of C57BL/6J mice infected with E. coli (1×10⁸ CFU) that were fed ad libitum (n=9) or previously subjected to 5 cycles of EODF (n=10) at 30°C. See also Figure S7.