Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation

Abstract

Acute infections are associated with a set of stereotypic behavioral responses, including anorexia, lethargy, and social withdrawal. Although these so-called sickness behaviors are the most common and familiar symptoms of infections, their roles in host defense are largely unknown. Here, we investigated the role of anorexia in models of bacterial and viral infections. We found that anorexia was protective while nutritional supplementation was detrimental in bacterial sepsis. Furthermore, glucose was necessary and sufficient for these effects. In contrast, nutritional supplementation protected against mortality from influenza infection and viral sepsis, whereas blocking glucose utilization was lethal. In both bacterial and viral models, these effects were largely independent of pathogen load and magnitude of inflammation. Instead, we identify opposing metabolic requirements tied to cellular stress adaptations critical for tolerance of differential inflammatory states. VIDEO ABSTRACT.

Figure 1. Glucose caloric supplementation during Listeria monocytogenes infection worsens survival, while 2DG promotes survival

(A) Food consumption after infection with 5 × 10⁴ and 5 × 10⁵ CFU wild-type L. monocytogenes. (B) Survival after infection with 5 × 10⁴ L. monocytogenes. Mice were per os (PO) gavaged with Abbott Promote (Food), glucose, or PBS vehicle, and injected intraperitoneally (IP) with 2DG or PBS. PO PBS/IP PBS n=20, PO Food/IP PBS n= 15 (p=0.0011 vs PO PBS/IP PBS), PO Glucose/IP PBS n=19 (p=0.004 vs PO PBS/IP PBS), PO PBS/IP 2DG n=10 (p=0.0085 vs PO PBS/IP PBS). (C-F) Mice were infected with 5 × 10⁴ CFU L. monocytogenes, then treated with IP PBS, glucose, or 2DG. n=5/group. (C) Survival after L. monocytogenes and indicated treatments. PBS vs Glucose p=0.0396, PBS vs 2DG p=0.0344, Glucose vs 2DG p=0.0017. (D) Plasma IL-6 and IFNγ 24 and 48 hours after 5 × 10⁴ L. monocytogenes infection. 24h plasma IL-6: PBS vs 2DG p=0.0005, Glucose vs 2DG p=0.0014; 24h plasma IFNγ: PBS vs Glucose, PBS vs 2DG, Glucose vs 2DG all p<0.0001. n=5/group. (E) Listeria CFUs from spleen and liver 4 days post-infection. (F) Flow cytometry analysis of CD45⁺ cells within the liver 4 days post-infection. (G) CFU growth of L. monocytogenes after incubation in brain heart infusion broth with or without 15 mM 2DG for 18 hours. (H) Bone marrow-derived macrophages (BMDM) were infected with 5 × 10⁵ CFU of L. monocytogenes in the presence or absence of 15 mM 2DG for 24 hours. CFU of L. monocytogenes grown from the BMDM cell media supernatant and cell lysate. Data are represented as mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001. See also Figure S1.

Figure 2. Glucose caloric supplementation during LPS sepsis worsens survival, while 2DG promotes survival

(A and B) Survival after 15 mg/kg IP LPS and PO gavage with Abbott Promote (Food), glucose, olive oil, casein, or PBS vehicle. PO PBS n=10, PO Food n=10 (p=0.0002 vs PO PBS), PO Glucose n=8 (p<0.0001 vs PO PBS), no gavage n=10 (p=0.0679 vs PO PBS). Panel A is a subset of panel B, separated for clarity (the same PBS-treated, food-treated, and no gavage groups are shown in (A and B)). (C) Survival after 15 mg/kg IP LPS and PO gavage with Food with IP injections of PBS or 2DG. p=0.0433 (D-F) Mice were given 15 mg/kg IP LPS, then treated with IP PBS, glucose, or 2DG. (D) Survival after LPS and indicated treatments. IP PBS n=16, IP Glucose n=10 (p<0.0001 vs IP PBS), IP 2DG n=10 (p=0.01 vs IP PBS). (E) Plasma TNFα and IL-6. (n=5-10/group) (F) Liver mRNA expression 4 hours after LPS and treatment with PBS (LPS-PBS), glucose (LPS-glucose), and 2DG (LPS-2DG) compared to PBS alone. n=3-5/group. Data are represented as mean ± SEM. See also Figure S2.

Figure 3. Caloric supplementation and glucose utilization is required for surviving influenza infection

(A) Food consumption after infection with 450 plaque forming units (PFU) of Influenza strain A/WSN/33. (B-C) Survival after infection with 800 PFU of influenza virus. Mice were gavaged with Abbott Promote (Food), glucose, or PBS vehicle. Mice gavaged with Food were also injected with IP PBS (Food) or 2DG (Food+2DG). PBS vs Food p=0.0047, PBS vs Glucose p=0.1058, Food vs Food+2DG p=0.0001, PBS vs Food+2DG p=0.0256. n=10/group. Panel B is a subset of panel C, separated for clarity (the same PBS-treated and food-treated groups are shown in (B and C)). (D) Survival after infection with 375 PFU of influenza virus, and treatment with IP PBS or 2DG. p<0.0001, n=10/group. (E-J) Mice were infected with 375 PFU influenza virus, and treated with IP PBS or 2DG. n=4-5/group (E) Lung and broncho-alveolar lavage (BAL) viral load 6 days after influenza infection by PFU and quantitative PCR for WSN nucleoprotein (NP). (F) mRNA expression of whole lung tissue at day 6. (G) Plasma IFNα measured by ELISA. (H and I) H&E staining of lung tissue 6 days post-infection and histologic scoring. Scale bar = 500 μm. For magnified views of insets please see Figure S3E. (J) Vital signs after influenza infection. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S3.

Figure 4. Inhibition of glucose utilization is lethal in Poly(I:C) inflammation

(A) Survival of mice after Poly(I:C) challenge and treatment with either IP PBS, glucose, or 2DG. IP PBS n=15, IP Glucose n=10 (p=0.2207 vs IP PBS), and IP 2DG n=15 (p<0.0001 vs IP PBS). (B) Survival of B6 wild-type (WT) mice and Ifnar^-/- mice after Poly(I:C) challenge and treatment with IP PBS, glucose, or 2DG. B6 vs Ifnar^-/- p=0.0027, n=5/group. (C-D) Mice were challenged with Poly(I:C), then treated with either IP PBS, glucose, or 2DG. (C) Plasma IFNα. n=5/group. (D) Vital signs measured 18 hours after Poly(I:C) administration. n=3-7/group. (E) Averaged brain PET images after PBS vehicle (baseline), LPS, and Poly(I:C) administration. “A” is the brainstem and “B” is the hypothalamus. Anatomic atlas of regions-of-interest (ROI) and T2-weighted magnetic resonance images provided for reference. CT, computed tomography. n=3/group. Data are represented as mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S4.

Figure 5. Inhibition of glucose utilization in Poly(I:C)-induced inflammation enhances ER stress

(A) B6 WT and Ifnar^-/- mice were challenged with Poly(I:C), then treated with either IP PBS or 2DG. Whole hindbrain lysates 24 hours after Poly(I:C) treatment immunoblotted for CHOP and β-Tubulin. (B) Hindbrain mRNA expression of Gadd34 18 hours after Poly(I:C) challenge and treatment with IP PBS, glucose, or 2DG in WT mice. n=5/group (one death in Poly(I:C) group). (C) Survival of B6 WT mice and Ddit3^-/- mice after Poly(I:C) challenge and treatment with IP PBS or 2DG. WT vs Ddit3^-/- p=0.0015 WT: Poly(I:C) + 2DG vs Ddit3^-/-: Poly(I:C) + 2DG, n=5/group. (D and E) B6 WT and Ddit3^-/- mice were challenged with Poly(I:C) and treated with 2DG. (D) Plasma IFNα after Poly(I:C) challenge and treatment with 2DG. n=5/group. (E) Vital signs measured 18 hours after Poly(I:C) + 2DG. n=3-7/group (vital sign values of B6 WT mice same as those in Figure 4D) (F-H) B6 WT mice and Ddit3^-/- mice were infected with 700 PFU of influenza virus and treated with 2DG. (F) Survival after influenza infection and treatment with 2DG. p=0.048 n=5/group (G) Plasma IFNα after influenza infection and treatment with 2DG. n=5/group. (H) Lung and BAL viral load 5 days post-infection. n=5/group. Data are represented as mean ± SEM. **p<0.01, ***p<0.001. See also Figure S5.

Figure 6. Role of ketogenic program in surviving bacterial, but not viral inflammation

(A) Plasma nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHOB), and fibroblast growth factor 21 (FGF21) after LPS, with treatment with IP PBS or glucose. n=10/group. (B) Survival after 8 mg/kg IP LPS and treatment with glucose. Mice were treated with vehicle, valproic acid (VA) or levetiracetam (Keppra) starting 6 hours after LPS. p<0.0001 for Glucose+VA vs Glucose and Glucose+VA vs Glucose+Keppra. n=15 Glucose, n=15 Glucose+VA, n=4 Glucose+Keppra. (C) Survival after 12.5 mg/kg IP LPS in B6 WT, Fgf21^-/- and Ppara^-/- mice. n=10/group. p<0.0001 for WT vs Fgf21^-/-; p=0.0003 for WT vs Ppara^-/-. (D) Plasma BHOB after 12.5 mg/kg IP LPS in WT, Fgf21^-/- and Ppara^-/- mice. n=4-6/group. (E) Plasma TNFα and IL-6 in B6 WT, Fgf21^-/-, and Ppara^-/- mice after LPS. n=5/group *p<0.05. (F) Survival after 12.5 mg/kg IP LPS in Fgf21^-/- and Ppara^-/- mice, treated with i.v. 5 ng recombinant mouse FGF21 (rmFGF21) twice daily starting 6 hours after LPS injection. n=5-6/group. p=0.0491 Fgf21^-/- VEH vs Fgf21^-/- rmFGF21. p=0.5767 Ppara^-/- VEH vs Ppara^-/- rmFGF21. (G) Survival after 8 mg/kg IP LPS in B6 WT and Ppara^-/- mice. 2DG treatment was initiated one hour after LPS. Valproic acid (VA) was initiated 6 hours after LPS. n=4-5/group. p=0.0177 Ppara^-/- VEH vs Ppara^-/- VA. (H-I) WT and Ppara^-/- mice were infected with 400 PFU of influenza virus. (H) Survival after influenza infection. p=0.0074 WT n=6, Ppara^-/- n=8, representative of three independent experiments. (I) Lung and BAL viral load 5 days post-infection. n=6-7/group. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figures S6 and S7.

Figure 7. Model of glucose utilization during viral and bacterial-mediated inflammation supporting unique tissue tolerance mechanisms

(A) Glucose inhibits PPARα-dependent ketogenesis and cellular adaptation programs required for tissue tolerance during LPS and Listeria-mediated (bacterial) inflammation. Ketones act as fuel source and as HDACi allowing for cellular and tissue adaptation. Inhibition of glucose utilization during bacterial inflammation with 2DG protects against tissue dysfunction and organismal mortality. (B) Glucose utilization is required for adaptation to Poly(I:C) and influenza mediated (viral) sepsis. Viral inflammation activates ER stress and the unfolded protein response (UPR) downstream of type I interferon signaling through IFNaR. Inhibition of glucose utilization in viral inflammation with 2DG enhances ER stress through a CHOP-dependent pathway leading to tissue dysfunction and death.