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.

Here, we report that, whereas nutritional supplementation increased mortality of Listeria monocytogenes infection, it protected against lethality of influenza virus infection. The causative component of food was determined to be glucose, and this effect was largely independent of inflammation or pathogen burden. To study the differential effects of glucose metabolism in bacterial and viral inflammation and sepsis generally, we utilized lipopolysaccharide (LPS) and poly(I:C) models of sepsis and found that, whereas therapeutic blockade of glucose utilization with 2-deoxy-D-glucose (2DG) protected against LPS-mediated sepsis, it was uniformly lethal with poly(I:C) sepsis. We found that, whereas glucose was necessary for adaptation to and survival from the stress of antiviral inflammation by preventing initiation of endoplasmic reticulum (ER) stress-mediated apoptotic pathways, glucose prevented adaptation to the stress of bacterial inflammation by inhibiting ketogenesis, which was necessary for limiting reactive oxygen species (ROS) induced by anti-bacterial inflammation. Our study elucidates how specific metabolic programs are coupled to different types of inflammation to regulate tolerance to inflammatory damage.

There are several examples where organismal metabolism has been shown to be regulated in order to deprive necessary substrates for pathogen viability as a strategy of enhancing host resistance (), but how organismal metabolic states, such as the fasted state of anorexia, contribute to host defense is not understood. Indeed, the role of metabolic homeostasis during sepsis, which is directly impacted by nutritional status, is becoming increasingly recognized as critical in surviving sepsis in the clinical setting. In addition to the well-studied but contentious role of glucose homeostasis in managing sepsis in the intensive care units (), the largest proteomic and metabolomic screen of patients with sepsis to date identified fatty acid, glucose, and beta-oxidation pathways as being discriminatory between survivors and non-survivors (). Bacterial sepsis leads to a pro-lipolytic state, which affects the ability of target tissues to utilize glucose via glycolysis and alternative fuel sources, such as ketone bodies (KBs) and free fatty acids (FFAs) via oxidative phosphorylation (). Growing evidence suggests that a shift from glucose to KB/FFA utilization is protective in bacterial sepsis, and these studies largely rely on pharmacologic targeting of peroxisome proliferator-activated receptor alpha (PPARα), the master regulator of the ketogenic program and fasting metabolism (). The role of substrate utilization in viral infections is even less understood (). These metabolic changes are presumed to be protective, but their mechanism of protection remains obscure.

An important new paradigm that can help explain at least some benefits of sickness behavior is the notion of resistance and tolerance: survival of infections can be promoted by either reducing pathogen burden or by increasing host tolerance to the damage of infection (). During an infection, multiple physiological processes undergo dramatic alterations with often unknown rationale. Depending on the infection, these can include profound changes in metabolism and alterations in hepatic, renal, and cardiovascular functions. In principle, these responses can be either beneficial, induced as part of a general defense program, or they can be unintended and detrimental but unavoidable consequences of infections. Furthermore, the physiological changes that are beneficial for host survival can contribute to elimination of pathogens (resistance) or to mitigation of tissue damage caused by infection (tolerance). These physiological responses are poorly understood aspects of infection biology, where most of the focus has been devoted to the immune response and microbial pathogenesis.

Anorexia of infection is an aspect of sickness behavior that is conserved from humans to insects (), and much effort has been dedicated to understanding the role of nutrition in septic critical illness (). In the 1970s, it was demonstrated that interfering with the anorexia induced by Listeria infection using caloric supplementation led to pronounced mortality in mice (). Importantly,found that, in Drosophila, anorexia was beneficial in some, but not all, infections. Similarly, it was recently shown that the fasting metabolic state was critical to surviving bacterial sepsis (). The mechanism remains elusive, and clinically, the role of nutrition in managing patients with sepsis is unclear.

Sickness behaviors are a collection of prominent symptoms of acute illness that include anorexia, lethargy, fever, altered sleep patterns, depression, lack of grooming, and social withdrawal. It has been long appreciated that sickness behaviors are motivational states rather than a result of debilitation of physiological functions (). Furthermore, sickness behaviors have been conceptualized as adaptive programs that promote survival of acute infections (). However, the mechanisms whereby different sickness behaviors contribute to survival remain largely unknown. This issue is particularly important because a popular treatment for acute infections is the use of non-steroidal anti-inflammatory drugs, which work primarily by suppressing the symptoms of sickness behaviors (). Understanding the biology of sickness behaviors is also important for improved management of critically ill patients suffering from conditions with poorly defined pathogenesis, such as sepsis. Indeed, sepsis remains a largely intractable clinical problem with high mortality rates even in modern medical facilities (). It is now increasingly appreciated that the mortality of sepsis can stem from at least three distinct phases of the disease: hemodynamic shock; multiple organ failure; and prolonged immunosuppression (). Furthermore, different forms of sepsis can result from bacterial, fungal, and viral infections, each creating a distinct challenge and potentially requiring different management strategies.

We hypothesized that, because our viral and bacterial models had opposite dependencies on glucose, they would also have opposite dependencies on ketogenesis. Therefore, we subjected PPARα-deficient mice to influenza challenge. Whereas PPARα deficiency was lethal following LPS challenge, it was protective in influenza infection in a manner independent of pathogen control ( Figures 6 H and 6I). This protective effect was not observed in FGF21-deficient animals ( Figure S7 D). Together, these data show that, whereas impairment of ketogenesis, whether through genetic deletion of PPARα or glucose administration, was lethal in bacterial inflammation, it was protective in viral inflammation, in a manner independent of the magnitude of inflammation.

Because ketotic pre-conditioning has been shown to improve other neurologic conditions, such as epilepsy (), we tested whether it would improve survival in sepsis. We found that mice pre-fasted for 24 hr or mice fed ketogenic diets for 3 days displayed enhanced sensitivity to LPS despite generating adequate levels of KBs ( Figures S7 A and S7B). We excluded the possibility that ketoacidosis was driving death ( Figure S7 C). These data indicate that the activation of the ketogenic program must be temporally coupled to the course of the inflammatory challenge.

To test the role of ketogenesis in bacterial and viral inflammation, we subjected mice deficient in PPARα and FGF21 to LPS or influenza infection. Both PPARα- and FGF21-deficient mice displayed enhanced mortality after LPS administration ( Figure 6 C). We verified that PPARα-deficient mice have severely impaired ketogenesis following LPS challenge and did not observe significant changes in the level of BHOB in FGF21-deficient animals ( Figure 6 D), consistent with findings observed in the fasting state (). We also did not detect an increase in systemic cytokines in PPARα-deficient mice ( Figure 6 E). We hypothesized that either the lack of FGF21 or the lack of alternative fuel sources, which were both suppressed after glucose supplementation, was the cause of mortality. Because FGF21 is a known downstream target of PPARα (), we tested whether defective FGF21 production was the causative lesion in PPARα deficiency. We reconstituted PPARα-deficient and FGF21-deficient mice with recombinant FGF21 and found that, whereas FGF21 was sufficient to rescue FGF21-deficient mice, it was not sufficient to rescue PPARα-deficient mice ( Figure 6 F), arguing that other aspects of the fasting program were necessary to mediate survival of LPS sepsis. Finally, VA, but not 2DG, was able to rescue PPARα mice challenged with LPS ( Figure 6 G), indicating that some aspect of VA action—likely its HDACi activity—was sufficient to rescue the lack of KBs in PPARα mice and also that the protective effects of 2DG required an intact ketogenic program.

We observed that the administration of glucose to LPS-challenged mice potentiated seizures, implicating neurotoxicity as a mechanism for death. We therefore asked whether anti-epileptic drugs would be sufficient to rescue glucose-mediated death and found that administration of valproic acid (VA), but not levetiracetam (two commonly used anti-epileptic agents), was able to completely rescue LPS-challenged mice treated with glucose ( Figure 6 B). The anti-epileptic effects of VA are incompletely understood but appear to impact HDAC-inhibition (HDACi), GABA transduction, and PI3K and calcium handling (). KBs have also been implicated as HDACi of the same class as VA and have recently been shown to coordinate gene expression programs that confer resistance to ROS-mediated damage (). We thus hypothesized that the suppression of KBs by glucose administration may be inhibiting these HDACi-mediated ROS adaptation pathways. To test this, we utilized dihydroethidium staining to measure ROS in situ in the brains of LPS-challenged mice treated with glucose and observed increased ROS in the brains of these mice ( Figure S6 A). We also observed more TUNEL-positive nuclei in sections of mouse brain in glucose-treated mice compared to 2DG- or PBS-treated mice challenged with LPS ( Figure S6 B). All groups had TUNEL-positive nuclei in areas of lymphoid cell death (thymus and spleen), but there were no TUNEL-positive nuclei in any other tissues surveyed (heart, lung, liver, and kidney). The brain was the only tissue where differences in TUNEL-positive nuclei were seen between groups ( Figures S6 C and S6D); however, the amount of cell death, which was assessed in the hypothalamus—given PET localization—was not dramatically different, indicating that cellular dysfunction, and not necessarily cell death, was being potentiated by glucose administration. Together, these data suggest that the enhanced lethality caused by glucose supplementation in endotoxemia is likely mediated through increased ROS and neuronal dysfunction.

Prolonged fasting results in hypoglycemia accompanied by lipolysis and followed by ketogenesis. In anorexia following acute LPS challenge, there was a decrease in blood glucose and an increase in plasma FFA, plasma beta-hydroxybutyrate (BHOB), and the fasting hormone FGF21 ( Figures 6 A and S2 B). The switch to this fasting metabolic profile was ablated by glucose administration ( Figure 6 A). There was no appreciable difference in the kinetics of blood glucose subsequent to treatment with glucose or 2DG ( Figure S2 B).

(H and I) WT and Ppara −/− mice were infected with 400 PFUs of influenza virus. (H) Survival after influenza infection is shown. p = 0.0074; WT n = 6; Ppara −/− n = 8, representative of three independent experiments. (I) Lung and BAL viral load 5 days post-infection is shown. n = 6–7/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 hr after LPS. p < 0.0001 for glucose+VA versus glucose and glucose+VA versus glucose+Keppra. n = 15 glucose; n = 15 glucose+VA; n = 4 glucose+Keppra.

Because we found that mice displayed signs of neuronal damage in both influenza infection and poly(I:C)-induced viral inflammation, we further investigated the effect of inhibition of glucose utilization in viral inflammation. We reasoned that ER-stress-mediated apoptotic pathways, which are integral to the cellular response to viral infection, might link viral inflammation to neuronal damage (). In particular, we focused on the ER-stress-induced transcription factor CHOP, which can induce apoptosis upon prolonged or excessive activation (). We found that expression of CHOP protein and its target gene Gadd34 was elevated in the hindbrains of mice treated with poly(I:C) and 2DG, an effect that was IFNaR dependent ( Figures 5 A and 5B ). Moreover, CHOP-deficient mice (Ddit3) were completely protected from poly(I:C) and 2DG challenge in a manner independent of inflammatory magnitude ( Figures 5 C and 5D). Autonomic dysfunction was also completely abrogated in CHOP-deficient mice challenged with poly(I:C) and 2DG ( Figure 5 E). To test the contribution of CHOP to host tolerance and resistance, we utilized the influenza infection model. We found that CHOP-deficient mice were significantly protected from influenza and 2DG challenge in a manner independent of pathogen burden or degree of inflammation ( Figures 5 F–5H and S5 A). This is in contrast to models of bacterial inflammation, where CHOP deficiency promotes tissue injury and morbidity (). Interestingly, 2DG and IFNα together led to sustained and elevated CHOP expression and apoptosis in mouse embryonic fibroblasts (MEFs) ( Figures S5 B and S5C). These data together suggest that glucose utilization is critical to tissue tolerance of virally induced inflammation through maintenance of an appropriate ER stress response.

(C) MEFs treated with vehicle, IFNα, Poly(I:C), and Thapsigargin (Thaps), in the presence of vehicle, glucose or 2DG for 24 hr. Flow cytometric analysis for Annexin V. Two-three replicates per group. Data representative of three independent experiments.

(B) Mouse embryonic fibroblasts (MEFs) treated with vehicle, IFNα, 2DG, or IFNα and 2DG. mRNA expression at 0, 4, and 24 hr after treatment. n = 3 replicates per group. Data representative of two independent experiments.

(F–H) WT mice and Ddit3 −/− mice were infected with 700 PFUs of influenza virus and treated with 2DG. (F) Survival after influenza infection is shown. p = 0.048; n = 5/group. (G) Plasma IFNα is shown. n = 5/group. (H) Lung and BAL viral load 5 days post-infection is shown. n = 5/group.

(D and E) WT and Ddit3mice were challenged with poly(I:C) and treated with 2DG. (D) Plasma IFNα is shown. n = 5/group. (E) Vital signs measured 18 hr after poly(I:C) + 2DG are shown. n = 3–7/group (vital sign values of WT mice same as those in Figure 4 D).

(A) WT and Ifnar −/− mice were challenged with poly(I:C) and then treated with either IP PBS or 2DG. Whole-hindbrain lysates 24 hr after poly(I:C) treatment immunoblotted for CHOP and β-tubulin are shown.

These data suggest that the lethal effect of poly(I:C) and 2DG co-administration was likely independent of the magnitude of inflammation but rather was dependent on tissue tolerance to immunopathology in the hindbrain, downstream of IFNaR signaling.

To globally assess 2DG and glucose uptake and distribution following poly(I:C) or LPS challenge, we subjected mice to 2-deoxy-2-[F] fluorodeoxy-D-glucose-positron emission tomography-computed tomography (F-FDG-PET-CT). We found that glucose was actively taken up by the brainstem after poly(I:C), but not with LPS challenge. In contrast, LPS induced more glucose uptake in the hypothalamic area ( Figure 4 E). There were no other differences in glucose compartmentalization between poly(I:C) and LPS ( Figure S4 B). Increased 2DG uptake in the hindbrain in poly(I:C)-treated mice was associated with decreased levels of Il1b, Il6, and Tnfa and no difference in Mx1 in the hindbrain, indicating that neuronal dysfunction in brainstem was not due to increased inflammation ( Figure S4 C). Consistent with the PET data, we did not detect differences in blood glucose or measures of cardiac, liver, or renal dysfunction ( Figure S4 D).

To examine whether the lethal effects of 2DG were mediated by differences in the magnitude of the inflammatory response, we assessed plasma cytokines and did not find significant differences in circulating IFNα ( Figure 4 C). Histopathologic analyses showed no differences between treatment groups. To identify the cause of mortality, we performed vital sign monitoring and found that mice challenged with poly(I:C) and 2DG, like flu-infected mice, exhibited profound defects in the control of body temperature and respiratory and heart rate, but not oxygen saturation ( Figure 4 D). Thus, we reasoned that neuronal dysfunction and loss of autonomic control was responsible for mortality.

To generalize the findings from influenza infection, we next utilized intravenous poly(I:C) injection as a model of systemic viral inflammation (). Co-administration of poly(I:C) and 2DG or DMH was uniformly lethal ( Figures 4 A and S4 A). Because we were unable to generate a dose of poly(I:C) that caused lethality by itself, we were unable to assess whether glucose supplementation would be protective. To test whether type I IFN was required for the effect of 2DG on poly(I:C)-induced inflammation, we subjected IFNα-receptor (IFNaR)-deficient (Ifnar1) mice to poly(I:C) and 2DG challenge. Ifnar1mice were completely protected ( Figure 4 B), indicating that IFNaR signaling was required for mediating the lethal effects of 2DG.

(C and D) Mice were challenged with Poly(I:C), then treated with either IP PBS, glucose, or 2DG. (C) Hindbrain mRNA expression 4 hr after Poly(I:C) administration and indicated treatments. n = 3-5/group. (D) Blood glucose measured at 2, 6, and 18 hr after Poly(I:C) and indicated treatments. Plasma troponin-I, ALT and creatinine measured at 24 hr after Poly(I:C).

(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 (ROIs) and T2-weighted magnetic resonance images are provided for reference. CT, computed tomography. n = 3/group.

(C and D) Mice were challenged with poly(I:C) and then treated with IP PBS, glucose, or 2DG. (C) Plasma IFNα is shown. n = 5/group. (D) Vital signs measured 18 hr after poly(I:C) administration are shown. n = 3–7/group.

Mortality from influenza infection is often linked to development of pneumonia (). To determine whether 2DG impacted the extent of lung damage, we assessed the pathological outcomes of PBS versus 2DG treatment in influenza infection. Histopathologic examination of lung showed no difference in edema, hemorrhage, or inflammatory cell infiltrates ( Figures 3 H, 3I, and S3 D). To identify alternative causes of death, we assayed vital signs of PBS- or 2DG-treated mice over the course of influenza infection. We found that 2DG-treated mice had no difference in blood Olevel but had decreased heart rate, respiratory rate, and body temperature ( Figure 3 J). These findings are consistent with a derangement of central autonomic control. To verify that 2DG was not itself causing neuronal dysfunction and lethality, we administered the identical 2DG regimen utilized in influenza to mice infected with another pulmonary pathogen, Legionella pneumophila, and this did not cause mortality, indicating that the lethal effects of 2DG occurred only in the context of the viral inflammation induced by influenza infection ( Figure S3 E). Collectively, this suggests that the effect of caloric supplementation on influenza infection is mediated through availability of glucose utilization and its impact on tissue tolerance mechanisms, which are likely impaired in the brain.

Next, we assessed whether the effect of caloric supplementation on influenza infection was mediated through immune resistance of virus or tissue tolerance. Differences in viral burden and/or immune activation would implicate an effect of caloric supplementation on host resistance. Six days post-infection, we performed plaque assays using both lung homogenate and bronchoalveolar lavage fluid (BAL) from mice treated with PBS or 2DG. Additionally, we assayed gene expression of the influenza gene NP from lung tissue homogenate. In all three cases, we found no differences in viral load between groups ( Figure 3 E). We then assayed antiviral inflammatory mediators to determine whether immunopathology could account for the lethality caused by 2DG treatment. We found that expression of interferon-inducible genes as well as Cxcl1 and Il6 were similar between PBS- and 2DG-treated groups ( Figures 3 F and S3 B). Likewise, we found no difference in plasma IFNα levels or immune cell infiltration into the lung after infection ( Figures 3 G and S3 C).

We next asked whether viral infections, which induce a different type of immune response compared to bacterial infections, were also affected by caloric supplementation. We employed an influenza model in which mice are infected intranasally with influenza virus A/WSN/33. We observed that mice infected with influenza also exhibited anorexia, albeit less severely than in L. moncytogenes infection ( Figure 3 A). Surprisingly, we found that gavage of 1 kilocalorie BID of enteral nutrition starting 8 hr post-infection protected mice from influenza-associated mortality ( Figure 3 B). Gavage of isocaloric isovolumetric glucose partially recapitulated the effect of enteral nutrition, whereas i.p. injection of 2DG concurrently with feeding completely ablated the survival benefit ( Figure 3 C). Caloric supplementation with casein and olive oil provided little to no survival benefit ( Figure S3 A). With a lower dose of influenza infection, we found that 2DG alone was able to uniformly kill flu-infected mice compared to vehicle control ( Figure 3 D). These data together indicate that glucose availability and utilization are critical to surviving influenza infection.

(D) H&E staining of lung tissue 6 days after infection with 375 PFU influenza virus and treatment with IP PBS or 2DG. Letters correspond to areas of the lung annotated in Figure 3 . Scale bar = 50 μm.

(C) Flow cytometric analysis of lung and BAL on day 6 after infection with 700 PFU of influenza virus and treatment with IP PBS or 2DG.

(B) mRNA expression of whole lung tissue on day 6 after infection with 375 PFU of influenza virus and treatment with IP PBS or 2DG.

(A) Survival after infection with 800 PFU of influenza virus. Mice were gavaged with Abbott Promote (Food), casein, olive oil, or PBS vehicle. Figure 3 B and 3C are a subset of Figure S3A, separated for clarity (the same PBS-treated and food-treated groups are shown).

(E–J) Mice were infected with 375 PFUs influenza virus and treated with IP PBS or 2DG. n = 4–5/group. (E) Lung and bronchoalveolar lavage (BAL) viral load 6 days post-infection by PFU and qPCR for WSN nucleoprotein (NP) is shown. (F) mRNA expression of whole lung tissue at day 6 is shown. (G) Plasma IFNα is shown. (H and I) H&E staining of lung tissue 6 days post-infection and histologic scoring is shown. The scale bar represents 500 μm. For magnified views of insets, please see Figure S3 E. (J) Vital signs after influenza infection are shown.

(B and C) Survival after infection with 800 PFUs of influenza virus. Mice were gavaged with food, glucose, or PBS vehicle. Mice gavaged with food were also injected with IP PBS (food) or 2DG (food+2DG). PBS versus food p = 0.0047; PBS versus glucose p = 0.1058; food versus food+2DG p = 0.0001; PBS versus food+2DG p = 0.0256. n = 10/group. (B) is a subset of (C), separated for clarity (the same PBS-treated and food-treated groups are shown in B and C).

We observed that mice challenged with LPS and glucose displayed symptoms consistent with tonic-clonic seizure. Prior to death, animals would develop high-amplitude convulsions followed by decerebrate posturing. This is consistent with studies that have demonstrated neurologic deficits, including seizure, and neuronal apoptosis in animals suffering from endotoxic shock (). Hypoglycemia is a common cause of seizure, but we found that blood glucose did not differ significantly between groups over time ( Figure S2 B). We performed vital sign monitoring (body temperature, blood Osaturation, respiratory rate, and heart rate) in endotoxemic mice treated with PBS, glucose, or 2DG 24 hr post-LPS injection and found that 2DG-treated mice maintained their body temperature significantly better than glucose- and PBS-treated mice ( Figure S2 C). To assess other evidence of end-organ damage, we measured plasma markers of tissue injury (troponin-I, alanine aminotransferase, and creatinine), which were largely unchanged between treatments, with the exception that glucose-treated mice had higher levels of plasma creatinine ( Figure S2 D). Finally, detailed histopathologic analysis of H&E-stained slides of brain, heart, lung, liver, kidney, pancreas, stomach, bone marrow, thymus, and spleen was performed to identify any pathologic changes, including edema, hemorrhage, inflammation, necrosis, and apoptosis. The only difference in histopathologic changes was a decreased presence of dark, shrunken neurons in the brains of LPS mice given 2DG, compared to mice given LPS and PBS or glucose. Together, these data implicate neuronal dysfunction as a possible proximal cause of death in LPS endotoxemia.

The advantage of the LPS sepsis model is that it isolates the inflammatory response, as opposed to direct pathogen toxicity, as the source of tissue damage. In this model, nutrients can affect survival by altering the magnitude of inflammatory response or the tissue’s ability to tolerate it. We found no difference in circulating levels of tumor necrosis factor alpha (TNFα) and IL-6 or hepatic expression of acute phase-response genes in endotoxemic mice treated with PBS, glucose, or 2DG ( Figures 2 E and 2F). These findings suggest that glucose utilization does not affect the magnitude of the inflammatory response in endotoxic shock but rather the ability of the tissues to tolerate inflammatory damage.

To examine the role of tissue tolerance in mediating the protective effects of anorexia in bacterial sepsis, we used the LPS sepsis model, where mortality results entirely from a systemic inflammatory response. We found that gavaging mice with enteral nutrition starting 1 hr post-LPS injection led to significantly increased mortality, whereas fluid resuscitation improved survival ( Figure 2 A). To dissect the nutritional components that contribute to mortality, we gavaged mice with isocaloric isovolumetric amounts of glucose, olive oil, or casein and found that only glucose significantly increased mortality ( Figure 2 B). We then tested whether the effects of food intake on susceptibility to sepsis could be reversed with concurrent 2DG treatment. i.p. injection of 2DG concurrently with gavage of enteral nutrition in endotoxemic mice significantly improved survival ( Figure 2 C). As was the case in L. monocytogenes infection, i.p. injection of glucose was sufficient to uniformly kill endotoxemic mice whereas i.p. injection of 2DG was sufficient to fully rescue them ( Figure 2 D). To exclude contributions from carbohydrate-responsive element-binding protein (ChREBP) signaling, which would still be activated with 2DG, we utilized a glucose utilization inhibitor, D-mannoheptulose (DMH), which does not activate downstream ChREBP signaling (), and observed the same protective effects as 2DG ( Figure S2 A). Together, these data suggest that the component of nutritional intake that increased susceptibility to endotoxic shock was glucose and that inhibition of glucose utilization during sepsis was protective.

(C and D) Mice were given 15 mg/kg IP LPS, then treated with IP PBS, glucose, or 2DG given IP. (C) O 2 saturation, heart rate, respiratory rate, and body temperature 24 hr after LPS. (D) Plasma troponin-I, ALT, and creatinine levels measured at 24 hr after LPS.

(B) Blood glucose at baseline, 2, 6 and 24 hr after 15 mg/kg IP LPS with PO gavage of PBS vehicle (LPS-PBS), glucose (LPS-Glucose), or Abbott Promote (LPS-Food) BID; or with IP 2DG (LPS-2DG). Blood glucose of mice treated with only 2DG IP are also shown.

(D–F) Mice were given 15 mg/kg IP LPS and then treated with IP PBS, glucose, or 2DG. (D) Survival after LPS and indicated treatments is shown. IP PBS n = 16; IP glucose n = 10 (p < 0.0001 versus IP PBS); IP 2DG n = 10 (p = 0.01 versus IP PBS). (E) Plasma TNFα and IL-6 are shown. n = 5–10/group. (F) Liver mRNA expression 4 hr after LPS and treatment with PBS (LPS-PBS), glucose (LPS-glucose), and 2DG (LPS-2DG) compared to PBS alone is shown. n = 3–5/group.

(A and B) Survival after 15 mg/kg IP LPS and PO gavage with food, glucose, olive oil, casein, or PBS vehicle. PO PBS n = 10; PO food n = 10 (p = 0.0002 versus PO PBS); PO glucose n = 8 (p < 0.0001 versus PO PBS); no gavage n = 10 (p = 0.0679 versus PO PBS). (A) is a subset of (B), separated for clarity (the same PBS-treated, food-treated, and no gavage groups are shown in A and B).

We found that glucose treatment did not significantly affect bacterial burden or immune infiltration but did increase plasma interferon gamma (IFNɣ) 24 hr post-infection ( Figures 1 D–1F and S1 ). In contrast, 2DG-treated mice had significantly decreased bacterial load compared to controls ( Figure 1 E). The decreased bacterial burden was not due to heightened immune response, as plasma IFNɣ, plasma interleukin-6 (IL-6), and liver immune infiltrate were all decreased in 2DG-treated animals ( Figures 1 D–1F and S1 ). These data raised the possibility that 2DG was mediating clearance of L. monocytogenes through direct inhibition of bacterial growth. To address this, we tested the effect of 2DG on the growth of L. monocytogenes and on the antimicrobial activity of macrophages infected with L. monocytogenes. In both cases, 2DG administration did not affect bacterial growth ( Figures 1 G and 1H). Because the protective effect of anorexia during L. monocytogenes infection was neither mediated through conventional immune clearance mechanisms, nor was it due to inhibition of bacterial proliferation, we hypothesized that tissue-protective mechanisms may be at play.

To assess the role of anorexia in infection, we revisited the model of listeriosis used in prior studies (). We confirmed that, upon infection with L. monocytogenes, mice exhibited a dose-dependent decrease in their food intake ( Figure 1 A) and that enteral supplementation by gavage of 1 kilocalorie twice daily (bis in die [BID]) starting 8 hr post-infection uniformly killed L. monocytogenes-infected mice ( Figure 1 B). The caloric content supplemented was one-fifth of healthy mouse daily food intake. Additionally, we found that gavage with an isocaloric isovolumetric amount of glucose alone was sufficient to cause 100% mortality in infected mice ( Figure 1 B). To exclude contributions from enteroendocrine incretin signaling, we injected glucose intraperitoneally (i.p.) at a dose iso-osmolar to PBS, which provided about 2% of normal daily caloric intake, and found that this was sufficient to recapitulate the lethal effects of enteral glucose ( Figure 1 C). To assess whether glucose was necessary for lethality, we injected L. monocytogenes-infected mice with 2DG and found that 2DG fully rescued mice from listeriosis-induced mortality ( Figure 1 C), consistent with previous work (). Collectively, these data suggest that glucose is the component of food that is necessary and sufficient to mediate lethality in listeriosis when anorexia is blocked by force feeding.

(H) Bone-marrow-derived macrophages (BMDMs) were infected with 5 × 10 5 CFUs of L. monocytogenes in the presence or absence of 15 mM 2DG for 24 hr. CFUs of L. monocytogenes grown from the BMDM cell media supernatant and cell lysate.

(C–F) Mice were infected with 5 × 10 4 CFUs L. monocytogenes and then treated with IP PBS, glucose, or 2DG. n = 5/group. (C) Survival after L. monocytogenes infection and indicated treatments is shown. PBS versus glucose p = 0.0396; PBS versus 2DG p = 0.0344; glucose versus 2DG p = 0.0017. (D) Plasma IL-6 and IFNγ 24 and 48 hr after 5 × 10 4 L. monocytogenes infection are shown. 24-hr plasma IL-6: PBS versus 2DG p = 0.0005; glucose versus 2DG p = 0.0014; 24-hr plasma IFNγ: PBS versus glucose; PBS versus 2DG; glucose versus 2DG all p < 0.0001. (E) Listeria CFUs from spleen and liver 4 days post-infection are shown. (F) Flow cytometry analysis of CD45 + cells within the liver 4 days post-infection is shown.

(B) Survival after infection with 5 × 10 4 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 versus PO PBS/IP PBS); PO glucose/IP PBS n = 19 (p = 0.004 versus PO PBS/IP PBS); PO PBS/IP 2DG n = 10 (p = 0.0085 versus PO PBS/IP PBS).

Discussion

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O’Neill L.A. Metabolic reprograming in macrophage polarization. Figure 7 Model of Glucose Utilization during Viral- and Bacterial-Mediated Inflammation Supporting Unique Tissue Tolerance Mechanisms Show full caption (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. Here, we addressed the effect of anorexia during acute infection and uncovered a surprising differential role for fasting metabolism in maintaining tissue tolerance in different infectious states. It is increasingly appreciated that inflammatory responses must be coupled to specific metabolic programs to support their energetic demands (). In this study, we observed that systemic metabolism appears to be coordinated to support tolerance to different inflammatory states. We found that, whereas glucose utilization was required for survival in models of viral inflammation, it was lethal in models of bacterial inflammation. Concordantly, we found that, whereas ketogenesis was required for survival in bacterial inflammation, it was dispensable in the case of viral inflammation. Unexpectedly, we found that these effects on mortality were largely independent of the degree of inflammation and pathogen clearance. In the case of viral inflammation, lethality subsequent to inhibition of glucose utilization appeared to be mediated by type I IFN signaling on target tissues—likely the brain—which require glucose to mitigate the ER stress response and CHOP-mediated cellular dysfunction. In the case of bacterial inflammation, lethality subsequent to glucose administration appeared to be mediated by suppression of ketogenesis, which led to impaired resistance to ROS-mediated damage in the brain ( Figure 7 ). Thus, our results suggest that distinct inflammatory responses may be coupled with specific metabolic programs in order to support unique tissue tolerance mechanisms that, when uncoupled, lead to enhanced immunopathology, leading to death.

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Song B.J. Activation of PPARα by Wy-14643 ameliorates systemic lipopolysaccharide-induced acute lung injury. Liu et al., 2016 Liu L.

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et al. Proinflammatory signal suppresses proliferation and shifts macrophage metabolism from Myc-dependent to HIF1α-dependent. Yang et al., 2014 Yang L.

Xie M.

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et al. PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis. We found that interfering with the normal ketogenic state following LPS-mediated inflammation was lethal, likely by interfering with ROS adaption programs in the brain. Our findings are consistent with observations that PPARα agonism and inhibition of glucose utilization are generally protective in bacterial sepsis models (). However, unlike many of these studies, we did not observe large differences in the magnitude of inflammation, likely because we administered 2DG and glucose after and not before infectious or inflammatory challenge. Thus, our observations are likely unrelated to the body of literature that supports a role for HIF1α, PKM2, and aerobic glycolysis in generating the LPS inflammatory response (). Consistent with our inability to detect differences in inflammation in sterile inflammatory models, we did not detect differences in pathogen burden in live infection models where glucose administration led to lethality in L. monocytogenes infection in the absence of increased pathogen burden or bolstered immune response.

Hoetzenecker et al., 2012 Hoetzenecker W.

Echtenacher B.

Guenova E.

Hoetzenecker K.

Woelbing F.

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et al. ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression. Kolls, 2006 Kolls J.K. Oxidative stress in sepsis: a redox redux. Shimazu et al. (2013) Shimazu T.

Hirschey M.D.

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et al. Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor. ROS-mediated cytotoxicity is a well-appreciated phenomenon in bacterial sepsis (), and ROS-detoxification pathways have been implicated in mitigating tissue damage and mortality.recently reported that BHOB functioned as an HDAC-1 inhibitor and that this led to transcription of ROS-detoxification pathways. Furthermore, we found that the timing of ketogenesis, an adequately nourished host, or both are necessary for the protective effect of fasting that occurs as a coordinated response to bacterial inflammation. Instead of rescuing mice from LPS mortality, fasting and ketogenic pre-conditioning potentiated death. Taken together, we present evidence that the fasting response that occurs as part of the inflammatory response is required to maintain resistance to oxidative stress in LPS sepsis.

Janssens et al., 2014 Janssens S.

Pulendran B.

Lambrecht B.N. Emerging functions of the unfolded protein response in immunity. Tabas and Ron, 2011 Tabas I.

Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Burke et al., 2014 Burke J.D.

Platanias L.C.

Fish E.N. Beta interferon regulation of glucose metabolism is PI3K/Akt dependent and important for antiviral activity against coxsackievirus B3. Jamieson et al., 2013 Jamieson A.M.

Pasman L.

Yu S.

Gamradt P.

Homer R.J.

Decker T.

Medzhitov R. Role of tissue protection in lethal respiratory viral-bacterial coinfection. Although anorexia is a common response in both bacterial and viral infections, we find the opposite consequence of fasting metabolism in our models of bacterial and viral inflammation. Whereas in bacterial infection and LPS-induced inflammation, we found a detrimental effect of glucose, a protective effect of 2DG, and a requirement for ketogenesis in order to maintain tolerance, in viral infection and poly(I:C)-induced inflammation, these effects were opposite. 2DG administration led to lethality in poly(I:C)-induced inflammation in a manner that was independent of the magnitude of IFNα expression but dependent on IFNaR signaling. Manipulation of fasting metabolism did not affect viral burden in influenza infection. Viral infections are known to stimulate the unfolded protein response mediated, in part, via the PERK-eIF2a-ATF4-CHOP pathway (). When this pathway is engaged, cells can either adapt to ER stress or induce apoptosis through CHOP if ER stress cannot be managed (). Our data suggest that glucose utilization is required for the cytoprotective response in neurons in the setting of viral inflammation, as inhibition of glucose utilization led to death, which was CHOP dependent. Indeed, our PET studies indicate that glucose redistribution is largely the same between the early phases of bacterial and viral inflammatory challenge with the exception of sub-regions in the brain, where LPS and poly(I:C) appear to regulate glucose uptake differentially. The purpose and mechanism underlying this observation remain to be elucidated. The precise mechanism whereby IFN signaling converges with glucose utilization programs also remains to be fully resolved, but recent studies demonstrated that interferon signaling leads to changes in glucose uptake, which is important for the antiviral response (). Thus, whereas alternative fuel substrate availability is coupled to and necessary for adaptation to bacterial inflammation, glucose availability is coupled to and necessary for cellular adaptation to viral inflammation. The logic of their coupling is likely related to the substrate dependence of the cellular adaptation programs that are engaged. These findings are consistent with our previous study, where we found that synergistic lethality in mice co-infected with influenza and Legionella occurred in a manner independent of pathogen burden (), and it is interesting to speculate here that perhaps one cause of lethality in this co-infection model is a result of metabolic incompatibility in the setting of both a viral and bacterial infection.

Arabi et al., 2015 Arabi Y.M.

Aldawood A.S.

Haddad S.H.

Al-Dorzi H.M.

Tamim H.M.

Jones G.

Mehta S.

McIntyre L.

Solaiman O.

Sakkijha M.H.

et al. PermiT Trial Group

Permissive underfeeding or standard enteral feeding in critically ill adults. Casaer and Van den Berghe, 2014 Casaer M.P.

Van den Berghe G. Nutrition in the acute phase of critical illness. Seron-Arbeloa et al., 2013 Seron-Arbeloa C.

Zamora-Elson M.

Labarta-Monzon L.

Mallor-Bonet T. Enteral nutrition in critical care. Given the conservation of cellular adaptation and metabolic programs in mouse and human, our findings likely have clinical implications. The role of nutrition in managing patients with sepsis is unclear at best, and multiple studies have failed to show differences in survival from feeding, including the most-recent study, which asked whether lower caloric supplementation would improve outcomes (). There have been a series of studies exploring different feeding formulations with different caloric or micronutrient contents (). However, we could not find an example where different feeding formulations were targeted to different types of infections, as opposed to different types of organ failure (), or where post hoc analyses were directed at pathogen class. Our study implicates a differential need for metabolic fuels as a function of infection (or inflammation) class and sheds light on the biology behind the old adage “starve a fever, stuff a cold.” Much work will need to be done to identify how organismal metabolism is coordinated in other infectious and inflammatory states and whether or not these findings can be extended to humans in the management of inflammatory diseases and critical illness.