Abstract Preterm infants are susceptible to bloodstream infection by coagulase-negative staphylococci (CONS) that can lead to sepsis. Glucose-rich parenteral nutrition is commonly used to support the infants’ growth and energy expenditure but may exceed endogenous regulation during infection, causing dysregulated immune response and clinical deterioration. Using a preterm piglet model of neonatal CONS sepsis induced by Staphylococcus epidermidis (S. epidermidis) infection, we demonstrate the delicate interplay between immunity and glucose metabolism to regulate the host infection response. Circulating glucose levels, glycolysis, and inflammatory response to infection are closely connected across the states of tolerance, resistance, and immunoparalysis. Furthermore, high parenteral glucose provision during infection induces hyperglycemia, elevated glycolysis, and inflammation, leading to metabolic acidosis and sepsis, whereas glucose-restricted individuals are clinically unaffected with increased gluconeogenesis to maintain moderate hypoglycemia. Finally, standard glucose supply maintaining normoglycemia or pharmacological glycolysis inhibition enhances bacterial clearance and dampens inflammation but fails to prevent sepsis. Our results uncover how blood glucose and glycolysis control circulating immune responses, in turn determining the clinical fate of preterm infants infected with CONS. Our findings suggest further refinements of the current practice of parenteral glucose supply for preterm infants during infection. Keywords: Infectious disease, Inflammation Keywords: Bacterial infections, Cellular immune response Introduction Among millions of infants born preterm (i.e., <37 weeks of gestation) every year, 25%–50% of those with very low birth weight experience serious neonatal infection, leading to sepsis ([31]1). Up to 80% episodes of late-onset sepsis (i.e., >3 days after birth) are caused by coagulase-negative staphylococci (CONS), especially Staphylococcus epidermidis (S. epidermidis) ([32]2–[33]4). Although CONS-associated sepsis is not as life-threatening as Gram-negative bacterial sepsis, it is of serious concern as a predisposing factor to multiple morbidities occurring later in life ([34]5, [35]6). Currently, the only therapeutic option for neonatal infection is antibiotics, which are empirically used for almost all preterm infants ([36]7, [37]8) despite the risk of disturbing immune development and causing antimicrobial resistance ([38]9). The interplay between immune cell energy metabolism and function has emerged as a key mechanism in many adult diseases, but its role in neonatal infection is largely unknown. Initial theories ([39]10, [40]11) and in vitro reports ([41]12, [42]13) suggest that the low energy reservoir in newborns programs their immune system to disease tolerance — a strategy avoiding fast ATP production for inflammatory responses in immune cells via inhibition of the Warburg effect switching from oxidative phosphorylation (OXPHOS) to glycolysis. This may explain how newborns tolerate 10–100 times higher systemic bacterial loads ([43]10) and have diminished blood cytokine responses to in vitro infection challenge ([44]14, [45]15), relative to adults. However, it is unclear how this disease tolerance in preterm infants is connected to their high susceptibility to neonatal sepsis, a pathological state associated with an early hyperinflammatory phase followed by immunoparalysis or death ([46]16). During the first few weeks of life, a majority of preterm infants receive parenteral nutrition (PN) to maintain adequate nutrition, and international guidelines recommend a parenteral supplement containing a high concentration of glucose (~14–17 g/kg/d) to avoid hypoglycemia (i.e., blood glucose level <2.6 mM) and related brain injury ([47]17–[48]20). However, prolonged high parenteral glucose intake may lead to hyperglycemia (i.e., blood glucose level >6.9 mM) ([49]21), which is detected in up to 80% of preterm infants ([50]22). Notably, there are no specific guidelines for using parenteral glucose during neonatal infection, although PN-related hyperglycemia is associated with longer hospitalization of infected infants ([51]18). We postulate that high parenteral glucose provision to infected newborns may accelerate blood immune cell glycolysis, leading to excessive inflammation and sepsis. Detailed understanding of this mechanism may shed light on novel infection therapies (e.g., reduced parenteral glucose supply or glycolysis inhibition). Numerous animal models of infection and sepsis have been established e.g., cecal ligation and puncture ([52]23) and oral ([53]24, [54]25) or systemic bacterial challenge ([55]26), but no rodent models can address the contributing effects of PN. The preterm pig is a unique model because it allows PN administration via umbilical catheter ([56]27) and has multiple organ immaturities and infection susceptibility ([57]26, [58]28–[59]30), as found in preterm infants. Furthermore, systemic S. epidermidis administration to newborn preterm pigs can induce clinical and cellular responses (e.g., fever, inflammation, blood platelet and leukocyte depletion) that may progress to septic shock (acidemia and hypotension) 12–24 hours after infection, similar to sepsis caused by CONS and other bacteria in preterm infants ([60]26). Here, we further used this CONS sepsis model and showed that the infection response in preterm newborns was tightly related to circulating glycolysis and glucose levels. We found that high parenteral glucose supply predisposed to hyperglycemia, excessive inflammation, reduced bacterial clearance, and extreme sensitivity to sepsis following neonatal infection, whereas restricted glucose provision caused hypoglycemia but protected against sepsis. We also showed that a standard glucose supply maintaining normoglycemia, with or without using a glycolysis inhibitor, dichloroacetate (DCA), enhanced bacterial clearance and alleviated systemic inflammation and metabolic acidosis but did not prevent sepsis. Parenteral glucose restriction may be an effective therapy for preterm infants infected with CONS. Results S. epidermidis thresholds determine the host immunometabolic responses in vitro and in vivo. Preterm infants can presumably withstand higher circulating bacterial levels than can adults and term infants prior to mounting resistant responses and later becoming immunoparalyzed ([61]10). Here, we first tested the threshold switching among those phases by measuring in vitro immunometabolic responses of cord blood from preterm pigs to increasing doses of S. epidermidis ([62]Figure 1). When increasing bacterial doses, inflammatory (TNF-α) and antiinflammatory (IL-10) cytokine responses at both the gene and protein levels switched from an immune-tolerant state at low doses (5 × 10^1 to 5 × 10^4 CFU/mL) to resistance at the dose of 5 × 10^5 CFU/mL ([63]Figure 1, A–C, and [64]Supplemental Figure 1, A–C; supplemental material available online with this article; [65]https://doi.org/10.1172/jci.insight.157234DS1). Of note, the ratio of TNFA to IL10 (Th1 and Th2 cytokines, respectively) also peaked at 5 × 10^5 CFU/mL but decreased again at higher doses, indicating another switch to immunoparalysis. The same trends applied to other parameters, including elevated mRNA levels of targets related to inflammation (IL6, TLR2), Th1 responses (IFNG and IFNG/IL4), and the glycolysis–mTOR pathway (HIF1A); and decreased levels of genes related to OXPHOS (COX1) and Foxp3^+CD4^+ lymphocytes (a Treg marker) at the bacterial dose of 5 × 10^5 CFU/mL but not lower or higher doses ([66]Figure 1, D–I, and [67]Supplemental Figure 1, D and E). In parallel, cellular glucose uptake, measured by the differences in supernatant glucose levels with versus without bacterial challenge, was gradually elevated with increasing bacterial doses, then reached a plateau level at the bacterial dose of 5 × 10^5 CFU/mL ([68]Supplemental Figure 1F). These data revealed clear dose-dependent switches of immunometabolic response to S. epidermidis from tolerance to resistance and, later, immunoparalysis. Figure 1. In vitro immunometabolic response to S. epidermidis. [69]Figure 1 [70]Open in a new tab (A–H) mRNA levels of TNFA, IL10, TNFA to IL10 ratio, HIF1A, COX1, TLR2, IL6, and IFNG of cord blood from preterm piglets in responses to an increasing bacterial dose (5 × 10^1 to 5 × 10^7 CFU/mL, stimulated for 2 hours at 37°C and 5% CO[2]; n = 5–6). (I) Frequency of Foxp3^+ cells within the CD4^+ lymphocyte population in S. epidermidis–stimulated cord blood (2 hours at 37°C and 5% CO[2]; n = 4). All data are presented as violin dot plots with median (solid line) and IQR (dotted lines) and were analyzed using a linear mixed-effect model followed by Tukey post hoc comparisons. Values not sharing the same letters are significantly different (P < 0.05). We then tested clinical and metabolic responses to increasing S. epidermidis doses in vivo, using newborn preterm pigs (at 90% gestation) nourished by PN with a standard glucose (STG) level. The animals were clinically and metabolically unaffected by the 2 lowest doses (10^6 and 10^8 CFU/kg; disease tolerance). At a dose of 10^9 CFU/kg, survival was 75% with dysregulated glucose and lactate at 24-hour follow-up (i.e., disease resistance), whereas the highest dose of 5 × 10^9 CFU/kg decreased the 24-hour survival rate to less than 20% and induced glucose and lactate dysregulation already at 12 hours ([71]Figure 2, A–C). Plasma IL-6 and IL-10 at 24 hours after infection showed increased levels with increasing doses of inoculated bacteria ([72]Figure 2, D and E). Thus, clinical responses were clearly intertwined with perturbed glucose homeostasis and followed a severity spectrum dictated by bacterial dose and cytokine responses. Furthermore, to confirm the metabolic shifts following infection, we performed a follow-up experiment and collected liver of infected (10^9 CFU/kg) and control animals for transcriptomic analysis. The liver of infected animals clearly showed activation of both inflammatory pathways (i.e., TNF and IL-17 signaling, Th1 and Th1 differentiation) and sugar metabolic pathways (i.e., glycolysis, galactose, fructose and mannose metabolism) ([73]Figure 2F; [74]Supplemental Figure 1, G and H; and [75]Supplemental Table 1, A and B). Both in vitro and in vivo data showed that preterm immune cells had a propensity to undergo a metabolic shift toward aerobic glycolysis when activated, whereby glucose availability may determine the potency of cellular and cytokine responses with potential clinical implications. Figure 2. In vivo immunometabolic response to S. epidermidis. [76]Figure 2 [77]Open in a new tab (A) Survival rate, (B) blood glucose, (C) lactate, (D) plasma IL-6, and (E) plasma IL-10 levels of preterm newborn piglets 24 hours after infection with S. epidermidis (10^6 to 5 × 10^9 CFU/kg) via the intra-arterial catheter. (F) Gene set enrichment analysis (GSEA) of liver transcriptome from control and S. epidermidis–infected preterm pigs (10^9 CFU/kg, 12 hours after infection) revealing the top enriched pathways activated and suppressed by infection. Data are presented in a cumulative hazard curve (A) or violin dot plots with median (solid line) and IQR (dotted lines, B–E), and analyzed by Mantel-Cox test or linear model followed by Tukey post hoc comparisons. Values at a time point not sharing the same letters are significantly different (P < 0.05). ***P < 0.001, compared with the uninfected control. Transcriptomics was performed by DESeq2 with FDR adjusted by BH correction using α = 0.1 as the threshold. GSEAs were based on DEGs between infected and control groups, and pathways with adjusted (adjust.) P < 0.05 are considered significantly regulated pathways. Gene ratio (from 0 to 1) shows the fraction of the number of enriched genes relative to the total number of genes in the gene set. The size of the circle reflects the number of DEGs enriched in each pathway. Parenteral glucose determines sepsis susceptibility during S. epidermidis infection. We next investigated clinical, metabolic, and immune responses to neonatal systemic infection on the background of experimentally extreme differences in glucose provision to mimic hyperglycemic and hypoglycemic conditions in infected preterm infants. Newborn preterm piglets were nourished exclusively with PN containing either a high glucose (HG; 21% glucose) or a very low glucose (LG; 1.4% glucose) level, and we systemically challenged the piglets with 10^9 CFU/kg S. epidermidis, the dose leading to clinical symptoms but moderate acute mortality from the dose-finding study (experimental design in [78]Figure 3A). Based on the criteria for sepsis and the humane euthanasia endpoint for this study (i.e., arterial blood pH < 7.1 and clinical symptoms of extreme lethargy, discoloration, and tachypnea), animals receiving HG PN (hereafter, HG animals) at the end of the study (12 hours) had substantially higher sepsis incidence and mortality relative to those of animals receiving LG (hereafter, LG animals) (HG vs. LG: n = 10 of 11 vs. n = 2 of 10; P < 0.01) ([79]Figure 3B). Importantly, these findings were accompanied by impaired blood bacterial clearance dynamics from 3 to 12 hours in the HG animals (P for glucose levels [P[glu]] < 0.01) ([80]Figure 3C), whereas LG PN prevented respiratory and metabolic acidosis by preserving blood acid-buffering capacity (for all, P[glu] < 0.01) ([81]Figure 3, D–F). Moreover, HG piglets had a quicker meconium passage than did LG animals, a common physiological stress response in the perinatal period ([82]Figure 3I). Furthermore, plasma albumin levels by the end of the study were 2 times lower in the HG piglets relative to LG piglets (P < 0.01), indicating stress-induced changes in liver protein synthesis, vascular permeability, or renal dysfunction. Taken together, glucose restriction during neonatal S. epidermidis infection provided acute clinical benefits. Figure 3. Parenteral glucose restriction protects S. epidermidis–infected preterm piglets from sepsis. [83]Figure 3 [84]Open in a new tab (A) Preterm newborn piglets were nourished exclusively with PN containing HG (21%; 30 g/kg/d) or LG (1.4%; 2 g/kg/d) concentrations (n = 10–11 per group), intra-arterially infected with 10^9 CFU/kg S. epidermidis, and cared for 12 hours after infection or until clinical signs of sepsis. Uninfected animals (n = 3) receiving LG PN served as a reference and were not included in the statistics. (B) Survival curve, based on sepsis diagnosis and humane euthanasia endpoint (i.e., blood pH < 7.1 and presence of septic shock symptoms). (C) S. epidermidis density from blood collected by jugular venous (3–6 hours) or heart (12 hours) puncture, by counting CFUs after plating onto tryptic soy agar containing 5% sheep’s blood and incubated for 24 hours at 37°C. (D–H) Blood-gas parameters derived from arterial blood samples collected via the umbilical arterial catheter at 3, 6, and 12 hours. (I) Time of first passaged meconium after infection. (J–M). Blood biochemical parameters measured in heparinized plasma from arterial blood collected at 12 hours. Data are presented as cumulative hazard curves (B and I) or violin dot plots including median (solid line) and IQR (dotted lines) (C–H and J–M). Data were analyzed using a Mantel-Cox test (B and I) or a linear mixed-effects model (C–H and J–M), including an interaction between group and time after infection (C–H). All analyzed data represent 2 independent litters. P for time (P[time]), P[glu], and P for interaction (P[int]) denote probability values for effects over time across the HG and LG groups, group effect (HG vs. LG) over time, and interaction effect between time and group in the linear mixed effects interaction model, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with HG group at the same time point. ALAT, alanine aminotransferase; BUN, blood urea nitrogen; CON, control; pCO[2], partial pressure of CO[2]. Panel A was created using Biorender.com. Unsurprisingly, HG piglets were hyperglycemic (blood glucose level of 10–20 mM) with an increasing trend over time, whereas the LG nourishment paradigm led to hypoglycemia, with blood glucose levels of approximately 2 mM and a decreasing time trend ([85]Figure 3G). A similar pattern was observed for blood lactate: 40% of animals in the HG group had levels greater than 10 mM (P < 0.01 at 12 hours vs. LG group) ([86]Figure 3H), indicating accelerated circulating glycolysis and lactic acidosis, whereas lactate levels in the LG group decreased over time, likely because it was used for gluconeogenesis. Despite a large difference in plasma glucose levels, ATP and pyruvate levels only showed minor or no differences between the HG and LG groups ([87]Figure 3, J and K). However, blood urea levels at 12 hours were markedly increased in LG relative to HG animals ([88]Figure 3L), suggesting conversion of exogenous glucogenic amino acids to fuel endogenous glucose production. In parallel, the plasma activity of alanine aminotransferase, the enzyme responsible for deaminating alanine to pyruvate as an initial step in gluconeogenesis, was decreased in the LG group ([89]Figure 3M). In summary, high parenteral glucose provision during infection facilitated extensive circulating glycolysis, whereas acute metabolic adaptation to exogenous glucose restriction appeared to maintain adequate cellular energy. During the 12-hour course of infection, glucose infusion levels massively interfered with the fate of blood cell subsets. An overall decreasing trend in cell numbers was observed over time for leukocytes, erythrocytes, and thrombocytes ([90]Figure 4, A–C), with HG PN leading to a greater loss of total leukocytes and more severe thrombocytopenia (P[glu] < 0.01). Importantly, HG PN induced a robust depletion of lymphocytes, neutrophils, and monocytes at 3–6 hours with partial replenishment at 12 hours, which was not observed in the LG group ([91]Figure 4, D–F). Interestingly, this was associated with distinct temporal changes in plasma cytokine response to infection. TNF-α, IL-10, and IL-6 levels were elevated over time during infection, but HG animals had more pronounced TNF-α and IL-6 responses and lower IL-10 levels relative to LG group (P[glu] < 0.01) ([92]Figure 4, G–I). Collectively, the HG nourishment paradigm induced a more rapid immune response with greater cell loss and evidence of emergency hematopoiesis, prioritizing release of leukocytes but not erythrocytes and thrombocytes from the bone marrow. This may have compromised the regulatory response shown by reduced IL-10 secretion. Figure 4. Parenteral glucose restriction protects S. epidermidis–infected preterm piglets from excessive inflammation and immune cell loss. [93]Figure 4 [94]Open in a new tab (A–F) Numbers of hematopoietic cells and major leukocyte subsets in blood samples collected 3–12 hours after S. epidermidis infusion. (G–I). Cytokine levels measured in heparinized plasma from the same blood samples. Data are presented as violin dot plots with median and IQR and were analyzed using a linear mixed-effects model including interaction between group and time after infection. All analyzed data represent 2 independent experiments using separate litters. P for time (P[time]), P[glu], and P for interaction (P[int]) denote probability values for time effects across HG and LG groups, group effect (HG vs. LG) over time and interaction effect between time and group in the linear mixed-effects model, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with HG group at the same time point. Although glucose restriction has acute clinical benefits with reduced mortality and clinical signs of sepsis, glycolysis, and systemic inflammation, this practice led to hypoglycemia and may have negative effects on the preterm brain, which relies on steady glucose supplies for proper development. Therefore, an alternative strategy to manipulate the immunometabolic response to infection in a normo- or hyperglycemic state was investigated. Glycolysis inhibition decreases inflammatory response to S. epidermidis in vitro. Having shown that circulating glycolysis is closely connected to inflammation and clinical fate during neonatal S. epidermidis infection, we aimed to identify a clinically relevant treatment to prevent sepsis and exaggerated aerobic glycolysis beyond glucose restriction. First, we tested the well-known glycolysis inhibitors at their commonly used doses found in the literature for capacity to reduce inflammation in preterm pig cord blood challenged with S. epidermidis. Rapamycin (which targets the mTOR pathway), DCA (which targets pyruvate dehydrogenase kinase), and FX11 (which targets lactate dehydrogenase) all reduced bacteria-induced TNF-α response, but DCA seemed to have higher inhibitory potency across the 2 tested bacterial doses ([95]Figure 5A). We proceeded with a dose-finding test for DCA (0.1–10 mM), a water-soluble molecule with a short half-life and widely used for patients with cancer and diabetes ([96]31) to suppress inflammation, with limited adverse effects ([97]32). Relative to lower doses, DCA at 10 mM was more effective to decrease S. epidermidis–induced TNF-α secretion ([98]Figure 5B) and expression of hexokinase 2 (an enzyme facilitating the first reaction of glycolysis pathway) and CXCL8 (a proinflammatory chemokine) ([99]Supplemental Figure 2, A–C). Importantly, preterm cord blood treated with DCA had increased neutrophil phagocytic capacity under both normo- and hyperglycemic conditions ([100]Figure 5C), but DCA did not exert direct bacterial growth inhibitory effect in bacterial culture medium ([101]Figure 5D), nor did it decrease overall bacterial density in S. epidermidis–stimulated cord blood ([102]Figure 5E). Figure 5. Glycolysis inhibition decreases inflammation in S. epidermidis–challenged preterm cord blood. [103]Figure 5 [104]Open in a new tab (A and B) TNF-α levels in cord blood of preterm piglets (n = 6) following stimulation with S. epidermidis, with and without presence of a glycolysis inhibitor: rapamycin (500 nM), DCA (10 mM), and FX11 (100 μM) at 37°C and 5% CO[2] incubation (n = 6) (A); and DCA (0.1–10 mM) (B). (C) Cord blood neutrophil phagocytosis (n = 10, from 2 independent litters) measured by fraction of neutrophils having phagocytic capacity in cord blood with and without 10 mM DCA added at normo- or hyperglycemic conditions. In vitro phagocytosis assay was performed by incubating samples with pHrodo-conjugated E. coli for 30 minutes at 37°C and 5% CO[2] and analyzed by flow cytometry. (D) Bacterial density in preterm cord blood following stimulation with S. epidermidis (theoretical dose from 10^5 to 10^7 CFU/mL) for 30 minutes or 1 hour with and without preincubation with 10 mM DCA (n = 6). (E) Effects of DCA (10 mM) on S. epidermidis growth (n = 3). (F–J) Heatmaps from transcriptomic analyses of cord blood samples with or without S. epidermidis (5 × 10^5 CFU/mL) and DCA incubation. (F) The top 30 DEGs from the comparison between control (CON) and S. epidermidis–challenged samples. (G–J) Selective DEGs related to inflammation (G), OXPHOS (H), antiinflammatory effect (I), and phagocytosis and endocytosis (J) and obtained from the comparison between S. epidermidis–stimulated samples without versus with DCA addition. For each DEG (row), z-scores of the expression levels are depicted in colors from blue (low) to red (high). (A–E) Data are presented as violin dot plots with median and IQR and were analyzed using a linear mixed-effect model with inhibitor treatment as a fixed factor and pig ID as the random factor. *P < 0.05, ***P < 0.001. (B) Values not sharing the same letters are significantly different (P < 0.05). SE, S. epidermidis. We further performed RNA-Seq analysis of S. epidermidis–stimulated cord blood with and without DCA addition ([105]Figure 5, F–J; [106]Supplemental Table 2, A–H; and [107]Supplemental Figure 3) and observed clear effects of bacterial stimulation (n = 90 differentially expressed genes [DEGs]) and DCA treatment in stimulated samples (n = 239 DEGs). The bacterial challenge upregulated genes and pathways related to innate and adaptive immune activation and downregulated genes involved in OXPHOS ([108]Figure 5, G and H). Conversely, comparing the 2 bacteria-stimulated groups, DCA upregulated antiinflammatory and OXPHOS pathways, and downregulated inflammatory pathways ([109]Figure 5, G–I). DCA also increased expression of genes related to endocytosis and phagocytosis ([110]Figure 5J). Collectively, DCA appeared capable of inhibiting infection-induced immune cell glycolysis and inflammation and, therefore, was selected as our drug candidate for preventing neonatal sepsis under normo- and hyperglycemic conditions. STG supply and DCA reduce inflammation and improve bacterial clearance but do not prevent sepsis. Having identified glycolysis inhibition by DCA as a potential alternative to glucose restriction, we again used the preterm pig S. epidermidis infection model to test the ability of both parenteral glucose supply maintaining normoglycemia and DCA to reduce sepsis incidence and severity, relative to HG supply, in a 2 × 2 factorial experimental design. The animals were provided PN with STG level (10% glucose) or high parenteral glucose levels (21% glucose), as well as DCA treatment (50 mg/kg) or saline control shortly after S. epidermidis infusion (experimental design in [111]Figure 6A). The selected DCA dose was aimed to match the effective dose in vitro and the recommended dose in adult patients with lactic acidosis ([112]33). Figure 6. The impact of parenteral glucose levels and glycolysis inhibition by DCA on clinical response to S. epidermidis infection. [113]Figure 6 [114]Open in a new tab (A) Preterm newborn piglets were nourished exclusively with PN containing HG (21%; 30 g/kg/d) or STG (10%; 14.4 g/kg/d) concentrations, intra-arterially infected with 10^9 CFU/kg S. epidermidis, followed by saline or DCA treatment (50 mg/kg) 30 minutes after infection (n = 9–15/group). Uninfected animals receiving either HG or STG PN (n = 4 and 7, respectively) served as references and were