Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β, a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis, we performed metabolic and functional studies on human alveolar macrophages, human monocyte-derived macrophages, and murine bone marrow–derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2, increased levels of anti-inflammatory IL-10, and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival, demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies.
The innate immune response to Mycobacterium tuberculosis infection in the human lung is crucial in determining the outcome of infection (1). Mice deficient in the innate cytokine IL-1β signaling display increased susceptibility to infection (2, 3), supporting a critical role for this cytokine in the host response to the bacterium. IL-1β also induces differentiation of human monocytes into macrophages, with enhanced phagocytic and Ag-presentation functions in M. tuberculosis infection (4).
Recently, metabolic reprogramming in stimulated host immune cells was implicated in the regulation of innate immune function (5, 6). Macrophages stimulated with the TLR4 agonist LPS undergo a shift in glucose metabolism, similar to that seen in cancer cells, termed the Warburg effect (or aerobic glycolysis) (6). This involves increased rates of glycolysis and reduced oxidative phosphorylation via the tricarboxylic acid cycle, with concurrent accumulation of tricarboxylic acid intermediates, such as succinate. Increased succinate results in impaired activity of prolylhydroxylases, allowing stabilization of the transcription factor hypoxia inducible factor-1α and leading to transcription of Il-1b and production of mature IL-1β (6, 7). Molecular determinants of this significant change in metabolism are being investigated as potential targets for manipulation in this important pathway (7).
In this work, we demonstrate for the first time, to our knowledge, that M. tuberculosis infection induces a metabolic shift toward aerobic glycolysis in primary human alveolar macrophages (AM) and monocyte-derived macrophages (MDM) and that this shift is required for optimal production of IL-1β, as well as for suppression of the anti-inflammatory cytokine IL-10. We further demonstrate that this alteration in host glucose metabolism, through its induction of IL-1β, is required for control of bacillary intracellular replication. This work highlights host AM glucose metabolism as a potential target for host-directed adjunctive therapies.
Materials and Methods
Human AM were retrieved at bronchoscopy after informed consent, as approved by the Research Ethics Committee of St. James’s Hospital (Dublin, Ireland), and prepared and cultured as previously reported (8, 9). Cells were cultured for 24 h prior to experimentation.
Human MDM were isolated from peripheral blood buffy coats (obtained from the Irish Blood Transfusion Services) by density gradient centrifugation and adherence to plastic or immune-magnetic separation using CD14-positive magnetic beads (STEMCELL Technologies). Cells were cultured for 7–10 d to allow differentiation prior to experimentation.
CS57BL/6 (Harlan) mice and IL-1R−/− mice (CS57BL/6 background; The Jackson Laboratory), kindly donated by Prof. Kingston Mills (School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin), were bred under specific pathogen–free conditions, and BMDM were isolated and cultured as previously described (6, 7). Cells were differentiated until day 5, after which they were lifted using 10 mM EDTA and plated for experimentation. All experiments were carried out with prior ethical approval from the Trinity College Dublin Animal Research Ethics Committee.
Human cells were cultured in glucose-deprived RPMI 40 (Sigma-Aldrich) supplemented with 10 mM glucose or 10 mM galactose for 24 h prior to, and for the duration of, infection. Murine BMDM were treated 3 h prior to, and for the duration of, infection with 5 mM 2-deoxyglucose (2DG; Sigma-Aldrich).
Recombinant human IL-1β/IL-1F2 Ab (R&D Systems) was used at 0.1 μg/ml.
Mycobacterial culture and infection of macrophages
M. tuberculosis strains H37Ra (ATCC 2517) and H37Rv (ATCC 25618) (American Type Culture Collection, Manassas, VA) were propagated in Middlebrook 7H9 medium to log phase. M. tuberculosis strain H37Rv, γαμμα-irradiated whole cells, NR-49098 (BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health) was prepared according to the supplier’s instructions. Bacteria were resuspended, and multiplicity of infection (MOI) was determined for each donor as previously described (8, 9). Cells were infected at MOI 1–5 bacilli/cell for 3 h, extracellular bacteria were removed, and fresh media were applied to allow incubation for specified times.
Lactate concentration in supernatants was measured by colorimetric assay (Sigma-Aldrich) and read at 570 nm using an Epoch Microplate Spectrophotometer with Gen5 Data Analysis software (BioTek Instruments).
The bioenergetic profile of cells was determined using an XF24 Extracellular Flux analyzer (Seahorse Biosciences). Oxygen consumption rate and extracellular acidification rate were assessed in Seahorse Media supplemented with 10 mM glucose and 2 mM L-glutamine. Results were normalized to cell number using a Crystal Violet dye extraction growth assay.
RNA extraction was performed using the RNeasy Mini Kit (QIAGEN), and cDNA was generated using the Applied Biosystems High-Capacity cDNA Archive Kit. Real-time PCR was performed using TaqMan primers. Data were normalized to GAPDH, and mRNA expression fold-change relative to uninfected was calculated using the 2−ΔΔCt method.
Cytokine concentration in supernatants was measured by ELISA (TNF-α, IL-10 [both from eBioscience]; IL-1β [R&D Systems]).
Intracellular bacillary survival assay
Following a 3-h wash to remove extracellular bacteria, as described above, infected macrophages were lysed in 0.1% Triton-X at 3 or 72 h postinfection (p.i.), and serial dilutions were performed and plated on 7H10 Middlebrook Agar in triplicate. CFU were enumerated at 14–28 d.
Results are expressed as mean ± SEM. The data were analyzed by GraphPad Prism (La Jolla, CA) using the unpaired Student t test for all murine experiments and the paired Student t test for all human experiments. A p value < 0.05 was considered statistically significant.
First, we undertook to establish the impact of M. tuberculosis infection on macrophage glucose metabolism. Lactate, generated from the reduction of pyruvate by the enzyme lactate dehydrogenase, represents the end-product of the glycolytic pathway. Lactate exits the cell via the monocarboxylate transport protein system (10); therefore, increases in secreted lactate reflect increases in intracellular glycolytic activity. Human AM, human MDM, and murine BMDM demonstrated increased lactate production when infected with M. tuberculosis, and this was dependent on the MOI (Fig. 1A, 1B, Supplemental Fig. 1A). Extracellular flux analyses also demonstrated a shift toward glycolytic metabolism in iH37Rv-infected AM, with similar kinetics as observed for lactate secretion (Fig. 1C). Use of gamma-irradiated M. tuberculosis strain iH37Rv allowed examination of macrophage metabolism in isolation, and results were confirmed using live H73Ra and H37Rv in human MDM (Fig. 1D) and human AM (Fig. 1E, 1F).
The metabolic shift toward glycolysis is required for optimal production of IL-1β in murine macrophages (6); thus, we next sought to determine the effect of this metabolic shift on human macrophage cytokine profiles. Cells grown in glucose-deprived galactose-containing media are forced to generate ATP from glutamine-driven oxidative metabolism, effectively inhibiting the glycolytic pathway (11, 12). Inhibition of glycolysis resulted in decreased levels of IL-1β, but not IL-1α, at the transcriptional and mature protein levels (Fig. 2A, 2B). Interestingly, inhibiting glycolysis increased production of the anti-inflammatory cytokine IL-10, despite low levels of IL10 mRNA at the times points analyzed (Fig. 2B). Similar to earlier observations with LPS-stimulated murine macrophages, TNF-α production was unchanged (6). Inhibition of glycolysis did not impact IL-18 or IL-12 transcription (Supplemental Fig. 1B, 1C). Similar results were seen in murine BMDM treated with glycolytic inhibitor 2DG (Fig. 2C).
IL-1β–dependent host resistance to M. tuberculosis infection was linked to production of eicosanoids through induction of the enzyme cyclooxygenase-2 (gene known as Ptgs2) (13). We confirmed that blockade or absence of IL-1R signaling in murine and human macrophages is associated with reduced PTGS2 induction (Fig. 2D, 2E) and further demonstrated a trend towards reduced PTGS2 transcription following inhibition of glycolytic metabolism in M. tuberculosis–infected human MDM (Fig. 2F).
To investigate the functional implications of this metabolic shift, we measured intracellular bacillary killing in the presence and absence of glycolytic inhibitors described above. Inhibition of glycolysis impaired bacillary killing by macrophages, suggesting that this metabolic shift is required for optimal control of the bacillus (Fig. 3). The presence or absence of glycolytic inhibitors did not impact M. tuberculosis replication in macrophage-free culture (Supplemental Fig. 1D). Bacillary killing was impaired to a similar extent in murine BMDM cultured in the presence of a glycolytic inhibitor and those deficient in IL-1R (IL-1R−/−); the impact of inhibiting glycolysis was not seen in macrophages from IL1R−/− mice (Fig. 4A), suggesting that this effect of glycolysis is mediated through its induction of IL-1β. Similarly, in human MDM, pretreatment with anti-IL-1R–IgG impaired bacillary killing to a similar extent as galactose treatment (Fig. 4B).
Immunometabolism is an exciting new field with the potential for novel therapeutic targets for infection and autoimmune disease (14). Key innate immunity defense mechanisms in M. tuberculosis infection, such as proinflammatory cytokine secretion and autophagy, as well as development of trained immunity, have been linked to intracellular metabolism (6, 15, 16). Aerobic glycolysis, long described in cancer cells, recently was observed to occur in innate immune cells stimulated with TLR agonists (5, 6). Our data demonstrate a shift toward aerobic glycolysis in primary human cells infected with M. tuberculosis, including primary human AM that represent the first line of defense against M. tuberculosis in vivo.
We further demonstrate that this shift toward glycolysis is crucial for the production of proinflammatory IL-1β, and suppression of anti-inflammatory IL-10, a cytokine that assists the bacillus in evading host immune responses (8). We previously reported similar alterations in IL-1β and IL-10 through manipulation of the glycolytic enzyme PKM2 in TLR-stimulated murine BMDM (7). Our observations are supported by increased cytokine production in response to M. tuberculosis lysate in PBMC cultured in media with high glucose concentration (17). Although our results demonstrate that TNF-α production is not directly impacted by glycolytic reprogramming, IL-1β was shown to augment TNF-α signaling in M. tuberculosis infection (18); thus, glycolysis may also affect this cytokine indirectly at later time points. Taken together, these data suggest that host macrophage glucose metabolism plays a demonstrable role in the cytokine signature generated in response to infection with the bacillus.
Although direct alterations in extracellular glucose concentration do not impact upon bacillary replication in macrophages (17), we show that the metabolic pathway through which intracellular glucose is used by the macrophage plays a major role in facilitating elimination of M. tuberculosis in primary human cells. Although other investigators suggested that glycolysis may facilitate macrophage survival and, hence, permit bacillary replication, these studies were performed in the primarily glycolytic THP1 cell line (19). As our data illustrate, the metabolic shift induced in primary human cells (which are not dependent upon glycolysis for survival, in contrast to the THP1 cell line) is crucial for key innate immune functions, specifically those mediated through IL-1β. Sher and colleagues (13) reported that IL-1β–dependent resistance to M. tuberculosis infection was mediated, in part, through induction of Ptgs2. We confirmed, in primary human cells, that optimal PTGS2 transcription requires IL-1 signaling in M. tuberculosis infection. Furthermore, inhibition of the infection-induced switch to glycolysis results in decreased PTGS2, in parallel with decreased IL-1β production. Interestingly, Chen et al. (20) reported reduced PTGS2 induction in response to virulent H37Rv compared with avirulent H37Ra, which, combined with our observations, may implicate reduced efficacy of the downstream sequelae of glycolytic reprogramming in increased susceptibility to the virulent strain.
We also demonstrate that the impact of glycolysis on intracellular bacillary survival is lost when IL-1β signaling is impeded using IL-1R–deficient mice or anti–IL-1R Ab. This corroborates, in human cells, the observation that IL-1β plays a nonredundant role in the innate immune response to M. tuberculosis in mice, with IL-1β–deficient and IL-1R–deficient mice displaying increased susceptibility to infection (2, 3).
These findings might offer a partial explanation for the increased susceptibility to M. tuberculosis infection observed in type II diabetes mellitus, a disease characterized by impaired glucose utilization due to defective insulin signaling. Insulin is reported to enhance glycolytic activity in rat macrophages (21), and attenuated cytokine production was described in AM from hyperglycemic diabetic rats (22), suggesting that defects in macrophage insulin signaling may contribute to reduced glycolysis-mediated antimycobacterial functions.
This novel role for intracellular glucose metabolism in the innate immune response to M. tuberculosis infection suggests that metabolic manipulation could facilitate early enhanced clearance of the bacillus by the macrophage. A number of U.S. Food and Drug Administration–approved agents in clinical use have been identified as inducers of this shift in cellular metabolism toward aerobic glycolysis, such as the antiemetic agent meclizine; thus, they may warrant investigation as potential therapeutic adjuncts in the setting of M. tuberculosis infection (23). Additionally, reported links between immunometabolism and trained immunity may be specifically relevant to enhancing M. tuberculosis vaccination strategies in the future (16).
This work was supported by the Health Research Board, a Health Professionals fellowship, and the Royal City of Dublin Hospital Trust.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.