Tuberculosis is the single biggest infectious killer in the world and presents a major global health challenge. Antimicrobial therapy requires many months of multiple drugs and incidences of drug resistant tuberculosis continues to rise. Consequently, research is now focused on the development of therapies to support the function of infected immune cells. HIF1α-mediated induction of aerobic glycolysis is integral to the host macrophage response during infection with Mtb, as this promotes bacillary clearance. Some iron chelators have been shown to modulate cellular metabolism through the regulation of HIF1α. We examined if the iron chelator, desferrioxamine (DFX), could support the function of primary human macrophages infected with Mtb. Using RT-PCR, we found that DFX promoted the expression of key glycolytic enzymes in Mtb-infected primary human MDMs and human alveolar macrophages. Using Seahorse technology, we demonstrate that DFX enhances glycolytic metabolism in Mtb-stimulated human MDMs, while helping to enhance glycolysis during mitochondrial distress. Furthermore, the effect of DFX on glycolysis was not limited to Mtb infection as DFX also boosted glycolytic metabolism in uninfected and LPS-stimulated cells. DFX also supports innate immune function by inducing IL1β production in human macrophages during early infection with Mtb and upon stimulation with LPS. Moreover, using hypoxia, Western blot and ChIP-qPCR analyses, we show that DFX modulates IL1β levels in these cells in a HIF1α-mediated manner. Collectively, our data suggests that DFX exhibits potential to enhance immunometabolic responses and augment host immune function during early Mtb infection, in selected clinical settings.

In order to mount an appropriate immune response to infection, the macrophage must alter its metabolism by increasing aerobic glycolysis and concomitantly decreasing oxidative phosphorylation; a process known as the Warburg effect. Consequently, lactate, the end-product of glycolysis, accumulates in the extracellular environment. The subsequent effect of lactate on surrounding macrophages is poorly understood. Mycobacterium tuberculosis (Mtb), the causative organism of Tuberculosis (TB), is phagocytosed by macrophages in the airways. Mtb infected macrophages upregulate aerobic glycolysis and effector functions to try to kill the bacteria. Our lab has previously shown that human macrophages produce lactate in response to infection with Mtb. Although lactate has largely been considered a waste product of aerobic glycolysis, we hypothesised that the presence of extracellular lactate would impact subsequent immunometabolic responses and modulate macrophage function. We demonstrate that the presence of exogenous lactate has an immediate effect on the cellular metabolism of resting human macrophages; causing a decrease in extracellular acidification rate (ECAR; analogous to the rate of glycolysis) and an increase in the oxygen consumption rate (OCR; analogous to oxidative phosphorylation). When lactate-treated macrophages were stimulated with Mtb or LPS, glycolysis proceeds to increase immediately upon stimulation but oxidative phosphorylation remains stable compared with untreated cells that display a decrease in OCR. This resulted in a significantly reduced ECAR/OCR ratio early in response to stimulation. Since altered metabolism is intrinsically linked to macrophage function, we examined the effect of lactate on macrophage cytokine production and ability to kill Mtb. Lactate significantly reduced the concentrations of TNF and IL-1β produced by human macrophages in response to Mtb but did not alter IL-10 and IL-6 production. In addition, lactate significantly improved bacillary clearance in human macrophages infected with Mtb, through a mechanism that is, at least in part, mediated by promoting autophagy. These data indicate that lactate, the product of glycolysis, has a negative feedback effect on macrophages resulting in an attenuated glycolytic shift upon subsequent stimulation and reduced pro-inflammatory cytokine production. Interestingly, this pro-resolution effect of lactate is associated with increased capacity to kill Mtb.

Mycobaterium tuberculosis (Mtb) kills more people than any other infectious agent worldwide. Our group has shown the importance of glycolysis in controlling intracellular Mtb growth in human macrophages1, which leads to increased lactate production. Therefore, we examined if lactate had an immunomodulatory effect on human monocyte derived macrophages (hMDM) infected with Mtb. hMDM were adherence purified from healthy donor buffy coats with human serum for 6-8 days. Sodium L-Lactate was added 3 hours prior to infection with Mtb or stimulation with LPS (100 ng/ml). Macrophage phenotype and function was assessed by ELISA, flow cytometry and Seahorse metabolic-flux analysis. Bacillary killing was determined by colony forming units (CFU) after lysing infected hMDM, plating on middlebrook agar and counting 21 days later. 25 mM of lactate improves hMDM ability to control intracellular Mtb growth with a 55% reduction in CFU at day 5 post infection. The glycolytic response seen immediately following infection with Mtb or LPS stimulation was decreased with lactate. LPS-induced upregulation of activation markers was diminished by lactate. Lactate is a product of aerobic glycolysis induced by infection and supports the intracellular killing of Mtb. Further research is required to elucidate the mechanism of this effect. 1. Gleeson, L. E. et al. Cutting Edge: Mycobacterium tuberculosis Induces Aerobic Glycolysis in Human Alveolar Macrophages That Is Required for Control of Intracellular Bacillary Replication. J. Immunol. 196, 2444–2449 (2016).