Lactate Alters Metabolism in Human Macrophages and Improves Their Ability to Kill Mycobacterium tuberculosis.

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-1ß 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.

Desferrioxamine Supports Metabolic Function in Primary Human Macrophages Infected With Mycobacterium tuberculosis.

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 IL1ß 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 IL1ß 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.

Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.

STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6-GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.