All-trans Retinoic Acid Augments Autophagy during Intracellular Bacterial Infection.

Vitamin A deficiency strongly predicts the risk of developing tuberculosis (TB) in individuals exposed to Mycobacterium tuberculosis (Mtb). The burden of antibiotic-resistant TB is increasing globally; therefore, there is an urgent need to develop host-directed adjunctive therapies to treat TB. Alveolar macrophages, the niche cell for Mtb, metabolize vitamin A to all-trans retinoic acid (atRA), which influences host immune responses. We sought to determine the mechanistic effects of atRA on the host immune response to intracellular bacterial infection in primary human and murine macrophages. In this study, atRA promoted autophagy resulting in a reduced bacterial burden in human macrophages infected with Mtb and Bordetella pertussis, but not bacillus Calmette-Guérin (BCG). Autophagy is induced by cytosolic sensing of double-stranded DNA via the STING/TBK1/IRF3 axis; however, BCG is known to evade cytosolic DNA sensors. atRA enhanced colocalization of Mtb, but not BCG, with autophagic vesicles and acidified lysosomes. This enhancement was inhibited by blocking TBK1. Our data indicate that atRA augments the autophagy of intracellular bacteria that trigger cytosolic DNA-sensing pathways but does not affect bacteria that evade these sensors. The finding that BCG evades the beneficial effects of atRA has implications for vaccine design and global health nutritional supplementation strategies. The ability of atRA to promote autophagy and aid bacterial clearance of Mtb and B. pertussis highlights a potential role for atRA as a host-directed adjunctive therapy.

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.

Inhibiting Histone Deacetylases in Human Macrophages Promotes Glycolysis, IL-1ß, and T Helper Cell Responses to Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious killer in the world. Mycobacterium tuberculosis (Mtb), the bacteria that causes the disease, is phagocytosed by alveolar macrophages (AM) and infiltrating monocyte-derived macrophages (MDM) in the lung. Infected macrophages then upregulate effector functions through epigenetic modifications to make DNA accessible for transcription. The metabolic switch to glycolysis and the production of proinflammatory cytokines are key effector functions, governed by epigenetic changes, that are integral to the ability of the macrophage to mount an effective immune response against Mtb. We hypothesised that suberanilohydroxamic acid (SAHA), an FDA-approved histone deacetylase inhibitor (HDACi), can modulate epigenetic changes upstream of the metabolic switch and support immune responses during Mtb infection. The rate of glycolysis in human MDM, infected with Mtb and treated with SAHA, was tracked in real time on the Seahorse XFe24 Analyzer. SAHA promoted glycolysis early in the response to Mtb. This was associated with significantly increased production of IL-1ß and significantly reduced IL-10 in human MDM and AM. Since innate immune function directs downstream adaptive immune responses, we used SAHA-treated Mtb-infected AM or MDM in a co-culture system to stimulate T cells. Mtb-infected macrophages that had previously been treated with SAHA promoted IFN-γ, GM-CSF, and TNF co-production in responding T helper cells but did not affect cytotoxic T cells. These results indicate that SAHA promoted the early switch to glycolysis, increased IL-1ß, and reduced IL-10 production in human macrophages infected with Mtb. Moreover, the elevated proinflammatory function of SAHA-treated macrophages resulted in enhanced T helper cell cytokine polyfunctionality. These data provide an in vitro proof-of-concept for the use of HDACi to modulate human immunometabolic processes in macrophages to promote innate and subsequent adaptive proinflammatory responses.