The ability of Mycobacterium tuberculosis (Mtb) to persist inside host cells relies on metabolic adaptation, like the accumulation of lipid bodies (LBs) in the so-called foamy macrophages (FM), which are favorable to Mtb. The activation state of macrophages is tightly associated to different metabolic pathways, such as lipid metabolism, but whether differentiation towards FM differs between the macrophage activation profiles remains unclear. Here, we aimed to elucidate whether distinct macrophage activation states exposed to a tuberculosis-associated microenvironment or directly infected with Mtb can form FM. We showed that the triggering of signal transducer and activator of transcription 6 (STAT6) in interleukin (IL)-4-activated human macrophages (M(IL-4)) prevents FM formation induced by pleural effusion from patients with tuberculosis. In these cells, LBs are disrupted by lipolysis, and the released fatty acids enter the β-oxidation (FAO) pathway fueling the generation of ATP in mitochondria. Accordingly, murine alveolar macrophages, which exhibit a predominant FAO metabolism, are less prone to become FM than bone marrow derived-macrophages. Interestingly, direct infection of M(IL-4) macrophages with Mtb results in the establishment of aerobic glycolytic pathway and FM formation, which could be prevented by FAO activation or inhibition of the hypoxia-inducible factor 1-alpha (HIF-1α)-induced glycolytic pathway. In conclusion, our results demonstrate that Mtb has a remarkable capacity to induce FM formation through the rewiring of metabolic pathways in human macrophages, including the STAT6-driven alternatively activated program. This study provides key insights into macrophage metabolism and pathogen subversion strategies.

Highlights d Tuberculous pleural effusion (TB-PE) shifts glycolysis to OXPHOS in M1 macrophages d Metabolic shift is accompanied by low HIF-1a activity and high M. tuberculosis burden d HIF-1a stabilization reverts the metabolic shift and inhibitory effects of TB-PE d Host-derived lipids (eicosanoid-enriched fraction) in TB-PE drive the metabolic shift

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1The ability of Mycobacterium tuberculosis (Mtb) to persist inside host cells relies on 2 metabolic adaptation, like the accumulation of lipid bodies (LBs) in the so-called 3 foamy macrophages (FM). Indeed, FM are favorable to Mtb. The activation state of 4 macrophages is tightly associated to different metabolic pathways, such as lipid 5 metabolism, but whether differentiation towards FM differs between the 6 macrophage activation profiles remains unclear. Here, we aimed to elucidate if 7 distinct macrophage activation states exposed to a tuberculosis-associated 8 microenvironment can accumulate LBs, and its impact on the control of infection. 9We showed that signal transducer and activator of transcription 6 (STAT6) 10 activation in interleukin (IL)-4-activated human macrophages (M(IL-4)) prevents FM 11 formation induced by pleural effusion from patients with tuberculosis. In these cells, 12 LBs are disrupted by lipolysis, and the released fatty acids enter the -oxidation 13 (FAO) pathway fueling the generation of ATP in mitochondria. We demonstrated 14 that inhibition of the lipolytic activity or of the FAO drives M(IL-4) macrophages into 15 FM. Also, exhibiting a predominant FAO metabolism, mouse alveolar macrophages 16 are less prone to become FM compared to bone marrow derived-macrophages. 17 Upon Mtb infection, M(IL-4) macrophages are metabolically re-programmed 18 towards the aerobic glycolytic pathway and evolve towards a foamy phenotype, 19which could be prevented by FAO activation or inhibition of the hypoxia-inducible 20 factor 1-alpha (HIF-1α)-induced glycolytic pathway. In conclusion, our results 21 demonstrate a role for STAT6-driven FAO in preventing FM differentiation, and 22

Dendritic cells (DCs) are a key player in the host response to the tuberculosis (TB) agent, Mycobacterium tuberculosis (Mtb), which interfere with DC functions. However, how metabolic pathways influence DC function and activation in TB is unknown. We found that Mtb triggers both glycolysis and to a lesser extent mitochondrial respiration in human monocyte-derived DCs. This was accompanied by an increase in expression of the transcription factor HIF-1α and several glycolytic genes, dependent on TLR2 engagement by Mtb. Inhibition of either HIF-1α or glycolysis resulted in a strong alteration of the DCs migration capacities. In addition, DCs derived from monocytes of TB patients are deficient in inducing glycolysis and cell migration in response to Mtb, rescuable by induction of HIF-1α activity. Indeed, monocytes from TB patients are already highly glycolytic, differentiating into poorly migratory DCs. The data uncover a crucial role for glycolysis in DC migration and suggest that its modulation may be a promising avenue to better control TB.

Monocytes and macrophages play a central role in chronic brucellosis. Brucella abortus (Ba) is an intracellular pathogen that survives inside these cells. On the other hand, macrophages could be differentiated into classical (M1), alternative (M2) or other less-identified profiles. We have previously shown that Ba RNA (a bacterial viability-associated PAMP or vita-PAMP) is a key molecule by which Ba can evade the host immune response. However, we did not know if macrophages could be polarized by this vita-PAMP. To assess this, we used two different approaches: we evaluated if Ba RNA per se was able to differentiate macrophages to M1 or M2 or, given that Ba survives inside macrophages once a Th1 response is established (i.e., in the presence of IFN-γ), we also analysed if Ba RNA could interfere with M1 polarization. We found that Ba RNA alone does not polarize to M1 or M2 but activates human macrophages instead. However, our results show that Ba RNA does interfere with M1 polarization while they are being differentiated. This vita-PAMP diminished the M1-induced CD64, and MHC-II surface expression on macrophages at 48 h. This phenomenon was not associated with an alternative activation of these cells (M2), as shown by unchanged CD206, DC-SIGN and CD163 surface expression. When evaluating glucose metabolism, we found that Ba RNA did not modify M1 glucose consumption or lactate production. However, production of Nitrogen Reactive Species (NRS) did diminish in Ba RNA-treated M1 macrophages. Overall, our results show that Ba RNA could alter the proper immune response set to counterattack the bacteria that could persist in the host establishing a chronic infection.