The cytokine IFN-γ coordinates macrophage activation and is essential for control of pathogens including Mycobacterium tuberculosis. However, the mechanisms by which IFN-γ controls M. tuberculosis infection are only partially understood. Here, we show that the transcription factor HIF-1α is an essential mediator of IFN-γ dependent control of M. tuberculosis infection both in vitro and in vivo. M. tuberculosis infection of IFN-γ activated macrophages results in a synergistic increase in HIF-1α protein levels. This increase in HIF-1α levels is functionally important, as macrophages lacking HIF-1α are defective for IFN-γ dependent control of infection. RNA-seq profiling demonstrates that HIF-1α regulates nearly half of all IFN-γ inducible genes during infection of macrophages. In particular, HIF-1α regulates production of important immune effectors including inflammatory cytokines and chemokines, eicosanoids, and nitric oxide (NO). In addition, we find that during infection HIF-1α coordinates a metabolic shift to aerobic glycolysis in IFN-γ activated macrophages. We find that this enhanced glycolytic flux is crucial for IFN-γ dependent control of infection in macrophages. Furthermore, we identify a positive feedback loop between HIF-1α and aerobic glycolysis that amplifies macrophage activation. Finally, we demonstrate that HIF-1α is crucial for control of infection in vivo as mice lacking HIF-1α in the myeloid lineage are strikingly susceptible to infection, and exhibit defective production of inflammatory cytokines and microbicidal effectors. In conclusion, we have identified HIF-1α as a novel regulator of IFN-γ dependent immunity that coordinates an immunometabolic program essential for control of M. tuberculosis infection in vitro and in vivo.

Lipid droplet (LD) formation occurs during infection of macrophages with numerous intracellular pathogens, including Mycobacterium tuberculosis. It is believed that M. tuberculosis and other bacteria specifically provoke LD formation as a pathogenic strategy in order to create a depot of host lipids for use as a carbon source to fuel intracellular growth. Here we show that LD formation is not a bacterially driven process during M. tuberculosis infection, but rather occurs as a result of immune activation of macrophages as part of a host defense mechanism. We show that an IFN-γ driven, HIF-1α dependent signaling pathway, previously implicated in host defense, redistributes macrophage lipids into LDs. Furthermore, we show that M. tuberculosis is able to acquire host lipids in the absence of LDs, but not in the presence of IFN-γ induced LDs. This result uncouples macrophage LD formation from bacterial acquisition of host lipids. In addition, we show that IFN-γ driven LD formation supports the production of host protective eicosanoids including PGE2 and LXB4. Finally, we demonstrate that HIF-1α and its target gene Hig2 are required for the majority of LD formation in the lungs of mice infected with M. tuberculosis, thus demonstrating that immune activation provides the primary stimulus for LD formation in vivo. Taken together our data demonstrate that macrophage LD formation is a host-driven component of the adaptive immune response to M. tuberculosis, and suggest that macrophage LDs are not an important source of nutrients for M. tuberculosis.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.

IFN-γ is essential for control of Mycobacterium tuberculosis infection in vitro and in vivo. However, the mechanisms by which IFN-γ controls infection remain only partially understood. One of the crucial IFN-γ target genes required for control of M. tuberculosis is Inducible nitric oxide synthase (iNOS). While nitric oxide (NO) produced by iNOS is thought to have direct bactericidal activity against M. tuberculosis, the role of NO as a signaling molecule has been poorly characterized in the context M. tuberculosis infection. Here, we find that iNOS broadly regulates the macrophage transcriptome during M. tuberculosis infection, activating antimicrobial pathways while also limiting inflammatory cytokine production. The transcription factor Hypoxia inducible factor-1α (HIF-1α) was recently shown to be critical for IFN-γ mediated control of M. tuberculosis infection. We find that HIF-1α function requires NO production, and that HIF-1α and iNOS are linked by a positive feedback loop that amplifies macrophage activation. Furthermore, we find that NO inhibits NF-kB activity to prevent hyper-inflammatory responses. Thus, NO activates robust microbicidal programs while at the same time limiting damaging inflammation. IFN-γ signaling must carefully calibrate an effective immune response that does not cause excessive tissue damage, and this work identifies NO as a key player in establishing this balance during M. tuberculosis infection.

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