SUMMARYNeutrophils are the most abundant cell type in airways of tuberculosis patients. Recent investigations reported induction of neutrophil extracellular traps (NETs) duringMycobacterium tuberculosis(Mtb) infection, however, the molecular regulation and impact of NETosis onMtbpathogenesis is unknown. We find that in response toMtbinfection in neutrophils, PAD4 citrullinates histones to decondense chromatin that gets packaged into vesicles for release as NETs in a manner that can maintain neutrophil viability and promoteMtbreplication. Type I interferon, which has been associated with NETosis in numerous contexts but without a known mechanism, promotes formation of chromatin-containing vesicles and NET release. Analysis of nonhuman primate granulomas supports a model where neutrophils are exposed to type I interferon from macrophages as they migrate into the granuloma, where they release NETs that contribute to necrosis and caseation. Our data reveals NETosis as a promising target to inhibitMtbreplication and granuloma caseation.
Granulomas are the hallmark of Mycobacterium tuberculosis (Mtb) infection. Cytokine-mediated signaling can modulate immune function; thus, understanding the cytokine milieu in granulomas is critical for understanding immunity in tuberculosis (TB). Interferons (IFNs) are important immune mediators in TB, and while type 1 and 2 IFNs have been extensively studied, less is known about type 3 IFNs (IFNλs) in TB. To determine if IFNλs are expressed in granulomas, which cells express them, and how granuloma microenvironments influence IFNλ expression, we investigated IFNλ1 and IFNλ4 expression in macaque lung granulomas. We identified IFNλ expression in granulomas, and IFNλ levels negatively correlated with bacteria load. Macrophages and neutrophils expressed IFNλ1 and IFNλ4, with neutrophils expressing higher levels of each protein. IFNλ expression varied in different granuloma microenvironments, with lymphocyte cuff macrophages expressing more IFNλ1 than epithelioid macrophages. IFNλ1 and IFNλ4 differed in their subcellular localization, with IFNλ4 predominantly localizing inside macrophage nuclei. IFNλR1 was also expressed in granulomas, with intranuclear localization in some cells. Further investigation demonstrated that IFNλ signaling is driven in part by TLR2 ligation and was accompanied by nuclear translocation of IFNλR1. Our data indicate that IFNλs are part of the granuloma cytokine milieu that may influence myeloid cell function and immunity in TB.
Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), a leading cause of infectious disease-related mortality around the world. Myeloid cells are important in TB as Mtb host cells and anti-Mtb effector cells but the factors that differentiate these roles are not clearly defined. Immunometabolism is a driver of immune function and may be an important determinant of this interaction and represents a potential target for host-directed therapies against TB. To better understand how immunometabolism relates to TB, we incubated macrophages and neutrophils from Mtb-infected macaques with a panel of metabolic inhibitors and measured how this affected cellular responses to Mtb infection. Specifically, we targeted glycolysis, oxidative phosphorylation, and fatty acid oxidation and measured how this affected macrophage and neutrophil phagocytosis, macrophage control over Mtb viability and replication, and neutrophil production of extracellular traps. We found that targeting glycolysis by inhibiting PFKFB3, a rate-limiting enzyme in glycolysis, reduced macrophage anti-Mtb activity and neutrophil phagocytosis, while inhibition with the glucose analog 2-DG did not. In contrast, incubating cell-free mycobacteria with PFKFB3 inhibitors severely inhibited their growth, suggesting that these bacteria use different metabolic pathways in cells and culture to survive. Inhibiting oxidative phosphorylation with metformin or fatty acid oxidation with etomoxir did not change how macrophages or neutrophils responded to Mtb or affect mycobacterial growth in culture. These results indicate a role for immunometabolism in myeloid responses to Mtb and may inform efforts for development of metabolism-targeted therapies for TB.
Supported by grants from NIH (R01AI134183)
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