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Summary To establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease., Graphical Abstract, Highlights • MTb infection induces RNA sensor (RIG-I, MDA5, PKR) mRNA levels and activities • RIG-I, MDA5, MAVS, and PKR restrict intracellular MTb growth in human cells • NTZ enhances MTb-driven RNA sensor mRNA levels and RLR activities • NTZ and NTZ derivatives inhibit intracellular MTb growth in primary human cells, Biological Sciences; Immunology; Immune Response; Microbiology; Molecular Microbiology

Summary Here, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV., Graphical Abstract, Highlights • NTZ amplifies RNA sensor and type I interferon activities and induces GADD34 expression • NTZ inhibits infectious Ebola virus (EBOV) via RIG-I and PKR, but not GADD34 • NTZ inhibits a second negative-strand RNA virus, VSV, via RIG-I and GADD34, but not PKR • NTZ holds promise as an oral therapy against EBOV, Mechanism of Action; Pathogenic Organism; Immune Response; Viral Microbiology

SUMMARY The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1–3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity., Graphical abstract

Summary: Interferon γ (IFN-γ) priming sensitizes monocytes and macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF. Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of interferon regulatory factor 1 (IRF1), and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming, but does not affect LPS induction of the gene. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary toll-like receptor (TLR) stimulus. : Interferon γ (IFN-γ) priming is a critical immune event that enhances the monocyte and macrophage response, particularly expression of the TNF gene, to toll-like receptor (TLR) signaling. Chow et al. demonstrate that IFN-γ priming requires a distal enhancer element within the TNF/LT locus, thereby expanding the role of distal regulatory elements in the innate immune response.

Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand results in the rapid induction of TNF-alpha gene expression. The study presented here shows that autoregulation of TNF-alpha gene transcription by selective signaling through tumor necrosis factor receptor 1 (TNFR1) requires p38 mitogen-activated protein (MAP) kinase activity and the binding of the transcription factors ATF-2 and Jun to the TNF-alpha cAMP-response element (CRE) promoter element. Consistent with these findings, TNFR1 engagement results in increased p38 MAP kinase activity and p38-dependent phosphorylation of ATF-2. Furthermore, overexpression of MADD (MAP kinase-activating death domain protein), an adapter protein that binds to the death domain of TNFR1 and activates MAP kinase cascades, results in CRE-dependent induction of TNF-alpha gene expression. Thus, the TNF-alpha CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-alpha gene transcription.

The tumor necrosis factor-alpha (TNF alpha) gene is an immediate early gene in activated T cells, in that it is rapidly induced without a requirement for protein synthesis. Maximal induction of TNF alpha mRNA can be induced by treatment of T cells with calcium ionophores alone, via a calcineurin-dependent process that is blocked by cyclosporin A. We have previously identified a promoter element, kappa 3, that is required for calcium-stimulated, cyclosporin A-sensitive induction of the TNF alpha gene in activated T cells. Here, we demonstrate that the kappa 3 binding factor contains NFATp, a cyclosporin-sensitive DNA-binding protein required for interleukin-2 gene transcription. NFATp binds to two sites within the kappa 3 element, and occupancy of both sites is required for TNF alpha gene induction. Thus, although the kappa 3 element has little sequence similarity to other NFATp-binding sites, it appears to function as a cyclosporin-sensitive promoter element in T cells by virtue of its ability to bind NFATp. The involvement of NFATp in transcriptional activation of both the interleukin-2 and TNF alpha genes suggests that this factor plays an important role in the coordinate induction of multiple cytokine genes, starting at the earliest stages of T cell activation.