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Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection (STI) in the USA. As an STI, C. trachomatis infections can cause inflammatory damage to the female reproductive tract and downstream sequelae including infertility. No vaccine currently exists to C. trachomatis , which evades sterilizing immune responses in its human host. A better understanding of this evasion will greatly benefit the production of anti- Chlamydia therapeutics and vaccination strategies. This minireview will discuss a single branch of the immune system, which activates in response to genital Chlamydia infection: so-called "cell-autonomous immunity" activated by the cytokine interferon-gamma. We will also discuss the mechanisms by which human and mouse-adapted Chlamydia species evade cell-autonomous immune responses in their native hosts. This minireview will examine five pathways of host defense and their evasion: (i) depletion of tryptophan and other nutrients, (ii) immunity-related GTPase-mediated defense, (iii) production of nitric oxide, (iv) IFNγ-induced cell death, and (v) RNF213-mediated destruction of inclusions. [ABSTRACT FROM AUTHOR]

Macrophages use an array of innate immune sensors to detect intracellular pathogens and to tailor effective antimicrobial responses. In addition, extrinsic activation with the cytokine IFN‐γ is often required as well to tip the scales of the host‐pathogen balance toward pathogen restriction. However, little is known about how host‐pathogen sensing impacts the antimicrobial IFN‐γ‐activated state. It was observed that in the absence of IRF3, a key downstream component of pathogen sensing pathways, IFN‐γ‐primed macrophages more efficiently restricted the intracellular bacterium Legionella pneumophila and the intracellular protozoan parasite Trypanosoma cruzi. This effect did not require IFNAR, the receptor for Type I IFNs known to be induced by IRF3, nor the sensing adaptors MyD88/TRIF, MAVS, or STING. This effect also did not involve differential activation of STAT1, the major signaling protein downstream of both Type 1 and Type 2 IFN receptors. IRF3‐deficient macrophages displayed a significantly altered IFN‐γ‐induced gene expression program, with up‐regulation of microbial restriction factors such as Nos2. Finally, we found that IFN‐γ‐primed but not unprimed macrophages largely excluded the activated form of IRF3 from the nucleus following bacterial infection. These data are consistent with a relationship of mutual inhibition between IRF3 and IFN‐γ‐activated programs, possibly as a component of a partially reversible mechanism for modulating the activity of potent innate immune effectors (such as Nos2) in the context of intracellular infection. [ABSTRACT FROM AUTHOR]