Delivery of loaded MR1 monomer results in efficient ligand exchange to host MR1 and subsequent MR1T cell activation.

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

GSK3α/ß Restrain IFN-γ-Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation.

Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFN-γ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFN-γ-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages. Our findings reveal that IFN-γ robustly activates both macrophage types; however, the profile of activated IFN-γ-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFN-γ. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3α/ß alters the IFN-γ response. GSK3α/ß controlled distinct IFN-γ responses, and in AM-like cells, we found that GSK3α/ß restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFN-γ is restricted in a GSK3α/ß-dependent manner and that IFN-γ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

Crystalline silica-induced proinflammatory eicosanoid storm in novel alveolar macrophage model quelled by docosahexaenoic acid supplementation.

Introduction: Phagocytosis of inhaled crystalline silica (cSiO2) particles by tissue-resident alveolar macrophages (AMs) initiates generation of proinflammatory eicosanoids derived from the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (ARA) that contribute to chronic inflammatory disease in the lung. While supplementation with the ω-3 PUFA docosahexaenoic acid (DHA) may influence injurious cSiO2-triggered oxylipin responses, in vitro investigation of this hypothesis in physiologically relevant AMs is challenging due to their short-lived nature and low recovery numbers from mouse lungs. To overcome these challenges, we employed fetal liver-derived alveolar-like macrophages (FLAMs), a self-renewing surrogate that is phenotypically representative of primary lung AMs, to discern how DHA influences cSiO2-induced eicosanoids. Methods: We first compared how delivery of 25 µM DHA as ethanolic suspensions or as bovine serum albumin (BSA) complexes to C57BL/6 FLAMs impacts phospholipid fatty acid content. We subsequently treated FLAMs with 25 µM ethanolic DHA or ethanol vehicle (VEH) for 24 h, with or without LPS priming for 2 h, and with or without cSiO2 for 1.5 or 4 h and then measured oxylipin production by LC-MS lipidomics targeting for 156 oxylipins. Results were further related to concurrent proinflammatory cytokine production and cell death induction. Results: DHA delivery as ethanolic suspensions or BSA complexes were similarly effective at increasing ω-3 PUFA content of phospholipids while decreasing the ω-6 PUFA arachidonic acid (ARA) and the ω-9 monounsaturated fatty acid oleic acid. cSiO2 time-dependently elicited myriad ARA-derived eicosanoids consisting of prostaglandins, leukotrienes, thromboxanes, and hydroxyeicosatetraenoic acids in unprimed and LPS-primed FLAMs. This cSiO2-induced eicosanoid storm was dramatically suppressed in DHA-supplemented FLAMs which instead produced potentially pro-resolving DHA-derived docosanoids. cSiO2 elicited marked IL-1α, IL-1ß, and TNF-α release after 1.5 and 4 h of cSiO2 exposure in LPS-primed FLAMs which was significantly inhibited by DHA. DHA did not affect cSiO2-triggered death induction in unprimed FLAMs but modestly enhanced it in LPS-primed FLAMs. Discussion: FLAMs are amenable to lipidome modulation by DHA which suppresses cSiO2-triggered production of ARA-derived eicosanoids and proinflammatory cytokines. FLAMs are a potential in vitro alternative to primary AMs for investigating interventions against early toxicant-triggered inflammation in the lung.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

GSK3α/ß restrains IFNγ-inducible costimulatory molecule expression in alveolar macrophages, limiting CD4+ T cell activation.

Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFNγ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFNγ-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages (iBMDMs). Our findings reveal that IFNγ robustly activates both macrophage types; however, the profile of activated IFNγ-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFNγ. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3α/ß alters the IFNγ response. GSK3α/ß controlled distinct IFNγ responses, and in AM-like cells, we found GSK3α/ß restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFNγ is restricted in a GSK3α/ß-dependent manner and that IFNγ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

High Lethality of Mycobacterium tuberculosis Infection in Mice Lacking the Phagocyte Oxidase and Caspase1/11.

Immune networks that control antimicrobial and inflammatory mechanisms have overlapping regulation and functions to ensure effective host responses. Genetic interaction studies of immune pathways that compare host responses in single and combined knockout backgrounds are a useful tool to identify new mechanisms of immune control during infection. For disease caused by pulmonary Mycobacterium tuberculosis (Mtb) infections, which currently lacks an effective vaccine, understanding the genetic interactions between protective immune pathways may identify new therapeutic targets or disease-associated genes. Previous studies have suggested a direct link between the activation of NLRP3-Caspase1 inflammasome and the NADPH-dependent phagocyte oxidase complex during Mtb infection. Loss of the phagocyte oxidase complex alone resulted in increased activation of Caspase1 and IL-1ß production during Mtb infection, resulting in failed disease tolerance during the chronic stages of disease. To better understand this interaction, we generated mice lacking both Cybb, a key subunit of the phagocyte oxidase, and Caspase1/11. We found that ex vivo Mtb infection of Cybb-/-Caspase1/11-/- macrophages resulted in the expected loss of IL-1ß secretion but an unexpected change in other inflammatory cytokines and bacterial control. Mtb infected Cybb-/-Caspase1/11-/- mice rapidly progressed to severe TB, succumbing within 4 weeks to disease characterized by high bacterial burden, increased inflammatory cytokines, and the recruitment of granulocytes that associated with Mtb in the lungs. These results uncover a key genetic interaction between the phagocyte oxidase complex and Caspase1/11 that controls protection against TB and highlight the need for a better understanding of the regulation of fundamental immune networks during Mtb infection.

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction of Listeria monocytogenes infection.

Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised individuals. Macrophages play a dual role during Lm infection by both promoting dissemination of Lm from the gastrointestinal tract and limiting bacterial growth upon immune activation. Despite the relevance of macrophages to Lm infection, the mechanisms underlying phagocytosis of Lm by macrophages are not well understood. To identify host factors important for Lm infection of macrophages, we performed an unbiased CRISPR/Cas9 screen which revealed pathways that are specific to phagocytosis of Lm and those that are required for internalization of bacteria generally. Specifically, we discovered the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, we found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, we show that PTEN-dependent phagocytosis is important for host protection during oral Lm infection. Overall, this study provides a comprehensive identification of macrophage factors involved in regulating Lm uptake and characterizes the function of one factor, PTEN, during Lm infection in vitro and in vivo. Importantly, these results demonstrate a role for opsonin-independent phagocytosis in Lm pathogenesis and suggest that macrophages play a primarily protective role during foodborne listeriosis.

Mycobacterium tuberculosis Evasion of Guanylate Binding Protein-Mediated Host Defense in Mice Requires the ESX1 Secretion System.

Cell-intrinsic immune mechanisms control intracellular pathogens that infect eukaryotes. The intracellular pathogen Mycobacterium tuberculosis (Mtb) evolved to withstand cell-autonomous immunity to cause persistent infections and disease. A potent inducer of cell-autonomous immunity is the lymphocyte-derived cytokine IFNγ. While the production of IFNγ by T cells is essential to protect against Mtb, it is not capable of fully eradicating Mtb infection. This suggests that Mtb evades a subset of IFNγ-mediated antimicrobial responses, yet what mechanisms Mtb resists remains unclear. The IFNγ-inducible Guanylate binding proteins (GBPs) are key host defense proteins able to control infections with intracellular pathogens. GBPs were previously shown to directly restrict Mycobacterium bovis BCG yet their role during Mtb infection has remained unknown. Here, we examine the importance of a cluster of five GBPs on mouse chromosome 3 in controlling Mycobacterial infection. While M. bovis BCG is directly restricted by GBPs, we find that the GBPs on chromosome 3 do not contribute to the control of Mtb replication or the associated host response to infection. The differential effects of GBPs during Mtb versus M. bovis BCG infection is at least partially explained by the absence of the ESX1 secretion system from M. bovis BCG, since Mtb mutants lacking the ESX1 secretion system become similarly susceptible to GBP-mediated immune defense. Therefore, this specific genetic interaction between the murine host and Mycobacteria reveals a novel function for the ESX1 virulence system in the evasion of GBP-mediated immunity.

Rapid lethality of mice lacking the phagocyte oxidase and Caspase1/11 following Mycobacterium tuberculosis infection.

Immune networks that control antimicrobial and inflammatory mechanisms have overlapping regulation and functions to ensure effective host responses. Genetic interaction studies of immune pathways that compare host responses in single and combined knockout backgrounds are a useful tool to identify new mechanisms of immune control during infection. For disease caused by pulmonary Mycobacterium tuberculosis infections, which currently lacks an effective vaccine, understanding genetic interactions between protective immune pathways may identify new therapeutic targets or disease-associated genes. Previous studies suggested a direct link between the activation of NLRP3-Caspase1 inflammasome and the NADPH-dependent phagocyte oxidase complex during Mtb infection. Loss of the phagocyte oxidase complex alone resulted in increased activation of Caspase1 and IL1ß production during Mtb infection, resulting in failed disease tolerance during the chronic stages of disease. To better understand this interaction, we generated mice lacking both Cybb , a key subunit of the phagocyte oxidase, and Caspase1/11 . We found that ex vivo Mtb infection of Cybb -/- Caspase1/11 -/- macrophages resulted in the expected loss of IL1ß secretion but an unexpected change in other inflammatory cytokines and bacterial control. Mtb infected Cybb -/- Caspase1/11 -/- mice rapidly progressed to severe TB, succumbing within four weeks to disease characterized by high bacterial burden, increased inflammatory cytokines, and the recruitment of granulocytes that associated with Mtb in the lungs. These results uncover a key genetic interaction between the phagocyte oxidase complex and Caspase1/11 that controls protection against TB and highlight the need for a better understanding of the regulation of fundamental immune networks during Mtb infection.

A Genome-Wide Screen in Macrophages Defines Host Genes Regulating the Uptake of Mycobacterium abscessus.

The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies. Mycobacterium abscessus (Mab) is a highly antibiotic resistant, rapidly growing nontuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3, and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but not Escherichia coli or latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins, suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease. IMPORTANCE Pathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given that M. abscessus is broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required for M. abscessus uptake. We identified new macrophage uptake regulators during M. abscessus infection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions during M. abscessus infection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.

Differential Requirement for IRGM Proteins during Tuberculosis Infection in Mice.

Mycobacterium tuberculosis (Mtb) is a bacterium that exclusively resides in human hosts and remains a dominant cause of morbidity and mortality among infectious diseases worldwide. Host protection against Mtb infection is dependent on the function of immunity-related GTPase clade M (IRGM) proteins. Polymorphisms in human IRGM associate with altered susceptibility to mycobacterial disease, and human IRGM promotes the delivery of Mtb into degradative autolysosomes. Among the three murine IRGM orthologs, Irgm1 has been singled out as essential for host protection during Mtb infections in cultured macrophages and in vivo. However, whether the paralogous murine Irgm genes, Irgm2 and Irgm3, play roles in host defense against Mtb or exhibit functional relationships with Irgm1 during Mtb infection remains undetermined. Here, we report that Irgm1-/- mice are indeed acutely susceptible to aerosol infection with Mtb, yet the additional deletion of the paralogous Irgm3 gene restores protective immunity to Mtb infections in Irgm1-deficient animals. Mice lacking all three Irgm genes (panIrgm-/-) are characterized by shifted lung cytokine profiles at 5 and 24 weeks postinfection, but control disease until the very late stages of the infection, when panIrgm-/- mice display increased mortality compared to wild-type mice. Collectively, our data demonstrate that disruptions in the balance between Irgm isoforms is more detrimental to the Mtb-infected host than total loss of Irgm-mediated host defense, a concept that also needs to be considered in the context of human Mtb susceptibility linked to IRGM polymorphisms.

GarD-ing the pathogen-containing vacuole from destruction.

The human pathogen Chlamydia trachomatis evades killing by IFNγ-activated mechanisms, yet how this occurs remains unclear. In this issue of Cell Host & Microbe, Walsh et al. identify an IFNγ-dependent antimicrobial mechanism mediated by the host ubiquitin ligase RNF213 that is evaded by the Chlamydia effector GarD.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

Fetal Liver-Derived Alveolar-like Macrophages: A Self-Replicating Ex Vivo Model of Alveolar Macrophages for Functional Genetic Studies.

Alveolar macrophages (AMs) are tissue-resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. Although the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics, as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. In this study, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGF-ß. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation.

Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNγ) is a key cytokine in protective immunity that induces major histocompatibility complex class II molecules (MHCII) to amplify CD4+ T cell activation and effector function. Despite its central role, the dynamic regulation of IFNγ-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages, we identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase three beta (GSK3ß) or the mediator complex subunit 16 (MED16). Both pathways control distinct aspects of the IFNγ response and are necessary for IFNγ-mediated induction of the MHCII transactivator Ciita, MHCII expression, and CD4+ T cell activation. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses.

Mitochondrial respiration contributes to the interferon gamma response in antigen-presenting cells.

The immunological synapse allows antigen-presenting cells (APCs) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While pathways downstream of toll-like receptors rely on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFNγ). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in murine macrophages to identify the regulators of IFNγ-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multiscreen approach enabled us to identify novel pathways that preferentially control functionally distinct proteins. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFNγ signaling pathway. We report that the IFNγ response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFNγ and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity.

Host immunity increases Mycobacterium tuberculosis reliance on cytochrome bd oxidase.

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, the presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFNγ), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that ΔcydA mutants were hypersusceptible to the low pH encountered in IFNγ-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs.

Macrophage Infection Models for Mycobacterium tuberculosis.

Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages, HoxB8 conditionally immortalized myeloid cells, Max Planck Institute alveolar macrophage-like cells, and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.

Fighting Persistence: How Chronic Infections with Mycobacterium tuberculosis Evade T Cell-Mediated Clearance and New Strategies To Defeat Them.

Chronic bacterial infections are caused by pathogens that persist within their hosts and avoid clearance by the immune system. Treatment and/or detection of such pathogens is difficult, and the resulting pathologies are often deleterious or fatal. There is an urgent need to develop protective vaccines and host-directed therapies that synergize with antibiotics to prevent pathogen persistence and infection-associated pathologies. However, many persistent pathogens, such as Mycobacterium tuberculosis, actively target the very host pathways activated by vaccination. These immune evasion tactics blunt the effectiveness of immunization strategies and are impeding progress to control these infections throughout the world. Therefore, it is essential that M. tuberculosis immune evasion-related pathogen virulence strategies are considered to maximize the effectiveness of potential new treatments. In this review, we focus on how Mycobacterium tuberculosis infects antigen-presenting cells and evades effective immune clearance by the adaptive response through (i) manipulating antigen presentation, (ii) repressing T cell-activating costimulatory molecules, and (iii) inducing ligands that drive T cell exhaustion. In this context, we will examine the challenges that bacterial virulence strategies pose to developing new vaccines. We will then discuss new approaches that will help dissect M. tuberculosis immune evasion mechanisms and devise strategies to bypass them to promote long-term protection and prevent disease progression.

mSphere of Influence: Combining Host and Pathogen Genetics To Disrupt Chronic Infections.

Andrew J. Olive works in the field of host responses to chronic infections. In this mSphere of Influence article, he reflects on how "Tryptophan biosynthesis protects mycobacteria from CD4 T-cell-mediated killing" (Y. J. Zhang, M. C. Reddy, T. R. Ioerger, A. C. Rothchild, et al., Cell 155:1296-1308, 2013, https://doi.org/10.1016/j.cell.2013.10.045) impacted his own work using genetic approaches to dissect the interface between host and pathogen.