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.

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.

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.

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.

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.

Pathology after Chlamydia trachomatis infection is driven by nonprotective immune cells that are distinct from protective populations.

Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis-induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4+ and CD8+ T cells at later time points. To determine what draws these cells to C. trachomatis-infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis-induced pathology without exacerbating infection or transmission.

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.

The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis Infection.

Protection from infectious disease relies on two distinct strategies: antimicrobial resistance directly inhibits pathogen growth, whereas infection tolerance protects from the negative impact of infection on host health. A single immune mediator can differentially contribute to these strategies in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase (Phox) complex produces antimicrobial superoxide and protects from tuberculosis (TB) in humans. However, Phox-deficient mice display no sustained resistance defects to Mycobacterium tuberculosis, suggesting a more complicated role for NADPH Phox complex than strictly controlling bacterial growth. We examined the mechanisms by which Phox contributes to protection from TB and found that mice lacking the Cybb subunit of Phox suffered from a specific defect in tolerance, which was caused by unregulated Caspase-1 activation, IL-1ß production, and neutrophil influx into the lung. These studies imply that a defect in tolerance alone is sufficient to compromise immunity to M. tuberculosis and highlight a central role for Phox and Caspase-1 in regulating TB disease progression.

Integrin α4ß1 is necessary for CD4+ T cell-mediated protection against genital Chlamydia trachomatis infection.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection in the United States and a significant health burden worldwide. Protection from Chlamydia infection in the genital mucosa is dependent on IFN-γ derived from CD4(+) Th1 cells. These CD4(+) T cells must home successfully to the genital tract to exert their effector function and decrease C. trachomatis burden. Although adhesion receptors expressed by CD4(+) T cells in the genital tract have been characterized, the integrin receptor required for Chlamydia-specific CD4(+) T cell-mediated protection has not been explored. In this study, we demonstrate that C. trachomatis infection of the upper genital tract results in recruitment of Chlamydia-specific CD4(+) T cells robustly expressing the integrin α4ß1. Interfering with α4ß1, but not α4ß7, function resulted in defective CD4(+) T cell trafficking to the uterus and high bacterial load. We conclude that integrin α4ß1 is necessary for CD4(+) T cell-mediated protection against C. trachomatis infection in the genital mucosa. By identifying homing molecules required for successful CD4(+) T cell trafficking to C. trachomatis-infected tissues, we will be better equipped to design vaccines that elicit sterilizing, long-lasting immunity without inducing immune pathologies in the upper genital tract.

Attachment of Chlamydia trachomatis L2 to host cells requires sulfation.

Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3, B4GALT7, and SLC35B2, which encode glucuronosyltransferase I, galactosyltransferase I, and the 3'-phosphoadenosine 5'-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.

CD4+ T cells are necessary and sufficient to confer protection against Chlamydia trachomatis infection in the murine upper genital tract.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, mouse models in which the vaginal vault is inoculated with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not ascend efficiently to the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce significant CD4⁺ T cell infiltration. In this article, we describe a noninvasive transcervical infection model in which we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4⁺ T cell response that is both necessary and sufficient to clear infection and provide protection against reinfection.

Compared to other NHEJ factors, DNA-PK protein and RNA levels are markedly increased in all higher primates, but not in prosimians or other mammals.

The DNA dependent protein kinase (DNA-PK) initiates non-homologous recombination (NHEJ), the predominate DNA double-strand break (DSBR) pathway in higher vertebrates. It has been known for decades that the enzymatic activity of DNA-PK [that requires its three component polypeptides, Ku70, Ku80 (that comprise the DNA-end binding Ku heterodimer), and the catalytic subunit (DNA-PKcs)] is present in humans at 10-50 times the level observed in other mammals. Here, we show that the high level of DNA-PKcs protein expression appears evolutionarily in mammals between prosimians and higher primates. Moreover, the RNAs encoding the three component polypeptides of DNA-PK are present at similarly high levels in hominids, new-, and old-world monkeys, but expression of these RNAs in prosimians is ∼5-50 fold less, analogous to the levels observed in other non-primate species. This is reminiscent of the appearance of Alu repeats in primate genomes -- abundant in higher primates, but present at much lower density in prosimians. Alu repeats are well-known for their capacity to promote non-allelic homologous recombination (NAHR) a process known to be inhibited by DNA-PK. Nanopore sequence analyses of cultured cells proficient or deficient in DNA-PK revealed an increase of inter-chromosomal translocations caused by NAHR. Although the high levels of DNA-PK in primates may have many functions, we posit that high levels of DNA-PK may function to restrain deleterious NAHR events between Alu elements.

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.

Mycobacterium tuberculosis virulence lipid PDIM inhibits autophagy in mice.

Mycobacterium tuberculosis (Mtb) infects several lung macrophage populations, which have distinct abilities to restrict Mtb. What enables Mtb survival in certain macrophage populations is not well understood. Here we used transposon sequencing analysis of Mtb in wild-type and autophagy-deficient mouse macrophages lacking ATG5 or ATG7, and found that Mtb genes involved in phthiocerol dimycocerosate (PDIM) virulence lipid synthesis confer resistance to autophagy. Using ppsD mutant Mtb, we found that PDIM inhibits LC3-associated phagocytosis (LAP) by inhibiting phagosome recruitment of NADPH oxidase. In mice, PDIM protected Mtb from LAP and classical autophagy. During acute infection, PDIM was dispensable for Mtb survival in alveolar macrophages but required for survival in non-alveolar macrophages in an autophagy-dependent manner. During chronic infection, autophagy-deficient mice succumbed to infection with PDIM-deficient Mtb, with impairments in B-cell accumulation in lymphoid follicles. These findings demonstrate that PDIM contributes to Mtb virulence and immune evasion, revealing a contributory role for autophagy in B-cell responses.

TGFß primes alveolar-like macrophages to induce type I IFN following TLR2 activation.

Alveolar macrophages (AMs) are key mediators of lung function and are potential targets for therapies during respiratory infections. TGFß is an important regulator of AM differentiation and maintenance, but how TGFß directly modulates the innate immune responses of AMs remains unclear. This shortcoming prevents effective targeting of AMs to improve lung function in health and disease. Here we leveraged an optimized ex vivo AM model system, fetal-liver derived alveolar-like macrophages (FLAMs), to dissect the role of TGFß in AMs. Using transcriptional analysis, we first globally defined how TGFß regulates gene expression of resting FLAMs. We found that TGFß maintains the baseline metabolic state of AMs by driving lipid metabolism through oxidative phosphorylation and restricting inflammation. To better understand inflammatory regulation in FLAMs, we next directly tested how TGFß alters the response to TLR2 agonists. While both TGFß (+) and TGFß (-) FLAMs robustly responded to TLR2 agonists, we found an unexpected activation of type I interferon (IFN) responses in FLAMs and primary AMs in a TGFß-dependent manner. Surprisingly, mitochondrial antiviral signaling protein and the interferon regulator factors 3 and 7 were required for IFN production by TLR2 agonists. Together, these data suggest that TGFß modulates AM metabolic networks and innate immune signaling cascades to control inflammatory pathways in AMs.

Immunometabolic cues recompose and reprogram the microenvironment around implanted biomaterials.

Circulating monocytes infiltrate and coordinate immune responses in tissues surrounding implanted biomaterials and in other inflamed tissues. Here we show that immunometabolic cues in the biomaterial microenvironment govern the trafficking of immune cells, including neutrophils and monocytes, in a manner dependent on the chemokine receptor 2 (CCR2) and the C-X3-C motif chemokine receptor 1 (CX3CR1). This affects the composition and activation states of macrophage and dendritic cell populations, ultimately orchestrating the relative composition of pro-inflammatory, transitory and anti-inflammatory CCR2+, CX3CR1+ and CCR2+ CX3CR1+ immune cell populations. In amorphous polylactide implants, modifying immunometabolism by glycolytic inhibition drives a pro-regenerative microenvironment principally by myeloid cells. In crystalline polylactide implants, together with arginase-1-expressing myeloid cells, T helper 2 cells and γδ+ T cells producing interleukin-4 substantially contribute to shaping the metabolically reprogrammed pro-regenerative microenvironment. Our findings inform the premise that local metabolic states regulate inflammatory processes in the biomaterial microenvironment.

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.

Compensatory T cell responses in IRG-deficient mice prevent sustained Chlamydia trachomatis infections.

The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of bacterial sexually transmitted diseases in the United States. In women C. trachomatis can establish persistent genital infections that lead to pelvic inflammatory disease and sterility. In contrast to natural infections in humans, experimentally induced infections with C. trachomatis in mice are rapidly cleared. The cytokine interferon-γ (IFNγ) plays a critical role in the clearance of C. trachomatis infections in mice. Because IFNγ induces an antimicrobial defense system in mice but not in humans that is composed of a large family of Immunity Related GTPases (IRGs), we questioned whether mice deficient in IRG immunity would develop persistent infections with C. trachomatis as observed in human patients. We found that IRG-deficient Irgm1/m3((-/-)) mice transiently develop high bacterial burden post intrauterine infection, but subsequently clear the infection more efficiently than wildtype mice. We show that the delayed but highly effective clearance of intrauterine C. trachomatis infections in Irgm1/m3((-/-)) mice is dependent on an exacerbated CD4(+) T cell response. These findings indicate that the absence of the predominant murine innate effector mechanism restricting C. trachomatis growth inside epithelial cells results in a compensatory adaptive immune response, which is at least in part driven by CD4(+) T cells and prevents the establishment of a persistent infection in mice.

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.