Biology subjects

Olive, A. J.

Publications and source records attributed to Olive, A. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Effective macrophage clearance of Klebsiella pneumoniae requires the inducible nitric oxide synthase iNOS and is independent of reactive oxygen species generated by NADPH oxidase

Klebsiella pneumoniae is a leading cause of pneumonia and bacteremia and is especially dangerous in healthcare settings. Despite massive clinical significance, the mechanisms used by macrophages to kill K. pneumoniae are largely unknown. Macrophages are critical for controlling K. pneumoniae, as mice lacking monocyte-derived or alveolar macrophages have higher bacterial tissue burdens and mortality. Two prominent mechanisms used by macrophages to kill bacteria are the production of reactive oxygen species (ROS) via the NADPH oxidase NOX2 and reactive nitrogen species (RNS) via the inducible nitric oxide synthase iNOS. Previously, we found that a subset of K. pneumoniae factors which enhance bacteremia pathogenesis also support intracellular survival. The degree to which these factors enhance intracellular fitness correlated with resistance against RNS, not ROS. Here, we aimed to define the extent to which macrophage ROS and RNS contribute to K. pneumoniae clearance. Using wild-type, Cybb-/-, and Nos2-/- cells, we measured K. pneumoniae survival in macrophages lacking such defenses. NOX2 was dispensable for K. pneumoniae elimination, and ROS was undetectable in K. pneumoniae-infected macrophages. We confirmed that ROS was also undetectable within alveolar-like macrophages, indicating that ROS is dispensable for anti-K. pneumoniae defense across macrophage subsets. Instead, iNOS contributed to macrophage clearance of K. pneumoniae and likely enhances killing by RNS induction and coordinating a normal cytokine response. Activation of pathways upstream of iNOS may be the most relevant to supporting effective macrophage control of K. pneumoniae. This study defines unexpected differential roles for ROS and RNS in macrophage clearance of K. pneumoniae.

immunology↗

bioRxiv2026

Trans-Kingdom dsRNA Sensing: Aspergillus fumigatus Mycovirus Activates MDA5/MAVS Immunity and Limits Allergic Bronchopulmonary Aspergillosis

MDA5 is a cytosolic pattern-recognition receptor (PRR) that binds to double-stranded RNA (dsRNA) and subsequently interacts with the signaling adaptor protein MAVS to initiate the antiviral interferon (IFN) response. Our group previously demonstrated that MDA5 is essential for host resistance against the fungal pathogen, Aspergillus fumigatus. Although fungal dsRNA was sufficient to activate MDA5 signaling, the precise source of A. fumigatus dsRNA responsible for this MDA5-stimulating function remains unknown. Here, we demonstrate that the magnitude of the IFN-dependent antifungal response is A. fumigatus strain dependent. Unexpectedly, we found that A. fumigatus isolates infected with dsRNA mycoviruses triggered a more robust MAVS-dependent inflammatory response within alveolar macrophages. Furthermore, dsRNA mycovirus infection increased fungal susceptibility to antifungal killing without altering other A. fumigatus growth characteristics. Although dsRNA mycovirus infection did not alter virulence in an acute bronchopneumonia model of A. fumigatus infection, it significantly impaired virulence and improved disease parameters in a chronic model of allergic bronchopulmonary aspergillosis (ABPA). Collectively, these findings reveal a novel role for trans-kingdom interactions in driving the host antifungal IFN response and modulating virulence in chronic aspergillosis models.

immunology↗

bioRxiv2026

ULK1 drives NDP52-mediated selective autophagic degradation of MHC-I to promote immune evasion in HPV-positive head and neck cancer

Antigen presentation by major histocompatibility complex class I (MHC-I) is critical for tumor cell killing by CD8+ T cells. Accordingly, tumor cells downregulate MHC-I expression through multiple mechanisms, thereby evading the immune response. Importantly, lower levels of MHC-I are associated with poor responses to immune checkpoint inhibitor therapy. Our recent study has shown that the human papillomavirus (HPV) oncoproteins induce MHC-I protein ubiquitination by membrane-associated Ring-CH-type finger 8 (MARCHF8) in HPV-positive head and neck cancer (HPV+ HNC). However, the mechanism by which ubiquitinated MHC-I is degraded remains elusive. By performing genome-wide CRISPR screens, we identified components of the ULK1 and PIK3C3 complexes for autophagy initiation complexes among the top negative regulators of cell-surface MHC-I expression in HPV+ HNC cells. We show that MHC-I is recruited from the ER to autophagosomes by the cargo receptor NDP52, decreasing MHC-I levels. Further, inhibiting the initiation or nucleation steps of autophagy before autophagosome formation is critical for restoring MHC-I levels on the cell surface. Finally, genetic inhibition of autophagy initiation suppresses HPV+ HNC tumor growth in vivo and enhances the CD8+ T cell-mediated antitumor response. Our findings suggest that autophagic degradation of MARCHF8-ubiquitinated MHC-I is a key immune evasion mechanism in HPV+ HNC.

cancer biology↗

bioRxiv2025

Intracellular C. neoformans infection stimulates increased glycolytic activity in fetal liver-derived alveolar-like macrophages

Alveolar macrophages (AMs) serve as a first line of defense against respiratory pathogens, including Cryptococcus neoformans, the primary causative agent of cryptococcosis, a deadly pulmonary mycosis which commonly afflicts immunocompromised individuals. While these innate immune cells are thought to play a pivotal role in controlling the outcome of C. neoformans infections, this critical host-pathogen interaction is more commonly studied in vitro using bone marrow-derived macrophages (BMDM) or immortalized macrophage cell lines that differ in ontogeny and phenotype from AMs. In this work, we characterized fetal liver-derived alveolar-like macrophages (FLAMs) as an alternate model to study the earliest stages of C. neoformans infection. Here, we show that the FLAM steady state transcriptome is more similar to primary AMs than peritoneal macrophages and the macrophage cell lines, RAW264.7 and J774, and that FLAMs exhibit distinct transcriptional responses to IFN{gamma} stimulation and C. neoformans infection compared to J774 cells. Specifically, transcriptome profiling and gene ontology analysis indicate that C. neoformans infection of FLAMs, but not J774 cells, increases the expression of canonical glycolytic genes, including Slc2a1, Pgk1, and Ldha, which is accompanied by a metabolic shift favoring glycolysis. Furthermore, activation or inhibition of hypoxia inducible factor 1 (HIF1) activity utilizing dimethyloxalylglycine (DMOG) and echinomycin, respectively, indicates that the expression of select glycolytic genes in C. neoformans-infected FLAMs is HIF1-dependent. Collectively, our results suggest that FLAMs serve as an appropriate tool for modeling AM:C. neoformans interactions and investigating the effects of this pathogen on host AM immunometabolism.

immunology↗

bioRxiv2025

Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can be sensed by T cells, which are essential for the control of infection. In comparison to viral infections, Mtb antigens are relatively limited and hence, challenging to sample. Specialized antigen presentation pathways enable the presentation of such scarce antigens to CD8+ T cells, which are, thus, uniquely poised to survey intracellular environments. A subset of CD8+ T cells prevalent in the airways, known as mucosal-associated invariant T (MAIT) cells, can be activated through the presentation of Mtb antigens via the MHC class I-related protein 1 (MR1) molecule. Prior work demonstrates that endosomal calcium signaling is critical for MR1-mediated presentation of Mtb-derived antigens. Here, we show that the calcium-sensing trafficking proteins Synaptotagmin (Syt) 1 and Syt7 specifically promote MAIT cell activation in response to Mtb-infected cells. In bronchial epithelial cells, Syt1 and Syt7 localize to late endo-lysosomes and MR1 vesicles. Loss of Syt1 and Syt7 results in enlarged MR1 vesicles and an increased number of MR1 vesicles in close proximity to Mtb-containing vacuoles during infection. This study identifies a specialized pathway in which Syt1 and Syt7 facilitate the translocation of MR1 from Mtb-containing vacuoles, potentially to the cell surface for antigen presentation.

immunology↗

bioRxiv2025

Omega-3 Fatty Acid Synergy with Glucocorticoid in Lupus Macrophages: Targeting Pathogenic Pathways to Reduce Steroid Dependence

IntroductionSystemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by aberrant inflammation, type I IFN-stimulated gene (ISG) expression, and autoantibody production. Glucocorticoids (GCs) like dexamethasone (DEX) are standard long-term SLE treatments but cause significant side effects, highlighting the need for safer steroid-sparing options. Preclinical and clinical studies suggest that dietary supplementation with omega-3 fatty acids (O3FAs), particularly docosahexaenoic acid (DHA), suppress inflammation and autoimmunity associated with SLE disease progression. We explored the steroid-sparing potential of DHA to influence suppressive effects of DEX on pathogenic gene expression. MethodsMacrophages from SLE-prone NZBWF1 mice were first subjected to DHA (5, 10, or 25 {micro}M), DEX (1, 10, 100, or 1000 nM), or DHA+DEX cotreatment. Following pretreatment, cells were exposed to lipopolysaccharide (LPS; 20 ng/mL) to model SLE hyperinflammation. Effects on gene expression were analyzed by qRT-PCR and RNA-seq. ResultsqRT-PCR indicated that subinhibitory concentrations of DHA (5-10 {micro}M) potentiated the efficacy of low-dose DEX (1-100 nM) in suppressing LPS-induced ISG expression (e.g., Irf7, Oasl1, Rsad2), amplifying the effects of DEX monotherapy by 10- to 100-fold. SynergyFinder analysis confirmed that DHA and DEX interacted synergistically in suppressing ISG expression, with significant inhibition observed at concentrations as low as 1 nM DEX and 5 {micro}M DHA. RNA-seq revealed that combining suboptimal DHA (10 M) and DEX (100 nM) induced 247 differentially expressed genes (DEGs) at 4 hr and 347 DEGs at 8 hr post-LPS, dramatically surpassing the effects of each treatment alone. Functional enrichment analysis indicated that DHA+DEX cotreatment robustly suppressed immune and inflammatory pathways while promoting proliferative and metabolic processes, reflecting a shift from inflammatory (M1) to pro-resolving (M2) macrophage phenotypes. DHA and DEX countered LPS effects by i) downregulating common transcription factors (TFs) canonically associated with inflammation (e.g., NF-{kappa}B, AP-1, STATs, and IRF1), ii) upregulating shared regulatory factors involved in inflammation resolution (e.g., YBX1, EGR1, and BCL6), and iii) selectively influencing other regulatory factors. DiscussionAltogether, DHA and DEX synergistically suppress inflammation by targeting common and unique molecular pathways in SLE macrophages, favoring the pro-resolving M2 phenotype. O3FA-GC cotreatment might facilitate reducing requisite steroid dosages for SLE management.

immunology↗

bioRxiv2025

Differential activation of NF-κB and HIF-1α between alveolar-like macrophages and myeloid-derived macrophages drive inflammatory differences following Mycobacterium abscessus infection.

Pulmonary infections caused by Mycobacterium abscessus (Mab), a rapidly growing nontuberculous mycobacterium (NTM), are on the rise in patients with chronic or acquired lung disease. In contrast to immunocompetent individuals, these patient cohorts exhibit abnormal pulmonary function that result from chronic inflammation and mucus build-up. Treatment regimens rely on multi-drug cocktails yet Mabs natural recalcitrance to common antibiotics extends treatment timelines and increases the frequency of treatment failures. Thus, it is important to understand the mechanisms by which immunocompetent individuals clear Mab with relative ease while susceptible individuals do not. In the lungs, macrophages are the first immune cell Mab encounters following infection, with both resident alveolar macrophages and recruited myeloid derived macrophages playing important roles during infection control. However, the specific role of these distinct macrophage populations in regulating control and inflammatory responses during Mab remains limited due to a lack of ex vivo models that recapitulate the functions of different macrophage subsets. Here, we leverage a fetal-liver derived alveolar macrophage (FLAM) model to define early inflammatory responses occurring at the Mab-macrophage interface compared to bone-marrow derived macrophages (BMDMs). Even though both FLAMs and BMDMs similarly control intracellular Mab, the inflammatory response between these macrophage populations is significantly different. BMDMs robustly activated NF-{kappa}B transcriptional targets that include important chemokines and inflammatory cytokines like TNF, FLAMs transiently induced these genes following Mab infection. While activation of FLAMs or BMDMs with IFN{gamma} prior to Mab infection did not alter Mab intracellular dynamics, it did drive FLAMs to be more inflammatory, yet important differences remained compared to BMDMs, including the lower expression of the inducible nitric oxide synthase. This was reversed with chemical activation of HIF1. We conclude that FLAMs and BMDMs differentially respond to Mab infection due to differences in signaling networks activated following innate immune sensing, with FLAMs being more hypoinflammatory than BMDMs. More broadly our results highlight a need to better understand the initial interactions with Mab and distinct macrophage populations to define pathways that contribute to pulmonary protection or disease.

immunology↗

bioRxiv2024

TGFβ 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. TGF{beta} is an important regulator of AM differentiation and maintenance, but how TGF{beta} 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 TGF{beta} in AMs. Using transcriptional analysis, we first globally defined how TGF{beta} regulates gene expression of resting FLAMs. We found that TGF{beta} 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 TGF{beta} alters the response to TLR2 agonists. While both TGF{beta} (+) and TGF{beta} (-) FLAMs robustly responded to TLR2 agonists, we found an unexpected activation of type I interferon (IFN) responses in FLAMs and primary AMs in a TGF{beta}-dependent manner. Surprisingly, mitochondrial antiviral signaling protein and the interferon regulator factors 3 and 7 were required for IFN production by TLR2 agonists and the IFN response was dependent on mitochondrial reactive oxygen species. Together, these data suggest that TGF{beta} modulates AM metabolic networks and innate immune signaling cascades to control inflammatory pathways in AMs.

immunology↗

bioRxiv2023

GSK3α/β 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 IFN{gamma} 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{gamma}-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 IFN{gamma} robustly activates both macrophage types; however, the profile of activated IFN{gamma}-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{gamma}. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3/{beta} alters the IFN{gamma} response. GSK3/{beta} controlled distinct IFN{gamma} responses, and in AM-like cells, we found GSK3/{beta} 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{gamma} is restricted in a GSK3/{beta}-dependent manner and that IFN{gamma} 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.

immunology↗

bioRxiv2023

Immunometabolic cues recompose and reprogram the microenvironment around biomaterials

Circulating monocytes infiltrate and coordinate immune responses in various inflamed tissues, such as those surrounding implanted biomaterials, affecting therapeutic, diagnostic, tissue engineering and regenerative applications. Here, we show that immunometabolic cues in the biomaterial microenvironment govern CCR2- and CX3CR1-dependent trafficking of immune cells, including neutrophils and monocytes; ultimately, this affects the composition and activation states of macrophage and dendritic cell populations. Furthermore, immunometabolic cues around implants orchestrate the relative composition of proinflammatory, transitory and anti-inflammatory CCR2+, CX3CR1+ and CCR2+CX3CR1+ immune cell populations. Consequently, modifying immunometabolism by glycolytic inhibition drives a pro-regenerative microenvironment in part by myeloid cells around amorphous polylactide implants. In addition to, Arginase 1-expressing myeloid cells, T helper 2 cells and {gamma}{delta}+ T-cells producing IL-4 significantly contribute to shaping the metabolically reprogramed, pro-regenerative microenvironment around crystalline polylactide biomaterials. Taken together, we find that local metabolic states regulate inflammatory processes in the biomaterial microenvironment, with implications for translational medicine.

bioengineering↗

bioRxiv2023

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 IL1{beta} 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 IL1{beta} 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.

immunology↗

bioRxiv2022

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 non-tuberculous 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. IMPORTANCEPathogen 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.

microbiology↗

bioRxiv2022

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. Author SummaryListeria monocytogenes (Lm) is a bacterial pathogen that causes the foodborne illness listeriosis primarily in immunocompromised, elderly, and pregnant individuals. Listeriosis is one of the deadliest bacterial infections known, with a mortality rate of ~30% even when treated with antibiotics. The high mortality rate of listeriosis is due to inefficient restriction of Lm by the immune system, and subsequent spread of bacteria beyond the gastrointestinal tract to internal organs such as the liver and brain. Macrophages are important for immune clearance of Lm but are also hypothesized to promote dissemination of intracellular Lm; thus, studies of Lm-macrophage interactions are critical for understanding the balance between bacterial growth and restriction by these phagocytes. We performed a forward genetic screen in macrophages and discovered that the tumor suppressor PTEN promotes phagocytosis of Lm by enhancing adherence to macrophages. These results demonstrate a novel function of macrophage PTEN, which canonically acts as a repressor of phagocytosis. In addition, we found that PTEN protects mice from severe disease and lowers bacterial burdens following oral inoculation of Lm. Our results demonstrate for the first time that macrophage phagocytosis is an important immune defense against invasive Lm during the foodborne route of infection.

microbiology↗

bioRxiv2022

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 4 and 24 weeks post infection, 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.

microbiology↗

bioRxiv2020

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

The outcome of an encounter with Mycobacterium tuberculosis depends on the pathogens 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 associate 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 mutant fitness across the CC panel revealed that many virulence pathways are only in specific host microenvironments, identifying the large fraction of the pathogens genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, identifying the specific host-pathogen genetic interactions that influence pathogenesis.

genetics↗

bioRxiv2020

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

The immunological synapse allows antigen presenting cells (APC) 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 toll-like receptor ligation relies 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 (IFN{gamma}). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in macrophages to identify the regulators of IFN{gamma}-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multi-screen approach enabled us to identify novel pathways that control these functionally distinct markers. 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 IFN{gamma} signaling pathway. We report that the IFN{gamma} 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 IFN{gamma} and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity.

immunology↗

bioRxiv2020

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, 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 (IFN{gamma}), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that{Delta} cydA mutants were hypersusceptible to the low pH encountered in IFN{gamma}-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. Author SummaryTuberculosis, caused by Mycobacterium tuberculosis (Mtb) is a serious global health problem that is responsible for over one million deaths annually, more than any other single infectious agent. In the host, Mtb can adapt to a wide variety of immunological and environmental pressures which is integral to its success as a pathogen. Accordingly, the respiratory capacity of Mtb is flexible. The electron transport chain of Mtb has two terminal oxidases, the cytochrome bc1/aa3 super complex and cytochrome bd, that contribute to the proton motive force and subsequent production of energy in the form of ATP. The bc1/aa3 super complex is required for optimal growth during infection but the role of cytochrome bd is unclear. Here we report that the cytochrome bd oxidase is required for resisting the adaptive immune response, in particular, acidification of the phagosome induced by lymphocyte-derived IFN{gamma}. We found that the cytochrome bd oxidase is specifically required under acidic conditions, where the bc1/aa3 complex is preferentially inhibited. Additionally, we show that acidic conditions increased the potency of Q203, a cytochrome bc1/aa3 inhibitor and candidate tuberculosis therapy. This work defines a new link between the host immune response and the respiratory requirements of Mtb that affects the potency of a potential new therapeutic.

microbiology↗

bioRxiv2020

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 IFN{gamma}. While the production of IFN{gamma} 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 IFN{gamma}-mediated antimicrobial responses, yet what mechanisms Mtb resists remains unclear. The IFN{gamma}-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.

microbiology↗