Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection.

During mycobacterial infections, pathogenic mycobacteria manipulate both host immune and stromal cells to establish and maintain a productive infection. In humans, non-human primates, and zebrafish models of infection, pathogenic mycobacteria produce and modify the specialized lipid trehalose 6,6'-dimycolate (TDM) in the bacterial cell envelope to drive host angiogenesis toward the site of forming granulomas, leading to enhanced bacterial growth. Here, we use the zebrafish-Mycobacterium marinum infection model to define the signaling basis of the host angiogenic response. Through intravital imaging and cell-restricted peptide-mediated inhibition, we identify macrophage-specific activation of NFAT signaling as essential to TDM-mediated angiogenesis in vivo. Exposure of cultured human cells to Mycobacterium tuberculosis results in robust induction of VEGFA, which is dependent on a signaling pathway downstream of host TDM detection and culminates in NFATC2 activation. As granuloma-associated angiogenesis is known to serve bacterial-beneficial roles, these findings identify potential host targets to improve tuberculosis disease outcomes.

An ancestral mycobacterial effector promotes dissemination of infection.

The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.

mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity.

Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.

Decoding the tuberculous granuloma.

In this issue of Immunity,Gideon et al. (2022) couple sophisticated single-cell analyses with detailed in vivo measurements of Mycobacterium tuberculosis granulomas to define the cellular and transcriptional properties of a successful host immune response during tuberculosis.

Inhibition of infection-induced vascular permeability modulates host leukocyte recruitment to Mycobacterium marinum granulomas in zebrafish.

Mycobacterial granuloma formation involves significant stromal remodeling including the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as antiangiogenic or vascular normalizing therapies are beneficial host-directed therapies in preclinical models of tuberculosis across host-mycobacterial pairings. Using the zebrafish-Mycobacterium marinum infection model, we demonstrate that vascular normalization by inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) decreases granuloma hypoxia, the opposite effect of hypoxia-inducing antiangiogenic therapy. Inhibition of VE-PTP decreased neutrophil recruitment to granulomas in adult and larval zebrafish, and decreased the proportion of neutrophils that extravasated distal to granulomas. Furthermore, VE-PTP inhibition increased the accumulation of T cells at M. marinum granulomas. Our study provides evidence that, similar to the effect in solid tumors, vascular normalization during mycobacterial infection increases the T cell:neutrophil ratio in lesions which may be correlates of protective immunity.

A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

Elevated cerebrospinal fluid cytokine levels in tuberculous meningitis predict survival in response to dexamethasone.

Adjunctive treatment with antiinflammatory corticosteroids like dexamethasone increases survival in tuberculosis meningitis. Dexamethasone responsiveness associates with a C/T variant in Leukotriene A4 Hydrolase (LTA4H), which regulates expression of the proinflammatory mediator leukotriene B4 (LTB4). TT homozygotes, with increased expression of LTA4H, have the highest survival when treated with dexamethasone and the lowest survival without. While the T allele is present in only a minority of the world's population, corticosteroids confer modest survival benefit worldwide. Using Bayesian methods, we examined how pretreatment levels of cerebrospinal fluid proinflammatory cytokines affect survival in dexamethasone-treated tuberculous meningitis. LTA4H TT homozygosity was associated with global cytokine increases, including tumor necrosis factor. Association between higher cytokine levels and survival extended to non-TT patients, suggesting that other genetic variants may also induce dexamethasone-responsive pathological inflammation. These findings warrant studies that tailor dexamethasone therapy to pretreatment cerebrospinal fluid cytokine concentrations, while searching for additional genetic loci shaping the inflammatory milieu.

Early cell-autonomous accumulation of neutral lipids during infection promotes mycobacterial growth.

Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of this process using the zebrafish-M. marinum larval model of infection. We find that neutral lipids accumulate cell-autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Treatment with ezetimibe, an FDA-approved drug, resulted in decreased levels of free cholesterol and neutral lipids, and a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell-autonomously at early timepoints of mycobacterial infection, and limitation of this process results in decreased bacterial burden.

Macrophage ACKRobatics: An atypical Cxcr3 keeps macrophages in check.

Discussion on an unusual role for a cxcr3 receptor, in which it antagonizes a paralogous receptor to limit macrophage migration.

Thrombocyte Inhibition Restores Protective Immunity to Mycobacterial Infection in Zebrafish.

BACKGROUND: Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. METHODS: In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. RESULTS: We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. CONCLUSIONS: Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.

Spotlight on zebrafish: the next wave of translational research.

Five years after the launch of the Disease Models & Mechanisms (DMM) Special Issue on zebrafish as a disease model, the field has progressed significantly. Zebrafish have been used to precisely model human genetic variants, to unpick the mechanisms of metabolic and other diseases, to study infection, inflammation and cancer, and to develop and test new therapeutic approaches. In this Editorial, we highlight recent research published in DMM that uses zebrafish to develop new experimental tools and to provide new insight into disease mechanism and therapy. The broad spectrum of subjects and approaches covered in these articles underscores the versatility of zebrafish in translational research. Further, it highlights the zebrafish community's ethos of creativity and collaboration in translating basic biological research into clinically relevant advances affecting how we understand and treat human disease.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas.

A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.

Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling.

Mycobacterial infection leads to the formation of characteristic immune aggregates called granulomas, a process accompanied by dramatic remodeling of the host vasculature. As granuloma angiogenesis favors the infecting mycobacteria, it may be actively promoted by bacterial determinants during infection. Using Mycobacterium marinum-infected zebrafish as a model, we identify the enzyme proximal cyclopropane synthase of alpha-mycolates (PcaA) as an important bacterial determinant of granuloma-associated angiogenesis. cis-Cyclopropanation of mycobacterial mycolic acids by pcaA drives the activation of host Vegf signaling within granuloma macrophages. Cyclopropanation of the mycobacterial cell wall glycolipid trehalose dimycolate is both required and sufficient to induce robust host angiogenesis. Inducible genetic inhibition of angiogenesis and Vegf signaling during granuloma formation results in bacterial growth deficits. Together, these data reveal a mechanism by which PcaA-mediated cis-cyclopropanation of mycolic acids promotes bacterial growth and dissemination in vivo by eliciting granuloma vascularization and suggest potential approaches for host-directed therapies.

Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol.

Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi (S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches.

Infection-Induced Vascular Permeability Aids Mycobacterial Growth.

Pathogenic mycobacteria trigger formation of organized granulomas. As granulomas mature, they induce angiogenesis and vascular permeability. Here, in a striking parallel to tumor pro-angiogenic signaling, we identify angiopoietin-2 (ANG-2) induction as an important component of vascular dysfunction during mycobacterial infection. Mycobacterial infection in humans and zebrafish results in robust induction of ANG-2 expression from macrophages and stromal cells. Using a small-molecule inhibitor closely related to one currently in clinical trials, we link ANG-2/TIE2 signaling to vascular permeability during mycobacterial infection. Targeting granuloma-induced vascular permeability via vascular endothelial-protein tyrosine phosphatase inhibition limits mycobacterial growth, suggesting a new strategy for host-directed therapies against tuberculosis.

Macrophage Epithelial Reprogramming Underlies Mycobacterial Granuloma Formation and Promotes Infection.

Mycobacterium tuberculosis infection in humans triggers formation of granulomas, which are tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. Here, using the zebrafish-Mycobacterium marinum model, we found that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. We identified fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function resulted in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden, and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. Granuloma macrophages in humans with tuberculosis were similarly transformed. Thus, during mycobacterial infection, granuloma macrophages are broadly reprogrammed by epithelial modules, and this reprogramming alters the trajectory of infection and the associated immune response.

Macrophage form, function, and phenotype in mycobacterial infection: lessons from tuberculosis and other diseases.

Macrophages play a central role in mycobacterial pathogenesis. Recent work has highlighted the importance of diverse macrophage types and phenotypes that depend on local environment and developmental origins. In this review, we highlight how distinct macrophage phenotypes may influence disease progression in tuberculosis. In addition, we draw on work investigating specialized macrophage populations important in cancer biology and atherosclerosis in order to suggest new areas of investigation relevant to mycobacterial pathogenesis. Understanding the mechanisms controlling the repertoire of macrophage phenotypes and behaviors during infection may provide opportunities for novel control of disease through modulation of macrophage form and function.

Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.

CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.