Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPß within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Plexin D1 negatively regulates zebrafish lymphatic development.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.

3,4-Difluorobenzocurcumin Inhibits Vegfc-Vegfr3-Erk Signalling to Block Developmental Lymphangiogenesis in Zebrafish.

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vasculature, plays critical roles in disease, including in cancer metastasis and chronic inflammation. Preclinical and recent clinical studies have now demonstrated therapeutic utility for several anti-lymphangiogenic agents, but optimal agents and efficacy in different settings remain to be determined. We tested the anti-lymphangiogenic property of 3,4-Difluorobenzocurcumin (CDF), which has previously been implicated as an anti-cancer agent, using zebrafish embryos and cultured vascular endothelial cells. We used transgenic zebrafish labelling the lymphatic system and found that CDF potently inhibits lymphangiogenesis during embryonic development. We also found that the parent compound, Curcumin, does not inhibit lymphangiogenesis. CDF blocked lymphatic and venous sprouting, and lymphatic migration in the head and trunk of the embryo. Mechanistically, CDF impaired VEGFC-VEGFR3-ERK signalling in vitro and in vivo. In an in vivo pathological model of Vegfc-overexpression, treatment with CDF rescued endothelial cell hyperplasia. CDF did not inhibit the kinase activity of VEGFR3 yet displayed more prolonged activity in vivo than previously reported kinase inhibitors. These findings warrant further assessment of CDF and its mode of action as a candidate for use in metastasis and diseases of aberrant lymphangiogenesis.

Zebrafish facial lymphatics develop through sequential addition of venous and non-venous progenitors.

Lymphatic vessels are known to be derived from veins; however, recent lineage-tracing experiments propose that specific lymphatic networks may originate from both venous and non-venous sources. Despite this, direct evidence of a non-venous lymphatic progenitor is missing. Here, we show that the zebrafish facial lymphatic network is derived from three distinct progenitor populations that add sequentially to the developing facial lymphatic through a relay-like mechanism. We show that while two facial lymphatic progenitor populations are venous in origin, the third population, termed the ventral aorta lymphangioblast (VA-L), does not sprout from a vessel; instead, it arises from a migratory angioblast cell near the ventral aorta that initially lacks both venous and lymphatic markers, and contributes to the facial lymphatics and the hypobranchial artery. We propose that sequential addition of venous and non-venous progenitors allows the facial lymphatics to form in an area that is relatively devoid of veins. Overall, this study provides conclusive, live imaging-based evidence of a non-venous lymphatic progenitor and demonstrates that the origin and development of lymphatic vessels is context-dependent.

Liposome-Mediated Drug Delivery in Larval Zebrafish to Manipulate Macrophage Function.

Chemical interventions are regularly used to examine and manipulate macrophage function in larval zebrafish. Given chemicals are typically administered by simple immersion or injection, it is not possible to resolve whether their impact on macrophage function is direct or indirect. Liposomes provide an attractive strategy to target drugs to specific cellular compartments, including macrophages. As an example, injecting liposomal clodronate into animal models, including zebrafish, is routinely used to deliver toxic levels of clodronate specifically to macrophages for targeted cell ablation. Here we show that liposomes can also target the delivery of drugs to zebrafish macrophages to selectively manipulate their function. We utilized the drugs etomoxir (a fatty acid oxidation inhibitor) and MitoTEMPO (a scavenger of mitochondrial reactive oxygen species [mROS]), that we have previously shown, through free drug delivery, suppress monosodium urate (MSU) crystal-driven macrophage activation. We generated poloxamer 188 modified liposomes that were readily phagocytosed by macrophages, but not by neutrophils. Loading these liposomes with etomoxir or MitoTEMPO and injecting into larvae suppressed macrophage activation in response to MSU crystals, as evidenced by proinflammatory cytokine expression and macrophage-driven neutrophil recruitment. This work reveals the utility of packaging drugs into liposomes as a strategy to selectively manipulate macrophage function.

Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model.

Tumour angiogenesis has long been a focus of anti-cancer therapy; however, anti-angiogenic cancer treatment strategies have had limited clinical success. Tumour-associated myeloid cells are believed to play a role in the resistance of cancer towards anti-angiogenesis therapy, but the mechanisms by which they do this are unclear. An embryonic zebrafish xenograft model has been developed to investigate the mechanisms of tumour angiogenesis and as an assay to screen anti-angiogenic compounds. In this study, we used cell ablation techniques to remove either macrophages or neutrophils and assessed their contribution towards zebrafish xenograft angiogenesis by quantitating levels of graft vascularisation. The ablation of macrophages, but not neutrophils, caused a strong reduction in tumour xenograft vascularisation and time-lapse imaging demonstrated that tumour xenograft macrophages directly associated with the migrating tip of developing tumour blood vessels. Finally, we found that, although macrophages are required for vascularisation in xenografts that either secrete VEGFA or overexpress zebrafish vegfaa, they are not required for the vascularisation of grafts with low levels of VEGFA, suggesting that zebrafish macrophages can enhance Vegfa-driven tumour angiogenesis. The importance of macrophages to this angiogenic response suggests that this model could be used to further investigate the interplay between myeloid cells and tumour vascularisation.

Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation.

Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB-driven production of IL-1ß and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.

The innate immune cell response to bacterial infection in larval zebrafish is light-regulated.

The circadian clock, which evolved to help organisms harmonize physiological responses to external conditions (such as the light/dark cycle, LD), is emerging as an important regulator of the immune response to infection. Gaining a complete understanding of how the circadian clock influences the immune cell response requires animal models that permit direct observation of these processes within an intact host. Here, we investigated the use of larval zebrafish, a powerful live imaging system, as a new model to study the impact of a fundamental zeitgeber, light, on the innate immune cell response to infection. Larvae infected during the light phase of the LD cycle and in constant light condition (LL) demonstrated enhanced survival and bacterial clearance when compared with larvae infected during the dark phase of the LD cycle and in constant dark condition (DD). This increased survival was associated with elevated expression of the zebrafish orthologues of the mammalian pro-inflammatory cytokine genes, Tumour necrosis factor-α, Interleukin-8 and Interferon-γ, and increased neutrophil and macrophage recruitment. This study demonstrates for the first time that the larval zebrafish innate immune response to infection is enhanced during light exposure, suggesting that, similar to mammalian systems, the larval zebrafish response to infection is light-regulated.

Transient inflammatory response mediated by interleukin-1ß is required for proper regeneration in zebrafish fin fold.

Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta (il1b). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration.

A whole animal chemical screen approach to identify modifiers of intestinal neutrophilic inflammation.

By performing two high-content small molecule screens on dextran sodium sulfate- and trinitrobenzene sulfonic acid-induced zebrafish enterocolitis models of inflammatory bowel disease, we have identified novel anti-inflammatory drugs from the John Hopkins Clinical Compound Library that suppress neutrophilic inflammation. Live imaging of neutrophil distribution was used to assess the level of acute inflammation and concurrently screen for off-target drug effects. Supporting the validity of our screening strategy, most of the anti-inflammatory drug hits were known antibiotics or anti-inflammatory agents. Novel hits included cholecystokinin (CCK) and dopamine receptor agonists. Using a pharmacological approach, we show that while CCK and dopamine receptor agonists alleviate enterocolitis-associated inflammation, receptor antagonists exacerbate inflammation in zebrafish. This work highlights the utility of small molecule screening in zebrafish enterocolitis models as a tool to identify novel bioactive molecules capable of modulating acute inflammation.

A Zebrafish Model for Chlamydia Infection with the Obligate Intracellular Pathogen Waddlia chondrophila.

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the Waddliacea which, in addition to epithelia, cellular targets for nearly all Chlamydia, can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with Waddlia chondrophila. By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that W. chondrophila is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to W. chondrophila, eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence in vivo and opens up new possibilities for investigation of other members of the PVC superphylum.

Inflammatory pathway network-based drug repositioning and molecular phenomics.

Inflammation is a protective biological response to body/tissue damage that involves immune cells, blood vessels and molecular mediators. In this work, we constructed the pathway network of inflammation, including 11 sub-pathways of inflammatory factors. Pathway-based network efficiency and network flux were adopted to evaluate drug efficacy. By using approved and experimentally validated anti-inflammatory drugs as training sets, a predictive model was built to screen potential anti-inflammatory drugs from approved drugs in DrugBank. This drug repositioning approach would bring a fast and cheap way to find new indications for approved drugs. Moreover, molecular phenomics profiles of the expression of inflammatory factors will provide new insight into the drug mechanism of action.

Lymphatics, Cancer and Zebrafish.

Many solid tumors are known to metastasize through the lymphatic vasculature. This process is facilitated by the generation of new lymphatic vessels (tumor lymphangiogenesis) and also by the remodelling of existing lymphatics. Together these processes enable the spread of tumor cells to distant sites. Currently our understanding of tumor lymphangiogenesis has been informed from mouse tumor models and from studies of developmental lymphangiogenesis. Since the discovery of bona fide lymphatic vessels in zebrafish in 2006, zebrafish have become a well-established model of developmental lymphangiogenesis. The attributes that make zebrafish such an important model of blood vessel development-the ability to live image developing vessels, genetic tractability and the conserved nature of development-also make fish an attractive model of lymphatic vessel development. In particular, zebrafish have made important contributions to our understanding of the processes of lymphatic vessel sprouting from veins and the mechanisms by which lymphatic precursors remodel into mature vessels. To date, zebrafish have not been used to directly model tumor lymphangiogenesis. In this chapter we will summarise the contributions zebrafish have made to our understanding of lymphangiogenesis and investigate the possibilities of combining zebrafish transgenic cancer lines or tumor transplantation models with existing lymphatic reporter lines, which could provide valuable insights into the process of tumor-induced lymphangiogenesis. In addition the utility of using the zebrafish lymphatic model as a platform to screen and develop novel anti-lymphatic therapeutics will also be discussed.

A COL17A1 Splice-Altering Mutation Is Prevalent in Inherited Recurrent Corneal Erosions.

PURPOSE: Corneal dystrophies are a genetically heterogeneous group of disorders. We previously described a family with an autosomal dominant epithelial recurrent erosion dystrophy (ERED). We aimed to identify the underlying genetic cause of ERED in this family and 3 additional ERED families. We sought to characterize the potential function of the candidate genes using the human and zebrafish cornea. DESIGN: Case series study of 4 white families with a similar ERED. An experimental study was performed on human and zebrafish tissue to examine the putative biological function of candidate genes. PARTICIPANTS: Four ERED families, including 28 affected and 17 unaffected individuals. METHODS: HumanLinkage-12 arrays (Illumina, San Diego, CA) were used to genotype 17 family members. Next-generation exome sequencing was performed on an uncle-niece pair. Segregation of potential causative mutations was confirmed using Sanger sequencing. Protein expression was determined using immunohistochemistry in human and zebrafish cornea. Gene expression in zebrafish was assessed using whole-mount in situ hybridization. Morpholino-induced transient gene knockdown was performed in zebrafish embryos. MAIN OUTCOME MEASURES: Linkage microarray, exome analysis, DNA sequence analysis, immunohistochemistry, in situ hybridization, and morpholino-induced genetic knockdown results. RESULTS: Linkage microarray analysis identified a candidate region on chromosome chr10:12,576,562-112,763,135, and exploration of exome sequencing data identified 8 putative pathogenic variants in this linkage region. Two variants segregated in 06NZ-TRB1 with ERED: COL17A1 c.3156C→T and DNAJC9 c.334G→A. The COL17A1 c.3156C→T variant segregated in all 4 ERED families. We showed biologically relevant expression of these proteins in human cornea. Both proteins are expressed in the cornea of zebrafish embryos and adults. Zebrafish lacking Col17a1a and Dnajc9 during development show no gross corneal phenotype. CONCLUSIONS: The COL17A1 c.3156C→T variant is the likely causative mutation in our recurrent corneal erosion families, and its presence in 4 independent families suggests that it is prevalent in ERED. This same COL17A1 c.3156C→T variant recently was identified in a separate pedigree with ERED. Our study expands the phenotypic spectrum of COL17A1 disease from autosomal recessive epidermolysis bullosa to autosomal dominant ERED and identifies COL17A1 as a key protein in maintaining integrity of the corneal epithelium.

A zebrafish model of inflammatory lymphangiogenesis.

Inflammatory bowel disease (IBD) is a disabling chronic inflammatory disease of the gastrointestinal tract. IBD patients have increased intestinal lymphatic vessel density and recent studies have shown that this may contribute to the resolution of IBD. However, the molecular mechanisms involved in IBD-associated lymphangiogenesis are still unclear. In this study, we established a novel inflammatory lymphangiogenesis model in zebrafish larvae involving colitogenic challenge stimulated by exposure to 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sodium sulphate (DSS). Treatment with either TNBS or DSS resulted in vascular endothelial growth factor receptor (Vegfr)-dependent lymphangiogenesis in the zebrafish intestine. Reduction of intestinal inflammation by the administration of the IBD therapeutic, 5-aminosalicylic acid, reduced intestinal lymphatic expansion. Zebrafish macrophages express vascular growth factors vegfaa, vegfc and vegfd and chemical ablation of these cells inhibits intestinal lymphatic expansion, suggesting that the recruitment of macrophages to the intestine upon colitogenic challenge is required for intestinal inflammatory lymphangiogenesis. Importantly, this study highlights the potential of zebrafish as an inflammatory lymphangiogenesis model that can be used to investigate the role and mechanism of lymphangiogenesis in inflammatory diseases such as IBD.

mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish.

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.

An inducible transgene reports activation of macrophages in live zebrafish larvae.

Macrophages are the most functionally heterogenous cells of the hematopoietic system. Given many diseases are underpinned by inappropriate macrophage activation, macrophages have emerged as a therapeutic target to treat disease. A thorough understanding of what controls macrophage activation will likely reveal new pathways that can be manipulated for therapeutic benefit. Live imaging fluorescent macrophages within transgenic zebrafish larvae has provided a valuable window to investigate macrophage behavior in vivo. Here we describe the first transgenic zebrafish line that reports macrophage activation, as evidenced by induced expression of an immunoresponsive gene 1(irg1):EGFP transgene. When combined with existing reporter lines that constitutively mark macrophages, we reveal this unique transgenic line can be used to live image macrophage activation in response to the bacterial endotoxin lipopolysaccharide and xenografted human cancer cells. We anticipate the Tg(irg1:EGFP) line will provide a valuable tool to explore macrophage activation and plasticity in the context of different disease models.

Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry.

To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system's design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.

Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis.

Crohn's disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion and confirm our previous findings, we performed transcriptomic analysis in macrophages and identified a subgroup of individuals with CD who had low expression of the autophagy receptor optineurin (OPTN). We then clarified the role of OPTN deficiency in: macrophage cytokine secretion; mouse models of bacteria-driven colitis and peritonitis; and zebrafish Salmonella infection. OPTN-deficient bone-marrow-derived macrophages (BMDMs) stimulated with heat-killed Escherichia coli secreted less proinflammatory TNFα and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors, suggesting that TNFα is mis-trafficked to lysosomes via bafilomycin-A-dependent pathways in the absence of OPTN. OPTN-deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis, and showed reduced levels of proinflammatory TNFα in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality, compared with wild-type mice. Optn-knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans might increase the risk of developing CD.

Interception of host angiogenic signalling limits mycobacterial growth.

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.