Content area
Full Text
Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis1,2. Here we examine the development and consequences of vascularization of the tuberculosis, granuloma in the zebrafish-Mycobacterium marinum attention model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis2. 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 populations3. 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 antiangiogenic 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.
The human tuberculous granuloma, a tightly cohesive cellular structure that houses infecting mycobacteria, develops hypoxic areas around its necrotic core4. In tumours, the development of hypoxia is tightly linked to angiogenesis and subsequent metastasis5. In tuberculosis, attention has focused on the possible consequences of granuloma hypoxia to bacterial physiology4, but relatively little attention has been paid to the functional significance of findings that tuberculous granulomas are extensively vascularized6'8.
In its natural ectothermic hosts, M. marinum, the closest relative of the M. tuberculosis complex, causes a disease called fish tuberculosis, a systemic wasting disease with organized epithelioid granulomas with necrotic cores. In zebrafish larvae, mycobacterium-infected macrophages form early granulomas, undergo a hallmark epithelioid transformation, and activate granuloma-specific gene expression programs2,9.
To monitor host vasculature in zebrafish, we used the Tg(kdrl:egfp)s843 line (referred to hereafter as Tg(flkl:eGFP))> in which vascular endothelial cells are fluorescently labelled with enhanced green fluorescent protein (eGFP)10. Injection of mycobacteria into the most commonly used caudal vein site results in granulomas in the immediate vicinity of the richly vascularized area of the caudal haematopoietic tissue (CHT) (Fig. la). To determine whether a different injection site with sparser and smaller...