New drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder... Show moreNew drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder disease to treat. This thesis is focused on using the in vivo whole animalzebrafish embryo model for TB to evaluate potential anti-TB host-directed therapeutics (HDTs) arising from in vitro screens. Although in vitro screens for HDTs using cellular models can be performed at high throughput, a limiting step is the validation in whole animal models and translation of results to clinical applications. Due to the complex infection dynamics of mycobacteria, the use of whole animal models is indispensable in research into TB and the zebrafish model has contributed key findings about host-pathogen dynamics during mycobacterial infection. One of the most promising host targets of HDTs is autophagy, which is recognized as an important host-protective pathway. Boosting autophagy levels using HDTs could be a way to overcome the pathogen’s autophagy evasion strategies and could therefore be a promising therapeutic route. For this thesis we took advantage of the possibilities of the zebrafish embryo model for TB and the zebrafish toolkit to study several autophagy-modulating HDTs as potential anti-TB drugs. Show less
The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of... Show moreThe zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real-time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models. Show less
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm)... Show moreTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells. Show less
Background: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify... Show moreBackground: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB.Methods: SERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts.Findings: SERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls.Interpretation: SERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed. Show less
Mycobacterium tuberculosis, the agent of TB, is one of the deadliest human pathogens, infecting one third of the global population. Establishment of infection by mycobacteria relies on complex... Show moreMycobacterium tuberculosis, the agent of TB, is one of the deadliest human pathogens, infecting one third of the global population. Establishment of infection by mycobacteria relies on complex interactions with host innate immune cells, especially macrophages. Once engulfed by macrophages, mycobacteria “usurp” the host cell machineries to facilitate dissemination and to establish an intracellular niche for survival and replication. To investigate how mycobacteria force the immune cells to support infection, we explored the chemokine pathway, best known for its capability to induce cell migration. To dissect the interplay between immune cells and the pathogen, we modelled human TB using the zebrafish-Mycobacterium marinum natural host-pathogen pair, which is attractive for the excellent optical accessibility of the zebrafish larvae and the possibility to apply genetic tools to impair the chemokine signaling. We show that depletion of either CXCR3 or CXCR4 axes are beneficial to the host. Exploitation of CXCR3 signaling leads to macrophage recruitment and to transcriptional changes in macrophages that make them more permissive for mycobacterial intracellular persistence. Activating CXCR4 signaling triggers instead vascularization of the nascent tuberculous granulomas, which in turn supports expansion of the infection. Therefore, inhibitions of these pathways represent promising host-directed therapeutic avenues to counteract mycobacterial diseases. Show less
This thesis is focused on the innate immune defence mechanisms responsible for controlling mycobacterial growth after infection. To provide a detailed description of the host__s innate immune... Show moreThis thesis is focused on the innate immune defence mechanisms responsible for controlling mycobacterial growth after infection. To provide a detailed description of the host__s innate immune response to M. marinum infection, zebrafish gene expression levels were analysed by RNA sequencing at various time points during infection and correlated with imaging data of the process of pathogenesis. We demonstrate that the scavenger receptor Marco (macrophage receptor with collagenous structure) is a key player in the rapid phagocytosis of M. marinum and we use gene expression analysis in combination with gene knockdown studies to show that it is also essential in the establishment of an initial transient pro-inflammatory response to M. marinum infection. Once phagocytosed, M. marinum is capable of avoiding killing mechanisms of the host cell and can continue to grow within macrophages. This is the period when Membrane Attack Complex/Perforin proteins are involved in killing intracellular bacteria by their pore-forming activities. We reveal the regulatory mechanisms and function of two macrophage specific genes, mpeg1 and mpeg1.2 (macrophage expressed gene 1.2). The results from this thesis complement knowledge obtained from other model organisms by providing new insights into both counteracting and supporting mechanisms underlying the innate immune response. Show less
Type I immune responses play an essential role in the control of mycobacterial infections. Mutations in the genes involved in the type I cytokine pathway were found in patients with Mendelian... Show moreType I immune responses play an essential role in the control of mycobacterial infections. Mutations in the genes involved in the type I cytokine pathway were found in patients with Mendelian susceptibility to mycobacterial diseases. These patients are highly susceptible to infections with non-tuberculous mycobacteria (NTM), which are usually poorly pathogenic. The first part of this thesis focuses on the relation between the genotype and phenotype in the cause of an impaired immunity leading to the susceptibility to NTM infections. The role of genetic factors in the control of infections with more virulent tuberculous mycobacteria is less evident. The second part of this thesis focuses on putative non-genetic causes of an impaired immunity in the control of tuberculous mycobacterial diseases. In tuberculosis patients type I immune responses regulated by interferon-_ are also repressed. Virally induced interferons, other than interferon-_, may be involved in this repression, thereby influencing the immunopathogenesis of tuberculosis. Show less
