Zebrafish Model for Studying the Disease Spectrum of Tuberculosis

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Arvo building, Yellow Hall F025, address: Arvo Ylpön katu 34

Milka Hammarén

Doctoral defence of M.Sc. Milka Hammarén

Zebrafish Model for Studying the Disease Spectrum of Tuberculosis

The field of science of the dissertation is Experimental immunology.

The opponent is professor Pentti Huovinen (University of Turku). Docent Mataleena Parikka acts as the custos.

The language of the dissertation defence is Finnish.

Zebrafish Model for Studying the Disease Spectrum of Tuberculosis

Tuberculosis (TB) remains one of the deadliest infections worldwide killing 1.8 million people every year. Mycobacterium tuberculosis, a pathogenic mycobacterium and the causative agent of TB, is transmitted through the lung but can infect almost any organ. Inhalation of a few tuberculous mycobacteria can lead to a spectrum of different outcomes including primary active and latent infection, the latter of which usually remains asymptomatic but has the potential to cause a severe, reactivated infection after decades of quiescence. The limited efficacy of the current TB vaccine (BCG) combined with safety concerns has led to limitations in its use. The lack of an efficient and safe vaccine together with the decreasing efficacy of antibiotic treatments substantiate the urgent need for more powerful weaponry to control this devastating disease.
Knowledge of the nature of protective immune responses furthers the development of prevention and treatment strategies against TB. The outcome of an infection is a product of the complex interplay between the bacteria and a multitude of host cells necessitating the use of animal models. In this thesis, I have developed new methodology to utilize the adult zebrafish (Danio rerio) and its natural pathogen Mycobacterium marinum as a model of human tuberculosis.

M. marinum is a close relative of M. tuberculosis and the causative agent of fish TB. In this study, the experimental M. marinum infection of zebrafish was found to well replicate human TB. Cell aggregates called granulomas and typically seen in TB formed in various internal organs of the fish. A small dose of M. marinum created a vast disease spectrum in the fish closely resembling that seen in human TB with bacterial clearance (~10 %), primary active (~10 %), stable latent (~65 %) and reactivated infection (~15 %). Dormant bacteria were detected in the latently infected individuals with our new methods developed during the study. The latent infection could be experimentally reactivated by reversible depletion of immune cells. The zebrafish model developed in the first part of this thesis can be used for studying the entire spectrum of TB disease and the environmental and genetic factors affecting its course and treatability.

We were able to show that adaptive T cells important for efficient and targeted immune responses were centrally important for controlling mycobacterial infection in the zebrafish. We showed that the total number of T cells and especially type 2 T helper cells (Th2) were associated with the ability of the individual to limit mycobacterial growth. The association between Th2 cells and the ability to control the infection was surprising, as traditionally, TB researchers have focused on another group of T cells, namely Th1 cells. High numbers of mycobacteria residing within the host during a latent mycobacterial infection are known to increase the risk of reactivated infection. However, as the direct measurement of bacterial numbers is not feasible in humans, measuring Th2 cells from blood samples of latently infected people could serve as a proxy for the risk of reactivation.  Such diagnostics would allow the allocation of antibiotic treatment only the patients with a high reactivation risk. Moreover, based on our results, the possible protective effects Th2 responses in the context of vaccine-induced immunity against tuberculosis should be reassessed.

The adult zebrafish-M. marinum model was also used to test immunoactivators to study, whether the host immune response could be directed to more efficiently kill mycobacteria. Injecting a preparation made of certain heat-inactivated bacteria prior to infection with M. marinum led to significantly lower mycobacterial loads and increased the occurrence of a sterilizing immune response. The treatment was also effective among immunocompromised fish. The effect was based on the increased ability of immune cells to kill intracellular mycobacteria.  These findings pave the way for the development novel TB treatments based on steering or boosting the immune response.

The adult zebrafish-M. marinum system is a convenient non-mammalian model for studying the entire spectrum of TB, including spontaneous latency with dormant mycobacteria. It can serve as an in vivo platform to screen experimental vaccination strategies against latent and reactivated infection or treatments targeted at dormant mycobacteria. Due to the small size of the zebrafish and the sensitive method developed for detecting both actively dividing and dormant mycobacteria, bacterial clearance can be assessed in the zebrafish more reliably than any other animal. Thus, the zebrafish is an indispensable model in the quest for sterilizing treatments and prevention strategies against TB.


The dissertation is published in the publication series of Acta Universitatis Tamperensis; 2337, Tampere University Press, Tampere 2017. The dissertation is also published in the e-series Acta Electronica Universitatis Tamperensis; 1842, Tampere University Press 2017.

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