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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MYCONEUTROPHILS (Elucidating the involvement of neutrophils in the pathogenesis of tuberculosis using a zebrafish model)

Teaser

Summary

After centuries of research, tuberculosis, caused by Mycobacterium tuberculosis, remains one of the greatest scourges to human health. The limited effectiveness of existing vaccines and treatments and the increase of drug-resistant M. tuberculosis are leading to the rise of the disease. The World Health Organization reported 10 million cases and 1.6 million deaths in 2017. Thus, a better understanding of the mechanisms of disease pathogenesis is essential to stop this epidemy.

Macrophages and neutrophils are the two main phagocytic immune cells that rapidly engulf and destroy the microorganisms that invade our organism. The role of macrophages in the response against tuberculosis is well known because they form the typical and intensively studied granuloma, but little is known about the role of neutrophils despite their presence in tuberculous tissues of humans. Neutrophils present a surprising unconventional behaviour in response to mycobacterial infections. While they are the first responders to most infections, they do not migrate at all to the mycobacterial initial infection sites, suggesting that mycobacteria might be somehow inhibiting neutrophil recruitment and function. Therefore, the main objective of this project is to uncover the mechanism used by mycobacteria to avoid neutrophil response, as well as to determine whether that mechanism can be counteracted so that neutrophils can migrate to the infection site and kill the mycobacteria. Moreover, we propose to study in detail how neutrophils might be fighting the infection if we are able to abrogate the inhibition that mycobacteria exert on them, focusing on the possibilities that they could release neutrophil extracellular traps, and/or that there may be different neutrophil subpopulations that behave different as previously observed in the context of the early granuloma. Shedding light on all this will be crucial for the development of new therapies for the treatment of tuberculosis.

Summarizing the final conclusions of this project, we have been able to describe the mechanism that mycobacteria use to avoid neutrophil response, and have demonstrated that abrogating it results in the attenuation of the infection through a novel neutrophil-macrophage-mediated mechanism never described before in the context of mycobacterial infections.

Work performed

The zebrafish-Mycobacterium marinum infection model has been used for the development of this project. My host laboratory has established the zebrafish as a model for tuberculosis by infecting it with M. marinum, its natural pathogen and a close genetic relative of M. tuberculosis. The zebrafish-M. marinum infection model presents both the biological advantage that they are a host-pathogen pair that has evolved together, and the logistical advantage that M. marinum can be handled more safely than M. tuberculosis. This recognised model has provided insights into human tuberculosis with important therapeutic implications.

The development of this project by using mainly phagocyte recruitment assays and high definition confocal microscopy, led us to discover that the first phagocytes recruited very early after infection are the resident macrophages, that rapidly engulf the mycobacteria. Then, mycobacteria actively induce resident macrophages to produce and release lipoxins, that are pro-resolving lipid mediators that inhibit neutrophil migration and function. Our research with lipoxin-deficient animals has demonstrated that after removing this inhibitory signal, neutrophils are able to migrate to the infection site and then mycobacterial infection is dramatically attenuated. Importantly, one of the drugs used to inhibit lipoxin production is zileuton, that has already been approved for the treatment of asthma in humans. We have confirmed that neither neutrophil extracellular traps nor different neutrophil subpopulations are playing any relevant roles at the mycobacterial initial infection site. Surprisingly, the infection attenuation observed in the absence of the inhibitory lipoxins is not due to the killing of mycobacteria by neutrophils themselves, but to the enhancement of macrophage microbicidal activity when neutrophils are close to them. Going further in the study of this striking mechanism, we have discovered that neutrophils recruited in the absence of lipoxins release the enzyme myeloperoxidase, that is able to bind the macrophage mannose receptors that are present in the macrophage membrane resulting in the enhancement of macrophage activity. The existence of this mechanism in response to mycobacteria has never been shown before, and its knowledge will be crucial for the development of new strategies to treat tuberculosis by avoiding the inhibition of neutrophil response in patients.

All these results have been showed and discussed with the scientific community in different international meetings and workshops, and are included in a manuscript that is now in preparation and that will be sent for publication as an open-source article in Immunity, an international and prestigious scientific journal.

Final results

Besides the relevant results obtained, the development of this project has also resulted in the creation and optimization of new methods to image and quantify mycobacterial infections in different regions of zebrafish larvae, such as the hindbrain ventricle. This cavity is structurally analogous to the human lung alveoli and, therefore, ideal to model what happens during the first stages of tuberculosis in humans. Furthermore, multiple zebrafish mutant and transgenic lines have been established and characterized, such as mutant fish deficient in lipoxins, myeloperoxidase, or macrophage mannose receptors. All these tools will be extremely useful for people working on this field in the future.

Therefore, both the scientific findings and the new techniques resulting from this project will be relevant for researchers in the fields of mycobacterial infections and host-pathogen interactions. Moreover, these results could have an important socio-economic impact since they can drive to the development of new strategies to treat tuberculosis patients.