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bioRxiv · 10.1101/2024.06.28.601189

The inflammasome adaptor pycard is essential for immunity against Mycobacterium marinum infection in adult zebrafish

Uusi-Mäkelä, M.·Harjula, S.-K. E.·Junno, M.·Sillanpää, A.·Nätkin, R.·Niskanen, M. T.·Nykter, M.·Rämet, M.
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Abstract

Inflammasome regulates the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of Mycobacterium marinum infection in adult zebrafish (Danio rerio) has similar features than the course of tuberculosis in human. In this study, we have investigated the role of the inflammasome adaptor pycard in M. marinum infection in zebrafish. We produced two zebrafish knock-out mutant lines for the pycard gene with CRISPR/Cas9 mutagenesis. While the zebrafish larvae devoid of pycard develop normally and have unaltered resistance against M. marinum, the loss of pycard led to impaired survival and increased bacterial burden in the adult zebrafish. Based on histological analysis, immune cell aggregates, granulomas, were larger in pycard deficient fish compared to wild type controls. Transcriptome analysis with RNA sequencing of a zebrafish haematopoietic tissue, kidney, suggests a role for pycard in neutrophil mediated defence as well as in haematopoiesis and myelopoiesis during infection. Transcriptome analysis of fluorescently labelled kidney neutrophils further supported the importance of pycard for neutrophil-mediated immunity against M. marinum. Genes associated with neutrophil degranulation, haematopoiesis and PI3K signalling were differentially expressed in the pycard deficient neutrophils when compared to wild type controls. All in all, our results indicate that pycard is essential for resistance against mycobacteria in adult zebrafish. Based on transcriptional profiling of pycard mutants, we postulate that pycard mutant phenotype is mediated in part via defects in neutrophil function including neutrophil degranulation. Author summaryInflammasome is a multiprotein complex which is a part of immune response especially against intracellular microbes. Tuberculosis, caused by Mycobacterium tuberculosis, is a global health problem causing 1.3 million deaths yearly. A natural fish pathogen, Mycobacterium marinum, causes a systemic infection in adult zebrafish resembling human tuberculosis including granuloma formation and an asymptomatic, latent state. Here, we have used zebrafish as a model to investigate the role of an inflammasome regulator pycard in resistance against M. marinum infection in zebrafish. We show that while pycard is dispensable for normal immunity against mycobacterial infection in larval stages, it has a central role in the immune defense against M. marinum in adult zebrafish. Based on our results, we suggest that this relates to blood cell formation, haematopoiesis and especially to the function of neutrophils. By analysing transcriptrional profile of pycard deficient neutrophils upon Mycobacterial challenge, it seems that pycard has a role in PI3K signalling and neutrophil degranulation. Our results indicate that pycard is essential for normal resistance against M. Marinum in adult zebrafish, and that it regulates neutrophil function during Mycobacterial infection.

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Uusi-Mäkelä, M., Harjula, S.-K. E., Junno, M., Sillanpää, A., Nätkin, R., Niskanen, M. T., Nykter, M., Rämet, M.. 2024-07-02. The inflammasome adaptor pycard is essential for immunity against Mycobacterium marinum infection in adult zebrafish. https://doi.org/10.1101/2024.06.28.601189

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