Investigating tuberculosis immunopathogenesis by studying clinical samples and 3-dimensional cell culture using unbiased methodology

• Main Author: Reichmann, Michaela T
• Format: Dissertation
• Language: English
• Published: University of Southampton 2021

The global burden of tuberculosis continues to be of pandemic proportions, currently with a quarter of the world's population infected with Mycobacterium tuberculosis (Mtb), and 1.4 million people dying from Mtb infection in 2019. Sarcoidosis is another human granulomatous condition primarily affecting the lung and lymph nodes. TB and sarcoidosis share histological and clinical features which can be indistinguishable, including immune-related phenomena which suggest shared immunological processes. Here, I investigate TB pathogenesis by studying untreated TB and sarcoidosis clinical samples, alongside control tissue. Twenty-four formalin-fixed paraffin-embedded biopsies were selected, and granulomas excised using laser capture microdissection, with an equivalent area of control tissue excised. Total RNA was isolated, and sequenced by Ion Torrent Sequencing. In parallel, three cell culture models of TB were studied; 2-dimensional (2D), 3-dimensional (3D) with Alginate, and 3-dimensional with Alginate-Collagen. Peripheral blood mononuclear cells from six healthy donors were infected overnight with the Mtb strain H37Rv. Total RNA was extracted and sequenced by Illumina HiSeq. For bioinformatic analysis, both RNA sequencing data sets were aligned to human reference genome GRCh38 using kallisto, and differentially expressed genes identified using a linear model (R: limma, TMM normalised voom transformed data) with false discovery rate less than 0.05 and absolute log2 fold change at least 1.5. The overall strategy was to analyse pathways common to TB and sarcoid, and those unique to each condition, and then investigate these in a cell culture model whose gene expression best reflects that of human TB. Using a systematic selection process, comparison of clinical TB samples with infected 3D Alginate-Collagen microspheres identified twelve potential targets, including sphingosine kinase 1 (SphK1). Inhibition of SphK1 resulted in control of Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemistry of clinical TB specimens confirmed the presence of SphK1, demonstrating a potential novel host-directed therapy target to improve outcomes in TB. Therefore, by combining unbiased analysis of human clinical samples with a biomimetic model, I have established a translational pipeline to identify new therapeutic approaches.