MicroRNAs as biomarkers in anti-tuberculosis drug-induced liver injury: a translational study from zebrafish to humans
Rupprechter, Sarah Ann Elisabeth
Drug-induced liver injury is one of the leading causes of acute liver failure and is a serious barrier to drug development. Furthermore, it is a frequent adverse reaction to the antimicrobials used to treat tuberculosis, resulting in reduced treatment effectiveness, incomplete treatment, relapse and the development of antimicrobial resistance. The aim of the studies in this thesis were threefold, to investigate biomarkers of liver injury in patients receiving anti-tuberculosis medication, to investigate miRNA changes in zebrafish larvae due to drug-induced liver injury, and to develop a novel point of care diagnostic test for miR-122, a miRNA biomarker of liver injury. The novel biomarkers, microRNA-122 (miR-122) and cytokeratin-18 (K18) have been found to be diagnostic and prognostic in patients who overdose on paracetamol. A key question is, do these biomarkers retain their efficacy in the presence of infection? The circulating concentrations of miR-122 and K18 were defined in the following populations: healthy volunteers, active tuberculosis patients, latent tuberculosis patients, patients with non-tuberculous mycobacterial infection and HIV-tuberculosis coinfected patients. Circulating concentrations of miR-122 and K18 were not significantly different across these groups, indicating infection does not affect concentrations of miR-122 and K18. Furthermore, concentrations of miR-122 and K18 did not rise upon starting treatment. There was a significant correlation between the gold standard marker alanine aminotransferase (ALT) and both miR-122 and K18 demonstrating these biomarkers rise with elevations in ALT. In two individuals who developed drug-induced liver injury, miR-122 and K18 rose with ALT, suggesting these biomarkers have diagnostic potential for anti-tuberculosis drug-induced liver injury. To reduce the need for rodent studies a zebrafish larvae model of liver injury was developed. Reducing the number of rodent studies carried out reduces experimental costs and the use of zebrafish larvae enables rapid high-throughput experiments. Furthermore, using zebrafish larvae is in line with the “3Rs” (reduce, refine, replace) approach of animal use in science, with the replacement of rodents, a higher-order mammal, with the lower-order zebrafish larvae. Zebrafish larvae exposed to isoniazid and pyrazinamide developed liver toxicity as determined by mortality, fluorescent imaging and histology. In order to investigate pathways altered in anti-tuberculosis drug-induced liver injury and identify potential novel biomarkers small RNA sequencing was undertaken. Pathways were altered in anti-tuberculosis drug induced liver injury, including those associated with the metabolism of xenobiotics. Building on previous work by my group, zebrafish larvae were exposed to triptolide, a Chinese herbal medicine which induces liver injury. A method was developed to collect specific cell populations, hepatocytes and immune cells, from transgenic zebrafish larvae using fluorescence activated cell sorting (FACS). The miRNA changes in isolated cells populations were determined using qRT-PCR, and in whole fish using small RNA sequencing. Novel miRNA biomarkers of liver injury were identified from the small RNA sequencing data and translated in plasma samples from patients with anti-tuberculosis drug-induced liver injury. Two of these miRNAs rose with ALT in liver injury. A point of care test is needed for drug-induced liver injury, suitable for use in resource poor settings. A novel RNA toehold switch sensor was combined with a fluorescent and colorimetric outputs to provide a quantitative response to miR-122 concentrations. Toehold switches with a range of structures were developed and tested. Switches demonstrated varying levels of sensitivity and specificity for miR-122.