Browsing by Author "Mishra, Abhilasha Madhvi"

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    2020-12-11 Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria
    (Stellenbosch : Stellenbosch University, 2019-12) Mishra, Abhilasha Madhvi; Baker, Bienyameen; Leisching, Gina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
    Background:Tuberculosis (TB) is a major cause of infection-related mortalityworldwide. In 2017 an estimated 1.3 million people who were HIV-negative died of TB. An estimated 5-10% of infected individual develop active TB during their lifetime, while the remaining90% (of infected population) successfully control the bacteria. Also, some of the close household contacts of TB patients remain uninfected and healthy. Studying host immune response towards Mycobacterium tuberculosis(M. tb) can unfold the reason behind this enigma. Methods:We conducted a detailed investigation of in vitrohost response from human monocyte derived macrophages(hMDMs)towards different strains of mycobacteria(grown in detergent-freemedia), i.e. pathogenic (M. tbR179) andnon-pathogenic (M. smegmatisand M. bovisBCG). The host response was measured post-infection (at mRNA and protein levels) using AmpliSeq, quantitative real time polymerase chain reaction (qRT-PCR), multiplex ELISA (Luminex), intracellular mycobacterial survivaland cytotoxicity assay. Biological network analysis (ingenuity pathway analysis IPA) was performed to understand the gene regulatory networkinvolved in the pathophysiology associated with the host-immune system.Based on false discovery rate (FDR) and biological functions, we selected an inter-related gene family of interferon induced protein with tetratricopeptides (IFIT1, IFIT2 andIFIT3) from the list of 19 potential differentially expressed genes(DEGs)for knock-up (vector-based over-expression)/down experiments. This gene family is known to form a protein complex during viral infection to act against the antigen. Studyencompassing their role against bacteria is not well established.Therefore, we performed knocking-up of IFITsvia vector-based transfection and knocking-down via small interferingRNA (siRNA) approach to investigate their effect upon mycobacteria inside the host macrophages. Results:AmpliSeqanalysis found 19 DEGs at 12 hours post-infection across all three strains. We observed lower number of mycobacterial CFUs and higher host response (at both RNA and protein level) in hMDMs infected with M. smegmatisas compared to other two strains. Biological network analysis revealed interferon-interleukin associated signalling pathways as most prominent among the 19 differentially expressed genes.We found a differed host response towardsall three strains, which mayattributeto their pathogenicity. Messenger RNA and protein level comparisons at different time points, depicted strong role of interferon and interleukin associated gene network. This network was able to successfully counter M. smegmatisbut succumb to M. bovisBCG andM. tbR179. Most importantly, across all three strains, intra-cellular bacterial growth and survival measured through colony forming units (CFUs)decreased significantly upon knocking up of IFITs(IFIT1, IFIT2 andIFIT3),while we recordedan increase in CFUs upon knocking down ofIFITsin the host macrophages. Using multiplex ELISA, we found higher expression of key pro-inflammatory cytokines (i.e. IDO1, IFN-γ, IL-6, and IL-23) during knock-up (vector-based over-expression)of IFITsresulting in reduction of mycobacteria. Conclusion:Differentially expressed IFITs showed a strong effect against mycobacteria, which can be used as a promising therapeutic targetadjunct to anti-TB therapy. This knowledge will broaden the scope of host drug targets for resistance free bacteriostatic immuno-therapy.