2020-12-11 Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria

Mishra, Abhilasha Madhvi (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

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.

Agtergrond:Tuberkulose (TB) is ‘n hoofoorsaak van infeksieverwante sterftes wêreldwyd. ‘n Benaderde 1.3 miljoen MIV-negatiewe mense is in 2017 dood aan TB. ‘n Benaderde 5-10% van geïnfekteerde individue ontwikkel aktiewe TB in hul leeftyd, terwyl die oorblywende 90%(van die geïnfekteerde bevolking) die bakterie suksesvol beheer. Sommige huisgenote van TB pasiënte bly ook ongeïnfekteerd en gesond. Die rede vir hierdie enigma kan ontbloot word deur die gasheer immuunreaksie te bestudeer. Metodes:Ons het deeglik ondersoek ingestel na die in vitrogasheerreaksie (van menslike monosiet-afgeleide makrofae) op verskillende stamme van mikobakterieë (opgegroei in ontsmettingsmiddelvrye media), d.i. patogenies (R179) en nie-patogenies (M..smegmatisen M. bovisBCG). Die gasheer reaksie is na infeksie gemeet (boodskapper RNS en proteïenvlakke) met AmpliSeq, reële tyd PKR, veelvuldige baan ELISA (Luminex), biologiese netwerk analise (Ingenuity Pathway Analysis), intrasellulêre mikobakteriële oorlewing en sitotoksisiteit eksperimente. Ons het ‘n onderling-verwante geenfamilie van interferon geïnduseerde proteïen met tetratrigopeptiede (IFIT1, IFIT2 enIFIT3)gekies uit die lys van 19 moontlike verskillend-uitgedrukte gene vir ons oor-uitdrukking, en onderdrukking eksperimente. Hierdie geenfamilie is bekend daarvoor om ‘n proteïenkompleks te vorm om gedurende virale infeksie teen die antigeen op te tree. Hul rol in bakteriële infeksie is nie goed bevestig nie. Daarom het ons oor-uitdrukking van IFITsdeur vektor-gebaseerde transfeksie en onderdrukking deur klein onderdrukkende RNS (siRNA) uitgevoer om hul effek op mikobakterieë in gasheer makrofae te ondersoek. Resultate:AmpliSeq analise het 19 verskillend-uitgedrukte gene teen 12 ure na infeksie oor al drie stamme gevind. Ons het ‘n laer getal kolonie-vormende eenhede en ‘n hoër gasheerreaksie (in beide RNS-en proteïenvlak) in menslike monosiet-afgeleide makrofae geïnfekteer met M. smegmatisopgemerk, as in die ander twee stamme. Biologiese netwerk analise het gewys dat interferon-interleuken verwante seinweë ‘n belangrike rol speel tussen die 19 verskillend-uitgedrukte gene.Ons het verskillende gasheerreaksies teenoor die 3 stamme gevind, wat moontlik aan elk se patogeniese aard verwant is. Boodskapper RNS en proteïenvlak vergelykings by verskillende tydpunte, het die belangrike rol van ‘ninterferon en interleuken verwante geennetwerk gewys. Hierdie netwerk kon M. smegmatissuksesvol beheer, maar nie M. bovisBCG en R179 nie.Interessant genoeg, het intra-sellulêre bakteriële groei en oorlewing, soos gemeet in kolonie-vormende eenhede, beduidend verminder wanneer IFITs oor-uitgedruk is, terwyl dit vermeerder het wanneer IFIT1, IFIT2 enIFIT3onderdruk is in gasheer makrofae. Intrasellulêre groei en oorlewing van mikobakterieë is bevestig deur boodskapper RNS uitdrukking met kwantifiserende PKR en proteïen uitdrukking met “Western blot”, te ondersoek. ‘n Paar sleutel-molekules is opgemerk in die sel sitokien uitdrukking wat ons met veelvuldige-baan ELISA ondersoek het. Dit het IDO1, IFN-γ, IL-6, en IL-23 ingesluit, wat bekend is as pro-inflammatoriese sitiokiene. Afsluiting:Hierdie uitslae sal ons kennis verbreed oor gasheer middel-teikens vir weerstandvrye bakteriostatiese immuunterapie aanvullend tot die huidige chemoterapie.

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