Identification of distinct bio-signatures in whole blood and in-tube quantiferon pellets of Tuberculosis (TB) close contacts (CCs) and TB patients with and without type 2 diabetes (T2D)

Tshivhula, Happy (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.


ENGLISH ABSTRACT: Tuberculosis (TB) remains difficult to control despite effective treatment and the presence of more sensitive diagnostic tools. Immunity against Mycobacterium tuberculosis (Mtb) infection can further be impacted by other concomitant infections (like human immunodeficiency virus) and non-communicable diseases (like Type 2 diabetes (T2D)) rendering individuals susceptible to TB. T2D patients are three times more likely to progress from latent to active TB.Therefore, it is crucial to better understand why people with T2D are at increased risk for TB progression. We hypothesize that distinct bio-signatures exist in whole blood of TB patients and close contacts (CCs) of TB patients with latent TB infection (LTBI) with T2D compared to those without T2D and that these bio-signatures correlate with impaired immune responses to Mtb in peripheral blood mononuclear cells (PBMCs) and monocytes (MNs) in CCs. RNA was extracted from whole blood stimulated with Mtb antigens using the Quantiferon TB Gold in-tube assay and NanoString analysis was performed to determine whether specific transcription signatures exist between CCs with LTBI with and without T2D. We characterized serum cytokines and endocrine signatures by luminex and enzyme-linked immunosorbent assay. PBMCs and MNs from the same individuals were isolated and stimulated with live H37Rv Mtb to determine Mtb uptake and killing. Bacterial uptake and killing was correlated with HbA1c to determine whether there was an association with glycaemic control. RNA from TB patients, TB patients with transient hyperglycaemia (TB-THG) and TB patients with T2D (TB-T2D) was also extracted from whole blood stimulated with Mtb antigens and analysed using NanoString. Interleukin-22 was also measured in serum from these patients. PBMCs from TB patients were further used to determine the frequency of mucosal associated invariant T (MAIT) cells before, during and after TB treatment. Fifteen gene transcripts were downregulated in TB-T2D compared to TB and TB-THG patients. We identified a gene signature that was able to differentiate between TB-THG and TB-T2D. We further identified a gene signature unique to the CCs with LTBI and T2D, which could be associated with an increased risk of developing active TB. Differences in cytokines, hormones, lipids, differential blood counts and Mtb uptake was observed when comparing CCs with and without T2D. Differential expression of IL-6, IL-18 and IL-22 in CCs with and without T2D highlights the differential regulation of the immune response during T2D. We showed that the frequency of MAIT cells in the periphery of TB-T2D was significantly lower compared to TB-THG at baseline, suggesting the MAIT cells in TB-T2D have redistributed to either the lung or adipose tissue. The increase of MAIT cells in the periphery at month 2 compared to baseline could mean that MAIT cells population was restored in the blood due to TB treatment. Our gene expression results suggests that downregulated gene transcripts may be involved in pathways that favour immunopathology in TB-T2D. In addition, gene signatures differentiating THG from T2D could be useful in preventing the unnecessary diagnosis of patients with THG as having T2D. Differential gene expression in CCs with LTBI with and without T2D shows potential to be involved in the mechanisms leading to susceptibility to active TB. Cytokine and hormone data confirms that during T2D, the immune response is differentially regulated which may influence the response to infections. Lastly, MAIT cell frequency could be useful for monitoring treatment responses in TB-T2D patients.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) bly moeilik om te beheer ten spyte van effektiewe behandeling en die teenwoordigheid van meer sensitiewe diagnostiese toetse. Immuniteit teen Mycobacterium tuberkulose (Mtb) kan verder beïnvloed word deur ander gepaardgaande infeksies (soos menslike immuniteitsgebrekvirus) en nie-oordraagbare siektes (soos tipe 2 diabetes (T2D)) wat individue vatbaar maak vir TB. Pasiënte met T2D het ʼn groter kans om van latent na aktiewe TB te vorder en is dit noodsaaklik om te verstaan waarom mense met T2D ‘n groter risiko vir TB-progressie het. Ons veronderstel dat daar afsonderlike bio-handtekeninge in heel bloed van TB pasiënte asook nabye kontakte (CCs) van TB pasiënte met latente TB-infeksie (LTBI) en T2D in vergelyking met dié sonder T2D bestaan en dat hierdie bio-handtekeninge met verswakte immuunresponse teenoor Mtb in mononukleêr selle in perifere bloed (PBMCs) en monosiete (MNs) van CCs korreleer. RNS is uit volbloed wat met Mtb-antigene (Quantiferon TB Gold toets) gestimuleer is onttrek. Nanostring-analise is uitgevoer om vas te stel of spesifieke transkripsie-patrone tussen CCs met LTBI en met of sonder T2D bestaan. Ons het serum sitokiene en endokriene handtekeninge identifiseer deur luminex en ensien-gekoppelde Immunosorbent-toetse. PBMCs en MNs van dieselfde individue is geïsoleer en met lewendige H37Rv Mtb gestimuleer om Mtb opname en beheer te bepaal. Bakteriese opname en dood was met HbA1c gekorreleer om vas te stel of daar 'n verband met bloedsuiker was. RNS van TB-pasiënte, TB-pasiënte met oorgang hiperglukemie (TB-THG) en TB-pasiënte met T2D (TB-T2D) was ook uit heelbloed wat met Mtb-antigene gestimuleers was onttrek en met behulp van die Nanostring tegnologie geanaliseer. IL-22 was ook in the serum van hierdie pasiënte gemeet. PBMCs van TB pasiënte is verder gebruik om die frekwensie van ‘mucosal associated invariant’ T (MAIT) selle voor, tydens en na TB behandeling te bepaal.Vyftien geen-transkripsies is in TB-T2D afwaarts gereguleer in vergelyking met TB- en TB-THG pasiënte. Ons het 'n geen-handtekening geïdentifiseer wat in staat was om tussen TB-THG en TB-T2D te onderskei. Ons het verder 'n handtekening van gene geïdentifiseer wat uniek is aan die CCs met LTBI en T2D wat met 'n verhoogde risiko om aktiewe TB te ontwikkeling geassosieer kan word. Verskille in sitokiene, hormone, lipiede, differensiële bloedtellings en Mtb opname is waargeneem tydens die vergelyking van CC's met en sonder T2D. Differensiële uitdrukking van IL-6, IL-18 en IL-22 in CCs met LTBI met en sonder T2D beklemtoon die differensiële regulering van die immuunrespons tydens T2D. Ons het getoon dat die frekwensie van MAIT selle in die sirkulasie van TB-T2D laer was as TB-THG by basislyn, wat daarop dui dat die MAIT-selle in TB-T2D na die long- of vetweefsel migreer. Die toename van MAIT selle in die bloed by maand 2 in vergelyking met basislyn kan beteken dat MAIT selpopulasies in die bloed herstel, as gevolg van TB behandeling.

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