Characterization of the innate and adaptive immune systems during active TB disease and during treatment

Kotze, Leigh Ann (2019-04)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Individuals presenting with symptoms of active tuberculosis (TB) disease currently undergo a lengthy diagnostic procedure, followed by an intense six-month treatment regimen. Delays in accurate diagnoses and severe treatment side effects contribute to low treatment adherence and drive the emergence of drug resistant Mycobacterium tuberculosis (M.tb) strains. Improvements in diagnostic technologies have allowed for same day diagnoses, however the roll-out of such devices are limited to settings with stable infrastructure. Additionally, many patients do not require the full treatment regimen when diagnosed early or when presenting with mild disease, however few reliable methods are available for identifying fast-responders. Immune biomarkers show great promise in addressing the need for improved diagnostic and treatment response prediction techniques. This study aimed to investigate multiple promising biomarkers, including (1) promising host diagnostic biomarkers for accurate discrimination of active TB disease from other diseases at point-of-care level; (2) promising host cell surface biomarkers for identifying treatment response shortly after treatment initiation; and also (3) functional host biomarkers for elucidating host-pathogen interaction responses for translation into diagnostic or treatment response biomarkers. Host diagnostic biomarkers were investigated (Chapter 2) in individuals presenting with symptoms suggestive of active TB disease, stratified according to an algorithm based on a combination of clinical, radiological and laboratory findings. Nine acute-phase proteins were investigated in participants' serum, and a biosignature comprising the biomarkers C-reactive protein (CRP) and serum amyloid A (SAA), accurately discriminated between participants with and without active TB disease with a sensitivity of 87.4% and specificity of 75.7%, irrespective of human immunodeficiency virus (HIV) co-infection. The validated biosignature performance in Ascaris lumbricoides sensitized participants remained relatively stable (Chapter 3; Sensitivity of 78%; Specificity of 75%). Validation of this biosignature in scenarios where both HIV- and Ascaris co-infection were considered, identified a robust and reliable biosignature suitable for use in high-burden settings. Individuals with confirmed active TB disease and healthy controls were recruited to investigate cell surface biomarkers of treatment response (Chapter 4). Markers of interest (CD126, CD120b, CD62L, CD197, and CD58) were investigated via flow cytometry on various immune cell subsets across three time points (diagnosis, month 1, end of treatment). No markers were differentially expressed at month 1, whereas CD120b and CD58 were upregulated the end of treatment on CD4+ and CD8+ T-cells, limiting their use as early treatment response biomarkers. Innate and adaptive response cytokines were then investigated from isolated cell subsets (neutrophils, monocytes, T-cells, combination) and compared to whole blood under unstimulated and antigen-stimulated conditions (Chapter 5). The downregulation of protective innate cytokines in the whole blood compared to the culture of monocytes with T-cells, suggested an active suppressive mechanism. A potential innate suppressive immune cell, myeloid-derived suppressor cells (MDSC), was then investigated in TB patients and healthy controls. MDSC were significantly upregulated in peripheral blood mononuclear cells (PBMC) from active TB patients, and cytokine production from MDSC co-cultured with T-cells displayed T-cell-specific downregulation of IFN- (Chapter 6). MDSC should be considered as a potential TB diagnostic biomarker, and future studies investigating frequency changes during treatment will inform on their value as treatment response biomarker.

AFRIKAANSE OPSOMMING: lndividue wat met simptome van aktiewe tuberkulose (TB)-siekte aanbied moet tans deur 'n lang diagnostiese prosedure ondergaan, gevolg deur 'n intensiewe ses-maande lank behandelingskema. Vertragings in akkurate diagnose en slegte behandeling newe-effekte dra by tot lae behandeling nakoming en verhoog die opkoms van middelweerstandige Mycobacterium tuberculosis (M.tb) stamme. Verbeterings in diagnostiese tegnologiee het vir dieselfde dag diagnose toegelaat, maar die uitrol van sulke toestelle is egter beperk tot instellings met stabiele infrastruktuur. Daarbenewens benodig baie pasiente nie die volle behandelingskema wanneer hulle vroeg gediagnoseer word, of wanneer hulle met sagte siekte aanbied nie, maar min betroubare metodes is beskikbaar vir die identifisering van vinnige genesing. lmmuun biomerkers toon 'n groot belofte om die behoefte aan verbeterde diagnostiese en behandelings reaksie voorspelling tegnieke aan te spreek. Hierdie studie het daarop gemik om kandidaatbiomerkers te ondersoek, insluitend (1) gasheer diagnostiek kandidaatbiomerkers vir die akkurate onderskeiding van aktiewe TB-siekte van ander siektes, op die punt van sorg; (2) sel-oppervlak kandidaatbiomerkers vir die identifisering van behandelings reaksie kort na die begin van behandeling; asook (3) funksionele kadidaatbiomerkers vir die verduideliking van gasheer-patogeen interaksie uitkomste. Kandidaatbiomerkers is ondersoek (Hoofstuk 2) in individue wat met simptome van aktiewe TB-siekte aan gebied het, wat gestratifiseer was volgens 'n algoritme gebaseer op 'n kombinasie van kliniese, radiologiese en laboratoriums bevindinge. Nege akute-fase prote'iene was ondersoek in die pasiente se serum, en 'n biosignature bestaande uit die biomerkers C-reactive prote'ien (CRP) en serum amyloid A (SAA) het akkuraat tussen deelnemers met en sonder aktiewe TB-siekte onderskei, met 'n sensitiwiteit van 87.4% en spesifisiteit van 75.7%, ongeag menslike immunogebreksvirus (MlV) mede- infeksie. Die prestasie van die biomerk in Ascaris lumbricoides sensitiseerde deelnemers was gevalideer en het relatief stabiel gebly (Hoofstuk 3; Sensitiwiteit van 78%; Spesifisiteit van 75%). Validering van hierdie biomerk in gebiede waar beide MlV- en Ascaris mede-infeksie oorweeg is, het 'n betroubare biomerk ge'identifiseer wat geskik is vir gebruik in hoe-las instellings. lndividue met bevestigde aktiewe TB-siekte en gesonde kontroles is gewerf om sel-oppervlak biomerkers van behandelings reaksie te ondersoek (Hoofstuk 4). Merkers van belangstel (CD126, CD120b, CD62L, CD197, en CD58) is deur middel van vloeisitometrie op verskeie immuunstelsel onderafdelings oor drie tydpunte (diagnose, maand 1, behandeling einde) ondersoek. Geen merkers is op maand 1 differensieel uitgedruk nie, terwyl CD120b en CD58 aan die einde van behandeling opgerig op CD4+ en CD8+ T selle is, wat hulle gebruik as behandelings reaksie biomerkers beperk. Aangebore en aanpasbare immuunstelsel-sitokiene is dan ondersoek uit ge'isoleerde sel onderafdelings (neutrofiele, monosiete, T selle, kombinasies) en met heelbloed onder nie-gestimuleerde en antigeen- gestimuleerde toestande vergelyk (Hoofstuk 5). Die af-regulering van beskermende aangebore sitokiene in die heelbloed, in vergelyking met die kultuur van monosiete met T selle, het 'n aktiewe onderdrukkende meganisme voorgestel. 'n Potensiele aangebore onderdrukkende immuun sel, myelo'ide-afgeleide onderdrukker selle, is dan ondersoek in TB-pasiente en gesonde kontroles. Myelo'ide-afgeleide onderdrukker selle was beduidend opgerig in perifere bloed mononukleere selle van aktiewe TB-pasiente, en sitokien produksie van myelo'ide-afgeleide onderdrukker selle gekultuur saam met T selle het T sel spesifieke af-regulering van IFN- vertoon (Hoofstuk 6). Myelo'ide-afgeleide onderdrukker selle behoort dus beskou as 'n potensiele TB-diagnostiese biomerker, en toekomstige studies wat die frekwensie veranderings tydens behandeling ondersoek, sal op hulle waarde as biomerkers vir behandelings reaksie inlig.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105613
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