The influence of mycobacterium tuberculosis on B cells during disease and treatment response

Du Plessis, Willem Jacques (2016-03)

Thesis (MSc)--Stellenbosch University, 2016

Thesis

ENGLISH ABSTRACT : The therapeutic depletion therapy of B cells in humans have led to the discovery that B cells have the potential to function within immunity in a non-humoral fashion. Although this attribute wasn’t fully appreciated, an ever growing body of evidence suggests otherwise. With Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), still endemic in many parts of the world it is crucial that previously under- appreciated cell types be studied for their potential participation in the disease to explore new avenues in curbing the disease. Whole blood was collected from TB patients (n=52), other-lung disease controls (OLD, n=24) and healthy community controls (HHC, n=20) at diagnosis of disease, week 1 (after start of treatment) and week 24 (end of treatment, EOT). Antibodies to cell surface markers were utilised to stain whole blood for B cell immunophenotyping. In a separate experiment, B cells were enriched from peripheral blood mononuclear cells (PBMCs) isolated from interferon gamma release assay (IGRA) positive healthy community controls (n=11) and stimulated overnight with six antigens including phytohaemagglutinin (PHA), lipopolysaccharides (LPS), Bacillus Calmette–Guérin (BCG), Toll-like receptor 9 agonist (TLR9-a) and purified protein derivative (PPD). Following stimulation, B cells were assessed for functionality by multiplex analysis and via polychromatic flow cytometry. Statistical analysis included non-parametric analysis with Mann-Whitney correction, one-way ANOVA and Wilcoxon rank sum tests with p-value adjustment done with the Bonferroni method. Comparisons were made between different groups (TB, OLD and HCC) and time points (diagnosis, week 1 and week 24) for the B cell immunophenotyping. Cytokine production was compared between the different stimulating conditions to assess functional capacity and we compared B cell phenotypes to identify the top contributing phenotype in terms of cytokine production. Our results revealed that memory-based B cells and marginal zone (MZ) B cells can distinguish between TB diagnosis and EOT, class-switched (CS) MZ B cells and non-class switched (NCS) mature B cells can distinguish TB from OLD. B cells readily produce cytokines in a stimulant dependent manner. BCG preferentially induce IL-1β. LPS and TLR9-a stimulation resulted in the highest cytokine- secretion upregulation. We also identified plasma-memory B cells (CD19+CD27+CD138+) as the phenotype predominantly contributing to cytokine production. Taken together these results not only indicate that TB disease has an influence on B cell frequencies, but that they also have the potential to function as effectors/regulators within the latent TB milieu. Future studies will investigate promising phenotypes as potential biomarkers and expand on B cell functional responses within the TB disease host-pathogen interaction.

AFRIKAANSE OPSOMMING : Die sistematiese uitputtings terapie van B selle in mense het gelei na die ontdekking dat B selle die potensiaal het om te funksioneer binne immuniteit in ‘n nie-humorale manier. Alhoewel hierdie eienskappe nie ten volle waardeer was nie, is daar ‘n toenemende groei in die literatuur met bewyse wat andersins toon. Met Mycobacterium tuberculosis (M.tb), die veroorsakende agent vir tuberkulose (TB), wat steeds endemies is in verskeie dele van die wêreld is dit van uiterse belang dat sel tipes wat voorheen nie hoog op waarde geplaas was nie te ondersoek om hulle bydrae te evalueer in die poging om die siekte in toom te hou. Heelbloed was versamel van TB pasiënte (n=52), ander-long siekte kontroles (OLD, n=24) en gesonde gemeenskap kontroles (HHC, n=20) by die diagnose van siekte, week 1 (na die begin van behandeling) en week 24 (einde van behandeling, EOT). Antiliggame teen sel oppervlak merkers was gebruik om heelbloed te vlek vir B sel immunofenotipering. In ‘n aparte eksperiment was B selle verryk vanaf perifere bloed mononukleêre limfosiete (PBMCs) wat verkry was van interferon gamma release assay (IGRA) positiewe gesonde gemeenskap kontroles (n=11), waarna dit oornag gestimuleer was met antigene wat onder andere phytohaemagglutinin (PHA), lipopolysaccharides (LPS), Bacillus Calmette–Guérin (BCG), Toll-like receptor 9 agonist (TLR9-a) and purified protein derivative (PPD) ingesluit het. Na die afloop van stimulasie was die B selle vir funksionele kapasiteit geëvalueer deur multiplex analise en via polichromatiese vloei sitometrie. Statistiese analise het nie-parametriese analises met Mann-Whitney korreksie, een-rigting ANOVA en Wilcoxon rank sum test met p-waarde aanpassing deur die Bonferroni metode ingesluit. Vergelykings was gemaak tussen verskillende groepe (TB, OLD en HCC) en tussen verskillende tyd-punte (diagnose, week 1 en week 24) vir die B sel immunofenotipering. Sitokien produksie was vergelyk tussen verskillende stimulasie kondisies om funksionele kapasiteit te assesseer en ons het B sel fenotipes onderlangs vergelyk om die top sitokien produserende fenotipe te identifiseer. Ons resultate het getoon dat geheue-gebaseerde B selle en marginal zone (MZ) B selle onderskeid kan tref tussen TB diagnose en EOT, klas-omgeskakelde (CS) marginal zone B selle en nie-klas omgeskakelde (NCS) volwasse B selle onderskeid tussen TB en OLD kan tref. B selle produseer geredelik sitokiene in ‘n stimulant afhanklike wyse. BCG induseer IL-1β by voorkeur. LPS en TLR9-a stimulasie lewer die hoogste sitokien-afskeiding opregulasie. Ons het ook plasma-geheue B selle (CD19+CD27+CD138+) geïdentifiseer as die fenotipe wat predominant bydrae tot sitokien produksie. Als saam gevat toon hierdie resultate nie net dat TB siekte ‘n invloed op B sel frekwensies het nie, maar ook dat B selle die potensiaal het om te funksioneer as effektore/regulatore binne die TB milieu. Toekomstige studies sal belowende fenotipes ondersoek as potensiële biomerkers en uitbrei op funksionele B sel response binne die TB siekte gasheer-patogeen interaksie.

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