The effect of Binding Immunoglobulin Protein on the induction of regulatory B-cells during Mycobacterium tuberculosis infection

Motaung, Bongani (2019-03)

Thesis (MMed)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Regulatory and killer function in B-cells have been described during tuberculosis (TB) disease, and these were shown to regulate inflammation through several mechanisms including both cytokine secretion (IL-10, IL-35, TGF-β, sFas-L and Granzyme-B) and cell surface expression of Fas-L, PD-L1 and FoxP3. During TB treatment, binding immunoglobulin protein (BiP) secretion was assessed through Enzyme Linked ImmunoSorbent (ELISA) assay. This included plasma samples from healthy controls (n=32), TB diagnosis (n=29), week-1 follow up (n=8), month-2 follow up (n=7), and month-6 follow up (n=19) with additional collection from 20 participants (month-6 total (n=39)). Increased detection of BiP in plasma was observed between TB diagnosis and week-1 follow up suggesting a metabolism shift due to cell stress. The effect of extracellular BiP on immune cell responses were determined by stimulating Peripheral Blood Mononuclear Cells (PBMCs) isolated from healthy controls (n=12) and Mycobacterium tuberculosis (M.tb) exposed participants (LTBI) (n=8) with human recombinant BiP at 20 μg/ml. This effect was compared to other antigenic material including Toll-like Receptor-9 agonist (TLR-9a), M.tb H37Rv, Isoniazid (INH), BiP+TLR-9a, pooled Bronchoalveolar Lavage (BAL) fluid at TB diagnosis (TBdx) and month 6 TB treatment (M6) with unstimulated PBMCs as a baseline control. From this, the cytokine profile was established which indicated an elevated level of sFas-L and IL-13 by BiP stimulation. Further, increased frequency of CD19+CD5+ B-cells co-expressing Fas-L and IL-5Rα was greatly induced by BiP in both healthy controls and LTBI participants. Kinase activity was assessed by Luminex multiplex assay between unexposed group, M.tb exposed and stimulation conditions. Taken together, this study shows BiP potential to aid in better M.tb control by upregulating a B-cell population with immune regulatory function through expression of Fas-L. This highlights the potential use of BiP in host directed therapies (HDT) during TB disease to aid in better infection response.

AFRIKAANSE OPSOMMING: Regulerings- en vernietigingsaksie in B-selle is tydens tuberkulose (TB) siekte beskryf, en daar is getoon dat ditinflammasie deur verskeie meganismes reguleer, insluitende sitokiensekresie (IL-10, IL-35, TGF-β, sFas-L en Granzyme -B) en seloppervlakte uitdrukking van Fas-L, PD-L1 en FoxP3. Tydens TB behandeling, is bindende immunoglobulien proteïen (BiP) sekresie geassesseer deur ‘n Enszyme Linked ImmunoSorbent Assay (ELISA) toets. Plasma monsters van gesonde pasiënte (n = 32), TB pasiënte tydens diagnose (n = 29), week 1 opvolg (n = 8), maand 2 opvolg (n = 7) en maand-6 opvolg n = 19) met die bykomende versameling van 20 deelnemers (maand-6 totaal (n = 39)) is ingesluit. Verhoogde BiP vlakke in plasma is waargeneem tussen TB diagnose en week 1 opvolg wat 'n metabolisme verskuiwing weens selspanning aandui. Die effek van ekstrasellulêre BiP op immuun sel response is bepaal deur die stimulering van perifere bloed mononukleêre selle (PBMCs) wat geïsoleer is van gesonde kontroles (n = 12) en Mycobacterium tuberculosis (M.tb) blootgestelde deelnemers (LTBI) (n = 8) met menslike rekombinante BiP van 20 μg/ml. Hierdie effek is in vergelyking met ander antigeenmateriaal, insluitend Toll-like Receptor-9-agonist (TLR-9a), M.tb H37Rv, Isoniazid (INH), BiP + TLR-9a, saamgevoegde Bronchoalveolêre spoeling (BAL) vloeistof by TB diagnose (TBdx ) en maand 6 TB behandeling (M6) met ongestimuleerde PBMCs as 'n basislynbeheer. Hieruit is die sitokienprofiel vasgestel wat 'n verhoogde vlak van sFas-L en IL-13 deur BiP-stimulasie aangedui het. Verder is gevind dat verhoogde frekwensie van CD19+CD5+ B-selle wat Fas-L en IL-5Rα terselfdetyd uitdrukveroorsaak word deur BiP in beide gesonde pasiënteen LTBI-deelnemers. Kinase-aktiwiteit is geassesseer deur Luminex-multipleks assessering tussen die nie-blootgestelde groep, M.tb blootgestel en stimulasie toestande. Hierdie studie het dus getoon dat BiP potensiaal het om M.tb beheer te verbeter deur 'n B-sel bevolking met immuunregulerende funksie te reguleer deur die uitdrukking van Fas-L. Hierdie resultatedui op diedie potensiële gebruik van BiP in gasheergerigte terapieë (HDT) tydens TB-siekte om beter infeksierespons te bevorder.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105590
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