Characterization of human alveolar macrophages and blood monocyte-derived macrophages responses to M.tb in TB close contacts with and without type 2 diabetes

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dc.contributor.advisorRonacher, Katharinaen_ZA
dc.contributor.advisorKleynhans, Leanieen_ZA
dc.contributor.authorKunsevi Kilola, Carineen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2022-10-24T12:14:07Zen_ZA
dc.date.accessioned2023-01-23T06:50:31Zen_ZA
dc.date.available2022-10-24T12:14:07Zen_ZA
dc.date.issued2022-12en_ZA
dc.descriptionThesis (PhD)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a global threat to human health and a challenge for TB control. Type 2 diabetes (T2D) is a known risk factor for TB, as people living with diabetes have an increased risk of developing TB. The chronic low-grade inflammation associated with T2D is likely to contribute to compromised immune responses to M.tb in the lung and the periphery. We hypothesize that lung macrophages from T2D patients have an impaired response to M.tb and reduced capacity to contain M.tb growth in vitro. Human alveolar macrophages (HAMs) and peripheral monocyte-derived macrophages (MDMs) from 23 close contacts (CCs) of active TB patients with and without T2D were isolated and stimulated with live H37Rv M.tb to determine M.tb uptake and killing. Circulating numbers of white blood cells and neutrophils were elevated in T2D compared to no T2D. Moreover, neutrophil counts in the lung was lower in T2D patients. HAMs of T2D patients have a reduced ability to control M.tb growth one day post infection, and the colony forming units (CFUs) in these cells at day one post infection was inversely correlated to the neutrophil count in this compartment. HbA1c and HDL was associated with M.tb killing in HAMs and MDMs, respectively. CFU fold change at day one in HAMs was correlated with HbA1c suggesting a high bacterial load in the lung is attributable to poor glycemic control. Furthermore, cytokines in HAMs and MDMs supernatants were determined using the Luminex platform. M.tb infection induced unique cytokine responses in MDMs and HAMs. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-1RA concentrations were significantly higher in the MDM culture supernatant of no T2D controls compared to T2D patients. HAMs of T2D patients produced more MIP-1α and IL-1β, than no T2D controls. We further identified unique pathways associated with individual cytokine clusters in MDMs and HAMs. Finally, we determined the phenotypes of HAMs and MDMs using Flow cytometry and Time-of-flight mass cytometry (CyTOF), respectively. The flow cytometry data revealed three different MDM subsets (CD11b+CD11c+, CD11b-CD11c+ and CD11b+CD11c-), with the CD11b+CD11c+ cell population being the most abundant. Diabetes affected the frequency of CD36, CD32, CD16, HLA-DR and CD206 in the MDMs subsets before and after infection. The CyTOF data revealed two dominant CD11b lineages in HAMs (CD11b+CD11c+ and CD11b++CD11c+) with CD11b+CD11c+ cells being the most predominant subpopulation. A trend suggested that the frequency of uninfected CD11b++CD11c+ expressing CD36 was increased in T2D group. Furthermore, the median metal intensity of CD32 was significantly lower in the HAMs T2D patients. During T2D, the immune response is differentially regulated in the two compartments (periphery vs site of infection), which may influence the response to infections. Alterations in macrophages phenotypes and function during diabetes, pre- and post-exposure to M.tb may result in dysregulated host defense functions in both HAMs and MDMs.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Tuberkulose (TB), wat deur Mycobacterium tuberculosis (M.tb) veroorsaak word, bly 'n wêreldwye bedreiging vir menslike gesondheid en 'n uitdaging vir TB-beheer. Tipe 2-diabetes (T2D) is 'n bekende risikofaktor vir TB, aangesien mense met diabetes 'n groter risiko het om TB te ontwikkel. Die chroniese laegraadse inflammasie wat met T2D geassosieer word, dra waarskynlik by tot ‘n gekompromitteerde immuunresponse teen M.tb in die long en die periferie. Ons hipotetiseer dat longmakrofage van T2D pasiënte 'n verswakte reaksie teenoor M.tb toon asook ’n verminderde kapasiteit het om M.tb groei in vitro te beheer. Menslike alveolêre makrofage (HAMs) en perifere monosiet-afgeleide makrofage (MDMs) van 23 nabye kontakte (CCs) van aktiewe TB pasiënte met en sonder T2D is geïsoleer en gestimuleer met lewende H37Rv M.tb om M.tb opname en beheer te bepaal. Sirkulerende witbloedsel en neutrofiel getalle was hoёr in T2D in vergelyking met geen T2D. Terselfde tyd was die neutrofieltellings laer in die longe van T2D-pasiënte. Een dag na infeksie het die HAMs van T2D pasiënte 'n verminderde vermoë om M.tb groei te beheer, en die kolonievormende eenhede (CFUs) in hierdie selle op dag 1 na infeksie was omgekeerd gekorreleer met die neutrofieltelling in dieselfde kompartement. HbA1c en HDL het met met M.tb doodmaak in HAMs en MDMs, onderskeidelik, gekorreleer. Die korrelasie tussen vou verandering op dag een in HAM CFU en HbA1c dui daarop dat 'n hoë bakteriële lading in die long moontlik toegeskryf kan word aan swak glukemiese beheer. Verder is sitokiene in HAMs en MDMs supernatante bepaal deur gebruik te maak van die Luminex platform. M.tb-infeksie het unieke sitokienreaksies in MDMs en HAMs geïnduseer. GM-CSF, IL-6 en IL-1RA konsentrasies was beduidend hoër in die MDM kultuur supernatant van geen T2D kontroles in vergelyking met T2D pasiënte. HAMs van T2D-pasiënte het meer MIP-1α en IL-1β geproduseer as geen T2D-kontroles. Ons het verder unieke weë geïdentifiseer wat geassosieer word met individuele sitokienklusters in MDM's en HAM's. Laastens het ons die fenotipes van HAM's en MDM's bepaal deur vloeisitometrie en CyTOF onderskeidelik te gebruik. Die vloeisitometrie-data het drie verskillende MDM-subpopulasies geïdentifiseer (CD11b+CD11c+, CD11b-CD11c+ en CD11b+CD11c-), waarvan die CD11b+CD11c+ selpopulasie die volopste was. Diabetes het die frekwensie van CD36, CD32, CD16, HLA-DR en CD206 in die MDMs subpopulasies voor en na infeksie beïnvloed. Die CyTOF-data het twee dominante CD11b-lyne in HAMs (CD11b+CD11c+ en CD11b++CD11c+) geïdentifiseer met CD11b+CD11c+-selle wat die mees oorheersende subpopulasie was. 'n Tendens het voorgestel dat die frekwensie van ongeïnfekteerde CD11b++CD11c+ wat CD36 uitdruk, verhoog is in die T2D-groep. Verder was die mediaan metaal intensiteit van CD32 beduidend laer in die HAMs T2D pasiënte. Tydens T2D word die immuunrespons differensieёl gereguleer in die twee kompartemente (periferie vs plek van infeksie), wat die reaksie op infeksies kan beïnvloed. Veranderinge in makrofage se fenotipes en funksie tydens diabetes, voor- en na-blootstelling aan M.tb kan lei tot wan gereguleerde gasheerverdedigingsfunksies in beide HAMs en MDMs.af_ZA
dc.description.versionDoctorateen_ZA
dc.embargo.lift2023-12-31en_ZA
dc.embargo.terms2023-12-31en_ZA
dc.format.extentxxiii, 224 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126287en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMacrophagesen_ZA
dc.subject.lcshTuberculosis -- Patientsen_ZA
dc.subject.lcshType 2 diabetes -- Alternative treatmenten_ZA
dc.subject.lcshUCTDen_ZA
dc.titleCharacterization of human alveolar macrophages and blood monocyte-derived macrophages responses to M.tb in TB close contacts with and without type 2 diabetesen_ZA
dc.typeThesisen_ZA
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Doctoral Degrees (Molecular Biology and Human Genetics)