Abstract
Co-infection with Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis (M.tb) is a well-known global issue, and South Africa continues to have one of the world's highest HIV/Tuberculosis (HIV/TB) loads. M.tb is contained within a granuloma in the latent stage and remains inactive with no clinical signs or symptoms. The granuloma is made up of several immune cells, including cluster of differentiation 4 positive T (CD4+ T) cells, which are the target of HIV infection. HIV/M.tb co-infection can cause granuloma dissemination and increased susceptibility to active TB. Vitamin D is required for the generation of antimicrobial peptides that kill M.tb and the active form of vitamin D activates the vitamin D receptor (VDR). The antimicrobial pathway is inhibited when vitamin D concentration is low. The VDR gene plays an important role in the immune response to tuberculosis. HIV-induced methylation downregulates VDR. The ability of M.tb to influence macrophages, granulomas, and host cell metabolism is critical to its pathogenesis. Metabolomic analysis of blood-based indicators can reliably predict rapid disease development or poor immune response. The mechanism of immune response to HIV/M.tb co-infection, principally in CD8+ T cells, has not been studied. A non-directional hypothesis was used to identify if the metabolic profile differed between the CD4+ only and CD4+/CD8+ T cell immune response to early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) (M.tb antigens) in a newly diagnosed HIV+ environment. ESAT-6 and CFP-10 antigens were used in the study because they are key components of the IGRA (Interferon-Gamma Release Assay) tubes, which are designed to detect immune responses specific to M.tb infection. Blood samples from newly diagnosed HIV+, treatment naïve individuals who were self-reported to have never been diagnosed with TB were collected at the Helen Joseph Hospital, Johannesburg, South Africa. Using the Quantiferon (QFT) Gold Plus IGRA M.tb antigens ESAT-6 and CFP-10, an immune response was elicited within the blood samples. Following immune stimulation, the plasma from the IGRA tubes were used to analyse latent tuberculosis (LTB) status, quantify vitamin D concentration, and the metabolic signatures of peripheral blood mononuclear cells (PBMCs) following CD4+ and CD8+ T cell stimulation using untargeted metabolomics analysis. The vitamin D receptor (VDR) CpG Island (CGI) 1060 methylation analysis was performed using DNA isolated from PBMCs from IGRA tubes. Multivariable multidimensional data analysis (MVMA) was used to identify metabolites that significantly differ between the CD4+ only and CD4+/CD8+ T cell
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response and assess the metabolic pathways involved in the immune response. The results showed that the immune response was higher in the overall CD4+/CD8+ T cell response versus CD4+ T cell only response. However, for the majority of the metabolites, the concentration in CD4+ T cells was higher than that in CD4+/CD8+ T cells. The majority of the samples were LTB+ (56.3%) and vitamin D deficient (79%). 70% of the individuals in this study showed methylation in the VDR CGI 1060 region. It was found that the T-cell response to M.tb antigens, especially in the Warburg effect, significantly differs between the CD4+ and CD4+/CD8+ T-cell response, indicating metabolic changes in co-infection. Key metabolites, including glucose, pyruvic acid, lactic acid, glutamic acid, and L-alanine, were identified as important, with correlations made between their levels, vitamin D status, and gene methylation patterns. The findings suggest a complex relationship between immune dysregulation and metabolic pathways in HIV+ individuals, highlighting the importance of both T-cell types in the immune response to M.tb. The study shows the potential of metabolomic profiling for discovering biomarkers and therapeutic targets in HIV/LTB co-infection, while also noting limitations such as small sample size and the need for more research into genetic and epigenetic factors.