Host response in tuberculosis and BCG vaccination

BackgroundRewiring cellular metabolism is important for activation of immune cells during host defense against Mycobacterium tuberculosis.Glutamine has been implicated as an immunomodulatory nutrient, but its role in response to M. tuberculosis is unknown. MethodsWe assessed expression of glutamine pathway genes in M. tuberculosis-infected macrophages and blood transcriptomic profiles of individuals with latent M. tuberculosis infection or tuberculosis.Subsequently, we studied the effect of blocking glutaminolysis on M. tuberculosis-induced cytokines.Finally, we examined whether polymorphisms in genes involved in the glutamine pathway influence M. tuberculosis-induced cytokines in a cohort of 500 individuals. ResultsGlutamine pathway genes were differentially expressed in infected macrophages and patients with tuberculosis.Human peripheral blood mononuclear cells stimulated with M. tuberculosis displayed decreased cytokine (i.e., interleukin 1, interferon , and interleukin 17) responses when medium was devoid of glutamine.Specific inhibitors of the glutamine pathway led to decreased cytokine responses, especially T-cell cytokines (i.e., interferon , interleukin 17, and interleukin 22).Finally, genetic polymorphisms in glutamine metabolism genes (including GLS2, SLC1A5, and SLC7A5) influenced ex-vivo cytokine responses to M. tuberculosis, especially for T-cell cytokines. ConclusionsCellular glutamine metabolism is implicated in effective host responses against M. tuberculosis.Targeting immunometabolism may represent new strategies for tuberculosis prevention and/or treatment.Chapter 2 Methods PBMC isolation and stimulationInformed consent from healthy volunteers was obtained for use of their blood for scientific purposes, as approved by the Ethics Committee of Radboud University Medical Centre (Nijmegen, the Netherlands).PBMCs were isolated from buffy coats obtained from healthy volunteers (Sanquin Bloodbank, Nijmegen, the Netherlands).Isolation was performed using Ficoll-Paque, involving separation by a density gradient followed by 3 wash steps in cold phosphate-buffered saline and resuspension in Roswell Park Memorial Institute 1640 (Life Technologies, Paisley, UK) supplemented with 10 g/mL gentamicin, 2 mM GlutaMAX (except for the glutamine depletion experiment), and 5.5 mmol/L glucose.After isolation, PBMCs (5 10 5 cells/well) were seeded in 96-well round-bottomed plates and stimulated with M. tuberculosis H37Rv lysate (1 g/mL), Candida albicans (1 10 6 copies/mL), Staphylococcus aureus (1 10 6 copies/mL), phytohemagglutinin (10 g/mL), or culture medium.The cultures were incubated for 24 hours or 7 days at 37C in 5% CO 2 and supplemented with 10% human pooled serum for cultures stimulated for 7 days, after which supernatants were collected and stored at -20C.In some experiments, cells were pre-incubated (before stimulation) for 1 hour with 600 M gamma-L-Glutamyl-p-Nitroanilide (GPNA), 10 M 6-Diazo-5-oxo-L-norleucine (DON), 30 M C968, 50 M BPTES (Sigma), or 5 mM aminooxyacetate. Cytokine and lactate measurementsEnzyme-linked immunosorbent assays were performed on supernatants from the PBMC stimulations following the manufacturer's protocols for measuring cytokines in supernatants.The concentrations of interleukin-1 (IL-1), tumor necrosis factor (TNF-; R&D Systems, Minneapolis, MN), IL-10 and IL-6 (Sanquin, Amsterdam, the Netherlands) were measured after incubation for 1 day, and the concentrations of IFN- (Sanquin), IL-17 and IL-22 (R&D Systems) were measured after 7 days.Lactate concentrations were measured after 1 day of incubation using fluorometric quantification assay its (Biovision, Milpitas, CA). Metabolite measurementsAfter cell culture, cells were lysed in 0.5% Triton-X in phosphate-buffered saline.Concentrations of fumarate, glutamate, pyruvate, -ketoglutarate, and malate were determined by commercial kits (Sigma), according to the instructions of the manufacturer.