Granulysin-Expressing CD4+ T Cells as Candidate Immune Marker for Tuberculosis during Childhood and Adolescence

BACKGROUND: Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB. METHODS: Peripheral blood mononuclear cells (PBMC) from children and adolescents (1-17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4(+) CD45RO(+) memory T cells. RESULTS: CD4(+) CD45RO(+) T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSE(low)CD4(+)CD45RO(+)) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4(+) T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells. CONCLUSIONS: Our data suggest granulysin expression by CD4(+) memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence

Biomarkers of Inflammation, Immunosuppression and Stress Are Revealed by Metabolomic Profiling of Tuberculosis Patients

Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHDFAPESP, CNP

CXCL9/Mig Mediates T cells Recruitment to Valvular Tissue Lesions of Chronic Rheumatic Heart Disease Patients

Rheumatic fever (RF) is an autoimmune disease triggered by Streptococcus pyogenes infection frequently observed in infants from developing countries. Rheumatic heart disease (RHD), the major sequel of RF, leads to chronic inflammation of the myocardium and valvular tissue. T cells are the main population infiltrating cardiac lesions; however, the chemokines that orchestrate their recruitment are not clearly defined. Here, we investigated the expression of chemokines and chemokine receptors in cardiac tissue biopsies obtained from chronic RHD patients. Our results showed that CCL3/MIP1α gene expression was upregulated in myocardium while CCL1/I-309 and CXCL9/Mig were highly expressed in valvular tissue. Auto-reactive T cells that infiltrate valvular lesions presented a memory phenotype (CD4(+)CD45RO(+)) and migrate mainly toward CXCL9/Mig gradient. Collectively, our results show that a diverse milieu of chemokines is expressed in myocardium and valvular tissue lesions and emphasize the role of CXCL9/Mig in mediating T cell recruitment to the site of inflammation in the heart

Demonstration of IDO1 expression and kynurenine production in response to <i>M. tuberculosis</i> infection and regulation of <i>M. tuberculosis</i>-specific T-cell responses by kynurenines.

<p>(<b>A</b>) Immunohistochemistry staining of formalin-fixed, paraffin-embedded tissue of a murine pulmonary TB lesion stained with anti-IDO polyclonal antibody; staining representative of lesions from five animals. Bar is equal to 200 nm. Human monocyte-derived dendritic cells (DCs) (<b>B</b>) and macrophages (<b>C</b>) were infected with <i>M. tuberculosis</i> H37Rv or stimulated with irradiated and heat-killed <i>M. tuberculosis</i> H37Rv for 24 h and indoleamine 2,3 dioxygenase 1 (IDO1) gene expression was measured by qPCR. Mean and standard deviation (SD) of fold-change IDO1 gene expression of one donor representative of four. Line indicates minimal significant fold change threshold equal to 1.5. DCs (<b>D</b>) and macrophages (<b>E</b>) were infected with <i>M. tuberculosis</i> H37Rv and cell culture supernatants were collected at indicated times for measurement of kynurenines by HPLC; kynurenine levels from uninfected controls were subtracted. Means and SD of four donors are depicted (C and D). Star indicates significance (p<0.05) in Friedman test.(<b>F</b>) Human monocyte-derived DCs were pulsed with purified protein derivative (PPD) and mannosylated lipoarabinomannan (ManLAM) and co-cultured for 4 days with autologous CFSE-labeled purified T cells (DC:T cell ratio 1∶20) in the presence or absence of 1-methyl-DL-tryptophan (1-MT-DL) and 3-OH-kynurenine (Kyn). Cell proliferation was assessed by CFSE dilution using flowcytometry. Figure representative of three independent experiments (ANOVA F = 4.1 for CD⁺CD4⁺ cells and F = 3.3 for CD3⁺CD8⁺ cells, p<0.05).</p

Abundance of cytokines and their correlation with selected metabolites in TB patients, healthy uninfected and latently infected individuals.

<p>(A) Strip charts showing abundances of eight cytokines that differed significantly between the study groups. Significance thresholds for a two-tailed t-test corrected for multiple testing: *, p<0.05; **, p<0.01; ***, p<0.001. Blue line indicates group median, red line indicates group mean. (B) Correlations between metabolic compounds and cytokines. Grey squares, healthy controls (TST^–); green circles, latently infected individuals (TST⁺); brown triangles, active TB patients (TB^active). Spearman correlation coefficient (rho) and p-values are given.</p

Heat map showing fold changes of small metabolic compounds in the three study groups, TB patients, healthy uninfected and latently infected individuals.

<p>Fold changes are relative to the average abundance in the TST^– group. Red indicates relative abundance higher than average in the TST^– group; blue indicates relative abundance lower than average in the TST^– group. Horizontal axis: samples belonging to the three study groups; vertical axis: top 50 compounds selected by variable importance in RF analysis, including compounds that could not be identified, but were strong predictors of sample status. Color bars above the heat map denote study groups: grey, TST^–; green, TST⁺; red, TB^active. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040221#pone.0040221.s002" target="_blank">Figure S2</a>.</p

Confusion matrices showing the results of external cross-validation for random forests models of the discrimination procedure among the tuberculosis patients (TB^active) and healthy uninfected (TST^–) and latently infected (TST⁺) individuals using the full set of metabolites.

<p>Confusion matrices showing the results of external cross-validation for random forests models of the discrimination procedure among the tuberculosis patients (TB^active) and healthy uninfected (TST^–) and latently infected (TST⁺) individuals using the full set of metabolites.</p