Mycobacterium Tuberculosis-Specific TNF-α Is a Potential Biomarker for the Rapid Diagnosis of Active Tuberculosis Disease in Chinese Population

<div><p>Interferon-gamma release assays (IGRAs) have proven to be useful to accurately detect <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infection, but they cannot reliably discriminate between active tuberculosis (TB) and latent tuberculosis infection (LTBI). This study aims to test whether <i>Mtb</i>-specific tumor necrosis factor-alpha (TNF-α) could be used as a new tool for the rapid diagnosis of active TB disease. The secretion of TNF-α by <i>Mtb</i>-specific antigen-stimulated peripheral blood mononuclear cells (PBMCs) of sixty seven participants was investigated in the study. Our results showed that the total measurement of TNF-α secretion by <i>Mtb</i>-specific antigen-stimulated PBMCs is not a good biomarker for active TB diagnosis. However, we found that calculation of <i>Mtb</i>-specific TNF-α not only distinguish between active and latent TB infection, but also can differentiate active TB from non-TB patients. Using the cutoff value of 136.9 pg/ml for <i>Mtb</i>-specific TNF-α, we were able to differentiate active TB from LTBI. Sensitivity and specificity were 72% and 90.91%. These data suggest that <i>Mtb</i>-specific TNF-α could be a potential biomarker for the diagnosis of active TB disease.</p></div

The percentages and absolute numbers of Tim-3⁺ NK and IFN-γ⁺ NK cells.

<p>Data are mean ± SEM of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with septic mice at 0 h after LPS injection; ^##<i>p</i><0.01, ^###<i>p</i><0.001 compared with septic mice at 12 h after LPS injection.</p><p>The percentages and absolute numbers of Tim-3⁺ NK and IFN-γ⁺ NK cells.</p

Tim-3 expression is inversely associated with NK cell activity.

<p>(A) The expression of Tim-3 and IFN-γ was analyzed in NK cells from septic mice at 24 h after LPS injection. The percentage of IFN-γ⁺ cells between Tim-3⁺ and Tim-3⁻ NK cell subsets was shown in the graphs. (B) Representative flow cytometry histograms of CD69 expression on NK cells from septic mice at 0, 12 and 24 h after LPS injection were shown. (C) The MFI of CD69 on NK cells was shown in the bar graphs. (D) The percentage of CD69⁺ NK cells between Tim-3⁺ and Tim-3⁻ NK cell subsets from septic mice at 24 h after LPS injection was shown. Data are mean ± SEM of at least three independent experiments. **<i>p</i><0.01, ***<i>p</i><0.001.</p

Expression of Tim-3 and IFN-γ in NK cells during the development of LPS-induced endotoxic shock.

<p>Peritoneal cavity cells were collected from normal and septic mice at different time points after LPS injection and were analyzed by flow cytometry. (A) NK cells were gated as the CD3⁻NKp46⁺ population. Representative flow cytometry histograms showed Tim-3 expression on NK cells from normal and septic mice at 24 h after LPS injection. The percentage of Tim-3⁺ NK cells was shown in the bar graphs. (B) The percentages of Tim-3⁺ NK cells and IFN-γ⁺ NK cells from septic mice at different time points after LPS injection were shown. Data are mean ± SEM of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with septic mice at 0 h after LPS injection; ^##<i>p</i><0.01, ^###<i>p</i><0.001 compared with septic mice at 12 h after LPS injection.</p

Blockade of Tim-3 pathway increased IFN-γ production and decreased apoptosis of NK cells in vitro.

<p>Spleen cells harvested from septic mice at 24 h after LPS injection were stimulated with LPS (1 µg/ml) in the presence of anti-Tim-3 Ab (1 µg/ml), Tim-3 Fc protein (5 µg/ml) or control IgG for 24 h. The bar graphs showed the percentages of (A) IFN-γ⁺ NK cells and (B) CD107a⁺ NK cells between the groups. (C) The apoptosis of NK cells (gated on CD3^–NKp46⁺ cells) was analyzed by Annexin V/PI double staining. The percentage of Annexin V⁺PI^– cells (representative of apoptosis cells) was compared between the groups. (D) The expression of surface galectin-9 on peritoneal macrophages (F4/80⁺ cells), neutrophils (CD11b⁺ cells), DCs (CD11c⁺ cells), CD4⁺ T cells, CD8⁺ T cells and NK cells from normal and septic mice at 24 h after LPS injection were analyzed. (E) The MFI of galectin-9 on F4/80⁺ and CD11b⁺ cell populations was shown in the bar graphs. Data are mean ± SEM of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

The relationship between Tim-3 expression and the cytotoxicity of NK cells.

<p>(A) Representative flow cytometric dot plots of CD107a expression on NK cells from septic mice at 0, 4, 12 and 24 h after LPS injection were shown. (B) The bar graphs showed the percentage and MFI of CD107a expression on NK cells at different time points. (C) The bar graphs showed the percentage of CD107a⁺ cells between Tim-3⁺ and Tim-3⁻ NK cell subsets from septic mice at 24 h after LPS injection. (D, F) Representative flow cytometry histograms showed granzyme B and perforin expression in NK cells from normal and septic mice at 24 h after LPS injection. The MFI of granzyme B and perforin expression in NK cells was shown. (E, G) The MFI of granzyme B and perforin expression in NK cells was compared between Tim-3⁺ and Tim-3⁻ NK cell subsets. Data are mean ± SEM of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Blocking Tim-3 pathway enhances the cytotoxic activity of NK cells.

<p>NK cells were purified from the spleen cells of septic mice. Purified NK cells treated with anti-Tim-3 Ab (1 µg/ml) or control IgG for 18 h were used as effector cells. The effeteor cells were cocultured with CFSE-labeled K562 target cells at the E: T of 0∶1 and 10∶1 for 6 h. The death of target cells was detected by flow cytometry using PI staining. (A) The purity of NK cells was assessed by flow cytometric analysis of cells stained with anti-CD3 and anti-NKp46. (B) Representative flow cytometric dot plots showing the percentages of dead target cells in different experimental groups. (C) The percentage of target cell lysis was shown in the bar graphs. Data are expressed as the mean ± SEM of at least three independent experiments. **<i>p</i><0.01.</p

The cytotoxicity of ESAT-6 to PHA-stimulated PBMCs of three non-TB patients.

<p>Note. PHA: phytohemagglutinin; TB: tuberculosis; AFS: acid fast staining.</p

Statistical analysis of ROC curve for <i>Mtb</i>-specific TNF-α to distinguish between active TB and LTBI.

<p>Note. TB: tuberculosis; LTBI: latent tuberculosis infection; AUC: area under the curve.</p

The secretions of TNF-α and <i>Mtb</i>-specific TNF-α by ESAT-6 or CFP-10-stimulated PBMCs.

<p>PBMCs obtained from active TB patients (n = 25), LTBI individuals (n = 22), healthy control subjects (n = 10) and non-TB patients (n = 10) were stimulated with ESAT-6 or CFP-10. PBMCs stimulated with medium alone were used as a background control. (A) After 16–20 h of incubation, the supernatant was collected and tested for concentrations of secreted TNF-α by ELISA. (B) <i>Mtb</i>-specific TNF-α was calculated by subtracting background TNF-α secreted by medium-stimulated PBMCs from TNF-α secreted by ESAT-6 or CFP-10-stimulated PBMCs. Median values for each group of participants are represented by a horizontal bar. *<i>p</i> < 0.05, **<i>p</i> < 0.001.</p