Production of TNF-alpha, IL-12(p40) and IL-17 can discriminate between active TB disease and latent infection in a West African cohort

Abstract

Background: Mycobacterium tuberculosis (MTb) infects approximately 2 billion people world-wide resulting in almost 2 million deaths per year. Determining biomarkers that distinguish different stages of tuberculosis (TB) infection and disease will provide tools for more effective diagnosis and ultimately aid in the development of new vaccine candidates. The current diagnostic kits utilising production of IFN-gamma in response to TB antigens can detect MTb infection but are unable to distinguish between infection and disease. The aim of this study was to assess if the use of a longer term assay and the analysis of multiple cytokines would enhance diagnosis of active TB in a TB-endemic population.

Methods: We compared production of multiple cytokines (TNF-alpha, IFN-gamma, IL-10, IL-12(p40), IL-13, IL-17 and IL-18) following long-term (7 days) stimulation of whole-blood with TB antigens (ESAT-6/CFP-10 (EC), PPD or TB10.4) from TB cases (n = 36) and their Mycobacterium-infected (TST+; n = 20) or uninfected (TST-; n = 19) household contacts (HHC).

Results and conclusions: We found that TNF-alpha production following EC stimulation and TNF-alpha and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-gamma and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation. Combined analysis of TNF-alpha, IL-12(p40) and IL-17 following TB10.4 stimulation resulted in 85% correct classification into TB cases or TST+ HHC. 74% correct classification into TST+ or TST- HHC was achieved with IFN-gamma alone following TB10.4 stimulation (69% following EC) and little enhancement was seen with additional cytokines. We also saw a tendency for TB cases infected with M. africanum to have increased TNF-alpha and IL-10 production compared to those infected with M. tuberculosis. Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.

Figure 1. Cytokine responses following PPD stimulation.

Whole blood from TB cases (n = 36), TST+ (n = 20) and TST− (n = 19) contacts was stimulated for 7 days with PPD and the supernatant assessed by multiplex cytokine analysis. A) IFN-γ. B) TNF-α, C) IL-10, D) IL-12(p40), E) IL-13, F) IL-17 and G) IL-18. Line indicates median. Data were analysed using Tweedie distributions with p-values indicating significant differences after adjustment for age and sex. H) Percentage of responders producing IFN-γ, IL-10, TNF-α, IL-12, IL-13, IL-17, IL-18. Dark bars = cases, grey bars = TST+ contacts, white bars = TST− contacts; p-values are indicated and refer to comparison with TST− contacts.

Figure 2. Cytokine responses following EC stimulation.

Whole blood from TB cases (n = 36), TST+ (n = 20) and TST− (n = 19) contacts was stimulated for 7 days with ESAT-6/CFP-10 fusion protein and the supernatant assessed by multiplex cytokine analysis. A) IFN-γ. B) TNF-α, C) IL-10, D) IL-12(p40), E) IL-13, F) IL-17 and G) IL-18. Line indicates median. Data were analysed using Tweedie distributions with p-values indicating significant differences after adjustment for age and sex. H) Percentage of responders (prior to adjustment for age and sex) producing IFN-γ, IL-10, TNF-α, IL-12, IL-13, IL-17, IL-18. Dark bars = cases, grey bars = TST+ contacts, white bars = TST− contacts; p-values are indicated and refer to comparison with TST− contacts.

Figure 3. Cytokine responses following TB10.4 stimulation.

Whole blood from TB cases (n = 36), TST+ (n = 20) and TST− (n = 19) contacts was stimulated for 7 days with TB10.4 and the supernatant assessed by luminex technology for secretion of cytokines. A) IFN-γ. B) TNF-α, C) IL-10, D) IL-12(p40), E) IL-13, F) IL-17 and G) IL-18. Line indicates median. Data were analysed using Tweedie distributions with p-values indicating significant differences after adjustment for age and sex. H) Percentage of responders (prior to adjustment for age and sex) producing IFN-γ, IL-10, TNF-α, IL-12, IL-13, IL-17, IL-18. Dark bars = cases, grey bars = TST+ contacts, white bars = TST− contacts; p-values are indicated and refer to comparison with TST− contacts.

Figure 4. Flow chart demonstrating how cytokine production following TB10.4 stimulation will improve diagnosis of active TB.

At the moment, patients with suspected TB may be confirmed by analysis of chest x-ray (CXR) plus sputum culture positivity in laboratories which have these capabilities. Analysis of TNF-α, IL-12(p40) and IL-17 production following TB10.4 stimulation should enhance classification into active disease or latent infection in laboratories without culture facilities and also for patients with suspected TB but smear/culture negative results (such as HIV-positive patients or extrapulmonary TB patients). These findings should be further validated in a short-term assay and in difficult to diagnose patient groups.