Combination of cytokine responses indicative of latent TB and active TB in Malawian adults

Abstract

Background: An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods: Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results: Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions: CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.

Figure 1. The proportion of cytokine responders to ESAT-6 at baseline and follow-up time points.

The proportion of responders producing IFN-γ (A), IL-13 (B), IL-10 (C), CXCL10 (D), IL-17 (E) and TNF-α (F) in response to ESAT-6 was analysed in 40 spouses at baseline, 49 at 6 months, 39 at 12 months and 22 at 24 months follow-up. There were no significant differences in the proportions of IFN-γ responders between baseline and 24 months follow-up. Most of spouses did not produce IL-13 in responses to ESAT-6 at baseline, and this did not change over the 24 months follow-up. The proportions of IL-10 and TNF-α responders to ESAT-6 gradually declined at 6, 12 and 24 months.

Figure 2. The proportion of cytokine responders to PPD at baseline and follow-up time points.

The proportion of responders producing IFN-γ (A), IL-13 (B), IL-10 (C), CXCL10 (D), IL-17 (E) and TNF-α (F) in response to PPD was analysed in 40 spouses at baseline, 49 at 6 months, 39 at 12 months and 22 at 24 months follow-up. The majority of the spouses did not produce IL-13 and IL-10 in response to PPD at baseline or 24 months later while high levels of IFN-γ, CXCL10, IL-17 and TNF-α were detected. There were no significant changes in the proportions of cytokine responders throughout the follow-up period.

Figure 3. Cytokine responses in spouses and TB patients.

The cytokine responses to ESAT-6 (A), PPD (B) and PHA (C) were analysed in the spouses who showed negative IFN-γ responses to ESAT-6 (<62.5 pg/mL, n = 102; indicated as S1), the spouses with positive IFN-γ responses (>62.5 pg/mL, n = 48; indicated as S2) and TB patients (n = 15; indicated as T). The S2 contacts and TB patients showed significantly higher production of CXCL10, IL-17 and TNF-α production in response to ESAT-6 compared with the S1 contacts. In response to PPD, IL-17 and TNF-α production was also significantly higher in the 2 groups compared with the group of S1. However, only IL-10 and IL-17 differentiated the S2 spouses from TB patients by showing higher production in the S2 group than TB patients. In response to PHA, all cytokines were highly detected in 3 groups. Median responses are marked with horizontal bars. (* P<0.05, ** P<0.01, *** P<0.001)

Figure 4. The ratio of cytokine responses in spouses and TB patients.

The ratio of cytokine responses to ESAT-6 (A) and PPD (B) were analysed in the spouses who showed negative IFN-γ responses to ESAT-6 (<62.5 pg/mL, n = 102; indicated as S1), the spouses with positive IFN-γ responses (>62.5 pg/mL, n = 48; indicated as S2) and TB patients (n = 15; indicated as T). The ratio of IFN-γ/IL-10, CXCL10/IL-10, TNF-α/IL-17, IFN-γ/IL-17 and IL-17/IL-10 differentiated the S2 and TB groups from S1 in responses to both ESAT-6 and PPD. None of the ratios differentiated between the S2 and T groups upon ESAT-6 stimulation while IFN-γ/IL-10 and IFN-γ/IL-17 in response to PPD distinguished S2 from TB patients. Median ratio of cytokine responses is marked with horizontal bars. (* P<0.05, ** P<0.01, *** P<0.001)

Figure 5. TST indurations and association between TST size and cytokine responses.

A. TST sizes were obtained from 34 spouses at baseline, 37 spouses at 6 months, 22 spouses at 12 months and 10 spouses at 24 months follow-up time point. More than 75% of spouses already had above 10 mm of TST induration at baseline. The proportion of those who had more than 10 or 15 mm of TST induration was increased according to the follow-up time while the proportion of those with below 10 mm of TST induration was decreased. None of the subjects showed a TST induration below 10 mm at the 24 months follow-up but about 80% of these had more than 15 mm of TST induration. B–C. None of the cytokine production in response to ESAT-6 (B) and PPD (C) was correlated with TST size in 101 spouses by Spearman's rank correlation test. Spearman's rank correlation coefficients (r) and P values are indicated in each graph.