Pro- and anti-inflammatory cytokines against Rv2031 are elevated during latent tuberculosis: a study in cohorts of tuberculosis patients, household contacts and community controls in an endemic setting

Abstract

Tuberculosis (TB) is among the leading causes of morbidity and mortality. The causative agent, Mycobacterium tuberculosis (Mtb), has evolved virulent factors for entry, survival, multiplication and immune evasion. Rv2031 (also called alpha crystallin, hspX, 16-kDa antigen), one of the most immunogenic latency antigens, is believed to play a key role in long-term viability of Mtb. Here, we report the dynamics of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokines against Rv2031 using whole blood assay in human cohorts in a TB endemic setting. Cytokine responses to ESAT-6-CFP-10 were also measured for comparison. Blood samples were collected from smear positive pulmonary TB patients and their contacts at baseline, 6 and 12 months, and from community controls at entry. At baseline, 54.4% of controls and 73.2% of contacts were QFT-GIT test positive. Baseline IFN-γ, TNF-α and IL-10 responses to Rv2031 were significantly higher in controls compared to contacts and untreated patients (p<0.001). Furthermore, untreated patients had significantly higher TNF-α and IL-10 responses to Rv2031 compared to contacts (p<0.001). In contacts and treated patients, IFN-γ, TNF-α and IL-10 responses to Rv2031 significantly increased over 12 months (p<0.0001) and became comparable with the corresponding levels in controls. There was a positive and significant correlation between Rv2031 and ESAT-6-CFP-10 specific cytokine responses in each study group. The fact that the levels of IFN-γ, TNF-α and IL-10 against Rv2031 were highest during latent TB infection may indicate their potential as markers of protection against TB. Taken together, the findings of this study suggest the potential of IFN-γ, TNF-α and IL-10 against Rv2031 as biomarkers of the host response to Mtb during convalescence from, and the absence of, active tuberculosis.

Fig 1. Comparison of IFN-γ response to Rv2031 among groups at baseline, 6 and 12 months.

IFN-γ response at baseline (a), 6 months (b) and 12 months (c) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent patients, filled diamonds represent contacts and filled triangles represent controls. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Kruskal-Wallis test with Dunn’s multiple comparisons (a) and Mann-Whitney test (b and c) were used to compare groups. ****p<0.0001.

Fig 2. Changes in IFN-γ response to Rv2031 over 12 months in patients and contacts.

IFN-γ response over 12 months in patients (a) and contacts (b) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent baseline levels, filled diamonds represent 6 month levels and filled triangles represent 12 month levels. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Freidman test with Dunn’s multiple comparisons was used to compare IFN-γ response over time. ****p<0.0001.

Fig 3. Comparison of TNF-α response to Rv2031 among groups at baseline, 6 and 12 months.

TNF-α response at baseline (a), 6 months (b) and 12 months (c) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent patients, filled diamonds represent contacts and filled triangles represent controls. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Kruskal-Wallis test with Dunn’s multiple comparisons (a) and Mann-Whitney test (b and c) were used to compare groups. ***p<0.001, ****p<0.0001.

Fig 4. Changes in TNF-α response to Rv2031 over 12 months in patients and contacts.

TNF-α response over 12 months in patients (a) and contacts (b) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent baseline levels, filled diamonds represent 6 month levels and filled triangles represent 12 month levels. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Freidman test with Dunn’s multiple comparisons was used to compare TNF-α response over time. ***p<0.001, ****p<0.0001.

Fig 5. Comparison of IL-10 response to Rv2031 among groups at baseline, 6 and 12 months.

IL-10 response at baseline (a), 6 months (b) and 12 months (c) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent patients, filled diamonds represent contacts and filled triangles represent controls. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Kruskal-Wallis test with Dunn’s multiple comparisons (a) and Mann-Whitney test (b and c) were used to compare groups. ***p<0.001, ****p<0.0001.

Fig 6. Changes in IL-10 responses to Rv2301 over 12 months in patients and contacts.

IL-10 response over 12 months in patients (a) and contacts (b) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent baseline levels, filled diamonds represent 6 month levels and filled triangles represent 12 month levels. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Freidman test with Dunn’s multiple comparisons was used to compare IL-10 response over time. ****p<0.0001.

Fig 7. Comparison of IFN-γ/IL-10 ratio among groups at baseline, 6 and 12 months.

IFN-γ/IL-10 ratio at baseline (a), 6 months (b) and 12 months (c) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent patients, filled diamonds represent contacts and filled triangles represent controls. Results are individual responses. Horizontal bars are medians. Kruskal-Wallis test with Dunn’s multiple comparisons (a) and Mann-Whitney test (b and C) were used to compare groups. ****p<0.0001.

Fig 8. Comparison of TNF-α/IL-10 ratio among groups at baseline, 6 and 12 months.

TNF-α/IL-10 ratio at baseline (a), 6 months (b) and 12 months (c) following whole blood stimulation with Rv2031 for 48 hours. Filled circles represent patients, filled diamonds represent contacts and filled triangles represent controls. Results are individual responses. Horizontal bars are medians. Kruskal-Wallis test with Dunn’s multiple comparisons (a) and Mann-Whitney test (b and C) were used to compare groups. *p = 0.03, **p<0.01, ****p<0.0001.

Fig 9. Comparison of baseline cytokine responses to E6C10 among groups.

Baseline IFN-γ (a), TNF-α (b) and IL-10 (c) responses to E6C10 in patients, contacts and controls following whole blood stimulation for 48 hours. Filled circles represent patients, filled diamonds represent contacts and filled triangles represent controls. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Kruskal-Wallis test with Dunn’s multiple comparisons was used to compare groups. **p<0.01, ****p<0.0001.

Fig 10. Changes in cytokine responses to E6C10 over 12 months in patients.

IFN-γ (a), TNF-α (b) and IL-10 (c) responses to E6C10 in patients at baseline, 6 and 12 months following whole blood stimulation with E6C10 for 48 hours. Filled circles represent baseline levels, filled diamonds represent 6 month levels and filled triangles represent 12 month levels. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Freidman test with Dunn’s multiple comparisons was used to compare cytokine levels overtime.

Fig 11. Changes in cytokine responses to E6C10 over 12 months in contacts.

IFN-γ (a), TNF-α (b) and IL-10 (c) responses to E6C10 in contacts at baseline, 6 and 12 months following whole blood stimulation with E6C10 for 48 hours. Filled circles represent baseline levels, filled diamonds represent 6 month levels and filled triangles represent 12 month levels. Results are individual responses and cytokine levels are expressed as log₁₀ pg/ml. Horizontal bars are medians. Freidman test with Dunn’s multiple comparisons was used to compare cytokine levels overtime. **p<0.01, ****p<0.0001.