IL-10 dependent suppression of type 1, type 2 and type 17 cytokines in active pulmonary tuberculosis

Abstract

Background: Although Type 1 cytokine responses are considered protective in pulmonary tuberculosis (PTB), their role as well as those of Type 2, 17 and immunoregulatory cytokines in tuberculous lymphadenitis (TBL) and latent tuberculosis (LTB) have not been well studied.

Aim and methods: To identify cytokine responses associated with pulmonary tuberculosis (TB), TB lymphadenitits and latent TB, we examined mycobacterial antigen-specific immune responses of PTB, TBL and LTB individuals. More specifically, we examined ESAT-6 and CFP-10 induced Type 1, Type 2 and Type 17 cytokine production and their regulation using multiplex ELISA.

Results: PTB individuals exhibited a significantly lower baseline as well as antigen-specific production of Type 1 (IFNγ, TNFα and IL-2); Type 2 (IL-4) and Type 17 (IL-17A and IL-17F) cytokines in comparison to both TBL and LTB individuals. TBL individuals exhibited significantly lower antigen-specific IFNγ responses alone in comparison to LTB individuals. Although, IL-10 levels were not significantly higher, neutralization of IL-10 during antigen stimulation resulted in significantly enhanced production of IFNγ, IL-4 and IL-17A in PTB individuals, indicating that IL-10 mediates (at least partially) the suppression of cytokine responses in PTB.

Conclusion: Pulmonary TB is characterized by an IL-10 dependent antigen-specific suppression of Type 1, Type 2 and Type 17 cytokines, reflecting an important association of these cytokines in the pathogenesis of active TB.

Figure 1. PTB is associated with decreased spontaneous production of Type 1, 2, 17 and immunoregulatory cytokines.

Whole blood from PTB, TBL and LTB individuals was stimulated with media alone for 72 h, and levels of (A) Type 1 cytokines IFNγ, TNFα and IL-2; (B) Type 2 cytokines IL-4, IL-5, and IL-13; (C) Type 17 cytokines IL-17A, IL-17F and IL-22 and (D) Immunoregulatory cytokines IL-10 and TGFβ were measured by ELISA. Results are shown as scatterplots with each dot representing a single individuals. P values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (* p<0.05, ** p<0.01, *** p<0.001).

Figure 2. PTB is associated with decreased antigen-stimulated production of Type 1 cytokines.

Whole blood from PTB, TBL and LTB individuals was stimulated with (A) PPD (10 µg/ml) or (B) ESAT-6 (10 µg/ml) or (C) CFP-10 (10 µg/ml) or (D) anti-CD3 (5 µg/ml) for 72 h, and levels of Type 1 cytokines IFNγ, TNFα and IL-2 were measured by ELISA. Results are shown as net cytokine production over media control. The bars represent geometric means and 95% confidence intervals. P values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons comparisons (* p<0.05, ** p<0.01, *** p<0.001).

Figure 3. PTB is associated with decreased antigen-stimulated production of IL-4.

Whole blood from PTB, TBL and LTB individuals was stimulated with (A) PPD (10 µg/ml) or (B) ESAT-6 (10 µg/ml) or (C) CFP-10 (10 µg/ml) or (D) anti-CD3 (5 µg/ml) for 72 h, and levels of Type 2 cytokines IL-4, IL-5 and IL-13 were measured by ELISA. Results are shown as net cytokine production over media control. The bars represent geometric means and 95% confidence intervals. P values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons comparisons (* p<0.05, ** p<0.01, *** p<0.001).

Figure 4. PTB is associated with decreased antigen-stimulated production of Type 17 cytokines.

Whole blood from PTB, TBL and LTB individuals was stimulated with (A) PPD (10 µg/ml) or (B) ESAT-6 (10 µg/ml) or (C) CFP-10 (10 µg/ml) or (D) anti-CD3 (5 µg/ml) for 72 h, and levels of Type 17 cytokines IL-17A, IL-17F and IL-22 were measured by ELISA. Results are shown as net cytokine production over media control. The bars represent geometric means and 95% confidence intervals. P values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons comparisons (* p<0.05, ** p<0.01, *** p<0.001).

Figure 5. PTB is not associated with antigen – induced alterations in immunoregulatory cytokines.

Whole blood from PTB, TBL and LTB individuals was stimulated with (A) PPD (10 µg/ml) or (B) ESAT-6 (10 µg/ml) or (C) CFP-10 (10 µg/ml) or (D) anti-CD3 (5 µg/ml) for 72 h, and levels of immunoregulatory cytokines IL-10 and TGFβ were measured by ELISA. Results are shown as net cytokine production over media control. The bars represent geometric means and 95% confidence intervals. P values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons comparisons (* p<0.05, ** p<0.01, *** p<0.001).

Figure 6. Neutralization of IL-10 but not TGFβ significantly enhances cytokine production in PTB.

Whole blood from PTB individuals was stimulated with PPD (10 µg/ml) in the presence of anti-IL-10 Ab or anti-TGFβ Ab or isotype controls for 72 h and the levels of IFNγ, IL-4 and IL-17A were measured by ELISA. Results are shown as line graphs with each line representing a single PTB individual (n = 9). Results are shown as net cytokine production over media control. P values were calculated using the Wilcoxon signed rank test.