Comparison of interferon-γ-, interleukin (IL)-17- and IL-22-expressing CD4 T cells, IL-22-expressing granulocytes and proinflammatory cytokines during latent and active tuberculosis infection

Abstract

In this study, we investigated the role and expression of T helper type 17 (Th17) cells and Th17 cytokines in human tuberculosis. We show that the basal proportion of interferon (IFN)-γ-, interleukin (IL)-17- and IL-22-expressing CD4(+) T cells and IL-22-expressing granulocytes in peripheral blood were significantly lower in latently infected healthy individuals and active tuberculosis patients compared to healthy controls. In contrast, CD4(+) T cells expressing IL-17, IL-22 and IFN-γ were increased significantly following mycobacterial antigens stimulation in both latent and actively infected patients. Interestingly, proinflammatory IFN-γ and tumour necrosis factor (TNF)-α were increased following antigen stimulation in latent infection. Similarly, IL-1β, IL-4, IL-8, IL-22 and TNF-α were increased in the serum of latently infected individuals, whereas IL-6 and TNF-α were increased significantly in actively infected patients. Overall, we observed differential induction of IL-17-, IL-22- and IFN-γ-expressing CD4(+) T cells, IL-22-expressing granulocytes and proinflammatory cytokines in circulation and following antigenic stimulation in latent and active tuberculosis.

Fig. 1

The frequency of interferon (IFN)-γ-, interleukin (IL)-17- and IL-22-expressing CD4⁺ T cells are lower in tuberculosis (TB)-infected subjects. Left panel: the percentage of IFN-γ-, IL-17- and IL-22-expressing CD4⁺ T cells of each participant is shown in (a), (b) and (c), respectively. The broken horizontal lines represent the cut-off values of each test. The cut-off value is generated using receiver operating characteristic (ROC) curves for (a), (b) and (c), which are 2·5, 3 and 4, respectively. The ROC cut-off values are based on optimal sensitivity and specificity values between each group. In (a), the sensitivity values between healthy versus latent TB and healthy versus active TB subjects was 0·88 and 1, respectively, whereas the specificity value between both the groups was 0·5. In (b), the sensitivity values between healthy versus latent TB and healthy versus active TB subjects was 0·52 and 1, respectively, whereas the specificity value between both the groups was 0·4. In (c), the sensitivity values between healthy versus latent TB and healthy versus active TB subjects was 0·75 and 1, respectively, whereas the specificity value between both the groups was 0·5. Left panel group comparisons were performed with Wilcoxon tests. Right panel: based on the cut-off values, the percentage of subjects expressing IFN-γ, IL-17 and IL-22 on CD4⁺ T cells was compared for the healthy (n = 10), latent (n = 21) and active TB groups (n = 9). The P-values are calculated by Fisher's exact tests. *P < 0·05. All other combinations of groups are not significant, with P-values greater than 0·05.

Fig. 2

Circulating levels of interleukin (IL)-22-expressing granulocytes are lower in individuals with latent and active infection. Cells enriched in granulocytes were gated according to size and cytoplasmic structure (forward-scatter and side-scatter; a, left panel) and further gated on CD4^-CD8^- cells (a, middle panel). These cells express IL-22 as detected by flow cytometry (a, right panel). The percentage of IL-22-expressing CD4^-CD8^- granulocytes is shown in (b) (P = 0·01). The percentage of IL-22-producing granulocytes is greater in healthy controls than in latent and active tuberculosis (TB). IL-22 mRNA expression [quantitative polymerase chain reaction (qPCR)] in unstimulated and phorbol myristate acetate (PMA)-stimulated granulocytes (4, 24, 48 h) isolated from healthy controls (n = 3) (c). Data were analysed using the Kruskal–Wallis test and each possible pair was analysed further using Dunn's multiple comparison test. *P < 0·05. All other combinations of groups are not significant, with P-values greater than 0·05.

Fig. 3

Interleukin (IL)-17-, IL-22- and interferon (IFN)-γ-expressing CD4⁺ T cells are induced in individuals with active tuberculosis (TB) infection following stimulation with mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) (1 × 10⁶/ml) were cultured in the presence or absence of mycobacterial culture filtrate for 7 days. Intracellular IFN-γ (a), IL-17 (b) and IL-22 (c) expression in CD4⁺ T cells was detected by flow cytometry. (a,b,c) Percentage frequency of IFN-γ⁺ (n = 7), IL-17⁺ (n = 10) and IL-22⁺ (n = 8)-expressing CD4⁺ T cells, respectively. US, unstimulated group; ST, stimulated group. Data were analysed by the Kruskal–Wallis test. Each possible pair was analysed further by Dunn's multiple comparison tests. All other combinations of groups are not significant, with P-values greater than 0·05. *P < 0·05; **P < 0·01.

Fig. 4

Proinflammatory interleukin (IL)-17, IL-22 and interferon (IFN)-γ are up-regulated following mycobacterial stimulation in individuals with latent tuberculosis (TB) infection. Purified peripheral blood mononuclear cells (PBMCs) from healthy and Mycobacterium tuberculosis-infected subjects were cultured in the presence of M. bovis culture filtrate and supernatants were collected after 7 days. Cytokine levels were measured using human T helper type 1 (Th1)/Th2 cytokine assay kit by flow cytometry. A mean ± standard error of the mean value among each group has been shown. Left panel shows the cytokine levels from individual subjects among the groups. Right panel shows the fold increase over unstimulated controls in healthy controls (n = 11), latent (n = 21) and active TB patients (n = 9). Data are analysed using the Kruskal–Wallis test. Each possible pair is analysed further by Dunn's multiple comparison test. All other combinations of groups are not significant, with P-values greater than 0·05. *P < 0·05.

Fig. 5

Secretion of interleukin (IL)-8, IL-6, tumour necrosis factor (TNF)-α and IL-1β in culture supernatants upon stimulation with mycobacterium culture filtrate. Purified peripheral blood mononuclear cells (PBMCs) from healthy latent and active tuberculosis (TB) subjects were cultured in the presence of M. bovis culture filtrate and supernatant was collected after 7 days. Cytokine levels were measured using human T helper type 1 (Th1)/Th2 cytokine assay kit by flow cytometry. A mean ± standard error of the mean values among each group has been shown. Data are analysed using the Kruskal–Wallis test. Each possible pair is analysed further by Dunn's multiple comparison tests. US, unstimulated group; ST, stimulated group. All other combinations of groups are not significant, with P-values greater than 0·05. *P < 0·05.

Fig. 6

Serum levels of interferon (IFN)-γ, interleukin (IL)-17A, IL-22, IL-8, IL-6, tumour necrosis factor (TNF)-α, IL-1β and IL-4 in subjects with latent tuberculosis (TB), active TB and healthy controls. Cytokines were measured using human T helper type 1 (Th1)/Th2 cytokine assay kit by flow cytometry. Data are analysed using the Kruskal–Wallis test. Each possible pair is analysed further by Dunn's multiple comparison test or Mann–Whitney U-test. Results are shown as mean ± standard deviation. All other combination of groups are not significant, with P-values greater than 0·05. *P < 0·05; **P < 0·01; ***P < 0·001.