Constitutive STAT3 phosphorylation and IL-6/IL-10 co-expression are associated with impaired T-cell function in tuberculosis patients

Abstract

T-cells critically contribute to protection against Mycobacterium tuberculosis infection, and impaired T-cell responses can lead to disease progression. Pro-inflammatory and immunosuppressive cytokines affect T-cells, and fine-tuned regulation of cytokine signaling via the Jak/STAT signaling pathways is crucial for appropriate T-cell function. Constitutive STAT3 phosphorylation as a consequence of aberrant cytokine signaling has been described to occur in pathognomonic T-cell responses in inflammatory and autoimmune diseases. We characterized blood samples from tuberculosis patients (n=28) and healthy contacts (n=28) from Ghana for M. tuberculosis-specific T-cell responses, constitutive cytokine production, and SOCS3 and pSTAT3 expression. Lentiviral modulation of primary CD4⁺ T-cells was performed to determine the effects of SOCS3 on T-cell functions. T-cells from tuberculosis patients expressed higher levels of IL-10 and IL-6 and lower levels of T helper type (T_H)17 cytokines after M. tuberculosis-specific stimulation compared to healthy contacts. In addition, tuberculosis patients had higher IL-10 and IL-6 levels in the supernatants of non-stimulated immune cells and plasma samples compared to healthy contacts. Notably, aberrant cytokine expression was accompanied by high constitutive pSTAT3 levels and SOCS3 expression in T-cells. Multivariate analysis identified an IL-6/IL-10 co-expression-based principal component in tuberculosis patients that correlated with high pSTAT3 levels. SOCS3 contributed to a regulatory component, and tuberculosis patients with high SOCS3 expression showed decreased T_H1 cytokine expression and impaired IL-2-induced STAT5 phosphorylation. SOCS3 over-expression in primary CD4⁺ T-cells confirmed the SOCS3 inhibitory function on IL-2-induced STAT5 phosphorylation. We conclude that constitutive pSTAT3 and high SOCS3 expression are influential factors that indicate impaired T-cell functions in tuberculosis patients.

Keywords: Interleukin-10; Interleukin-6; SOCS3; STAT3; tuberculosis.

Figure 1

M. tuberculosis PPD-specific and constitutive cytokine expression and plasma cytokine concentrations from tuberculosis patients and healthy contacts. Cytokine concentrations of IFN-γ, TNF-α, IL-17F, IL-22, IL-21, IL-6, and IL-10 of supernatants from diluted whole blood after 72 h in vitro culture with (a and b) or without (c and d) M. tuberculosis PPD are depicted. Association of differentially expressed cytokines are shown in (b) for PPD-specific IL-17F and IL-22, and (d) for constitutive IL-6 and IL-10 values. (e) Plasma concentrations of IL-10, IL-21, IL-23, IL-27 are shown for healthy contacts and tuberculosis patients. The dotted line indicates the detection limit for the respective cytokines. Symbols placed on this line indicate values below the detection limit. All samples were measured in duplicate, and mean values are indicated as open circles for healthy contacts and grey squares for tuberculosis patients. Study group medians and percentiles (25, 75) are shown. Significant differences are indicated by asterisks. Nominal P-values for the Mann-Whitney U-test (two-tailed) were calculated and shown as * for P<0.05, ** for P<0.01, *** for P<0.001, and **** for P<0.0001. The Spearman Rank test was used to determine significant correlations for all donors and both study groups separately. Correlation coefficients (rho) and nominal P-values are given. ns: not significant, nd: not detectable.

Figure 2

Constitutive and IL-6-induced STAT3 phosphorylation and SOCS3 expression in CD4⁺ T-cells from tuberculosis patients and healthy contacts. (a–c) Phosphorylation of STAT3 with or without IL-6 in vitro stimulation and (d) SOCS3 expression of CD4⁺ T-cells from tuberculosis patients and healthy contacts. (a) Representative histograms indicating non-stimulated (w/o) and IL-6-induced pSTAT3 expression of samples from a tuberculosis patient (left graph) and a healthy contact (right graph) are shown. Dotted lines indicate similar mean fluorescence intensities (MFI) of non-stained control samples for both respective study groups. (b) Study group comparisons of non-stimulated (w/o) and IL-6-induced pSTAT3 levels are shown. (c) The ratios between non-stimulated pSTAT3 and pSTAT5 values calculated for each individual donor. (d) SOCS3 expression for all CD4⁺ T-cells (left graph) as well as for CD45RA_high and CD45RA_low (right graph) CD4⁺ T-cell subpopulations are shown. Tuberculosis patients are represented by grey squares, healthy contacts are depicted as open circles. Study group medians and percentiles (25, 75) are given. Significant differences are indicated by asterisks. Nominal P-values for the Mann-Whitney U-test (two-tailed) were calculated and shown as * for P<0.05, ** for P<0.01, *** for P<0.001, and **** for P<0.0001. ns: not significant. (e) Correlation plots for constitutive pSTAT3 and SOCS3 expression are shown for CD4⁺ T-cells. The Spearman Rank test was used to determine significant correlations for all donors and both study groups separately. Correlation coefficients (rho) and nominal P-values are given.

Figure 3

M.tuberculosis-specific activation and intracellular cytokine expression in association with SOCS3 expression in both study groups. (a) Representative graphs depicting the gating procedure for intracellular cytokine analysis are shown. (b) Proportions of CD40L/IL-2-positive CD4⁺ T-cells are shown for tuberculosis patients (grey squares) and healthy contacts (open circles). (c) Correlation plots between SOCS3 expression and CD40L/IL-2 (upper graph) as well as CD40L/IFN-γ (lower graph) are shown for CD4⁺ T-cells. The Spearman Rank test was used to determine significant correlations for all donors and both study groups separately. Correlation coefficients (rho) and nominal P-values are given. ns: not significant.

Figure 4

IL-2-induced pSTAT5 levels in T-cells from both study groups, and effects of lentiviral SOCS3 modulation on IL-2-induced pSTAT5. (a and b) IL-2-induced STAT5 phosphorylation of CD4⁺ T-cells is shown for tuberculosis patients (grey squares) and healthy contacts (open circles). (b) Correlation of IL-2-induced pSTAT5 with SOCS3 expression. The Spearman Rank test was used to determine significant correlations for all donors and both study groups separately. Correlation coefficients (rho) and nominal P-values are given. (c) Representative flow cytometry histogram indicating fluorescence (eBFP) expression of non-transduced, vector control-, SOCS3cDNA-, and SOCS3shRNA-transduced CD4⁺ T-cells are shown. (d) Flow cytometry-based quantification of CD4⁺ T-cells with SOCS3cDNA (dark grey) and SOCS3shRNA (black line, open) compared to vector control (bright grey). The results from seven experiments are shown. (e) STAT5 phosphorylation in CD4⁺ T-cells from three experiments with increased or reduced SOCS3 expression. Nominal P-values for the paired t Test (two-tailed) were shown. MFI, mean fluorecence intensity.

Figure 5

Multivariate analyses of influential factors and correlation with constitutive pSTAT3 in tuberculosis patients and healthy contacts. (a) Correlation of principal component (PC)-1 (left graph) and PC-2 (right graph) with constitutive pSTAT3 levels. The Spearman Rank test was used to determine significant correlations for all donors and both study groups separately. Correlation coefficients (rho) and nominal P-values are given. (b) Regression scores for PC-1 (left graph) and PC-2 (right graph) are compared between tuberculosis patients and healthy contacts. Data are presented as medians, and nominal P-values for the Mann-Whitney U-test (two-tailed) are shown.