HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection

Abstract

Background: To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI.

Methods: Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27).

Results: CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04).

Conclusion: Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

Keywords: HBHA; IFN-γ; IL-10; IL-17; LTBI; Mycobacterium tuberculosis (M.tb); TB.

Figure 1

Characteristics of cytokine responses during tuberculosis antigen stimulation in the TB and LTBI groups with flowcytometry. PBMCs isolated from subjects with TB or LTBI were stimulated with ESAT-6/CFP-10, Acr or HBHA. The percentages were obtained under stimulated conditions minus those obtained under unstimulated conditions for controls. (A–C) Representative FACS plots of IFN-γ (A), IL-10 (B), and IL-17 (C) expression in CD4+ cells stimulated with PMA. (D–F) Box plot with dot plot shows the concentrations of cytokines following each antigen stimulation of PBMC in TB (n=15) or LTBI (n=15) groups analyzed by flow cytometry. IFN-γ for (D), IL-10 for (E), IL-17 for (F). Statistical significance was determined using the Wilcoxon rank sum test. **p < 0.01.

Figure 2

Differentiation of memory T cells stimulated by various tuberculosis antigens. Statistical significance was determined by Wilcoxon rank sum test. The percentages of antigen-specific Tcm and Tem were obtained by subtracting the baseline values of NIL samples. (A) Contour and dot plots from flow cytometry analysis representing memory T cells (Tm) among CD3+CD4+ cells. Tm cells (right box) are defined as central memory T cells (Tcm, CD27+CD45RO+ cells) and effector memory T cells (Tem, CD27-CD45RO+ cells). (B) Box plot with dot plot shows the frequencies of Tcm in CD3+CD4+ T cells following antigen stimulation of PBMC in TB (n=15) and LTBI (n=15) groups. (C) Box plot with dot plot shows the frequencies of Tem in CD3+CD4+ T cells following antigen stimulation of PBMC in TB (n=15) or LTBI (n=15) groups.

Figure 3

The proportion of cytokine-expressing cells in Tcm and Tem subsets. (A–F) Frequencies of background-subtracted M.tb antigen-specific cytokine-expressing cells within Tcm (Central Memory T cells) for (A–C) and Tem (Effector Memory T cells) for (D–F) subsets. This figure illustrates the distribution of IFN-γ (A, D), IL-10 (B, E), and IL-17 (C, F) expressing cells in response to Mycobacterium tuberculosis antigen stimulation. PBMC collected from TB (n=15) and LTBI (n=15) groups were analyzed by flow cytometry. Statistical significance was determined by Wilcoxon rank sum test. *p < 0.05, **p < 0.01.

Figure 4

Characteristics of cytokine expressing cells during HBHA stimulation in the TB and LTBI groups with flowcytometry. Boxplot overlaid with dot plot illustrating the distribution of cytokine expressing cells following with HBHA stimulation in the TB and LTBI groups. (A) IL-10+FoxP3+CD25+ CD4 Tcells, (B) IL-17+FoxP3+CD25+ CD4+T cells and (C) CCR6+IL-17+ CD4+T cells. PBMC in TB (n=15) or LTBI (n=15) group was analyzed by flow cytometry.Statistical significance was determined by Wilcoxon rank sum test. *p < 0.05.

Figure 5

IFN-γ and IL-10/IL-17 double positive cells across different M.tb antigens. Boxplot overlaid with a dot plot showing the distribution of double-positive cells expressing both IFN-γ and IL-10 (above)/IFN-γ and IL-17 (below) in CD4+ T cells across different antigens (E/C, Acr and HBHA). The Y-axis is displayed on a logarithmic scale to accommodate a wide range of values, with the minimum value set to 0.1. PBMC in TB (n=15) or LTBI (n=15) group was analyzed by flow cytometry.Statistical significance was determined by Wilcoxon rank sum test. *p < 0.05, **p < 0.01.

Figure 6

Characteristics of cytokine responses during tuberculosis antigen stimulation in the TB and LTBI groups with ELISA. (A–C) Box plot with dot plot shows the cytokine following each antigen stimulation to PBMC in TB (n=26) or LTBI (n=27) group analysed by ELISA.IFN-γ for (A), IL-10 for (B), IL-17 for (C). (D, E) Scatter plot depicting the relationship between IL-10 and IFN-γ levels induced with HBHA (D) and ESAT-6/CFP-10 (E) stimulation for categories ‘TB’ (red) and ‘LTBI’ (blue). Each point represents a data point within a category.Y-axis represents IFN-γ levels and X-axis represents IL-10 levels. The plot includes regression lines fitted with a linear model that accounts for the interaction term between the cytokines. P-values indicate the significance of the interaction effect between the two cytokines (HBHA,p=0.020; ESAT-6/CFP-10,p=0.292). Points are colored and shaped according to their respective categories (TB; Red circle, LTBI; Blue triangle). Statistical significance was determined by Wilcoxon rank sum test. *p < 0.05, **p < 0.01.