Imbalanced IL10/TGF-β production by regulatory T-lymphocytes in patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis

Abstract

Background: Human T-cell lymphotropic virus type 1 (HTLV-1), also denominated Human T-cell leukemia virus-1, induces immune activation and secretion of proinflammatory cytokines, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Regulatory T lymphocytes (Tregs) may control of inflammation through the production of regulatory cytokines, including IL10 and TGF-β. In this study we determined the frequencies of CD4 + and CD8 + Tregs in a HAM/TSP population, compared to asymptomatic carriers and uninfected individuals, as well as investigated the profiles of regulatory and inflammatory cytokines.

Methods: Asymptomatic HTLV-1 carriers and HAM/TSP patients were matched by sex and age. The frequencies of IL10- and/or TGF-β-producing Tregs were quantified by flow cytometry. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify HTLV-1 proviral load and the mRNA expression of cytokines and cellular receptors in peripheral blood mononuclear cells.

Results: Total frequencies of CD4 + Tregs, as well as the IL10-producing CD4 + and CD8 + Treg subsets, were statistically higher in patients with HAM/TSP compared to asymptomatic HTLV-1-infected individuals. In addition, a positive correlation was found between the frequency of CD4 + IL10 + Tregs and proviral load in the HAM/TSP patients evaluated. A positive correlation was also observed between gene expression of proinflammatory versus regulatory cytokines only in HAM / TSP group.

Conclusions: A higher frequencies of IL10-producing Tregs were identified in patients with HAM/TSP. Imbalanced production of IL10 in relation to TGF-β may contribute to the increased inflammatory response characteristically seen in HAM/TSP patients.

Keywords: HTLV-1; HTLV-associated myelopathy; IL10; Regulatory T lymphocytes; TGF-β.

Fig. 1

Frequencies of CD4 + and CD8 + Treg cells in HAM/TSP, asymptomatic HTLV-1-infected and uninfected individuals. (A) Representative gating strategies to select FoxP3 + and CD25 + cells in CD4 + and CD8 + T cell subsets. (B) Frequency of CD4 + Treg cells in HAM/TSP patients (n = 15), asymptomatic HTLV-1-infected individuals (n = 17) and uninfected controls (n = 15). (C) Frequencies of CD8 + Treg cells in these same groups. Bars represent median cell frequencies and interquartile ranges. *Significant difference as calculated using the Kruskal-Wallis test, followed by Dunn’s multiple comparison

Fig. 2

Profile of IL10 and TGF-β production by CD4 + and CD8 + Treg cells in HAM/TSP, HTLV-1-infected and uninfected individuals. (A) Representative gating strategy for CD4 + FOXP3 + CD25 + cells (CD4 + Treg) and CD8 + FOXP3 + CD25+ (CD8 + Treg) cells. Frequency of CD4 + Treg cells (B) and CD8 + Treg cells (D) producing IL10 and/or TGF-β in HAM/TSP patients (n = 15), asymptomatic individuals (n = 17) and uninfected controls (n = 15). Pie charts with the distribution profile of cytokine producing CD4 + and CD8 + Treg cells (C and E). Data expressed as medians and interquartile ranges. *Significant differences as determined by the Kruskal-Wallis test, followed by Dunn’s multiple comparison post-test

Fig. 3

Correlation between proviral load (copies/10⁶ PBMCs) and frequency of IL10 + TGF-b- CD4 + Treg cells in HTLV-1-infected individuals diagnosed with HAM/TSP (n = 7) (Spearman’s correlation, p < 0.05)

Fig. 4

Evaluation of cytokine/chemokine and receptor gene expression in HAM/TSP patients and asymptomatic individuals. (A) Gene expression of CCR4 (2^dCt) in HAM/TSP patients (n = 6) and asymptomatic individuals (n = 10). *Significant differences as determined by the Mann-Whitney test. (B) Correlation between proviral load (copies/106 PBMCs) and TFG-b gene expression in asymptomatic HTLV-1-infected individuals. (C) Correlation between gene expression of inflammatory cytokines (IFN-g, IL1b and TNF) and regulatory cytokines (IL10 and TGF-b) in HTLV-1-infected individuals diagnosed with HAM/TSP. Spearman’s correlation, p < 0.05)