HTLV-1 bZIP Factor Induces Inflammation through Labile Foxp3 Expression.

Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4(+)Foxp3(+) T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4(+) T cells were enhanced in these mice. Foxp3(-)CD4(+) T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3(-)T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4(+) T cells is implicated in the pathogenesis of HTLV-1-associated inflammation

DNA methylation status in the promoter and intronic CpG island region of the <i>Foxp3</i> gene.

<p>(A) The purity of the isolated Treg cells, sorted from the spleens of male mice, was confirmed by staining the intracellular expression of Foxp3 and analysis by flow cytometry. (B) DNA methylation status in the indicated regions was determined by bisulfite sequencing. Each line represents one analyzed clone; open circles, unmethylated CpGs and filled circles, methylated CpGs.</p

Stability of Foxp3 expression during <i>ex vivo</i> culture.

<p>(A) Treg cells, sorted from HBZ-Tg or non-Tg mice, were cultured in the presence of IL-2 for 3 or 7 days. The expression of Foxp3 was analyzed by flow cytometry. (B) Sequential changes of the Foxp3⁻ population are shown. (C) IFN-γ production of <i>ex vivo</i> cultured Foxp3⁺ cells was evaluated by intracellular staining. Sorted Treg cells were cultured for 7 days, and then stimulated for 4 h with PMA/ionomycin and protein transport inhibitor. (D) Foxp3 expression of sorted CD4⁺CD25⁺GITR^highthymocytes from HBZ-Tg mice.</p

Expression of CD11a, CD18 and CD103 in CD4⁺T cells from spleen, lung and LN cells isolated from HBZ-Tg mice.

<p>(A) The expression of CD11a, CD18 and CD103 in CD4⁺ T cells from non-Tg (dashed line) and HBZ-Tg (solid line) mice was analyzed by flow cytometry. Histograms from one representative mouse splenocytes of each group are shown (top panels). The bottom panel shows the results of 4 or 6 mice in each group, each symbol representing an individual mouse. The small horizontal lines indicate the mean. Frozen sections of intestine (B) and lung (C) of non-Tg and HBZ-Tg mice were stained with HE and the indicated antibodies. Original magnification is ×20. Results from one representative mouse of each group are shown. (D) CD11a, CD18 and CD103 expressions are shown on CD4⁺ cells from HDs, CD4⁺Tax⁻ and CD4⁺Tax⁺ cells from HAM/TSP patients.</p

Production of cytokines in HBZ-Tg mice.

<p>(A) Splenocytes of HBZ-Tg mice or non-Tg mice were stimulated with PMA/ionomycin and protein transport inhibitor for 4 h. IFN-γ, IL-17, TNF-α, IL-4 or IL-2 production was analyzed in CD4⁺ T cells by flow cytometry. (B) Cytokine production was analyzed along with Foxp3 expression. (C) Production of cytokines was shown in CD4⁺Foxp3⁺ T cells and CD4⁺Foxp3⁻ T cells. (D) IFN-γ and Foxp3 expression gated on CD4⁺ T cells from PBMC or cells isolated from the lungs were analyzed by flow cytometry. Percentage of IFN-γ⁺ cells in CD4⁺ splenocytes, PBMC and lung cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean.</p