IL-10-mediated signals act as a switch for lymphoproliferation in Human T-cell leukemia virus type-1 infection by activating the STAT3 and IRF4 pathways

<div><p>Human T-cell leukemia virus type-1 (HTLV-1) causes two distinct diseases, adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since there are no disease-specific differences among HTLV-1 strains, the etiological mechanisms separating these respective lymphoproliferative and inflammatory diseases are not well understood. In this study, by using IL-2-dependent HTLV-1-infected T-cell lines (ILTs) established from patients with ATL and HAM/TSP, we demonstrate that the anti-inflammatory cytokine IL-10 and its downstream signals potentially act as a switch for proliferation in HTLV-1-infected cells. Among six ILTs used, ILTs derived from all three ATL patients grew much faster than those from three HAM/TSP patients. Although most of the ILTs tested produced IFN-γ and IL-6, the production of IL-10 was preferentially observed in the rapid-growing ILTs. Interestingly, treatment with exogenous IL-10 markedly enhanced proliferation of the slow-growing HAM/TSP-derived ILTs. The IL-10-mediated proliferation of these ILTs was associated with phosphorylation of STAT3 and induction of survivin and IRF4, all of which are characteristics of ATL cells. Knockdown of STAT3 reduced expression of IL-10, implying a positive-feedback regulation between STAT3 and IL-10. STAT3 knockdown also reduced survivin and IRF4 in the IL-10- producing or IL-10- treated ILTs. IRF4 knockdown further suppressed survivin expression and the cell growth in these ILTs. These findings indicate that the IL-10-mediated signals promote cell proliferation in HTLV-1-infected cells through the STAT3 and IRF4 pathways. Our results imply that, although HTLV-1 infection alone may not be sufficient for cell proliferation, IL-10 and its signaling pathways within the infected cell itself and/or its surrounding microenvironment may play a critical role in pushing HTLV-1-infected cells towards proliferation at the early stages of HTLV-1 leukemogenesis. This study provides useful information for understanding of disease mechanisms and disease-prophylactic strategies in HTLV-1 infection.</p></div

IL-10 induced STAT3-phosphorylation and mild suppression of NF-κB activity in ILTs.

<p><b>A.</b> Luciferase activities in ILT-H2 and ILT-294 cells containing NF-κB-luc or STAT3-luc and TK-RL reporter genes were measured following incubation with (closed bar) or without (open bar) IL-10 (20 ng/ml) for 48 h. The NF-κB and STAT3 activities were standardized with TK promoter activities, and the relative values against the sample without IL-10 are presented as the mean and SD of duplicate samples. <b>B.</b> The surface IL-10Rα expression (open histogram) on various ILTs was assessed by flow cytometry. The numbers represent relative MFI of IL-10Rα compared with isotype controls (closed histogram). <b>C.</b> ILT cells were cultured with or without IL-10 for two (ILT-294 and -441) or one (ILT-439, -22, -227, and -H2) weeks, and cell lysates were probed with antibodies to phospho-STAT3 (p-STAT3), STAT3, and β-actin in an immunoblotting assay. The numbers under each band represent the relative values of intensity of the band against β-actin. The ratio of the value of p-STAT3 normalized against total STAT3 was also indicated in the bottom of the panel. Representative results of two independent experiments are shown. * <i>p</i><0.05, ** <i>p</i><0.01.</p

Critical roles of IRF4 in expansion of ILTs.

<p><b>A.</b> Intracellular IRF4 expression (solid line) was analyzed in various ILT cells following incubation with or without IL-10 for 7–11 days. Closed histograms represent staining with control antibody. The numbers indicate the proportions (%) of cells with high IRF4 expression. <b>B.</b> ILT-294 (cultured with rhIL-10), ILT-22, and ILT-H2 cells were transfected with si-CTRL and si-IRF4, and the cell lysates were subjected to immunoblot assays for cleaved caspase-3, caspase-3, survivin, and β-actin 48 hours after electroporation. Approximate sizes of caspases are indicated. ^a, ^b, ^c, denote the same images for β-actin because the same membranes were used, respectively. <b>C.</b> Indicated ILTs and Jurkat cells were transfected with si-CTRL, si-STAT3, or si-IRF4, and the total viable cell count were evaluated 3 days after electroporation by trypan blue exclusion test. The data represent the mean and SD of duplicate or triplicate samples. The immunoblot confirming the knockdown efficiency of STAT3 in Jurkat cells is also indicated. IRF4 was not detectable in Jurkat. <b>D.</b> Intracellular IRF4 and Ki67 expression of the ILTs prepared in C were analyzed by flow cytometry. ILT-294 cells were cultured in the presence of IL-10 in B, C, D. Numbers in D indicate the proportion (%) of cells in each quadrant. Similar results were obtained in two independent experiments. * <i>p</i><0.05, ** <i>p</i><0.01.</p

STAT3 knockdown inhibited expression of <i>IL10</i>, <i>BIRC5</i> (<i>survivin</i>), <i>MYC</i>, and <i>IRF4</i>.

<p><b>A</b>. ILT cells were transfected with control siRNA (si-CTRL) (open bar) or si-STAT3 (black bar), and subjected to qRT-PCR 48 h after electroporation. Indicated gene expression levels were measured and standardized with <i>ACTB</i> (<i>β-actin</i>) mRNA levels in each sample. The relative values against the si-CTRL samples are presented as means and SD of duplicate samples. Representative results of two independent experiments are shown. * <i>p</i><0.05, ** <i>p</i><0.01. <b>B.</b> ILT-22 and ILT-294 cells cultured with (■) or without (□) rhIL-10 for at least 1 week, and <i>IL10</i> mRNA levels were measured. The normalized values against <i>ACTB</i> were indicated as means and SD of duplicate samples. N.D., not detected. <b>C.</b> ILT-H2 (●), ILT-22 (▲), and Jurkat (◯) cells were incubated with indicated concentrations of AS101 or vehicle control (0.13% ethanol) for 5 days and the viable cell number was assessed using Cell Counting Kit-8. Relative values against vehicle control were plotted as means and SD of duplicate samples. <b>D</b>. ILT-H2 (▲), ILT-22 (●), ILT-294 (+) (cultured with IL-10) (■), and ILT-294 (-) (cultured without IL-10) (□) cells were incubated with indicated concentrations of Cucurbitacin I or vehicle control (0.1% DMSO) for 2 days, and the cell number was evaluated by Cell Counting Kit-8. The relative values against vehicle controls indicate means and SD of duplicate samples. <b>E.</b> Cell lysates of ILTs were harvested 48 h (ILT-294, ILT-22) or 72 h (ILT-H2) after transfection with si-CTRL or si-STAT3, probed with antibodies to caspase-3, cleaved caspase-3, survivin, and IRF4 in immunoblotting assay, and presented together with the β-actin immunoblots of corresponding membranes. Approximate sizes of caspases are indicated. ^a, ^b, denote the same images for β-actin because the same membranes were used for the detection of caspases and IRF4, respectively. ILT-294 cells were cultured in the presence of IL-10 in <b>A</b> and <b>E</b>.</p

IL-10 enhanced expression of Ki67 and survivin in HAM/TSP-derived ILTs.

<p>ILTs were cultured with or without IL-10 for 7–11 days, and subjected to flow cytometry (A, B) and immunoblot assays (C, D). <b>A</b>. Permeabilized cells were stained with anti-Ki67 (solid line) or control (closed histogram) antibodies. Numbers indicate MFI of Ki-67 expression. <b>B</b>. Cells were stained with Annexin V, and the proportion of apoptotic cells (%) was indicated as the mean and SD of 2–3 independent experiments. The representative flow cytometry data are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006597#ppat.1006597.s003" target="_blank">S2 Fig</a>. <b>C.</b> ILTs cultured with or without IL-10 were treated with MG132 (10 μM) for 3 h, then subjected to immunoblot assays probed with antibodies to caspase-3, cleaved caspase-3, and β-actin. Approximate sizes (kDa) of caspases are indicated. The results of a similar experiment without MG132-treatment is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006597#ppat.1006597.s004" target="_blank">S3 Fig</a>. <b>D.</b> ILTs cultured with or without IL-10 were subjected to immunoblot assays to detect survivin. The number under each band represents the relative value of survivin expression normalized against β-actin. Similar results were obtained in two independent experiments.</p

CSF CXCL10, CXCL9, and Neopterin as Candidate Prognostic Biomarkers for HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis

<div><p>Background</p><p>Human T-lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare chronic neuroinflammatory disease. Since the disease course of HAM/TSP varies among patients, there is a dire need for biomarkers capable of predicting the rate of disease progression. However, there have been no studies to date that have compared the prognostic values of multiple potential biomarkers for HAM/TSP.</p><p>Methodology/Principal Findings</p><p>Peripheral blood and cerebrospinal fluid (CSF) samples from HAM/TSP patients and HTLV-1-infected control subjects were obtained and tested retrospectively for several potential biomarkers, including chemokines and other cytokines, and nine optimal candidates were selected based on receiver operating characteristic (ROC) analysis. Next, we evaluated the relationship between these candidates and the rate of disease progression in HAM/TSP patients, beginning with a first cohort of 30 patients (Training Set) and proceeding to a second cohort of 23 patients (Test Set). We defined “deteriorating HAM/TSP” as distinctly worsening function (≥3 grades on Osame's Motor Disability Score (OMDS)) over four years and “stable HAM/TSP” as unchanged or only slightly worsened function (1 grade on OMDS) over four years, and we compared the levels of the candidate biomarkers in patients divided into these two groups. The CSF levels of chemokine (C-X-C motif) ligand 10 (CXCL10), CXCL9, and neopterin were well-correlated with disease progression, better even than HTLV-1 proviral load in PBMCs. Importantly, these results were validated using the Test Set.</p><p>Conclusions/Significance</p><p>As the CSF levels of CXCL10, CXCL9, and neopterin were the most strongly correlated with rate of disease progression, they represent the most viable candidates for HAM/TSP prognostic biomarkers. The identification of effective prognostic biomarkers could lead to earlier detection of high-risk patients, more patient-specific treatment options, and more productive clinical trials.</p></div

Demographics and clinical characteristics of HAM/TSP patients (Training Set).

<p>In the Training set, deteriorating patients were significantly older, experienced disease onset later in life, had been living with the disease for shorter periods, and were more severely disabled (OMDS).</p>*<p>Stable HAM/TSP vs Deteriorating HAM/TSP.</p>**<p>Data are expressed as median [range].</p>†<p>By Mann-Whitney test.</p>‡<p>By Fisher's exact test.</p><p>OMDS = Osame's Motor Disability Score.</p

Demographics and clinical characteristics of HAM/TSP patients (Test Set).

<p>In the Test set, deteriorating patients experienced disease onset later in life and had been living with the disease for shorter periods, but there were no significant differences in current age or OMDS.</p>*<p>Stable HAM/TSP vs Deteriorating HAM/TSP.</p>**<p>Data are expressed as median [range].</p>†<p>By Mann-Whitney test.</p>‡<p>By Fisher's exact test.</p><p>OMDS = Osame's Motor Disability Score.</p

Inclusion and exclusion criteria for this study.

<p>CLEIA = chemiluminescent enzyme immunoassay.</p

Identification of biomarkers associated with clinical progression of HAM/TSP.

<p>(<b>A</b>) Five CSF marker candidates (CXCL10, CXCL9, neopterin, cell count, and anti-HTLV-1 antibody titer) and four blood marker candidates (proviral load in PBMCs, serum sIL-2R, plasma CXCL9, and plasma CXCL10) were compared among a cohort of patients called the Training Set (deteriorating HAM/TSP, n = 11; stable HAM/TSP, n = 14). Data is shown for the top eight CSF markers ranked according to the significance of the difference between the deteriorating and stable subjects. Black circles indicate patients with particularly rapidly progressive HAM/TSP. Horizontal bars indicate the median values. The Mann-Whitney <i>U</i>-test was used for statistical analysis. (<b>B</b>) ROC analysis was employed to assess the sensitivities and specificities of the nine markers listed above for discriminating deteriorating HAM/TSP patients from stable patients. AUC = area under the ROC curve; 95% CI = 95% confidence interval.</p