Induction of pro-inflammatory cytokines by human T-cell leukemia virus type-1 Tax protein as determined by multiplexed cytokine protein array analyses of human dendritic cells

Abstract

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by a hyperstimulated immune response, including elevated levels of inflammatory cytokines/chemokines and oligoclonal expansion of virus-specific CD8(+) T cells in the cerebrospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immune recognition by antigen presenting cells (APCs), such as dendritic cells (DCs). DCs are relevant to the pathogenesis of HAM/TSP because the presentation of Tax peptides by activated DCs to naïve CD8(+) T cells may play an important role in the induction of the Tax-specific immune response that is observed in HAM/TSP. In this study, a human cytokine protein array was used to study the secretion of cytokines by monocyte-derived DCs (MDDCs) exposed to Tax. Of the 16 cytokines analyzed, 6 cytokines were secreted in significantly high amounts (> or =2-fold), including Th1 cytokines (IFN-gamma, IL-12, and TNF-alpha) and C-C chemokines (Eotaxin, MCP-1, and MCP-3). Selected cytokines were further examined at two concentrations of Tax and at two time periods. Furthermore, a transient exposure to Tax did not result in any cytokine production when examined at three different time points after exposure, indicating that a prolonged presence of Tax is required for its activity. Finally, inhibition of the NF-kappaB signaling pathway by specific inhibitors, abrogated Tax-mediated cytokine secretion. Collectively, these findings suggest a role for Tax-induced cytokine secretion from MDDCs, which may be critical for the cellular activation and tissue damage that has been observed in HAM/TSP.

Fig. 1

HTLV-1 Tax induces the secretion cytokines from primary human MDDCs. MDDCs were exposed to mock fluid (negative control), LPS (positive control) or Tax (1 μg/ml) for 24 hr. Cell supernatants, harvested 24 hours after treatment, were analyzed for cytokine production using a human cytokine protein array as described in Materials and Methods. A: A grid layout of the antibodies as spotted on the slide. B: Normalized amounts (pg/ml) and fold-changes of cytokines secreted by Tax- or LPS-stimulated MDDCs.

Fig. 2

Validating Tax-induced cytokine expression profile. The expression of significantly expressed (≥ 2.0-fold) cytokines was further validated by exposing MDDCs to mock fluid, LPS or two doses of Tax (0.1 and 1 μg/ml) at two time periods (24 and 48 hours). Cell supernatants were analyzed using the cytokine protein array as described in Materials and Methods and it was found that Tax induced the expression of significantly expressed cytokines in a dose- and time-dependent manner.

Fig. 3

A transient exposure to Tax is not sufficient to induce cytokine secretion from MDDCs. MDDCs were either untreated or treated with Tax (1 μg/ml) for 4, 8, or 24 hours. Cells were then washed extensively with PBS to remove Tax and supplied with fresh medium without Tax. Supernatants were collected after 1, 4, and 24 hours from each data point and analyzed by ELISA. The results are represented by mean values in pg/ml ± standard deviation of duplicate samples analyzed in the ELISA. In case of both IL-12 and TNF-α, the presence of Tax was essential for sustained cytokine production.

Fig. 4

Role of NF-κB in Tax-mediated induction of cytokines. MDDCs were either directly treated with mock fluid or Tax or pre-incubated (30 minutes) with NF-κB inhibitors PDTC (10 μM) or TLCK (10 μM) followed by treatment with Tax (1 μg/ml). At the end of incubation period (24 hours post-treatment), supernatants were collected and analyzed by ELISA. The results are represented by mean values in pg/ml ± standard deviation of duplicate samples analyzed in the ELISA. Pre-incubation of MDDCs with PDTC or TLCK completely blocked the Tax-mediated induction of cytokines indicating a role for NF-κB signaling in DC activation by Tax.