Ascorbic Acid Has Superior Ex Vivo Antiproliferative, Cell Death-Inducing and Immunomodulatory Effects over IFN-α in HTLV-1-Associated Myelopathy

Abstract

Background

Clear therapeutic guidelines for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are missing due to the lack of randomized double-blind controlled clinical trials. Moderate yet similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (AA) monotherapy in a large open clinical trial. However, there is a lack of in vivo and in vitro studies exploring and comparing the effects of high-dose AA and IFN-α treatment in the context of HAM/TSP. Therefore, we performed the first comparative analysis of the ex vivo and in vitro molecular and cellular mechanisms of action of IFN-α and high-dose AA in HAM/TSP.

Principal Findings

Through thymidine incorporation and quantification of Th1/Th2/Th17 cytokines, we demonstrate that high-dose AA displays differential and superior antiproliferative and immunomodulatory effects over IFN-α in HAM/TSP PBMCs ex vivo. In addition, high-dose AA, but not IFN-α, induced cell death in both HAM/TSP PBMCs and HTLV-1-infected T-cell lines MT-2 and MT-4. Microarray data combined with pathway analysis of MT-2 cells revealed AA-induced regulation of genes associated with cell death, including miR-155. Since miR-155 has recently been demonstrated to up-regulate IFN-γ, this microRNA might represent a novel therapeutic target in HAM/TSP, as recently demonstrated in multiple sclerosis, another neuroinflammatory disease. On the other hand, IFN-α selectively up-regulated antiviral and immune-related genes.

Conclusions

In comparison to IFN-α, high-dose AA treatment has superior ex vivo and in vitro cell death-inducing, antiproliferative and immunomodulatory anti-HTLV-1 effects. Differential pathway activation by both drugs opens up avenues for targeted treatment in specific patient subsets.

Author Summary

HAM/TSP is a chronic and disabling neuroinflammatory disease, for which clinical management is mostly empirical and symptomatic rather than evidence-based, due to the lack of biomarkers and controlled clinical trials. Although similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (vitamin C) in one major open clinical trial with 200 patients, their cellular and molecular mechanisms of action remain unexplored in HAM/TSP. We demonstrate that high-dose ascorbic acid strongly inhibits lymphoproliferation of HAM/TSP mononuclear cells in ex vivo cultures, in contrast to IFN-α. Furthermore, high-dose ascorbic acid, but not IFN-α, significantly decreased ex vivo TNF-α and IFN-γ pro-inflammatory cytokine levels in supernatant of mononuclear cells from HAM/TSP patients. In addition, ascorbic acid, but not IFN-α, induced cell death in HTLV-1-infected T-cell lines, which was confirmed by gene expression profiling, revealing cell death-associated pathways activated by high-dose ascorbic acid, including miR-155. This microRNA has previously been shown up-regulated in HTLV-1-infected cells, as well as in blood and brain samples of multiple sclerosis patients, another neuroinflammatory disease. In addition, miR-155 has also been reported to up-regulate IFN-γ production in human natural killer cells, thus linking both cell death and cytokine signaling pathways, rendering it a potential therapeutic target in neuroinflammatory disorders. Thus, our findings reveal molecular mechanisms of action as well as candidate biomarkers for high-dose ascorbic acid therapy and provide a rational basis, rather than an empirical basis, for its use in HAM/TSP treatment.