Arsenic Trioxide Induces Apoptosis and Regulatory Functions of Plasmacytoid Dendritic Cells

Introduction

Arsenic trioxide (As₂O₃), a well-established drug for acute promyelocytic leukemia, has been recently shown to have therapeutic potential in a number of mouse models of autoimmune diseases such as, systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and chronic graft-versus-host disease. However, the therapeutic mechanism remains largely unknown.

Plasmacytoid dendritic cells (pDCs) are a unique subset of dendritic cells specialized in secreting high level of type I interferons and promote anti-viral responses. Clinically, pDCs have also been reported to be implicated in the pathogenesis of a series of autoimmune diseases characterized by a type I IFN signature, such as SSc.

Given the pathogenetic and therapeutic role of As₂O₃ and pDCs in the same spectrum of diseases, we examined the effects of As₂O₃ on pDCs viability and functions to better understand the therapeutic mechanism of As₂O₃ on pDC-related autoimmune diseases.

Materials and Methods

pDCs cells were isolated from fresh peripheral blood mononuclear cells (PBMCs) by magnetic selection. pDCs were cultured in presence of different concentrations of As₂O₃ to check for viability and functional changes. pDCs viability and apoptosis were checked by flow cytometry with Fixable Viability Dye-Annexin V staining. Phenotype evaluation was done with surface staining. Bcl-2 and Bax protein expressions, reactive oxygen species (ROS) level, and cytokine production of pDCs were measured by intracellular staining. IRF7 and phospho-IRF7 protein levels were measured with cell signaling staining. Cytokine concentrations in supernatants were quantified by ELISA, and IRF7 expression in pDCs was analyzed by real-time RT-PCR.

Naïve CD4+ T and CD19+ B cells were isolated from PBMCs. A pDC/T cell co-culture system was constructed to test As₂O₃ effect on pDCs in inducing T cell proliferation and polarization. A pDC/B cell co-culture system was used to test As₂O₃ effect on pDCs in inducing B cell differentiation.

Results

We first investigated the effect of As₂O₃ on the viability of human pDCs. We observed that As₂O₃ decreased dose-dependently the percentages of viable pDCs by inducing cell apoptosis. We then checked the protein expression levels of the apoptosis related Bcl-2 family proteins. As₂O₃ induced a dramatic upregulation of Bax, while slightly down-regulated the expression of Bcl-2, leading to a significantly decreased Bcl-2 to Bax ratio, which determined cell apoptosis.

As₂O₃ is a well-known inducer of oxidative stress, which is one of the major upstream mediators of cell apoptosis. We first demonstrated that As₂O₃ induced reactive oxygen species (ROS) within pDCs. However, the anti-oxidant NAC pre-treatment only slightly decreased the percentages of apoptotic cells, indicating that As₂O₃ could induce pDCs apoptosis independently of ROS generation.

Then, we investigated the functional effects of As₂O₃ on pDCs, and observed that culture for 6h with 5μM of As₂O₃ almost totally inhibited production of IFN-α from gated pDCs within PBMCs (IFN-α positive cells: 28.56±9,99% without As₂O₃ vs 1.43±1.65% with 5μM As₂O₃, p<0.05, n=4). ELISA results proved these results and revealed that As₂O₃ also restrained the secretion of TNF-α, IL-6 and CXCL10 from pDCs. We also demonstrated that As₂O₃ inhibited IFN-α secretion from pDCs of 12 untreated SSc patients (IFN-α positive cells: 22.48±12.71% without As₂O₃ vs 1.65±1.24% with 5μM As₂O₃, p=0.0001, n=12).

Further studies revealed that As₂O₃ inhibited the secretion of IFN-α from activated pDCs by potently down-regulating the total protein and mRNA expression, and also phosphorylation level of inteferon regulatory factor 7 (IRF7), a key adaptor of the type I inteferon pathway.

Finally, As₂O₃ inhibited pDCs maturation by decreasing the expressions of CD80, CD86 and HLA-DR, and increasing PD-L1 expression. As₂O₃ also down-regulated pDCs capacity to induce CD4+ T cell proliferation, Th1/Th22 polarization, and B cell differentiation towards plasmablasts.

Conclusion

Our study demonstrated that As₂O₃, at different concentrations, negatively affects the viability and functions of human pDCs, suggesting that pDCs could be a target for As₂O₃ treatment efficacy in autoimmune diseases with type-I interferon signature. We are currently running a prospective multicenter clinical trial testing As₂O₃ in the setting of chronic GVHD (ClinicalTrials.gov Identifier: NCT02966301).