Arsenic Trioxide Induces Apoptosis and Regulatory Functions of Plasmacytoid Dendritic Cells
Authors: Ye Yishan , Ricard Laure , Stocker Nicolas , Lamarthée Baptiste , De Vassoigne Frédéric , Brissot Eolia , Mekinian Arsène , Mohty Mohamad , Gaugler Béatrice , Malard Florent 
Published: October 2019

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).

Disclosures

Mohty: MaaT Pharma: Consultancy, Honoraria.