Suppression by Thimerosal of Ex-Vivo CD4+ T Cell Response to Influenza Vaccine and Induction of Apoptosis in Primary Memory T Cells

Emily Loison,Odile Launay,Vered Abitbol,Audrey Paoletti,Valérie Seffer,Marie-Lise Gougeon,Béatrice Poirier-Beaudouin,Eric Tartour

doi:10.1371/journal.pone.0092705

Abstract

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.

Highlights

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine [1] [2]
Thimerosal could cause S phase arrest followed by mitochondrial apoptosis in murine myoblast cells that occurred via inhibition of the PI3K/ Akt/survivin signaling pathway [12]
We found that Panenza was toxic when used ex-vivo on patients’ peripheral blood lymphocytes (PBMC) in T-cell assays, and this multidose vaccine-related toxicity was attributed to the preservative thimerosal

Summary

Introduction

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine [1] [2]. The deleterious effects of thimerosal were reported on HeLa S epithelial cells, inducing an oxidative stress and cell death that were completely suppressed by pretreating the cells with N-acetyll-cysteine (NAC), a radical scavenger [11]. Thimerosal could cause S phase arrest followed by mitochondrial apoptosis in murine myoblast cells that occurred via inhibition of the PI3K/ Akt/survivin signaling pathway [12]. Trompezinski et al found that it induced an oxidative stress in monocyte-derived dendritic cells [13], and Agrawal et al reported that thimerosal could exercise a Th2 promoting effect through modulation of functions of dendritic cells [14]. At the T cell level, thimerosal was reported to induce caspase-dependent mitochondrial apoptosis in the human leukemic Jurkat T cells [15,16]

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Conclusion