In vitro exposure to the herbicide atrazine inhibits T cell activation, proliferation, and cytokine production and significantly increases the frequency of Foxp3+ regulatory T cells.

Atrazine (ATR), a chlorotriazine herbicide (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) used to control annual broadleaf and grassy weeds, is one of the most widely applied herbicides in the United States with more than 80 million pounds applied annually (Grube et al., 2011). It is found in ∼70% of all surface and fresh ground water in the United States, making it the most common water contaminant (Bexfield, 2008; Solomon et al., 1999). Surveys conducted by the US Environmental Protection Agency (2003) have reported >88% of well water in agricultural areas is contaminated with ATR at levels above the maximum containment level. ATR is not acutely toxic and, owing to its relatively short half-life in the body, it does not bioaccumulate (Mcmullin et al., 2003; Ross et al., 2009). However, its prolonged persistence in the environment results in chronic exposure to tens of millions of people. Adverse effects on fetal growth and development (Ochoa-Acuna et al., 2009; Waller et al., 2010; Winchester et al., 2009), male fertility (Swan, 2006), and the nervous (Hossain and Filipov, 2008) and immune systems (Karrow et al., 2005; Rowe et al., 2006) have been described. At the biochemical level, ATR is a potent phosphodiesterase (PDE) inhibitor, leading to elevated levels of cAMP (Roberge et al., 2004). ATR is also classified as an Endocrine-Disrupting Compound (EDC), which at low concentrations increases levels of estrogen via induction of CYP19 (aromatase) transcription (Laville et al., 2006). Intriguingly, both cAMP (Averill et al., 1988; Tasken and Stokka, 2006) and estrogen (Tai et al., 2008) are implicated in control of T lymphocyte activation and effector functions, suggesting that ATR may be capable of modulating immune function. Previous studies have shown that ATR affects components of the immune system. Immunotoxic effects such as inhibition of natural killer cell degranulation (Rowe et al., 2007) and dendritic cell maturation (Pinchuk et al., 2007) have been associated with in vitro ATR exposure. ATR exposure also reduces pro-inflammatory cytokine secretion by mitogen-activated human peripheral blood mononuclear cells (PBMC) (Devos et al., 2003; Hooghe et al., 2000), induces mast cell degranulation (Mizota and Ueda, 2006) and modulates macrophage functions (Karrow et al., 2005). In vivo, ATR exposure has been shown to decrease tumor resistance (Karrow et al., 2005). Furthermore, in utero exposure to ATR has been shown to result in immune dysfunction of adult male offspring (Rooney et al., 2003; Rowe et al., 2006). Although these previous studies have provided important clues as to the effects of ATR on the immune system, much remains unknown. The previously published studies have predominantly focused on the interaction between ATR and innate immune cells. The focus of this study is to characterize how ATR exposure in vitro modulates adaptive immunity, in particular the activation and effector functions CD4+ T lymphocytes. To better understand how ATR may modulate CD4+ helper T cell activity we have exposed primary murine T cells to ATR during activation in vitro. We observed that ATR exposure significantly inhibited CD4+ T cell activation, proliferation, and pro-inflammatory effector cytokine production. We also found that ATR exposure caused a significant increase in the frequency of CD4+ Foxp3+ regulatory T cells (Tregs). ATR exposure significantly increased cAMP levels in CD4+ T cells and the ATR phenotype was partially mimicked by compounds that elevate cAMP, consistent with the ability of ATR to act as a PDE inhibitor. Interestingly, the effect was more pronounced in cells derived from male animals, suggesting that ATR-induced elevated estrogen levels may also play an important role in the observed immune modulation. Taken together, our results show that in vitro, ATR exposure leads to a significant increase in the frequency of Foxp3+ regulatory T cells (Treg), which suppress the activation and effector functions of conventional CD4+ T cells (Tconv). This may have important implications for the generation of protective immune responses by chronically exposed individuals.