Biomedical Toxicology BIOM*6440 Fall 2005

Immunotoxicity of Deoxynivalenol

George Girgis

Contents

Introduction .........................................................................................................................1 Immune modulation by trichothecenes is paradoxical in experimental models..................1 DON-induced IgA production.............................................................................................2 Induction and regulation of immune-related gene expression by DON..............................3 Ribotoxic stress response.....................................................................................................4 Apoptosis.............................................................................................................................5 Conclusion ..........................................................................................................................5 References ...........................................................................................................................6

Figures .................................................................................................................................7 Introduction Trichothecene-induced immune modulation is considered as a spectrum whereby low doses stimulate immune function but as the doses increase immunosuppression ensues, mainly due to apoptosis of leukocytes (Pestka, 2003). The capacity to inhibit protein synthesis has been known to be a central effect of trichothecenes. Now, it appears that toxic manifestations of trichothecenes are related to de-regulation of cell signaling and consequent alterations in downstream gene expression (Pestka et al., 2004). This minireview discusses recent advances in understanding the effect of trichothecenes on the immune response, with special reference to deoxynivalenol (DON). Immune modulation by trichothecenes is paradoxical in experimental models Early reports on the immunosuppressive effect of trichothecenes in the literature were anecdotal. Depending on the dose and exposure regimen, trichothecenes can be either immunosuppressive or immunostimulatory (Bondy and Pestka, 2000). It is documented that repetitive exposure to high doses of trichothecenes results in injury to actively dividing cells such as those present in bone marrow, intestinal mucosa, spleen, thymus and lymph nodes. This was evidenced by leucopenia, inhibition of immune response to model antigens, impaired delayed type hypersensitivity responses and decreased resistance to certain pathogens (Pestka et al., 2004). In addition, the decreased resistance to inflammatogenic stimuli such as lipopolysaccharide (LPS) in animals concurrently exposed to a trichothecene suggested that such impaired resistance to LPS

might contribute to the increased susceptibility to gram negative bacteria reported by some researchers (Bondy and Pestka, 2000). On the other hand, low doses of trichothecenes have been reported to increase the resistance to certain pathogens, elevate serum IgA levels and initiate rapid and transient up-regulation of some immune-related genes. The exposure to trichothecenes at levels that only partially inhibit translation up-regulates expression of immune-related genes such as proinflammatory, Th1 and Th2 cytokines as well as chemokines, cyclooxygenase-2 (COX-2) and inducible nitric synthase (Pestka et al., 2004). Nevertheless, it should be taken into consideration that the manifestations of immunomodulation by DON can overlap (Pestka, 2003). Dose-dependent decreases or increases in B and T cell responses after exposure to DON or other trichothecenes have been reported to occur both in vivo and in vitro where mitogen-induced lymphocyte proliferation was either impaired or enhanced. Such differences in lymphotoxicity appeared to be dependent on the degree of acylation in trichothecenes as well as differences in uptake and metabolism to less toxic compounds (Pestka et al., 2004). DON-induced IgA production One of the major effects of DON is the pronounced elevation in serum IgA and concurrent depression in IgM and IgG. This effect has been first described in mice exposed to the toxin with inductive threshold of 2 ppm and maximal effects occurring in the 10-25 ppm range (Bondy and Pestka, 2000). Peyers patches lymphocytes and to a lesser extent splenic lymphocytes isolated from mice fed DON produced significantly

high level of IgA, suggesting that DON enhances differentiation to IgA-secreting cells at the Peyers patch level which subsequently impacts the systemic compartment (Pestka, 2003). Resultant polyspecific autoreactive IgA may contribute to kidney pathogenesis via immune complex deposition or direct binding to the kidney mesangium. (Pestka et al., 2004). However, DON does not appear to exert adjuvant effects when orally administered with exogenous mucosal antigens. Rather, it polyclonally induces IgAs that are reactive with a variety of intestinal and self antigens, while simultaneously downregulating IgG and IgM reactive with the same antigens (Pestka, 2003). T cells and macrophages have been found to be implicated in the polyclonal expansion of IgA-secreting cells as evidenced by: (1) increased T cell numbers, CD4 + cells and CD4+:CD8+ ratio in Peyers patches and spleen, (2) increased terminal differentiation of cultured nave B cells from Peyers patches upon addition of T cells from Peyers patches of exposed mice, (3) increased secretion of IgA by B cells cocultured with CD4+ cells pulsed with DON, and (4) increased IL-2, IL-4, IL-5 and /or IL6 at the mRNA and protein levels in activated CD4 + cells exposed to DON (Pestka, 2003). The capacity of DON to polyclonally expand IgA-secreting cells is also mediated by increased cytokine production by macrophages, particularly IL-6 (Fig.1). It is well established, through the use of IL-6 knock-out mice, that IL-6 plays a critical role in driving differentiation of B cells to IgA production (Pestka et al., 2004). Induction and regulation of immune-related gene expression by DON

DON induces expression of immune-related genes such as interleukins, tumor necrosis factor and COX-2. It has been found that DON induction of immune-related genes reflects regulatory effect at the transcription level, as it increases the binding activity of transcription factors in leukocytes (Fig. 2), and at the post-transcription level by increasing the stability of mRNA (Pestka et al., 2004). DON has been shown to induce COX-2 gene expression in macrophages (Pestka, 2003). COX-2 is a rate limiting enzyme that catalyzes oxygenation of arachidonic acid to prostaglandin endoperoxides which are subsequently converted enzymatically into prostaglandins. Prostaglandin E2 (PGE2) regulates production of macrophage and T cell cytokines which can in turn impact cell-mediated responses as well as immunoglobulin induction by B cells (Pestka et al., 2004). Macrophage-generated PGE2 is now recognized to modulate proinflammatory cytokines such as IL-6 in an autocrine or paracrine fashion (Pestka, 2003) DON not only induces gene expression but also regulates the stability of mRNA of the induced genes, suggesting that DON-mediated gene up-regulation in macrophages and T cells can be explained, in part, by mRNA stability. This was evidenced by the finding that the effect of AU-rich element in the 3-untranslated region of mRNA, which targets the transcript for rapid degradation, was counteracted by DON (Pestka et al., 2004). Ribotoxic stress response The term ribotoxic stress response describes the effects which result from binding of translational inhibitors to ribosomes changing their structure or function. This

initiates a signal for rapid activation (through phosphorylation) of mitogen-activated protein kinases (MAPKs) which are involved in many physiological processes including cell growth, differentiation and apoptosis. MAPKs also are important transducers of immune response. Activation of members of MAPKs subfamilies by trichothecenes has been reported as a result of targeting the 60S ribosomal subunit, suggesting that the ribotoxic stress response might be a critical transduction step during trichothecene immunotoxicity (Pestka et al., 2004). DON-induced MAPKs drive activation of transcription factors that promote cytokine and COX-2 expression as well as induce apoptosis. Two possible upstream signal transducers for MAPKs activation are doublestranded RNA-activated protein kinase R (PKR) and hematopoietic cell kinase (Hck) (Fig. 3). However, the transduction mechanisms by which ribosome binding by DON might activate PKR and Hck are unknown so far (Pestka and Smolinski, 2005). Apoptosis Trichothecene-induced apoptosis has been described in a variety of tissues including thymus, spleen, bone marrow and liver. It also occurs in the gastric mucosa, gastric glandular epithelium and intestinal crypt epithelium, resulting in breakdown of nonspecific mucosal defense mechanisms such as the epithelial barrier and mucus secretion and thus results in increased translocation of bacteria and toxins in the gut (Bondy and Pestka, 2000). Moreover, high doses of trichothecenes promote rapid onset of leukocyte apoptosis that is manifested as immunosuppression. Activation of caspases leading to apoptosis appears to be related to the capacity of DON to inhibit translation and induce MAPKs phosphorylation (Pestka et al., 2004).

Conclusion Low doses of dietary DON promote polyclonal activation and expansion of IgAproducing cells at the Peyers patches level and up-regulate immune-related gene expressions at the transcriptional level through activating the binding of transcription factors, and at the post-transcriptional level through increasing the stability of mRNA. In contrast, high doses result in immunosuppression linked to apoptosis of leukocytes. Trichothecenes toxicity is partially explained by their capacity to bind to ribosomes and inhibit protein synthesis. Recent studies suggest that early alterations in cell signaling at the level of MAPKs are critical to trichothecene toxicity. Ribotoxic stress response occurs due to translation inhibition and is postulated to be an initial signal for MAPKs activation. MAPKs are important transducers of downstream signaling events related to immune response and apoptosis. Reference Bondy G. and Pestka J. 2000. Immunomodulation by fungal toxins. Journal of Toxicology and Environmental Health, Part B, 3:109-043. Pestka J. 2003. Deoxynivalenol-induced IgA production and IgA nephropathy-aberrant mucosal immune response with systemic repercussions. Toxicology Letters, 140:287-295. Pestka J. and Smolinski A. 2005. Deoxynivalenol: toxicology and potential effects on humans. Journal of Toxicology and Environmental Health, Part B, 8:39-69.

Pestka J., Zhou H., Moon Y. and Chung Y. 2004. Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox. Toxicology Letters, 153:61-73.

Figures

Fig.1. Cellular mechanisms involved in DON-induced IgA production and IgA nephropathy (Pestka, 2003).

Fig.3. Interactive molecular and cell signaling mechanisms involved in trichothecene9 induced cytotoxicity. PKR, Hck and MAPKs as molecular rheostats and Fig.2. Mechanisms and kinetics by which DON function induces gene expression in vivo. The define whether an immunostimulatory or immunosuppressive response will result involved MAPKs and DNA-binding proteins as well as timing are indicated (Pestka et (Pestka et al., 2004). al., 2004).