Toxicological Sciences 2011 Oct 9
Nyland JF, Fairweather D, Shirley DL, Davis SE, Rose NR, Silbergeld EK.
Source: Department of Pathology, Microbiology & Immunology, University of South Carolina School of Medicine, Columbia, SC USA 29209.
Mercury is a widespread environmental contaminant with neurotoxic impacts that have been observed over a range of exposures.
In addition, there is increasing evidence that inorganic mercury (iHg) and organic mercury (including methyl mercury) have a range of immunotoxic effects, including immune suppression and induction of autoimmunity. In this study, we investigated the effect of iHg on a model of autoimmune heart disease in mice induced by infection with coxsackievirus B3.
We examined the role of timing of iHg exposure on disease; in some experiments mice were pretreated with iHg (200μg/kg, every other day for 15 days) before disease induction with virus inoculation and in others they were treated with iHg after the acute (viral) phase of disease, but before the development of dilated cardiomyopathy (DCM). iHg alone had no effect on heart pathology.
Pretreatment with iHg before CVB3 infection significantly increased the severity of chronic myocarditis and DCM compared to control animals receiving vehicle alone. In contrast, treatment with iHg after acute myocarditis did not affect the severity of chronic disease. The increased chronic myocarditis, fibrosis, and DCM induced by iHg pretreatment were not due to increased viral replication in the heart, which was unaltered by iHg treatment. iHg pretreatment induced a macrophage infiltrate and mixed cytokine response in the heart during acute myocarditis, including significantly increased interleukin-12, IL-17, interferon-γ, and tumor necrosis factor-α levels. IL-17 levels were also significantly increased in the spleen during chronic disease.
Thus, we show for the first time that low-dose Hg exposure increases chronic myocarditis and DCM in a murine model.
Mercury (Hg) is a heavy metal with well-deﬁned neurotoxicity (National Research Council [U.S.] Committee on the Toxicological Effects of Methylmercury, 2000) and increasing evidence for immunotoxic effects (Schiraldi and Monestier, 2009; Silbergeld et al., 2005). Hg is of global public health importance because many populations worldwide are exposed at levels that exceed recommended guidelines of safe levels (Mahaffey et al., 2004; WHO, 2010). Exposure to Hg compounds can occur through occupation, diet, and consumption of contaminated ﬁsh. Exposures to inorganic mercury (iHg) are associated with the manufacture of ﬂuorescent light bulbs, pressure gauges, use of Hg-containing dental amalgams (Gochfeld, 2003), and artisanal gold mining (Silbergeld et al., 2005).
An association has been reported between self-reported occupational exposure to Hg (primarily in those working with dental amalgams) and diagnosis of systemic lupus erythematosus (SLE; Cooper et al., 2004).
We and others have reported increases in serum biomarkers of immune activation and autoantibodies in individuals exposed to Hg through artisanal gold mining and/or consumption of contaminated ﬁsh (Alves et al., 2006; Gardner et al., 2010; Nyland et al., 2011, forthcoming; Silva et al., 2004).
Many studies in rodents have demonstrated that Hg exposure activates the immune response in certain inbred strains resulting in lupus-like autoimmune disease, as well as accelerating pathology and death in the graft-versus-host model of SLE (Hultman and Nielsen, 2001; Monestier et al., 1994; Nielsen and Hultman, 2002; Via et al., 2003).
The mechanism(s) by which Hg induces autoimmune disease is still being elucidated. In rodent models, Hg treatment either in vivo or ex vivo induces elevated interleukin (IL)-4 production from spleen cells resulting in a Th2 response and proliferation of B cells and the Th2-associated antibodies IgG1 and IgE (Bagenstose et al., 1999; Schiraldi and Monestier, 2009). These animal model ﬁndings are consistent with the type of immune response known to be induced by Hg exposure in humans of increased autoantibodies and immune complex deposition (Gardner et al., 2010; Nyland et al., 2011; Schiraldi and Monestier, 2009). Although Hg increases a Th2 response, in reality, Hg activates a ”mixed” Th1 and Th2 response with increased interferon (IFN)-c production as well (Bagenstose et al., 1999; Fournie et al., 2002).
Myocarditis, or inﬂammation of the myocardium, leads to around half of all dilated cardiomyopathy (DCM) cases in the United States (Roger et al., 2011). DCM is the most common form of cardiomyopathy requiring a heart transplant due to heart failure (Drory et al., 1991; Feldman and McNamara, 2000; Roger et al., 2011).
Viral infection, particularly by coxsackievirus B3 (CVB3), is the most frequent cause of myocarditis in developed countries (Blauwet and Cooper, 2010; Guarner et al., 2007). CVB3 infection is believed to induce autoimmunity by viral-induced damage resulting in exposure of the immune system to the intracellular protein cardiac myosin and by activation of Toll-like receptors (TLRs) during the innate immune response similar to an adjuvant (Cihakova et al., 2004; Fairweather and Rose, 2007b; Zhang et al., 2009).
In a hybrid model where susceptible BALB/c mice are inoculated with heart-passaged CVB3 (i.e., infectious CVB3 and heart proteins), mice develop disease that is similar to that observed in humans, with the development of acute inﬂammatory myocarditis from day 7 to 14 postinfection (pi) followed by progression to chronic myocarditis and DCM that persists from day 35 to at least day 90 pi (Fairweather et al., 2001, Fairweather and Rose, 2007a).
In this model, inﬂammation is driven by the TLR-4–derived proinﬂammatory cytokines IL-1b and IL-18, and their levels are directly correlated with the extent of inﬂammation in the heart (Fairweather et al., 2003, 2005; Lane et al., 1992). Further, TLR-4 and IL-12 receptor b1 signaling are critical for progression of disease in susceptible male BALB/c mice (Frisancho-Kiss et al., 2006, 2007).
To investigate whether low-dose Hg exposure could alter acute or chronic myocarditis/DCM in this model (similar to its effects on other models of autoimmune disease), we treated mice with iHg prior to inoculation with CVB3 or in the interval between inoculation and the development of chronic myocarditis/DCM. Disease status was evaluated during the acute and chronic stages of the disease.
In this study, we report for the ﬁrst time that low-dose iHg pretreatment signiﬁcantly increases chronic CVB3- induced autoimmune myocarditis, ﬁbrosis, and DCM. iHg pretreatment signiﬁcantly altered cytokine levels in the heart during acute myocarditis. Although, there was no overall change in the severity of acute inﬂammation, the number of inﬁltrating macrophages (but not eosinophils or mast cells) was signiﬁcantly increased in the heart during acute myocarditis. Thus, iHg pretreatment shifts in immune cell subsets during acute myocarditis without changing overall inﬂammation levels, as we demonstrated previously (Fairweather et al., 2005). Future studies will need to determine whether these shifts in cardiac immune cell populations during acute myocarditis were accompanied by changes in functional or activation states. Shifts in immune cell populations and activation levels could explain the signiﬁcant changes in cytokines observed in the heart during acute myocarditis in Hg-treated mice. It is interesting that IL-17a was signiﬁcantly increased in the heart of Hg-treated compared with PBS-treated mice during acute myocarditis (Fig. 4a) and in the spleen during chronic myocarditis (Fig. 8). However, the increase in IL-17 was not accompanied by changes in other cytokines in the IL-17 regulatory axis including IL-23 and IL-6. Recently, IL-17 was found to not alter the severity of acute myocarditis in the experimental autoimmune myocarditis (EAM) model but to be necessary for chronic ﬁbrosis and progression to DCM (Baldeviano et al., 2010). Our data show that Hg increases cardiac and splenic IL-17 levels providing a potential mechanism for how Hg pretreatment could increase chronic ﬁbrosis and DCM in this study without altering the severity of acute myocarditis. These data are consistent with other studies of inﬂammatory heart disease leading to DCM in which both IFN-c and IL-17, along with other cytokines, were increased in a mixed cytokine milieu prior to development of DCM (Kania et al., 2009). Although IL-17 and IFN-c inhibit each other in vitro, this has not been observed in vivo, potentially because other cytokines and factors inﬂuence the relationship as well. Elevated IL-5, IL-10, and eotaxin in iHg-pretreated mice suggest that Hg pretreatment may have also increased immune responses associated with Th2 immunity. IL-5 and eotaxin are known to increase the number and activation of mast cells and eosinophils, although that was not observed in these studies. Mast cells have been proposed to skew immune responses to a more Th2 response following Hg treatment in other models of Hg-induced autoimmunity (Schiraldi and Monestier, 2009). Mast cells have also been found to play a role in exacerbating myocarditis, ﬁbrosis, and chronic DCM in our CVB3 model (Fairweather et al., 2004; Frisancho-Kiss et al., 2007).
Increased levels of IL-10 (and Th2 cytokines) in the heart during acute myocarditis may regulate the proinﬂammatory effects of elevated TNF-a, IL-12, IL-17, and IFN-c (Fig. 4a; Roether et al., 2002).
Overall, our ﬁndings in this study clearly demonstrate that low doses of iHg are able to affect the immune response in the heart, resulting in signiﬁcantly worse autoimmune heart disease.
The temporal relationship between iHg exposure and induction of disease appears to be critical for modulation of disease because interim iHg exposure did not signiﬁcantly alter chronic disease. These results are consistent with other studies that examined the effect of iHg pretreatment on EAM in mice (Silbergeld et al., 2005).
These results are also similar to our studies in the graft-versus-host disease model of lupus nephritis in which pretreatment with iHg exacerbated glomerulonephritis and hastened death (Via et al., 2003). Importantly, our ﬁndings in this study are consistent with those of Ilback et al. (1996, 2000) who found that pretreatment of mice with a high dose of MeHg (3.9 mg/kg) for 12 weeks prior to CVB3 infection did not signiﬁcantly alter acute myocarditis. Hg was found to increase proliferation of splenocytes to concanavalin A ex vivo. However, cytokine levels were not examined in the heart and viral replication in the heart was not quantitated in those studies. Although sera TNF-a and IFN-c levels were signiﬁcantly increased following CVB3 infection, they were not altered in HgþCVB3 compared with CVB3-treated mice (Ilback et al., 1996). We are the ﬁrst to examine the effect of iHg treatment on the chronic phase of myocarditis and DCM.
Recent ﬁndings have revealed that genes necessary for ﬁbrosis induction are upregulated during acute CVB3 myocarditis resulting in the gradual remodeling that produces ﬁbrosis during the chronic phase of disease (Fairweather, unpublished data). Thus, we only observe ﬁbrosis in this model of CVB3- induced autoimmune myocarditis by day 35 pi in susceptible mice (Fairweather and Rose, 2007a), which is exasperated with iHg pretreatment.
These ﬁndings are consistent with studies in mice and humans linking Hg exposure and increased antiﬁbrillarin antibodies in scleroderma, an autoimmune disease with a signiﬁcant induction of ﬁbrosis (Arnett et al., 2000; Hultman et al., 1989). Interestingly, cardiac ﬁbrosis associated with scleroderma tends to be patchy and distributed throughout the myocardium (Champion, 2008), as we ﬁnd in the CVB3- induced model of autoimmune myocarditis. Hg may also contribute to the progression to DCM by mechanisms other than a direct effect on the immune response.
Boyd Haley PhD explains mercury induced
idiopathic dilated cardiomyopathy.
The mechanism to generate these elevated Hg levels or even the sequence of events (infection vs. exposure) is unknown, but Hg may compete with selenium, decreasing the activity of seleniumdependent enzymes. Selenium is a trace element with both structural and enzymatic roles that are essential for normal cardiac physiology (Cooper et al., 2007).
Exposure to elevated levels of Hg may reduce selenium’s bioavailability (Hg has a high binding afﬁnity for selenium [Gailer et al., 2000], which is higher than that for sulfur), negatively impacting cardiac function. Selenium deﬁciency has been shown to convert an ”amyocarditic” strain of CVB3 (one that induces high levels of viral replication in the heart but low or no inﬂammation) to a myocarditic strain (one that induces high levels of viral replication in the heart and myocarditis; Beck et al., 1995, 2003a,b). However, the effect of selenium deﬁciency on autoimmune myocarditis has not been established, and we did not examine cardiac Hg or selenium levels in this study.
In an epidemiologic study of 1833 men with heart disease, men with elevated Hg levels (2 lg/g) in hair samples were at a 2.9-fold increased risk of cardiovasculardeath compared with those with lower hair Hg levels (Salonen et al., 1995).
Although that study did not speciﬁcally examine myocarditis/DCM patients, their ﬁndings are consistent with these experimental ﬁndings that Hg exposures can increase cardiovascular disease.
In this study, we present data that show that low-dose iHg exposure can induce signiﬁcant changes in the immune response resulting in increased autoimmune heart disease.
The interaction with viral infection events, related to cytokine production and iHg-induced alterations in the immune response in this model, is demonstrated by the importance of the timing of iHg exposure.