Ischemia and subsequent reperfusion leads to myocardial injury through a variety of mechanisms, one of which involves an inflammatory response in the damaged myocardium. Myocardial ischemia is a common phenomenon in patients with coronary artery disease and experimental models of myocardial ischemia provide a platform for investigating new modes of therapy. We show here that nasal vaccination with troponin is effective in reducing MI/R injury by inducing CD4+ T cells that act by secreting IL-10 and reducing inflammation at the site of myocardial injury.
Currently, therapies for ischemic heart disease are directed at the rapid restoration of blood flow to the ischemic region (7
). However, during reperfusion, the heart undergoes further damage due in large part to the generation of ROS, e.g. superoxide anion, elevated levels of which can be detected within minutes after the reintroduction of oxygen to ischemic tissues. ROS have been shown to be key mediators of cellular and myocardial injury, causing lipid peroxidation and apoptosis (7
). The innate immune response to ischemia–reperfusion injury is the most common cause of myocardial inflammation (8
). Although innate immune responses may preserve myocardial function in the short term, they may be maladaptive in chronic states leading to production of ROS (8
). In addition as demonstrated here, there is an adaptive immune response directed against specific myocardial antigens such as troponin that occurs after MI/R injury. Thus, modulation of the immune response following MI/R injury appears important in reducing of tissue damage.
Because we used porcine troponin for nasal administration to mice, we established that there was functional immunologic cross-reactivity between porcine and murine troponin by demonstrating that both murine and porcine troponin given nasally affected immune responses in animals immunized with porcine troponin. Furthermore, BLAST comparison of the sequences of porcine and murine troponin-I showed 95% homology.
The presence of recruited leukocytes at the site of inflammation is dependent upon the coordinated expression of adhesion molecules on inflammatory cells and the activated capillary endothelium. T cells are re-stimulated upon encounter with the target immunogen presented by local APCs (7
). Thus, several types of APCs are activated following heart injury, including dendritic and macrophage-like cells, which express MHC molecules and produce pro-inflammatory cytokines such as TNF-α and IL-12, which may enhance the appearance of adhesion molecules (32
). Furthermore, pro-inflammatory cytokines play a direct role in tissue damage following myocardial damage: IFN-γ halts collagen synthesis by smooth muscle cells; TNF-α contributes to post-ischemic myocardial dysfunction via induction of myocyte apoptosis (34
). In line with this, following MI/R injury, we found macrophage-type cells (CD11b) by immunostaining of heart sections at 24 h, which serve to enhance the destructive effect of infiltrating CD4+ T cells. In animals treated with nasal troponin, there was decrease of these CD11b cells.
Both cardiac and skeletal muscles are exquisitely controlled by changes in the intracellular calcium concentration. Troponin is part of thin filament (along with actin and tropomyosin) and is the protein to which calcium binds to accomplish this regulation. Troponin has three subunits, TnC, TnI and TnT. When calcium is bound to specific sites on TnC, the structure of the thin filament changes in such a manner that myosin attaches to thin filaments and produces force and/or movement. Troponin is synthesized exclusively in myocardial cells. While normal levels of cardiac troponin-I are ~10 ng ml−1
, in patients with acute myocardial infarction, serum cardiac troponin-I is elevated within 4–6 h, reaches a mean peak level of 112 ng ml−1
at 18 h and remains above normal for up to 6–8 days following infarction (36
). Moreover, it has been shown that an autoimmune response to troponin induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice (37
). We observed that increased cellular proliferative responses to troponin occur in the spleen 24 h after MI/R injury and that reduction in proliferation to troponin was associated with protection by nasal vaccination with troponin. Thus, high concentrations of troponin following cardiac ischemia may lead to an injurious adaptive inflammatory response directed at the ischemic site following ischemic reperfusion injury and measurement of cellular immune responses to troponin can serve as an indicator of protection by nasal troponin.
An important question is why does troponin given nasally 1 h after I/R induce protection whereas troponin released into the circulation at the same time after injury afford no protection? We believe that different immune mechanisms are induced following mucosal versus intravenous delivery of antigen. We previously found that mucosal (oral) but not intravenous alloantigen resulted in increased Th
2 cell activation in cardiac allografts (38
). Thus, we believe than troponin released from the circulation does not afford protection as it does not induce troponin-specific IL-10-secreting T cells whereas nasal troponin does. Troponin released into the circulation induces anergy of troponin-reactive T cells, whereas mucosal antigen induces IL-10-secreting T cells due to the unique immunologic properties of the nasal mucosa in inducing IL-10-specific immune responses (14
). Because such regulatory T cells are triggered in an antigen-specific fashion but suppress via cytokine release in an antigen-non-specific fashion, they mediate ‘bystander suppression’ when they encounter the nasal autoantigen at the target organ. Thus, mucosal tolerance can be used to treat inflammatory processes that are not autoimmune in nature via the secretion of cytokines such as IL-10 after antigen-specific triggering (16
). Furthermore, using CFSE labeling, we found endogenous reactivity of CD4+ T cells to troponin in naive animals. These findings, together with reports (18
) that showed elevation of IL-10 secreting T cells following MI/R injury in dog, suggest that underlying cellular endogenous reactivity to troponin is being boosted both by myocaridal damage and by nasal troponin.
Although we have studied troponin, it is known that immune responses have been reported to other contractile proteins such as myosin. Murine myocarditis may be induced by immunization with cardiac myosin (39
) and cellular responses to both mysosin and troponin have been observed in autoimmune myocarditis (37
). In addition, auto-antibodies to both troponin and myosin may induce myocarditis (41
) and cardiac auto-antibodies may play a role in dilated cardiomyopathy which has led to initial clinical testing of Ig-adsorption therapy (42
Anti-inflammatory cytokines may have a protective effect following the inflammation that occurs after MI/R injury. It has been suggested that IL-10 inhibits inducible nitric oxide synthase (iNOS) activity after MI/R and consequently exerts cardioprotective effects (18
). Studies have also shown that TGF-β can attenuate myocardial injury induced by I/R, though we did not find increased TGF-β in our studies (43
). In animals nasally vaccinated with troponin, we found enhanced expression of the anti-inflammatory cytokine IL-10 and reduced expression of the pro-inflammatory cytokine IFN-γ in the ischemic region. It has recently been shown that IFN-γ parallels iNOS activity during the course of ischemia following reduction in the blood flow (8
). IL-10 is the mediator of the protection we observed following nasal vaccination with troponin. Furthermore, it has been shown that IL-10−/− mice have larger ischemic injury as compared with wild type animal following MI/R injury (44
). We have previously shown that IL-10-secreting CD4+ T cells induced by nasal MOG (35–55) reduce injury ischemic injury following stroke (15
). In addition, we observed a dramatic reduction of CD11b(+) cells (macrophage) in nasal MOG-treated animals. CD11b(+) cells may contribute to secondary infarct expansion by enhancing NO synthesis that may be reduced by IL-10 levels. IL-10 has also been shown to reduce inflammation in autoimmune animal models including experimental autoimmune encephalomyelitis (25
). IL-10 may deactivate macrophage-like cells and thus limit their involvement in a secondary inflammatory process. Moreover, IL-10 targets the interface between heart and periphery by preventing adhesion and extravasation of leukocytes. Finally, we have demonstrated that IL-10 protects cardiac myocytes from oxidative stress in vitro
, using a myocardial cell culture system.
Administration of antigen by the mucosal route is known to induce regulatory T cells (16
). We have found that Tr1 type Tregs that primarily act via IL-10 are induced by the nasal route and it appears that this is the type of regulatory T cell induced by nasal troponin. In a recent study in which we induced Tregs by nasal anti-CD3 to suppress models of lupus, we induced an IL-10-secreting CD4+ CD25- LAP+ regulatory T cell which also shared the properties of Tr1 cells. (45
) We did not observe an increase in Foxp3 expression in these cells.
Given our finding that IL-10 is responsible for the effect observed, it is theoretically possible that intravenous infusion of IL-10 post-infarction might have a beneficial effect. However, large amounts of systemic IL-10 may be required for IL-10 to be active in the heart and there may be systemic side-effects. CD4+ T cells from nasal troponin-treated mice secrete IL-10 when they encounter troponin in the myocardium. Thus, it is targeted local delivery of IL-10. As discussed above, because regulatory T cells are triggered in an antigen-specific fashion but suppress in an antigen-non-specific fashion, they mediate bystander suppression when they encounter the nasal autoantigen at the target organ (16
). Nasal troponin also has the advantage that it could be given prophylactically to patients at risk for cardiac damage. Whether intravenous IL-10 would be a better therapeutic agent than nasal troponin given immediately after myocardial damage both in terms of efficacy and toxicity is not known.
We were surprised to find that nasal immunization with troponin 1 h after MI/R could lead to an effective immune response within a 24-h time period. Nonetheless, we found immune responses in the spleen to troponin 24 h after injury as measured by proliferation. Moreover, both in human and mouse, there is detectable level of troponin in the blood (36
) and since we found endogenous troponin-specific CD4+ T cells in naive animal, we believe this explain the protective effect of nasal immunization with troponin 1 h after MI/R. Consistent with this, we found immune changes as measured by RT–PCR both in the spleen and in the heart 24 h after MI/R injury and these changes were modulated by nasal troponin. Furthermore, this modulatory effect was lost in IL-10-deficient mice, clearly showing the role of IL-10 in the immunologic effects seen at 24 h and is supported by transfer of protection that is also dependent on IL-10. We believe that these findings suggest that there may be underlying endogenous reactivity to troponin that is being boosted both by myocardial damage and by nasal troponin even though we did not find measurable proliferative responses to troponin in naive animals.
In line with our results, other anti-inflammatory approaches are being investigated for cardioprotection in acute myocardial infarction. It has been shown that activation of macrophages by complement can exacerbate cardiac injury following ischemic insult. C-reactive protein (CRP) enhances heart failure by activation of complement and increasing myocardial and infarct size (46
), and administration of CRP inhibitors reduces heart infarct size and improves cardiac function (46
Our results not only demonstrate the presence of a cellular immune response to troponin in response to damage after MI/R injury but they also have clear clinical implications. First, our results suggest prophylactic treatment with nasal troponin may be of benefit to those at a risk for ischemic cardiac injury including subjects undergoing cardiac bypass surgery or coronary angioplasty. Nasal troponin treatment significantly improved heart function after MI/R injury, and this effect remained significant up to 6 weeks following heart ischemia. Second, we have shown that nasal vaccination 1 h following MI/R can significantly reduce ischemic damage, making this approach clinical applicable for patients in the immediate period following myocardial infarction. Thus, nasal vaccination with troponin is a novel therapeutic intervention for treatment of cardiac ischemia both in those in risk and in the period immediately following cardiac injury.