This study demonstrates for the first time that T. cruzi
modulates the expression of PD-1, which has been widely involved in T cell exhaustion and persistent infections (5
), features that have been associated with T. cruzi
infection. The expression of PD-1 in association with parasite persistence has been reported for filariasis (4
). In addition, increased expression of the ligands PD-L1 and PD-L2 has been reported to be induced by Taenia crassiceps
in macrophages, which is associated with the inhibition of T cell proliferation by this parasite (36
T. cruzi infection is characterized by acute parasitemia that is usually cleared out raising an intense cellular immune response against the parasite that usually causes extensive tissue damage, leading to fibrosis and dysfunction of the myocardium and other organs. However, tissue parasitism can persist, being responsible for continued tissue destruction. In this study, we show that T. cruzi infection upregulates the expression of PD-1 by CD8 and CD4 T cells migrating to the myocardium during the acute phase of this systemic infection. The high expression levels of PD-1 and its ligands reported in effectors T cells can be responsible for such pathogen persistence. In fact, the blockade of PD-1 and PD-L1 or deletion of the PD-1 gene ameliorated the control of parasite burden both systemically and in cardiac tissue.
Similar to our current results, we previously reported that T. cruzi
induces upregulated expression of another coinhibitor molecule, CTLA-4, in lymphocytes in vivo
and in vitro
, and the blockade of this inhibitory signaling pathway lead to increased inflammation and decreased tissue parasitism (23
appears to have evolved to manage the expression of costimulatory molecules as a strategy to persistently survive within the mammalian host. A recent study showed that the parasite is also able to exert immune evasion by downregulating CD28 ligands and major histocompatibility complex (MHC) molecules in dendritic cells (28
). In addition, it has been proposed that differential expression levels of these costimulator molecules induced by the parasite can be associated with the intensity of the inflammatory response, leading to different clinical forms of the disease in humans (28
). Whether differential expression of PD-1 and CTLA-4 would be a marker of clinical forms of the disease in humans is currently under investigation in our laboratory.
The modulation of expression of PD-1, PD-L1, and to a lesser extent, PD-L2, by lymphocytes in response to T. cruzi
infection in vivo
became clear by the time course cytometry study. A wave of increasing expression of PD-1 in T cells (both CD4 and CD8) obtained from the spleen was detected on days 10 and 15 p.i. By day 20 p.i., however, the expression of PD-1 fell. This may correspond to a migration effect from the spleen to the heart and to other tissues. It can be confirmed by an indirect fluorescent-antibody assay (IFA) of the spleen, as shown in Fig. S1 in the supplemental material, in which the expression of these molecules is lower in infected mice than in uninfected mice. These data clearly show that T. cruzi
infection lead to modulation in the normal patterns of expression of PD-1 and its ligands by lymphocytes and monocytes. In all cases, PD-L1 exhibited the most upregulated expression, which is known to participate in immune evasion in other microorganisms (6
). Previous reports showed that PD-1 and its ligands are induced in immune cells late after activation, and PD-1 is now considered a marker of late activation and cell exhaustion during chronic infections and tumor immune evasion (6
). The T cells present at the myocardium migrate in response to the parasite's presence and are known to exhibit a phenotype of activated cells, producing massive amounts of cytokines, predominantly those of the Th-1 immune response (13
). In addition, we demonstrated that nearly 100% of these cells express high levels of PD-1. Such high expression of PD-1 in T cells has already been described during viral infections (16
). The apparent discrepancy in the detection of PD-1 by IFA and flow cytometry is due to differential sensibility among the two methods. In addition, the effect of concentration is also evident, as flow cytometry takes into account cells found in a whole heart, while IFA focuses only on a small area of the heart. Our findings suggest the participation of a novel regulatory mechanism in the control of the inflammatory response in cardiac tissues during this parasitic infection.
We believe that the balanced expression of positive and negative coreceptors is what determines the final outcome of infection in terms of the intensity and clearance of the parasite. The pathogenic role of collateral destruction of cardiac tissue during this inflammatory reaction is also undeniable. It is mediated by cellular and soluble components of the immune response, which is poorly regulated by classic immune regulatory mechanisms. For instance, regulatory cells do not play a strong role in the modulation of this inflammatory response (17
). Our data demonstrate that PD-1 is crucial to reduce the intensity of myocarditis.
It has been suggested that the effector lymphocytes recruited in response to the presence of the parasite in the myocardium may display altered tolerance mechanisms, leading to self-damage in an autoreactive fashion (10
) because activated T cells are not properly cleared out from circulation or because an altered peripheral tolerance is induced by the pathogen. Thus, it is possible that the inflammatory damage of cardiomyocytes cause an imbalanced expression of PD-L1, known to maintain the tolerance to cardiac troponin I (26
), which is a protein exclusively expressed by cardiomyocytes. These hypotheses are currently under further investigation.
The role for PD-1 in maintaining immunological tolerance during acute myocarditis induced by T. cruzi was showed in this study by the treatment of mice with blocking antibodies against PD-1, PD-L1, or PD-L2. These treatments induced increased inflammation that was more remarkable in the cardiac tissues but not in other tissues where the presence of the parasites has been described during this infection. We also demonstrated that the blockade of PD-1 and PD-L1 was more effective in worsening myocarditis than the blockade of PD-L2. These data are in agreement with those from previous studies on the role of PD-1 in the immune response against intracellular pathogens and suggest that PD-L1 has a predominant regulatory role over PD-L2 during this parasitic infection. Although spontaneous cardiomyopathy has been described in BALB/c mice as a consequence of PD-1 deficiency, this autoimmune disorder has not been described in C57BL/c mice. We did not observe any myocarditis in uninfected C57BL/c PD-1−/− mice. In addition, we did not detect any cell migration to the heart tissue of normal C57BL/6 mice after treatment with anti-PD-1 MAb, indicating that PD-1 is one of the factors responsible for the deregulation of the immune response in the myocardium after T. cruzi infection.
The mechanism by which PD-1 regulates the immune response to T. cruzi
appears to involve regulation of T cell proliferation and apoptosis. The induction of PD-1 expression in lymphocytes is known to be induced through the signaling by common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21. We showed that deletion of PD-1 leads to increased Stat-5 phosphorylation, which is crucial to T cell proliferation. In addition, the blockade of PD-1 and PD-L1 restored the proliferative capacity of lymphocytes obtained from infected mice. This result is very important since it explains one of the mechanisms that causes the inhibition of T cell proliferation that is well known to be induced after T. cruzi
infection, which induces a large production of NO, which is responsible for induction of apoptosis (22
It has been shown that the blockade/deletion of PD-1 leads to an improved immune response to intracellular pathogens (2
), which correlates with our findings of reduced parasite load, an increased number of inflammatory infiltrates in the myocardium, and increased levels of the proinflammatory cytokines TNF-α and IFN-γ and chemokines CCL3, CCL5, and CCL2. These data support the regulatory role for PD-1 signaling, mainly through the PD-L1 ligand in the infected myocardium during the acute phase of T. cruzi
In spite of favoring parasite persistence, PD-1 signaling is important for the survival of the infected host, as mice receiving the anti-PD-1 treatment die earlier than the control group mice or the mice receiving other treatments. We hypothesized that the increased mortality rate of these mice could be associated with the uncontrolled, intense myocardial inflammation observed as a consequence of the PD-1 blockade. This reduced tolerance to myocardial inflammation results from increased lymphocyte proliferation, proinflammatory cytokine production, and reduced apoptosis of T cells. The participation of regulatory T cells in the mechanisms involving PD-1 regulation during this infection was not studied and is an interesting issue for future reports.
In conclusion, our data demonstrated that PD-1 and PD-L1 participate in T. cruzi-induced myocarditis and that they regulate the inflammatory immune response.
Finally, recent studies have started to suggest that immune therapy involving soluble PD-1 could be beneficial under inflammatory conditions, as is the case with Chagas' heart disease. Soluble PD-1 does not appear to be produced under normal conditions. However, an alternative splicing variant of the PD-1 gene (PD-1Deltaex3), which leads to production of soluble PD-1, has recently been described (39
). We did not perform any assays to test the presence of PD-1 or its ligands in mouse plasma. We hypothesize that a delicate manipulation of this signaling pathway on T cells of T. cruzi
-infected hosts could become a potential strategy to design future therapeutic approaches for Chagas' heart disease.