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. 2013;8(2):e57181.
doi: 10.1371/journal.pone.0057181. Epub 2013 Feb 22.

Elevated serum levels of macrophage migration inhibitory factor are associated with progressive chronic cardiomyopathy in patients with Chagas disease

Romina A Cutrullis  1 , Patricia B Petray, Edgardo Schapachnik, Rubén Sánchez, Miriam Postan, Mariela N González, Valentina Martín, Ricardo S Corral
Affiliations

Elevated serum levels of macrophage migration inhibitory factor are associated with progressive chronic cardiomyopathy in patients with Chagas disease

Romina A Cutrullis et al. PLoS One. 2013.

Abstract

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MIF is overexpressed in the heart of chronic chagasic mice and potentiates production of TNF-α and ROS by Trypanosoma cruzi-infected macrophages.
C3H/He mice (n = 5) were infected intraperitoneally with 106 blood trypomastigotes of the Sylvio X10/4 clone of T. cruzi (upper panel) or with 50 blood trypomastigotes of the Tulahuén strain of the parasite (central panel). Hearts from infected animals and uninfected controls (lower panel) were removed at 120 days p.i. (A) Immunohistochemical analysis was performed on cardiac muscle sections using murine MIF polyclonal antibodies. Microphotographs of myocardial tissues show a representative experiment of three performed. Bar = 50 µm. (B) MIF overexpression in the heart was closely accompanied by intense inflammatory cell infiltration. The leukocyte population invading the myocardium was isolated from 20 T. cruzi-infected mice at 120 days p.i. and digested with collagenase and hyaluronidase. The mononuclear cell fraction was incubated with anti-mouse CD11b/MAC-1- PerCP-Cy 5.5, CD3-FITC, CD4-PE and CD8-Alexa Fluor 647 antibodies. The labeled cells (at least 5×104) were fixed with 1% p-formaldehyde and analyzed by flow cytometry. (C), (D) Effect of exogenous MIF on T. cruzi-induced release of TNF-α and ROS in murine J774 macrophages. Adherent cells were infected for 24 h with culture trypomastigotes of T. cruzi (Tulahuén strain) at a 10∶1 parasite/cell ratio, in the presence (+ rMIF) or in the absence (− rMIF) of recombinant mouse MIF (1 µg/ml). TNF-α production was quantified in uninfected (Mock) and parasite-infected cell supernatants using a sandwich ELISA (C). For ROS measurement. J774 macrophages (106) were incubated with 10 µM DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) for 30 min and then infected for 24 h with T. cruzi trypomastigotes with or without addition of rMIF, as described above. Uninfected (Mock) and infected cells were then fixed and analyzed by flow cytometry (D). Data are the means ± S E M of three independent experiments, each performed in triplicate. * P<0.05 and ** P<0.01 versus cells not pre-treated with MIF; #P<0.05 between infected and uninfected MIF-stimulated cells.
Figure 2
Figure 2. Concentrations of MIF and TNF-α in serum samples collected from patients chronically infected with Trypanosoma cruzi.
Infected patients without evident cardiac involvement (▴indeterminate, n = 14) or with Chagas cardiomyopathy (▪ CCC, n = 12), and uninfected controls (▾; n = 15) were studied. MIF (A) and TNF-α (B) levels were measured by ELISA in three independent experiments, and individual results for each patient are given. The horizontal lines represent mean values in each group. * P<0.05; ** P<0.01.
Figure 3
Figure 3. Correlation between serum MIF level and parameters of cardiac dysfunction in chagasic patients.
(A) LV end-diastolic diameter (LVEDD), (B) LV end-systolic diameter (LVESD), (C) left atrial diameter (LAD), (D) aortic diameter (AD), (E) LV per cent fractional shortening (LVFS), and (F) serum concentration of MIF and high sensitivity C-reactive protein (HS-CRP) were analyzed in chagasic (both chronic indeterminate and cardiomyopathic) patients (n = 26). MIF concentration was measured by ELISA; LVEDD, LVESD, LAD, AD and LVFS were determined by echodopplercardiography; HS-CRP level was quantified by immunonephelometry. Individual results are shown for each patient; the line represents the linear regression for each comparison. Alpha level was adjusted to 0.01. The correlation coefficient (r) and P values for each association are indicated.
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Grants and funding

RSC, PBP, MP and VM are members of the Research Career Program from National Research Council (CONICET, Argentina). RAC and MNG thank CONICET for fellowship granted. The study was funded by CONICET and by a grant (PICT 2010-2148) to MP from FONCyT, Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.