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Role of SOCS2 in Modulating Heart Damage and Function in a Murine Model of Acute Chagas Disease

Open AccessPublished:May 31, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.03.042
      Infection with Trypanosoma cruzi induces inflammation, which limits parasite proliferation but may result in chagasic heart disease. Suppressor of cytokine signaling 2 (SOCS2) is a regulator of immune responses and may therefore participate in the pathogenesis of T. cruzi infection. SOCS2 is expressed during T. cruzi infection, and its expression is partially reduced in infected 5-lipoxygenase–deficient [knockout (KO)] mice. In SOCS2 KO mice, there was a reduction in both parasitemia and the expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-10, SOCS1, and SOCS3 in the spleen. Expression of IFN-γ, TNF-α, SOCS1, and SOCS3 was also reduced in the hearts of infected SOCS2 KO mice. There was an increase in the generation and expansion of T regulatory (Treg) cells and a decrease in the number of memory cells in T. cruzi–infected SOCS2 KO mice. Levels of lipoxinA4 (LXA4) increased in these mice. Echocardiography studies demonstrated an impairment of cardiac function in T. cruzi–infected SOCS2 KO mice. There were also changes in calcium handling and in action potential waveforms, and reduced outward potassium currents in isolated cardiac myocytes. Our data suggest that reductions of inflammation and parasitemia in infected SOCS2-deficient mice may be secondary to the increases in Treg cells and LXA4 levels. This occurs at the cost of greater infection-associated heart dysfunction, highlighting the relevance of balanced inflammatory and immune responses in preventing severe T. cruzi–induced disease.
      Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi. This infection results in acute myocarditis and chronic cardiomyopathy. Chagasic cardiomyopathy is the result of intense multifocal progressive inflammation of the myocardium
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      Once secreted in vivo, IL-12 induces natural killer (NK) cells to synthesize IFN-γ, which in turn induces increased levels of IL-12 that cycles back to enhance IFN-γ production by NK cells. IFN-γ production is also amplified by IFN-γ–induced production of macrophage TNF-α and IL-1β by a positive feedback loop. Importantly, these cytokines drive the generation of type 1 CD4+ and CD8+ T cells, which produce further IFN-γ, a key requirement for inducing and maintaining control of acute infection.
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      The down-regulatory cytokines IL-10 and transforming growth factor-β (TGF-β) are associated with susceptibility to infection
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      by inhibiting IFN-γ–mediated macrophage activation. Resistance to the initial infection is a result of a balance between proinflammatory IFN-γ and anti-inflammatory IL-10 production.
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      The immune response generated to T. cruzi is largely effective at controlling, but not eliminating, the infection, which is life-long unless treatment is provided.
      Suppressor of cytokine signaling 2 (SOCS2) is known to have important actions in the regulation of various cytokine/growth hormone-mediated processes, including somatic growth, metabolism, and response to infection.
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      Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent.
      Among the SOCS family, SOCS2 may regulate the expression of other members, including SOCS1 and SOCS3,
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      Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions.
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      SOCS2 can enhance interleukin-2 (IL-2) and IL-3 signaling by accelerating SOCS3 degradation.
      effects that may eventually result in enhanced function of certain cytokines. Importantly, SOCS2 is known to regulate the cytokine-dependent Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway in cells of the innate and adaptive immune systems, modulating the production of several mediators, including TNF-α and IL-12,
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      Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions.
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      Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent.
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      SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses.
      and favoring T helper 2 (Th2) differentiation and production of Th2 cytokines.
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      • Johnston J.A.
      SOCS2 regulates T helper type 2 differentiation and the generation of type 2 allergic responses.
      SOCS2 is involved in the regulation of immune response in T. gondii infection.
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      • Johndrow J.E.
      • Esper L.
      • Dias A.
      • Bafica A.
      • Serhan C.N.
      • Aliberti J.
      Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent.
      • Machado F.S.
      • Aliberti J.
      Impact of lipoxin-mediated regulation on immune response to infectious disease.
      Because SOCS2 plays a role in certain infections and has been shown to modulate cytokines that are crucial for the pathogenesis of T. cruzi infection, we conducted studies to evaluate the expression and potential role(s) of SOCS2 in the pathogenesis of T. cruzi–induced heart disease. We present evidence for a role for SOCS2 in the modulation of experimental T. cruzi infection and suggest how it may interfere with cardiac function. These data provide a new insight into the pathogenesis of chagasic heart disease as well as provide novel therapeutic targets to ameliorate disease progression.

      Materials and Methods

      Mice

      Wild-type (WT) C57BL/6 mice were obtained from CEBIO [Centro de Bioterismo–Universidade Federal de Minas Gerais (UFMG)]. 5-Lipoxygenase (5-LO) KO (129-Alox5tm1Fun/J; as described by Chen et al
      • Chen X.S.
      • Sheller J.R.
      • Johnson E.N.
      • Funk C.D.
      Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene.
      ) and SOCS2 KO (as described by Metcalf et al
      • Metcalf D.
      • Greenhalgh C.J.
      • Viney E.
      • Willson T.A.
      • Starr R.
      • Nicola N.A.
      • Hilton D.J.
      • Alexander W.S.
      Gigantism in mice lacking suppressor of cytokine signalling-2.
      ) mice were kindly provided by Dr. Fernando Queiroz Cunha (University of Sao Paulo, Ribeirao Preto, Brazil) and Dr. Warren S. Alexander (the Walter and Eliza Hall Institute of Medical Research, Australia), respectively. 5-LO KO strain–matched WT mice were kindly provided by Dr. Vanessa Pinho da Silva (Federal University of Minas Gerais). All animal lineages were bred and maintained under pathogen-free conditions at the Institute of Biological Sciences–UFMG animal facility. All animal experiments were approved by the local animal ethics committee.

      Parasites and Experimental Infection

      The Y strain of T. cruzi was maintained in C57BL/6 mice. Seven- to 8-week-old WT and KO mice were infected intraperitoneally with 1 × 103 trypomastigotes, and parasitemia determined daily in 5 μL of blood collected from a tail vein.
      • Machado F.S.
      • Martins G.A.
      • Aliberti J.C.
      • Mestriner F.L.
      • Cunha F.Q.
      • Silva J.S.
      Trypanosoma cruzi-infected cardiomyocytes produce chemokines and cytokines that trigger potent nitric oxide-dependent trypanocidal activity.
      For in vitro infection, trypomastigotes were grown and purified from a monkey kidney epithelial cell line (LLC-MK2).

      Macrophage Culture, Evaluation of NO2 Production, and Microbicidal Activity

      WT and SOCS2 KO inflammatory macrophages were harvested from peritoneal cavities 3 days after injection of 1 mL of 3% sodium thioglycollate. The cells were washed and cultured (1 × 106 cells/mL) in RPMI 1640 (Sigma-Aldrich, St. Louis, MO), supplemented with 5% fetal bovine serum, 50 μmol/L 2-mercaptoethanol, 2 mmol/L l-glutamine, and penicillin/streptomycin (all from GIBCO, Grand Island, NY). The adherent cells were obtained after 2 to 4 hours of incubation of single-cell suspensions in tissue culture plates at 37°C in 5% CO2. Nonadherent cells were removed, and trypomastigote forms were added at a 5:1 parasite-to-cell (macrophage) ratio for 3 hours, after which the extracellular parasites were removed, and the cells were incubated at 37°C in 5% CO2 in the presence or absence of 100 ng/mL of recombinant murine IFN-γ (GIBCO). The supernatants were harvested 24, 48, and 72 hours post infection and assayed for nitrite concentration by mixing 0.1 mL of culture supernatant with 0.1 mL of Griess reagent. Absorbance at 540 nm was read 10 minutes later, and NO2 concentration was determined. Macrophages were infected, the extracellular parasites were removed, and the cells were incubated with or without IFN-γ as described above. Parasite growth was evaluated by daily counting of the trypomastigotes in the supernatant of infected macrophages on days 3 to 7 post infection. The intracellular amastigote growth rate was evaluated in macrophages plated (5 × 105 cell/mL) onto chamber slides (Nalge Nunc, Naperville, IL), as described.
      • Silva J.S.
      • Vespa G.N.
      • Cardoso M.A.
      • Aliberti J.C.
      • Cunha F.Q.
      Tumor necrosis factor alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon-activated macrophages.
      • Machado F.S.
      • Martins G.A.
      • Aliberti J.C.
      • Mestriner F.L.
      • Cunha F.Q.
      • Silva J.S.
      Trypanosoma cruzi-infected cardiomyocytes produce chemokines and cytokines that trigger potent nitric oxide-dependent trypanocidal activity.

      Isolation of Left Ventricular Myocytes

      Left ventricular cardiac myocytes from age-matched mice were enzymatically isolated as previously described, with minor modifications.
      • Roman-Campos D.
      • Duarte H.L.
      • Sales Jr., P.A.
      • Natali A.J.
      • Ropert C.
      • Gazzinelli R.T.
      • Cruz J.S.
      Changes in cellular contractility and cytokines profile during Trypanosoma cruzi infection in mice.
      Hearts were quickly removed, mounted, and then perfused using a customized Langendorff apparatus for 5 minutes with nominally Ca2+-free solution containing 130 mmol/L NaCl, 5.4 mmol/L KCl, 0.5 mmol/L MgCl2, 0.33 mmol/L NaH2PO4, 3 mmol/L pyruvate, 22 mmol/L glucose, and 25 mmol/L HEPES (pH 7.4). Hearts were then perfused for 10 to 15 minutes at 37°C with a solution containing 1 mg/mL type II collagenase (Worthington Biochemical Corporation, Lakewood, NJ). The digested heart was removed from the cannula, and the left and/or right ventricular free wall was separated, gently agitated, centrifuged (180 × g, 10 seconds at room temperature) and stored in Dulbecco's modified Eagle's medium (Sigma-Aldrich). The isolated cardiac myocytes were stored at room temperature and used within 4 to 6 hours of isolation. Only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied.

      Whole-Cell Ruptured, Patch Clamp Technique

      Whole-cell voltage and current-clamp recordings were obtained using an EPC-9.2 patch clamp amplifier (HEKA Electronics, Lambrecht/Pfalz, Germany).
      • Roman-Campos D.
      • Campos A.C.
      • Gioda C.R.
      • Campos P.P.
      • Medeiros M.A.
      • Cruz J.S.
      Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats.
      On attaining the whole-cell configuration, the cells were equilibrated for 3 to 5 minutes in the pipette solution. The experiments were carried out at room temperature (24°C to 27°C). The recording electrodes had resistances of 0.5 to 1.5 MΩ. Current recordings were low-pass filtered (2.9 kHz) and digitized at 10 kHz before being stored on a computer. Myocytes showing series resistance (Rs) larger than 7 MΩ were not used in the analysis. Rs compensation was used at 40% to 70%. We used Tyrode's as a bath solution of 140 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L CaCl2, 10 mmol/L glucose, 10 mmol/L HEPES (pH 7.4). For measurements of ICa,L, recording pipettes were filled with internal solution containing 120 mmol/L CsCl, 20 mmol/L TEACl, 5 mmol/L NaCl, 10 mmol/L HEPES, 5 mmol/L EGTA, pH set to 7.2 with CsOH. ICa,L was recorded in the presence of 1.8 mmol/L extracellular Ca2+. Membrane potential was first stepped from a holding potential of −80 mV to −40 mV for 50 ms (to inactivate Na+ channels), and then stepped to different membrane voltages between −40 to 50 mV (300-ms duration). To measure action potentials (AP) and outward potassium current (Ito), pipette solution contained 130 mmol/L potassium aspartate, 20 mmol/L KCl, 10 mmol/L HEPES, 2 mmol/L MgCl2, 5 mmol/L NaCl, 5 mmol/L EGTA, pH set to 7.2 with KOH. With regular Tyrode's as a bath solution, we recorded 30 to 50 AP per cell (at 1 Hz), until a stable recording condition was achieved, and we used the last recorded AP to perform the complete analysis. Ito was elicited by depolarization steps from −40 to 70 mV (3-seconds duration) from a holding potential of −80 mV at a stimulation frequency of 0.066 Hz. We used a 50-ms pre-pulse to −40 mV from −80 mV to inactivate INa but still be able to record Ito.
      • Roman-Campos D.
      • Campos A.C.
      • Gioda C.R.
      • Campos P.P.
      • Medeiros M.A.
      • Cruz J.S.
      Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats.
      Current-density relationships were fitted using the following equation:
      I(V)=Gmax*(VmEi)1+exp(VmV0.5)S
      (1)


      where Gmax is the maximal conductance; Vm is the test membrane potential; Ei, electrochemical equilibrium potential for ion i; V0.5 is the membrane potential where 50% of the channels are activated; and S is the slope factor.
      • Roman-Campos D.
      • Duarte H.L.
      • Sales Jr., P.A.
      • Natali A.J.
      • Ropert C.
      • Gazzinelli R.T.
      • Cruz J.S.
      Changes in cellular contractility and cytokines profile during Trypanosoma cruzi infection in mice.
      • Roman-Campos D.
      • Campos A.C.
      • Gioda C.R.
      • Campos P.P.
      • Medeiros M.A.
      • Cruz J.S.
      Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats.

      Echocardiography

      Echocardiography (Visual Sonics, Toronto, ON, Canada) was performed on mice anesthetized with 1.5% isoflurane. Heart morphology was assessed using M-mode configuration. Cardiac output and ejection fraction were obtained from the B-mode according to Simpson's method. All parameters were evaluated following protocols of the American Society of Echocardiography.
      • Lang R.M.
      • Bierig M.
      • Devereux R.B.
      • Flachskampf F.A.
      • Foster E.
      • Pellikka P.A.
      • Picard M.H.
      • Roman M.J.
      • Seward J.
      • Shanewise J.
      • Solomon S.
      • Spencer K.T.
      • St John Sutton M.
      • Stewart W.
      American Society of Echocardiography' S Nomenclature and Standards Committee; Task Force on Chamber Quantification; American College of Cardiology Echocardiography Committee; American Heart Association; European Association of Echocardiography; European Society of Cardiology
      Recommendations for chamber quantification.

      Transient Ca2+ Recordings

      Adult ventricular myocytes were isolated as described above, and intracellular Ca2+ (Ca2+i) imaging experiments were performed as described
      • Guatimosim S.
      • Sobie E.A.
      • dos Santos Cruz J.
      • Martin L.A.
      • Lederer W.J.
      Molecular identification of a TTX-sensitive Ca(2+) current.
      with cardiac myocytes loaded with Fluo-4 AM (10 μmol/L; Invitrogen, Eugene, OR) for 25 minutes. Cells were electrically stimulated at 1 Hz to produce steady-state conditions. The confocal line-scan imaging was performed with a Zeiss LSM 510META confocal microscope (Carl Zeiss, Oberkochen, Germany) (Centro de Microscopia Eletronica, UFMG). Digital image processing was performed by using custom-devised routines created with the IDL programming language (Research Systems, Boulder, CO). The Ca2+ level was reported as F/F0, where F0 is the resting Ca2+ fluorescence.

      LipoxinA4 and Cytokine Determinations

      The concentration of LipoxinA4(LXA4) was measured using commercially available antibodies and according to the procedures supplied by the manufacturer (Neogen, Lexington, KY). IL-12 p40 and IFN-γ levels were measured using commercial ELISA kits (BD Biosciences/Pharmingen, San Jose, CA, and R&D Systems, Minneapolis, MN, respectively). For in vivo experiments, a minimum of five mice were infected as described above, and on different days post infection were bled for assessment of cytokine levels in the serum.

      Total RNA Extraction and cDNA Preparation by Reverse Transcription

      Total RNA was isolated from mouse cardiac and splenic tissue, homogenized in 1 mL of Brazol reagent (LGC Biotecnologia, Cotia, Brazil) according to the manufacturer's instructions, followed by the addition of 0.2 mL of chloroform (Sigma-Aldrich). Purified RNA (2 μg) was used to synthesize cDNA using Superscript II (GIBCO).

      Parasite Load, Cytokines, and SOCS mRNA Detection

      The expression of mRNA for cytokines (IL-6, IL-10, IL-12, TNF-α, and IFN-γ), SOCS1, SOCS2, SOCS3, and β-actin was analyzed by RT-PCR, with GoTaqGreen Master Mix (Promega, Madison, WI) in a PTC-100 thermal cycler (MJ Research, Alameda, CA). The reaction conditions were 34 cycles of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C, with a final extension step of 2 minutes at 72°C. The parasite load was detected using a T. cruzi–specific primer (rDNA 24S α). Reaction conditions were 30 cycles of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C, with a final extension step of 10 minutes at 72°C. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. The primer sequences and PCR products for cytokines and β-actin have been described before.
      • Machado F.S.
      • Johndrow J.E.
      • Esper L.
      • Dias A.
      • Bafica A.
      • Serhan C.N.
      • Aliberti J.
      Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent.
      The primer sequences for T. cruzi load were (forward: 5′-AAGGTGCGTCGACAGTGTGG-3′ and reverse: 5′-TTTTCAGAATGGCCGAACAGT-3′), 125 bp.

      Spleen Cell Cultures

      Suspensions of splenocytes from uninfected and chronically T. cruzi–infected mice (6 months post infection) were washed in Hanks' balanced salt solution and treated with lysis buffer [nine parts 0.16 mmol/L ammonium chloride and one part 0.17 mmol/L Tris-HCl (pH 7.5)] for 4 minutes. The erythrocyte-free cells were then washed three times in Hanks' balanced salt solution and adjusted to cells per mL of RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (Cultilab, Campinas, Brazil), 2-mercaptoethanol (Invitrogen), l-glutamine (GIBCO), and penicillin/streptomycin (all from GIBCO). The cell suspension was cultured in 24-well tissue-culture plates (1 mL/well; Nalge Nunc) for 48 hours at 37°C, 5% CO2 atmosphere, either in the presence or absence of trypomastigotes (five parasites per cell) or T. cruzi antigen (10 μg/mL). The supernatants were harvested, and IFN-γ levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).

      Flow Cytometry Analysis

      Splenocytes were purified from the T. cruzi–infected mice [0, 8, 15, 29, or 180 days post infection (dpi)] as described above. Splenocytes purified from 0 and 15 dpi were plated and incubated with brefeldin A (1 μg/mL) (Invitrogen). Four hours post treatment, the cells were fixed and stained with labeled antibodies against IFN-γ (fluorescein isothiocyanate), CD4 (phycoerythrin), or CD8 (peridinin chlorophyll protein complex) (BD Biosciences/Pharmingen). Cells harvested from spleen and thymus 0, 8, 15, and 29 days post infection were directly stained with the combination of CD4 (peridinin chlorophyll protein complex), CD25 (phycoerythrin), and FOXP3 (fluorescein isothiocyanate) specific antibodies. Viable cells were analyzed by flow cytometry using FlowJo software version 8.7 (Tree Star, Ashland, OR).

      Heart Histopathology, Morphometric Analysis, and Parasitism

      To compare the effects of SOCS2 expression on the influx of inflammatory cells in the heart, we analyzed the pattern and intensity of myocarditis by morphometry. For this experiment, the hearts from three mice from each group were removed at 15 days post infection and cut transversally. Each heart was washed in phosphate-buffered saline and fixed in 4% phosphate-buffered formaldehyde. After 24 hours of fixation, the tissues were paraffin-embedded, and three 5-μm-thick, semiconsecutive sections were stained by H&E for inflammation assessment or by Trichrome to evaluate fibrosis. Inflammation and fibrosis were assessed both in the atrial and ventricular free walls and in the interventricular septum, in five fields from each H&E- or Trichrome-stained section randomly chosen at a ×20 objective magnification, analyzing a myocardial area of 1.5 × 106 μm2 per field. Images were captured at a resolution of 1392 × 1040 pixels with a CoolSNAP-Pro cf color microcamera (Media Cybernetics, Bethesda, MD) and analyzed in Image-Pro Express, version 4.0 (Media Cybernetics) and KS300 software (Carl Zeiss, Jena, Germany). An automatic macro recorder assembler (an algorithm of the KS300 software) was elaborated for capture, image processing, and segmentation, definition of morphometry conditions and counts of all of the cells detected in each image. The nucleus from each cell presented in the analyzed fields were counted and expressed as cells per field. Fibrosis was assessed using the image segmentation function. All pixels with blue hues in the Trichrome sections were selected to build a binary image and subsequently to calculate the total area occupied by connective tissue per field (fibrosis area per field). Heart parasitism was qualitatively evaluated by counting the number of parasite nests in three semiconsecutive sections as visualized by light microscopy with an ×40 objective.

      Statistical Analysis

      The statistical significance of differences in mean values between experimental versus control or vehicle-treated samples was evaluated by Student's t-test, one-way analysis of variance, or two-way analysis of variance. Bonferroni corrections were used as needed for multiple comparisons. Differences were considered to be significant at P < 0.05.

      Results

      Expression of SOCS2 in Infected Mice Is Partially 5-LO Dependent

      5-LO activation and eicosanoid production, mainly LXA4, may be involved in the modulation of SOCS2 expression. SOCS2 expression was increased in the spleen and cardiac tissue of T. cruzi–infected WT mice (Figure 1, A and B). There was a partial reduction in the expression of SOCS2 in both organs of 5-LO KO mice, indicating that products of this enzyme contribute to SOCS2 expression.
      Figure thumbnail gr1
      Figure 1Parasitemia, tissue parasitism, and macrophage function of T. cruzi–infected SOCS2 KO mice. To determine the role of 5-LO in the expression of SOCS2, WT, SOCS2 KO, and 5-LO KO mice were infected intraperitoneally with 103 trypomastigotes of T. cruzi (Y strain), and the spleen and heart were examined 15 days post infection. WT and 5-LO KO mice were infected, and spleens (A) and hearts (B) were harvested and homogenized, mRNA extracted, and then RT-PCR performed using primers specific for the genes for SOCS2 and β-actin. Data were normalized to β-actin expression and to noninfected control mice. Note that there was a partial decrease in the expression of SOCS2 both in the heart and spleen of 5-LO KO mice. C: Parasitemia in WT and SOCS2 KO mice. Note the reduction in the SOCS2 KO mice. D: The parasite load was detected by PCR using a T. cruzi–specific prime (primer rDNA 24S α, TCr) in the heart. There were no differences in parasite load between WT and SOCS2 KO mice. Nitrite levels were assayed at (E) 48 hours and (F) as shown, in supernatants of T. cruzi–infected macrophages (Tc) and/or stimulated with IFN-γ (IFN). Uninfected macrophages cultured with medium (Md) alone were used as controls. WT and SOCS2 KO macrophages were infected (Tc) and treated with IFN-γ (Tc+IFN), cultured for (G) 48 hours, washed, fixed, and stained, and intracellular amastigotes were counted in 300 cells. H: The released trypomastigote forms were counted daily. AD: Data are shown as the mean ± SD of the number of the parasites per microliter of blood from one of two independent experiments (eight mice per group). *P < 0.001, for SOCS2 versus WT mice infected with T. cruzi. EG: Each point represents mean ± SD of triplicate samples and is representative of three separate experiments. *P < 0.05 versus values obtained in absence of parasites and/or cytokines.

      SOCS2 Expression and Its Effects on Parasitemia, Tissue Parasitism, and Macrophage Function During T. cruzi Infection in Mice

      Parasitemia was significantly reduced in SOCS2 KO mice compared with WT mice (Figure 1C). There was no significant difference in parasite load in the heart WT and SOCS2 KO mice (Figure 1D). Macrophages obtained from SOCS2 KO mice were hyperresponsive to IFN-γ stimulation, producing higher levels of NO (Figure 1, E and F) and increased parasite killing compared with WT macrophages (Figure 1, G and H). However, nonstimulated SOCS2 infected macrophages were more susceptible to infection than WT cells (Figure 1H).

      Inflammatory Cytokine Responses, Expansion of T Regulatory and Memory Cells Are Controlled by a Regulatory Pathway Dependent on SOCS2 in Vivo

      We evaluated the immune response to T. cruzi in SOCS2 KO mice. There was significantly decreased expression of IL-6, TNF-α, IL-12, IFN-γ, and IL-10 in the spleen (Figure 2A) and of TNF-α and IFN-γ in the heart (Figure 2B) of T. cruzi–infected SOCS2 KO mice when compared to those of infected WT mice. Infection of SOCS2 KO mice resulted in reduced expression of SOCS1 and SOCS3 in spleen and heart (Figure 2, A and B). Decreased systemic levels of IL-12 and IFN-γ were observed 9 days post infection in SOCS2 KO mice (Figure 3A). The number of TH1 IFN-γ–producing CD4+ as well CD8+ T cells was reduced in infected SOCS2 KO mice (Figure 3B).
      Figure thumbnail gr2
      Figure 2To determine whether there were changes in the expression of cytokines and SOCS1 and SOCS3 in the heart and spleen of infected SOCS2 KO mice. WT and SOCS2 KO mice were infected with 103 trypomastigotes Y (T. cruzi), and 15 days post infection, spleens (A) and hearts (B) were harvested, homogenized, mRNA extracted, and then RT-PCR performed using primers specific for the genes for IL-6, TNF-α, IL-12, IFN-γ, IL-10, SOCS1, and SOCS3. Data were normalized to β-actin expression and to control mice. Data are shown as the means ± SD and are representative of two independent experiments. *P < 0.05 for SOCS2 versus WT mice infected with T. cruzi. Note the significant reduction in SOCS1 and SOCS3 expression and several proinflammatory cytokines.
      Figure thumbnail gr3
      Figure 3To determine whether there was a modulation of the inflammatory response, generation of effector, memory, and regulatory T cells in T. cruzi–infected SOCS2 KO mice 15 days post infection was assessed. A: The levels of IL-12 p40 and IFN-γ were observed in the serum of infected SOCS2 KO mice as compared to infected WT controls by ELISA. B: Splenocytes from WT and SOCS2 KO mice were incubated with brefeldin for 4 hours, followed by intracellular detection of IFN-γ in CD4+ or CD8+ T cells by flow cytometry. There was a reduction in IFN-γ in splenocytes from infected SOCS2 KO mice. C: The levels of LXA4 were increased in the spleen of infected SOCS2 KO mice as compared to infected WT controls by ELISA. D: Similarly, at 0, 8, 15, and 29 days post infection, splenocytes and thymocytes were prepared for detection of CD4+CD25+FOXP3+ cells by flow cytometry. There was an increase in CD4+CD25hiFOXP3+ cells in the these cells obtained from infected SOCS2 KO mice. E: Splenocytes from WT and SOCS2 KO mice chronically infected with T. cruzi (6 months) were incubated with medium only (Md), T. cruzi trypomastigotes (Tc), or T. cruzi antigen (Ag) in vitro for 48 hours. Incubation was followed by the detection of IFN-γ in the supernatant by ELISA. There was a reduction in IFN-γ in the supernatant from infected SOCS2 KO mice. Data are shown as the means ± SD and are representative of two independent experiments. *P < 0.05 for SOCS2 versus WT mice infected with T. cruzi.
      To investigate the basis for these observations, we determined both LXA4 production and the generation/expansion of Treg during T. cruzi infection. There was an increase in LXA4 in the spleen (Figure 3C) and increased numbers of Treg cells in the thymus and spleen of T. cruzi–infected SOCS2 KO mice when compared with WT infected counterparts (Figure 3D). TGF-β levels were increased and IL-10 levels decreased in spleen of infected SOCS2 KO mice compared with WT mice (data not shown). Additionally, SOCS2 deficiency resulted in a reduction in the generation of memory T cells specific for T. cruzi. SOCS2-deficient lymphocytes harvested from chronically infected mice produced reduced levels of IFN-γ during recall responses in vitro when compared with WT infected mice (Figure 3E).

      Myocardial Inflammation Is Reduced in SOCS2 KO Mice

      The impact of SOCS2 deficiency on the development of infection-induced myocarditis was next examined. There was intense lymphocytic myocarditis and deposition of collagen in the myocardium of WT mice 15 days post infection (Figure 4). By contrast, there was only mild myocarditis and few nests of inflammatory cells in infected SOCS2 KO mice (Figure 4). However, no difference in the number of parasite nests was found between WT and SOCS2 KO mice (data not shown).
      Figure thumbnail gr4
      Figure 4The myocardial pathology was examined in infected WT and SOCS2 KO mice 15 days post infection. Sections of cardiac tissue from WT (A,C, and E) and SOCS2 KO (B, D, and F) mice were either uninfected (A and B) or infected (CF) with 103 trypomastigotes, fixed, and then stained with H&E and Gomori's Trichrome. Thin arrows indicate tissue inflammatory infiltrate (C and D), and thick arrows indicate the site of fibrosis (E and F) in the tissue. The inflammatory score (G) and the cardiac fibrosis (H) are shown. *P < 0.001 for SOCS2 versus WT mice infected with T. cruzi. Original magnification: ×4 (A and B); ×10 (C and D); ×20 (E and F); ×40 (CF insets). The myocardial inflammation was reduced in infected SOCS2 KO mice compared to infected WT mice. Data are shown as the means ± SD and are representative of two independent experiments. *P < 0.05 for SOCS2 versus WT mice infected with T. cruzi.

      SOCS2 Expression Is Critical for Cardiac Myocyte/Cardiac Function During T. cruzi Infection

      Declining cardiac function is a significant source of mortality in both experimental models and in patients suffering from Chagas disease. The effects of SOCS2 deficiency in the modulation of immune response and development of myocarditis during T. cruzi infection prompted the examination of these mice for any evidence of myocardial dysfunction. Echocardiography performed 15 days post infection demonstrated no significant differences in right ventricular area or ejection fraction (Figure 5, A and B) in infected WT or SOCS2 KO mice compared with uninfected mice. Infected SOCS2 KO mice displayed cardiac hypertrophy as determined by higher fraction of circumferential area and increased left ventricular mass when compared with WT animals (Figure 5, C and D).
      Figure thumbnail gr5
      Figure 5Evaluation of cardiac hypertrophy in infected SOCS2 KO mice. Cardiac function of WT and SOCS2 KO mice infected with 1000 trypomastigotes of T. cruzi as assessed by echocardiography. A: Right ventricular (RV) area, ejection fraction (EF) (B), fraction of circumferential area (FAC) (C), and left ventricular hypertrophy computed from LV mass (mg)/LV (D) area (mm2). *P < 0.05 versus WT uninfected, P < 0.05 versus WT infected, and P < 0.05 versus SOCS2 KO uninfected. Infected SOCS2 KO mice displayed cardiac hypertrophy as determined by higher FAC and increased LV mass when compared with WT animals (C and D). CT, control.
      Patch clamping and confocal microscopy were used to evaluate cardiac myocyte physiology because changes in electrophysiological properties and calcium handling are a major cause of heart failure.
      • Roman-Campos D.
      • Campos A.C.
      • Gioda C.R.
      • Campos P.P.
      • Medeiros M.A.
      • Cruz J.S.
      Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats.
      • Gomez A.M.
      • Valdivia H.H.
      • Cheng H.
      • Lederer M.R.
      • Santana L.F.
      • Cannell M.B.
      • McCune S.A.
      • Altschuld R.A.
      • Lederer W.J.
      Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure.
      Reduced L-type calcium and outward potassium currents in SOCS2 deficient cardiac myocytes were observed 15 days post infection compared to infected WT mice (Figure 6, A–D). However, in uninfected WT and SOCS2 KO mice, L-type calcium and outward potassium currents, and action potential repolarization were almost identical. Furthermore, infected SOCS2 KO mice displayed an increased time to action potential repolarization when compared with WT mice (Figure 6, E and F). Typical line-scan fluorescence images recorded from electrically stimulated Fluo-4 AM–loaded ventricular myocytes are shown in Figure 7, A and B. T. cruzi infection led to a significant increase in the amplitude of the calcium transient in cardiac myocytes isolated 15 days after infection (Figure 7) (peak Ca2+ F/F0, WT control: 3.362 ± 0.091, n = 84 isolated cells; WT infected: 3.986 ± 0.133, n = 82 isolated cells; P < 0.001). There was no difference in the kinetics of decay of the calcium transient (T50, ms) after infection of cardiac myocytes (Figure 7) (WT control: 266.1 ± 5.879, n = 84 isolated cells; WT infected: 261.4 ± 6.695, n = 82 isolated cells). In SOCS2-deficient cardiac myocytes, there was an increase in calcium transient after infection that was only slightly smaller than in infected WT mice (Figure 7) (peak Ca2+ F/F0, WT infected: 3.986 ± 0.133, n = 82 isolated cells; and SOCS2 KO infected: 3.835 ± 0.154, n = 54 isolated cells). In addition, the kinetics of decay of the calcium transient after infection of cardiac myocytes was slower in infected SOCS2-deficient cardiac myocytes than infected WT (Figure 7) (WT infected: 261.4 ± 6.695, n = 82 isolated cells; and SOCS2 KO infected: 294.9 ± 7.509, n = 54 isolated cells; P < 0.05).
      Figure thumbnail gr6
      Figure 6The consequence of the absence of SOCS2 alters the electrophysiological characteristics of cardiac myocytes during T. cruzi infection. A: Representative L-type calcium current (ICa,L) recordings from WT CT, WT 15 dpi, SOCS2 KO CT, SOCS2 KO 15 dpi. B: Voltage dependence of ICa,L, plotted as current density (pA/pF) from −40 to 50 mV for WT CT, WT 15 dpi, SOCS2 KO CT, SOCS2 KO 15 dpi). C: Representative transient outward potassium current (Ito) recordings from WT CT, WT 15 dpi, SOCS2 KO CT, SOCS2 KO 15 dpi. D: Voltage dependence of Ito, plotted as current density (pA/pF) from −40 to 70 mV for WT CT, WT 15, SOCS2 KO CT, SOCS2 KO 15 dpi). E: Representative action potential (AP) from WT CT, WT 15 dpi, SOCS2 KO CT, SOCS2 KO 15 dpi. F: Bar graphs comparing AP repolarization time at different repolarization levels. n = number of cells. B: P < 0.05 SOCS2 KO 15 dpi and WT 15 dpi versus WT and SOCS2 KO uninfected; §P < 0.05 SOCS2 KO 15 dpi versus WT 15 dpi. D: *P < 0.05 WT 15 dpi and SOCS2 15 dpi versus WT and SOCS2 KO uninfected. F: Parameters were significantly increased in SOCS2 infected mice. *P < 0.05 versus WT uninfected; P < 0.05 versus WT 15 dpi; and P < 0.05 versus SOCS2 KO uninfected. CT, control; dpi, days post infection.
      Figure thumbnail gr7
      Figure 7T. cruzi infection alters calcium handling in ventricular myocytes from SOCS2 KO mice. The T. cruzi infection increased the peak Ca2+ transient amplitude in cardiomyocytes obtained from WT and SOCS2 KO mice, but changed the decay kinetics of only the infected SOCS2 KO mice. A: Representative confocal images of electrically stimulated intracellular Ca2+ transient recordings in ventricular myocytes. B: Representative Ca2+ transient line-scan profile. CT, uninfected mice; dpi, days post infection.

      Discussion

      We have described, for the first time, the role of SOCS2 in the pathogenesis of an experimental model of Chagas disease. Our results can be summarized as follows: i) T. cruzi infection induced SOCS2 expression in the heart of mice that was, in part, dependent on the activation of the 5-LO pathway; ii) SOCS2 deficiency resulted in reduction in peripheral parasitemia, but not in heart parasitism, and in the down-modulation of proinflammatory cytokines, including TNF-α, IL-12, and IFN-γ, and reduced myocardial inflammation; iii) although IFN-γ was reduced, SOCS2-deficient macrophages were hyperresponsive to this cytokine and dealt with infection more efficiently; iv) mechanistically, enhanced number of Treg cells and enhanced levels of LXA4 were associated with decreased inflammatory responses and reduced production of proinflammatory mediators; and v) in the absence of SOCS2, there was myocardial (hypertrophy) and cardiac myocyte (altered calcium handling) dysfunction. Altogether, these results clearly show that SOCS2 expression facilitates inflammatory and immune responses in T. cruzi–infected mice at the cost of preventing myocardial dysfunction.
      Initial experiments demonstrated that SOCS2 expression induced by T. cruzi infection was partially dependent on the 5-LO pathway. This pathway has received attention in the field of inflammation research because of the association of infection and leukotriene B (LTB) synthesis. LTBs have been reported to be important in the control of infection by protozoan parasites.
      • Henderson Jr., W.R.
      • Chi E.Y.
      The importance of leukotrienes in mast cell-mediated Toxoplasma gondii cytotoxicity.
      • Machado E.R.
      • Ueta M.T.
      • Lourenco E.V.
      • Anibal F.F.
      • Sorgi C.A.
      • Soares E.G.
      • Roque-Barreira M.C.
      • Medeiros A.I.
      • Faccioli L.H.
      Leukotrienes play a role in the control of parasite burden in murine strongyloidiasis.
      In addition to LTBs, 5-LO induces LXA4 production that triggers the AhR receptor altering the expression of SOCS2.
      • Machado F.S.
      • Johndrow J.E.
      • Esper L.
      • Dias A.
      • Bafica A.
      • Serhan C.N.
      • Aliberti J.
      Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent.
      These results suggest that production of a 5-LO–derived molecule, presumably LXA4, contributes to SOCS2 expression during experimental T. cruzi infection. It is interesting to notice that the phenotype of 5-LO–deficient mice infected with T. cruzi
      • Pavanelli W.R.
      • Gutierrez F.R.
      • Mariano F.S.
      • Prado C.M.
      • Ferreira B.R.
      • Teixeira M.M.
      • Canetti C.
      • Rossi M.A.
      • Cunha F.Q.
      • Silva J.S.
      5-Lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection.
      is similar to those observed here. Indeed, absence of 5-LO was associated with decreased inflammatory infiltrate and production of proinflammatory cytokines,
      • Pavanelli W.R.
      • Gutierrez F.R.
      • Mariano F.S.
      • Prado C.M.
      • Ferreira B.R.
      • Teixeira M.M.
      • Canetti C.
      • Rossi M.A.
      • Cunha F.Q.
      • Silva J.S.
      5-Lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection.
      suggesting that in addition to absence of LTB, decreased expression of SOCS2 could account for the phenotype observed in 5-LO–deficient mice. Since there was still increased SOCS2 expression in the absence of 5-LO, our results also suggest that other pathways may be involved in SOCS2 expression on infection.
      SOCS2 deficiency resulted in a reduction in peripheral parasitemia, but not in heart parasitism, even in presence of reduced expression of IFN-γ. The in vitro observations suggest that resistance to infection (parasite replication) could be attributable to an increased ability of SOCS2-deficient macrophages stimulated with IFN-γ to kill intracellular amastigotes via increased production of NO. We found that SOCS2-deficient macrophages were hyperresponsive to IFN-γ stimulation, which could be attributed to reduced inhibition of IFN-γ receptor signaling. Interestingly, T. cruzi–infected SOCS2 KO macrophages that are not stimulated with cytokines release greater numbers of trypomastigotes in the culture supernatants compared with WT cells despite producing a similar concentration of NO. Further studies are required to understand the molecular mechanisms underlying these observations.
      Absence of SOCS2 was clearly associated with decreased levels of cytokines in spleen and heart of T. cruzi–infected mice and decreased inflammatory infiltrate. This was at first a unexpected finding, because SOCS2 is known to suppress the function of cytokines. Hence, absence of SOCS2 would presumably be associated with enhanced cytokine responses. However, two observations help to explain our results. In the absence of SOCS2, there were greater numbers of Treg cells and enhanced production of LXA4.
      The role of Treg in experimental T. cruzi infection is still somewhat controversial.
      • Tonelli R.R.
      • Torrecilhas A.C.
      • Jacysyn J.F.
      • Juliano M.A.
      • Colli W.
      • Alves M.J.
      In vivo infection by Trypanosoma cruzi: the conserved FLY domain of the gp85/trans-sialidase family potentiates host infection.
      Part of this controversy stems from the difficulty in depleting specifically Treg cells in vivo and in differences of experimental models used. However, CD4+CD25+ cells expressing FoxP3 have been shown to associate with better outcome in chagasic patients.
      • Sathler-Avelar R.
      • Vitelli-Avelar D.M.
      • Teixeira-Carvalho A.
      • Martins-Filho O.A.
      Innate immunity and regulatory T-cells in human Chagas disease: what must be understood?.
      Moreover, an increased frequency of Treg cells was found during the acute phase of experimental T. cruzi infection in mice.
      • Tonelli R.R.
      • Torrecilhas A.C.
      • Jacysyn J.F.
      • Juliano M.A.
      • Colli W.
      • Alves M.J.
      In vivo infection by Trypanosoma cruzi: the conserved FLY domain of the gp85/trans-sialidase family potentiates host infection.
      In mice, there is also evidence to show that Treg cells migrate to the heart of infected mice and may have a protective role.
      • Mariano F.S.
      • Gutierrez F.R.
      • Pavanelli W.R.
      • Milanezi C.M.
      • Cavassani K.A.
      • Moreira A.P.
      • Ferreira B.R.
      • Cunha F.Q.
      • Cardoso C.R.
      • Silva J.S.
      The involvement of CD4+CD25+ T cells in the acute phase of Trypanosoma cruzi infection.
      Taken together, these studies suggest that Treg cells likely control disease severity during T. cruzi infection. Therefore, the enhanced numbers of Treg cells in SOCS2 KO mice may explain the reduced tissue inflammation observed in these mice. Production of anti-inflammatory cytokines, including IL-10 and TGF-β, account for most of the immunosuppressive effects of Treg cells in various experimental systems.
      • Josefowicz S.Z.
      • Lu L.F.
      • Rudensky A.Y.
      Regulatory T cells: mechanisms of differentiation and function.
      In our model, SOCS2 was not associated with increased production of IL-10, but with enhanced expression of TGF-β, suggesting the latter is the major pathway by which Treg cells may alter immune function in SOCS2-deficient mice. It is not clear why absence of SOCS2 would result in greater numbers of Treg cells after T. cruzi infection. AhR, a lipoxin receptor that may trigger SOCS2 expression, modifies Treg balance by modifying the cytokine milieu.
      • Stevens E.A.
      • Mezrich J.D.
      • Bradfield C.A.
      The aryl hydrocarbon receptor: a perspective on potential roles in the immune system.
      Therefore, we are currently investigating the possibility of induction of SOCS2 expression by activation of AhR receptor during T. cruzi infection as a modulator of Treg generation and expansion.
      There was an increase in levels of LXA4 in SOCS2 KO mice after T. cruzi infection. Because LXA4 is an effective anti-inflammatory molecule,
      • Serhan C.N.
      Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways.
      • Machado F.S.
      • Aliberti J.
      Role of lipoxin in the modulation of immune response during infection.
      enhanced levels of this lipid could account, at least partially, for the reduced inflammation and production of proinflammatory cytokines. It must be noted that LXA4 may function in SOCS2-dependent and -independent manners. As the lipid was enhanced in the absence of SOCS2, it is clear that SOCS2-independent pathways could be operational in the latter animals to mediate any anti-inflammatory actions of LXA4.
      Severe chronic Chagas heart disease is characterized by dilation of chambers, decreased ejection fraction, and electrical instability.
      • Marin-Neto J.A.
      • Cunha-Neto E.
      • Maciel B.C.
      • Simoes M.V.
      Pathogenesis of chronic Chagas heart disease.
      In our system, evaluation of heart function in WT mice at 15 days after infection was not associated with any major changes in heart function. Only when experiments were conducted in SOCS2-deficient mice did we notice that LV mass was increased and the fraction of circumferential area enhanced, suggesting that there were compensatory mechanisms operating at this early stage in these mice to prevent dysfunction. Overall, these data are indicative that SOCS2 deficient mice are more likely to develop heart dysfunction on infection. Electrophysiological studies showed that infection caused reduction of the outward potassium current and in L-type calcium current amplitude. These changes were more marked in the absence of SOCS2 and the calcium transient was also prolonged. Altogether, these changes in SOCS2-deficient mice could lead to greater duration of the action potential that could provide an electrophysiological basis for the echocardiographic findings. It has been demonstrated that different cytokines can modulate ionic currents in isolated cardiac myocytes.
      • Fernandez-Velasco M.
      • Ruiz-Hurtado G.
      • Hurtado O.
      • Moro M.A.
      • Delgado C.
      TNF-alpha downregulates transient outward potassium current in rat ventricular myocytes through iNOS overexpression and oxidant species generation.
      • Hagiwara Y.
      • Miyoshi S.
      • Fukuda K.
      • Nishiyama N.
      • Ikegami Y.
      • Tanimoto K.
      • Murata M.
      • Takahashi E.
      • Shimoda K.
      • Hirano T.
      • Mitamura H.
      • Ogawa S.
      SHP2-mediated signaling cascade through gp130 is essential for LIF-dependent I CaL, [Ca2+]i transient, and APD increase in cardiomyocytes.
      A correlation between reduced cardiac myocyte shortening and increased levels of TNF-α and IFN-γ during T. cruzi infection has been previously demonstrated.
      • Roman-Campos D.
      • Duarte H.L.
      • Sales Jr., P.A.
      • Natali A.J.
      • Ropert C.
      • Gazzinelli R.T.
      • Cruz J.S.
      Changes in cellular contractility and cytokines profile during Trypanosoma cruzi infection in mice.
      It is still unclear how the profile of cytokines found in the absence of SOCS2 correlates with the altered cardiac myocyte electrical remodeling. However, results showing decreased expression of SOCS1 and SOCS3 may provide an explanation to our findings. SOCS1 and SOCS3 expression is known to counteract the actions of molecules, such as IL-6, which induce cardiac myocyte hypertrophy.
      • Takahashi N.
      • Saito Y.
      • Kuwahara K.
      • Harada M.
      • Tanimoto K.
      • Nakagawa Y.
      • Kawakami R.
      • Nakanishi M.
      • Yasuno S.
      • Usami S.
      • Yoshimura A.
      • Nakao K.
      Hypertrophic responses to cardiotrophin-1 are not mediated by STAT3, but via a MEK5-ERK5 pathway in cultured cardiomyocytes.
      For example, leukemia inhibitory factor (LIF), a member of the IL-6 family, has been reported to increased ICa,L thought activation of ERK, a pathway known to be blocked by SOCS.
      • Kato T.
      • Sano M.
      • Miyoshi S.
      • Sato T.
      • Hakuno D.
      • Ishida H.
      • Kinoshita-Nakazawa H.
      • Fukuda K.
      • Ogawa S.
      Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats.
      Because there was reduced SOCS1 and SOCS3 in SOCS2-deficient mice, one would expect greater ability of the inflammatory milieu found in the infected heart to induce more hypertrophy. However, the mediators of this hypertrophic response still need to be identified.
      In summary, this report demonstrates the importance of SOCS2 in the modulation of experimental T. cruzi infection. In the absence of SOCS2, there is decreased inflammation and parasitemia, which are associated with an increase in the number of Treg cells and the LXA4 levels. This occurs at the cost of greater infection-associated heart dysfunction. Therefore, the expression of SOCS2 during T. cruzi infection decreases heart dysfunction at the cost of greater parasitemia and tissue inflammation. These studies highlight the role of SOCS2 in balancing immune and physiological functions in the heart of animals subjected to an infections insult.

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