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Defective T-Lymphocyte Migration to Muscles in Dystrophin-Deficient Mice

      Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3500 males, is caused by mutations in the dystrophin gene. DMD leads to degeneration of skeletal and cardiac muscles and to chronic inflammation. The mdx/mdx mouse has been widely used to study DMD; this model mimics most characteristics of the disease, including low numbers of T cells in damaged muscles. In this study, we aimed to assess migration of T cells to the heart and to identify any alterations in adhesion molecules that could possibly modulate this process. In 6-week-old mdx/mdx mice, blood leukocytes, including T cells, were CD62L+, but by 12 weeks of age down-modulation was evident, with only approximately 40% of T cells retaining this molecule. Our in vitro and in vivo results point to a P2X7-dependent shedding of CD62L (with high levels in the serum), which in 12-week-old mdx/mdx mice reduces blood T cell competence to adhere to cardiac vessels in vitro and to reach cardiac tissue in vivo, even after Trypanosoma cruzi infection, a known inducer of lymphoid myocarditis. In mdx/mdx mice treated with Brilliant Blue G, a P2X7 blocker, these blood lymphocytes retained CD62L and were capable of migrating to the heart. These results provide new insights into the mechanisms of inflammatory infiltration and immune regulation in DMD.
      Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 1 in 3500 males and is caused by mutations in DMD, the dystrophin gene. DMD is the largest gene described in humans, with more than 2.5 million base pairs.
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      experiments using null mutants for both dystrophin and perforin showed that lack of perforin decreased dystrophic muscle pathology.
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      T cell infiltrates in the muscles of patients with dermatomyositis and polymyositis are dominated by CD28null T cells.
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      It is currently not known why few T cells are found in DMD muscles, or whether pre- or postendothelial interactions are lacking or malfunctional.
      Leukocyte recruitment into inflamed tissues is a multistep cascade of adhesive interactions between leukocytes and endothelial cells.
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      Leukocyte-endothelial adhesion molecules.
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      The second step is the integrin-dependent firm adhesion of leukocytes (expressing LFA-1 and VLA-4) to the endothelial layer (expressing ICAM and VCAM) and, finally, transmigration.
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      Integrin function in T-cell homing to lymphoid and nonlymphoid sites: getting there and staying there.
      A large number of cell types express CD62L (including monocytes, T and B lymphocytes, eosinophils, NK cells, and neutrophils), and a variety of ligands have been described (including GlyCAM-1, CD34, MadCAM-1, and PSGL-1).
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      Blood leukocyte recruitment and transmigration to tissues is essential for appropriate inflammatory responses against infection and injury.
      CD62L, also known as L-selectin, is a 90-kDa glycoprotein with two basic functions: lymphocyte homing to lymph nodes during normal blood/lymph recirculation and migration of leukocytes to inflammatory sites.
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      Some authors have believed that CD62L is important to T cells homing to lymph nodes, but not to nonlymphoid tissues.
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      However, it was shown by others that CD62L also plays a role in T-cell migration to inflamed tissues.
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      The binding of CD62L to its ligands on activated endothelial cells initiates a cascade of downstream intracellular events in the leukocyte, resulting in free cytosolic calcium increase and phosphorylation of targeted proteins, including mitogen-activated protein kinases (MAPKs).
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      The cytoplasmic domains of TNFalpha-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding.
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      CD21 and CD62L shedding are both inducible via P2X7Rs.
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      The P2X7 receptor is a ligand-gated ion channel that mediates nonselective cation conductance when stimulated with appropriate ligands, such as extracellular ATP (ATPe). However, prolonged exposure or high concentrations of ATPe lead to a pore formation that renders plasma membrane permeable to molecules such as propidium iodide (PI) and YO-PRO Yellow.
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      The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).
      A variety of cells express P2X7 receptor, including lymphocytes, granulocytes, osteoclasts, osteoblasts, microglia, monocytes, and macrophages,
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      P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells.
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      Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel.
      and it is known that inflammatory cytokines (eg, TNF and IFN-γ) can up-regulate the expression of P2X7.
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      Modulation of P2X7 nucleotide receptor expression by pro- and anti-inflammatory stimuli in THP-1 monocytes.
      Regarding CD62L shedding induced by P2X7 activity, Sengstake et al
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      CD21 and CD62L shedding are both inducible via P2X7Rs.
      showed that agonistic stimulation of the receptor resulted in proteolytic cleavage of CD62L from both naïve and memory B and T cells. Scheuplein et al
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      NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells.
      showed that endogenous sources of NAD+ and ATP released from lysed cells activate P2X7 on T lymphocytes, leading to CD62L shedding from cell surface.
      Despite the strong inflammatory process evident in DMD muscles, little is known about the mechanisms governing leukocyte trafficking into these tissues. To evaluate the ability of T cells to migrate to cardiac tissue, we sought to explore the role played by adhesion molecule-mediated T cell-endothelium interactions in mdx/mdx mice. Our results show a defective migration of T cells to the heart of these mice, which may be, at least in part, a result of CD62L shedding from T-cell surface. Furthermore, we show that this shedding is induced by P2X7 in vitro and in vivo, because the blockage of the receptor after Brilliant Blue G (BBG) treatment led T lymphocytes in mdx/mdx mice to retain functional CD62L on the membrane and allowed their migration to the heart.

      Materials and Methods

      Animals

      All experiments were conducted with mice obtained from the Center for Breeding of Laboratory Animals at Fundação Oswaldo Cruz. We used 6-, 9-, 12-, 24-, and 48-week-old male mdx/mdx mice and age-matched C57BL/10 control mice. Mice were housed for 7 to 10 days at the Center for Animal Experimentation under environmental factors and sanitation according to the Guide for the Care and Use of Laboratory Animals (8th edition, 2011). This project was approved by the Fiocruz Committee of Ethics in Research (0308/06), according to resolution 196/96 of the National Health Council of Brazilian Ministry of Health. To avoid possible interference from genetic and environmental drift in the mouse colony used, most experiments were repeated with C57BL/10 and mdx/mdx mice obtained from the Multidisciplinary Center for Biological Investigation (CEMIB/Unicamp, Sao Paulo, Brazil). This is an internationally certified animal breeding facility and a member of the International Council for Laboratory Animal Science (ICLAS). All results were reproducible using both colonies.

      Isolation of Cardiac Inflammatory Cells

      Hearts from mdx/mdx and C57BL/10 mice were collected and cut in fragments 1 to 2 mm thick in ice-cold PBS. Fragments were then transferred to a 0.1% solution of collagenase type IV (5.2 U/mg) (Sigma-Aldrich, St. Louis, MO) and submitted to four or five cycles of enzymatic digestion under gentle agitation for 20 minutes each at 37°C. Isolated cells were centrifuged and immediately transferred to ice-cold RPMI 1640 medium supplemented with 10% fetal calf serum (Sigma-Aldrich) and maintained in ice.

      Flow Cytometry

      For phenotypic labeling by flow cytometry, all cells were incubated for 30 minutes at 4°C with RPMI 1640 medium supplemented with 10% fetal calf serum and 10% inactivated normal sheep serum for FcγR blockage. Cells were incubated with previously titrated APC/Cy7-conjugated anti-CD3, fluorescein isothiocyanate-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD8, fluorescein isothiocyanate-conjugated anti-CD62L, PE/Cy7-conjugated anti-CD69, PE-conjugated anti-LFA-1 (all from eBioscience, San Diego, CA), PE-conjugated anti-CD49d (SouthernBiotech, Birmingham, AL), and/or PE-conjugated anti-CD43 (BD Pharmingen, San Diego, CA) for 30 minutes in ice. All samples were washed twice in RPMI 1640 medium and fixed using 2% formaldehyde (EMD Chemicals, Gibbstown, NJ) in PBS until acquisition in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) or a Dako Cyan ADP analyzer (Beckman Coulter, Houston, TX). Data analysis was performed using Dako Summit version software 4.3. For cytokine secretion, we used a cytometric bead array flex set, as recommended by the manufacturer (BD Pharmingen).

      Permeabilization Assay

      Blood was collected by cardiac puncture in the presence of heparin and peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Histopaque 1077 gradient (StemCell Technologies, Vancouver, BC). Cells were incubated for 30 minutes at 4°C with fluorescein isothiocyanate-conjugated anti-CD3, anti-CD4, or anti-CD8 (SouthernBiotech), washed in RPMI 1640 medium, resuspended in prewarmed RPMI 1640/HEPES 10 mmol/L for 5 minutes at 37°C, and incubated for 10 minutes in the presence or absence of ATPe (5 mmol/L; Sigma-Aldrich). During the last 5 minutes of incubation, propidium iodide (Sigma-Aldrich) was added at the final concentration of 0.1 μg/mL, and all samples were immediately acquired in a FACSCalibur flow cytometer (BD Biosciences). Peritoneal cells were collected using ice-cold RPMI 1640/HEPES 10 mmol/L; cells in a macrophage region based on forward scatter by side scatter (FSC × SSC) were used as a positive control.

      Purification of Blood T Lymphocyte Subsets

      PBMCs were obtained as described above, and 1 × 107 cells were incubated with bead-conjugated anti-CD4, anti-CD8, or anti-CD3 (Miltenyi Biotec, Auburn, CA) for 20 minutes at 4°C, resuspended in 500 μL of PBS, and applied to a MiniMACS column (Miltenyi Biotec) under a magnetic field, as recommended by the manufacturer. Samples were centrifuged and resuspended in RPMI 1640 medium and sort purity was determined by labeling the cells with fluorescein isothiocyanate-conjugated anti-CD4, anti-CD8, or anti-CD3 (SouthernBiotech) for flow cytometry analysis. We used samples of ≥95% enrichment for T-cell subsets.

      Dot Blotting

      Blood samples were obtained from mdx/mdx or C57BL/10 mice by cardiac puncture without heparin; samples were left at room temperature for 30 minutes. Serum was obtained by blood centrifugation at 500 × g for 5 minutes, and protein concentration was determined using a Lowry modified RC DC protein assay (Bio-Rad Laboratories, Hercules, CA). For dot-blotting assay, serum samples with 80, 40, or 20 μg of total proteins/100 μL of Tris buffer were added per well. Proteins were then immobilized in Tris buffer prehydrated nitrocellulose membranes for 15 minutes at room temperature, blocked for 60 minutes in Tris buffer supplemented with 5% bovine serum albumin (Sigma-Aldrich), and incubated with anti-CD62L or anti-LFA-1 (eBioscience) for 1 hour at room temperature. The membrane was then washed twice with Tris buffer and incubated for 1 hour with goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (SouthernBiotech). Primary antibody was suppressed in one of the samples for negative control. Detection was performed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium solution (BCIP/NBT; SouthernBiotech).

      Western Blotting

      Purified T lymphocytes were lysed, as described previously,
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      • Menna-Barreto R.F.
      • Coutinho-Silva R.
      • Persechini P.M.
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      P2X7 modulatory web in Trypanosoma cruzi infection.
      with extraction buffer (Tris-HCl 50 mmol/L, 1% NP-40, leupeptin 1 mmol/L, phenylmethylsulfonyl fluoride 100 mmol/L, pepstatin A 1 mmol/L, EDTA 100 mmol/L; all purchased from Sigma-Aldrich). Total proteins (50 μg/slot) were resolved using SDS-PAGE (12%). Proteins were then transferred to nitrocellulose membranes, blocked using nonfat dry milk (5%) in Tris buffer, and incubated with anti-P2X7 primary antibody (Alomone, Jerusalem, Israel) for 2 hours. The membrane was then rinsed in blocking buffer and incubated with goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (SouthernBiotech) for 1 hour. Detection was performed using BCIP/NBT (SouthernBiotech). For TCR αβ detection, a fragment of left ventriculus was extensively washed in ice-cold PBS and cut in fragments 1 to 2 mm thick. Total proteins were extracted using extraction buffer, centrifuged at 500 × g for 5 minutes and subjected to SDS-PAGE as described above. Nitrocellulose membranes were incubated with biotin-conjugated anti-TCR αβ (MyBioSource, San Diego, CA), rinsed in blocking buffer, and incubated with horseradish peroxidase-conjugated streptavidin (Dako, Carpinteria, CA) for 1 hour. Detection was performed by chemifluorescence using an ECL plus kit (GE Healthcare, Piscataway, NJ) in a Storm 860 imaging system (Amersham-GE Healthcare, Uppsala, Sweden).

      In Vitro Blockage of P2X7

      Blood CD3+ T lymphocytes purified from 9-week-old mdx/mdx and C57BL/10 mice were preincubated or not with KN-62 (100 nmol/L) for 30 minutes and then incubated with only medium or ATPe 100 μmol/L for 1 hour. We chose this concentration based on a kinetic study considering the lowest dose of ATP that induced CD62L shedding with no cell death or permeabilization, as ascertained by 7-aminoactinomycin D and PI, respectively (data not shown). Supernatants were collected and used in dot-blotting assays revealed with anti-CD62L and goat anti-rabbit alkaline phosphatase-conjugated secondary antibody.

      Adhesion Assay

      The ability of peripheral blood CD4+ and CD8+ T cells to attach to cardiac vessels was tested in a modified Stamper-Woodruff assay.
      • Stamper Jr, H.B.
      • Woodruff J.J.
      Lymphocyte homing into lymph nodes: in vitro demonstration of the selective affinity of recirculating lymphocytes for high-endothelial venules.
      Freshly cut, unfixed, frozen cardiac sections (15 μm thick) from 6- or 24-week-old mdx/mdx or C57BL/10 mice were incubated with 100 μL of a cell suspension containing 3 × 106 purified CD4 or CD8 T cells from 6- or 12-week-old mdx/mdx mice in RPMI 1640 medium with 10 mmol/L HEPES and 5% fetal calf serum. The incubation was performed under gentle circular agitation at 60 revolutions per minute for 40 minutes at room temperature. After extensive washings with prewarmed PBS, the slides were fixed and stained with a Romanowsky-based staining kit (Laborclin, Pinhais, Brazil), according to the manufacturer's instructions. The number of adhered cells per blood vessel was counted under a light microscope. To characterize cell surface molecules involved in T-cell adhesion to endothelial cardiac blood vessels, some experiments were conducted with purified T cell subsets pretreated with different concentrations of anti-CD62L or anti-LFA-1 monoclonal antibodies (eBioscience).

      BBG Treatment for in Vivo Blockage of P2X7

      BBG reagent (Sigma-Aldrich) was diluted at 3 mg/mL in vehicle solution (calcium- and magnesium-free PBS/0.2% dimethyl sulfoxide; Sigma-Aldrich). For in vivo treatment, mdx/mdx and C57BL/10 mice were weighed once a week during drug administration and received freshly prepared BBG (45.5 mg/kg) or vehicle via intraperitoneal injection every 48 hours. Mice were treated from 9 to 12 weeks of age.

      T. cruzi Infection

      Parasites were isolated from infected Swiss-Webster mice as described previously
      • Araújo-Jorge T.C.
      • Sampaio E.P.
      • De Souza W.
      • Meirelles Mde N.
      Trypanosoma cruzi: the effect of variations in experimental conditions on the levels of macrophage infection in vitro.
      and mdx/mdx or C57BL/10 mice were intraperitoneally injected with 1 × 104 blood trypomastigote forms of T. cruzi Y strain in 200 μL of PBS. Age-matched, uninfected control mice received 200 μL of PBS and were treated under the same conditions. Samples were collected on day 15 after infection.

      Histopathological Analysis

      Control and infected C57BL/10 and mdx/mdx mice were euthanized, and the hearts were removed, sagittally divided, and fixed using Millonig-Rosman solution (10% formaldehyde in PBS). Paraffin-embedded samples were further processed and stained with H&E in 5-μm-thick slices. In T. cruzi-infected mice, the number of inflammatory foci (defined as ≥10 inflammatory cells) and the number of inflammatory cells per focus were determined by scanning the whole tissue in approximately 30 individual microscopic fields per sample.

      Immunohistochemistry

      Cardiac muscles were collected, frozen using cold isopentane,
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      • Partridge T.
      • Hoffman E.P.
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      Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne muscular dystrophy.
      and stored in liquid nitrogen until use. Cardiac slices (8 μm thick) were then fixed with 4% formaldehyde for 10 minutes at room temperature and then were washed twice in PBS. Endogenous peroxidase was blocked with 3% H2O2 for 10 minutes, and then slices were washed twice more. FcγR was blocked, and the slices were incubated with anti-CD3ε chain primary antibody (SouthernBiotech) overnight at room temperature under agitation. Slices were washed with PBS and incubated with goat anti-rat biotin-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour and then with horseradish peroxidase-conjugated streptavidin (Dako) for 1 hour. Detection was performed using 3-amino-9-ethylcarbazole (Dako).

      Biochemical Analysis

      CK isoforms from cardiac (CK-MB) and skeletal (CK-NAC) muscles were assessed in mouse serum. Blood samples were collected from tail snips in heparinized capillary tubes, centrifuged, and analyzed using commercially available kits (Wiener Laboratories, Rosario, Argentina). This quantitative assay is used as a marker of muscle damage; data are expressed as a rate of NADPH increase (ΔE/min) in three sequential readings in a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 340 nm.

      Spontaneous Mouse Activity

      We used a video tracking system to measure spontaneous activity (EthoVision XT6; Noldus, Leesburg, Holland), with a recording camera placed at a distance of 1 m from the subjects. Each mouse was individually adapted in the arena for 10 minutes before the experimental measurement, which lasted 20 minutes.
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      • Takenaga R.
      • Hayata A.
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      • Hashimoto H.
      Lipopolysaccharide affects exploratory behaviors toward novel objects by impairing cognition and/or motivation in mice: possible role of activation of the central amygdala.

      Statistical Analysis

      Data were analyzed using Student's t-test (GraphPad Prism version 4.00 software; GraphPad Software, La Jolla, CA). A P value of ≤0.05 was considered significant.

      Results

      Cardiac T-Cell Numbers and CD62L Shedding to Blood

      Given that most previous studies have associated T cells with increased pathology in DMD, we evaluated by flow cytometry the presence of these cells in cardiac muscles from 6-, 12-, 24-, and 48-week-old mdx/mdx mice (Figure 1). CD3+ T cells progressively decreased in the time frame studied (Figure 1A), although with a predominance of CD3+/CD4+ T cells over CD3+/CD8+ T cells in 6- and 12-week-old animals (Figure 1, B and C). We found neither NK, NKT, nor Treg cells in the cardiac muscle of mdx/mdx mice (data not shown), and did not find T cells at >5% in C57BL/10 mice.
      Figure thumbnail gr1
      Figure 1Distribution of T lymphocytes in cardiac tissue from mdx/mdx and C57BL/10 mice. Cardiac tissue was enzymatically dissociated, and all cells were obtained for flow cytometry analysis. Mononuclear cells were gated by FSC × SSC parameters and analyzed according to the expression of CD3 (A), CD3/CD4 (B), and CD3/CD8 (C) at 6, 12, 24, and 48 weeks of age. Data are expressed as means ± SD from five independent experiments. *P ≤ 0.05 versus C57BL/10. n = 4 or 5 animals per group.
      Because blood T cells from mdx/mdx mice could be less competent to migrate to cardiac tissue, we checked the panel of adhesion molecules expressed by blood and cardiac T cells. We observed normal levels of CD62L expression in blood T lymphocytes from 6- and 12-week-old C57BL/10 mice and 6-week-old mdx/mdx mice. This means that very few T cells were CD62L (Figure 2A). However, many T cells from 12-week-old mdx/mdx mice became CD62L (Figure 2A). This down-regulation was transitory, because we observed that 24-week-old mdx/mdx mice express similar levels of selectin, compared with age-matched C57BL/10 mice (data not shown). Cardiac T lymphocytes from 6- and 12-week-old mdx/mdx mice expressed low levels of this molecule (Figure 2B), as expected after endothelial interaction.
      • Langer H.F.
      • Chavakis T.
      Leukocyte-endothelial interactions in inflammation.
      We observed no differences in the expression of LFA-1, CD43, and CD49d in blood T cells from C57BL/10 and mdx/mdx mice at all ages, and there were always normal levels of CD62L expression in blood granulocytes and monocytes (data not shown). To determine whether the CD62L down-regulation could be due to cleavage from the T-cell surface, we evaluated the levels of this soluble molecule in mouse serum by dot blotting. The 12-week-old mdx/mdx mice had higher levels of soluble CD62L, compared with 6-week-old mice (Figure 2, C and E). LFA-1 was used as a control, and we never observed increased levels of this molecule in blood (Figure 2, D and F).
      Figure thumbnail gr2
      Figure 2T-cell expression and blood soluble CD62L in mdx/mdx mice. PBMCs (A) and cardiac cells (B) were obtained from 6- and 12-week-old mdx/mdx and C57BL/10 mice and labeled with anti-CD3 and anti-CD62L. Shown is the percentage of CD3+/CD62L PBMCs (A) and CD3+/CD62L+ (B) or isotype labeling (A and B) as gated in the morphological region (FSC × SSC) of mononuclear cells. Serum concentrations of CD62L (C) and LFA-1 (D) in 6- and 12-week-old mdx/mdx mice was evaluated by dot blotting using 20, 40, and 80 μg of total proteins per slot. Representative membranes of CD62L (E) and LFA-1 (F) of 12-week-old mdx/mdx mice are shown. Membrane labeling was performed with primary antibody, then phosphatase alkaline-conjugated goat anti-rat secondary antibody, and revealed with NBT/BCIP. For negative control, 80 μg of total proteins were immobilized and primary antibody was omitted. Data are expressed as means ± SD from four independent experiments. *P ≤ 0.05. n = 4 or 5 animals per group.
      CD62L is a key molecule for naïve T cells homing to lymph nodes, where antigen interaction takes place for cell activation and effector function. However, despite the shedding of CD62L that we observed, immunosuppression has not been described in mdx/mdx mice, suggesting that naïve T-cell homing to lymph nodes is preserved. We then decided to evaluate whether the shedding of CD62L was from activated or naïve T-cell subpopulations. Using the early activation marker CD69, we found no reduction of CD62L expression in CD69+ or CD69 T cells from 6-week-old C57BL/10 mice (Figure 3A), 6-week-old mdx/mdx mice (Figure 3B), and 12-week-old C57BL/10 mice (Figure 3C). On the other hand, although there was no down-regulation of CD62L in CD69 T cells from 12-week-old mdx/mdx mice (Figure 3D), only approximately 40% of CD69+ T cells from these mice retained this selectin on cell surface (Figure 3D).
      Figure thumbnail gr3
      Figure 3CD62L expression and activation status of blood T cells. PBMCs from 6- and 12-week-old C57BL/10 and mdx/mdx mice were labeled with anti-CD3, anti-CD69, and anti-CD62L monoclonal antibody and analyzed by flow cytometry in FSC × SSC gated PBMCs. Percentages of CD3+/CD69+ and CD3+/CD69 cells expressing CD62L are defined by R6 and R7 markers, respectively. Data are representative of three independent experiments. n = 3 or 4 animals per group.

      Limited Ability of Blood CD62L T Cells to Firmly Adhere to Cardiac Vessels

      Because the reduction of CD62L expression in blood T lymphocytes coincides with reduction of T cells in cardiac tissue, we tested the adhesion of these blood T cells to cardiac vessels in vitro. Purified blood CD4+ or CD8+ T lymphocytes from 6- or 12-week-old mdx/mdx mice were placed to adhere over cardiac tissue sections from 6- or 24-week-old C57BL/10 or mdx/mdx mice. Blood lymphocytes from 6- and 12-week-old mdx/mdx mice did not adhere to cardiac vessels in sections from control C57BL/10 mice (Figure 4, A and D). Likewise, T lymphocytes from 12-week-old mdx/mdx mice did not adhere to cardiac vessels from 6- or 24-week-old mdx/mdx mice, although endothelial cells were probably activated (Figure 4, B and D), because we observed inflammatory foci through H & E staining. On the other hand, blood T lymphocytes from 6-week-old mdx/mdx mice were capable of adhering to cardiac vessels from either 6- or 24-week-old mdx/mdx mice (Figure 4, C and D). We chose 24-week-old mice because the onset of tissue inflammation in the heart is present in older mice, which involves primarily macrophages. We also observed that CD4+ and CD8+ T cells from 6-week-old mdx/mdx mice preferably adhered to vessels in proximity to inflamed areas of mdx/mdx cardiac slices (data not shown). Moreover, we observed no differences in cardiac endothelial cell expression of VCAM and ICAM between 6- and 12-week-old mdx/mdx and age-matched C57BL/10 mice (data not shown). Because we found few T cells from 12-week-old mdx/mdx mice bound to cardiac vessels (Figure 4D), we tested whether the weak adhesion might be associated with the lower levels of CD62L on T cells from these mice. Blood purified CD4+ lymphocytes from 6-week-old mdx/mdx mice were then incubated with anti-CD62L or anti-LFA-1 before the adhesion assay. We observed that, although the blockage of LFA-1 is more efficient in impairing lymphocyte/endothelial cell interaction, the blockage of CD62L also reduces T-cell adhesion to cardiac vessels (Figure 4E). At this point, it seemed to us that there are two different pathways for CD62L shedding in 12-week-old mdx/mdx mice: first, normal shedding of CD62L from leukocytes, including T cells, during endothelial interaction for transmigration to inflamed tissues; second, CD62L shedding from T cells in blood, before blood vessel interaction, reducing endothelial adhesion. We therefore decided to explore possible mechanistic pathways that might be leading to the second pathway for CD62L shedding.
      Figure thumbnail gr4
      Figure 4T lymphocytes binding to cardiac endothelium. A–C: CD4+ T cells from mdx/mdx mice were purified from blood by magnetic sorting at indicated 6 or 12 weeks of age (wko) and were incubated over fresh frozen cardiac tissue slices from C57BL/10 or mdx/mdx. Arrows indicate blood purified T lymphocytes adhering to cardiac vessels. D: Quantification of purified CD4+ or CD8+ T cells adhering to cardiac blood vessels in C57BL/10 and mdx/mdx mice at 6 or 24 weeks of age. E: Blood purified CD3+ T cells from 6-week-old mdx/mdx mice were preincubated with either medium alone or neutralizing antibodies against CD62Lor LFA-1 at three different concentrations. These cells were then incubated under agitation over fresh frozen cardiac slices from 6-week-old mdx/mdx mice and the number of cells adhering to cardiac blood vessels was quantified. Data are expressed as means ± SD from three independent experiments. *P ≤ 0.05 mdx/mdx versus control (D) or a given concentration versus preincubation with CD62L or LFA-1 (E); P ≤ 0.05 versus all other variables. n = 5–7 animals per group. Original magnification, ×400.

      P2X7-Induced CD62L Shedding from Blood T Cells

      Because P2X7 receptor can induce CD62L proteolytic cleavage, we evaluated the function and expression of this receptor in T cells from C57BL/10 and mdx/mdx mice. We first performed a permeabilization assay to evaluate P2X7 ability to induce pore formation on cell membrane on activation by ATPe. We observed an increase in ATPe-induced permeabilization in T lymphocytes from 6-week-old mdx/mdx mice, compared with 6- and 12-week-old C57BL/10 and 12-week-old mdx/mdx mice (Figure 5, A and B). To determine P2X7 ability to induce CD62L shedding, we performed an in vitro assay with purified blood T cells obtained from 9-week-old mdx/mdx and C57BL/10 mice incubated in the presence or absence of an antagonist of P2X7 (KN-62) and then stimulated with ATPe. We chose the age of 9 weeks because this is when the shedding process begins (data not shown). T cells from mdx/mdx mice were more susceptible to ATPe-induced CD62L shedding than C57BL/10 mice, and this shedding was blocked by KN-62 (Figure 5C). Once more, LFA-1 was used as a control and no shedding was observed (Figure 5D). We performed Western blotting to evaluate whether P2X7 expression was up-regulated in T lymphocytes from 12-week-old mdx/mdx mice; levels of P2X7 expression were similar in 6- and 12-week-old mdx/mdx and C57BL/10 mice (Figure 5E). Because IFN-γ and TNF are known to up-regulate P2X7 activity in monocytes, we evaluated whether these cytokines were increased in blood from 12-week-old mdx/mdx mice; however, no differences were observed between 6- and 12-week-old mdx/mdx mice (data not shown).
      Figure thumbnail gr5
      Figure 5In vitro P2X7 activity in blood T cells. A and B: PBMCs from 6- and 12-week-old C57BL/10 (A) and mdx/mdx (B) mice were labeled with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibody and incubated with ATPe (5 mmol/L) for 10 minutes in the presence of PI in the last 5 minutes. T-cell membrane permeabilization was then evaluated by PI incorporation using flow cytometry. C and D: P2X7 ability to induce CD62L and LFA-1 shedding from purified CD3+ T lymphocyte surface was evaluated by dot blotting. Purified blood CD3+ T lymphocytes from 9-week-old mdx/mdx or C57BL/10 mice were incubated either in the presence or absence of KN-62 (a P2X7 blocker) and then stimulated with ATPe (100 μmol/L). Supernatants were collected and used to determine CD62L (C) and LFA1 (D) levels. E: P2X7 expression by CD3+ T lymphocytes purified from 6- and 12-week-old mdx/mdx and C57BL/10 mice was evaluated by Western blotting. Ponceau red staining served as control for total protein applied in each sample. Each experiment was performed three times. *P < 0.05. n = 5–7 animals per group.
      To assess the in vivo relevance of our findings, we treated mdx/mdx and C57BL/10 mice for 3 weeks with BBG, another antagonist of P2X7, and then evaluated CD62L expression on blood T lymphocytes and the concentration of soluble CD62L in serum. First, we tested whether the treatment was efficient in blocking the receptor through an ATPe-induced permeabilization assay using peritoneal macrophages, cells that are greatly susceptible to ATP. We observed that BBG administration almost abolished ATPe-induced pore formation in cells from both mdx/mdx (reduction from 77.4% to 16.2%) and C57BL/10 mice (reduction from 90.8% to 13.3%) (Figure 6, A and B). Regarding CD62L, we observed that BBG treatment restored its expression in blood T lymphocytes from mdx/mdx mice (increase from 35.4% to 81.1%) (Figure 6C). On the other hand, blood T cells from BBG- or vehicle-treated C57BL/10 mice were CD62L+ (90.9% and 92.4%, respectively) (Figure 6D), although with a higher frequency of CD62L expression in vehicle-treated (MFI = 70), compared with BBG-treated mice (MFI = 32). Simultaneously, BBG treatment reduced soluble levels of CD62L only in blood from mdx/mdx mice (Figure 6, E and G).
      Figure thumbnail gr6
      Figure 6In vivo role of P2X7 in CD62L shedding from the T-cell surface. A and B: Peritoneal cells from mdx/mdx (A) and C57BL/10 (B) mice treated with BBG (45.5 mg/kg) or vehicle (from 9 to 12 weeks of age) were collected and incubated with ATPe (5 mmol/L) and PI for permeabilization assays by flow cytometry in gated macrophages (FSC × SSC). Percentage of PI positive cells is indicated in parentheses. C and D: PBMCs were collected from mdx/mdx mice (C) and C57BL/10 mice (D) treated with BBG or vehicle. Percentage of CD3+/CD62L+ cells is indicated in parentheses. Horizontal bars define fluorescence positivity. E and G: Soluble CD62L was evaluated in serum samples from all groups of mice using dot-blotting assays. F: Cardiac tissue extracts were used for αβ T-cell detection by Western blotting. Data are representative of at least three independent experiments. *P ≤ 0.05. n = 4 or 5 animals per group.
      We then decided to evaluate whether, after BBG treatment, more T cells would be able to reach the cardiac tissue of these mice. We performed Western blotting assays with cardiac extracts from BBG- and vehicle-treated mdx/mdx and C57BL/10 mice using anti-TCR αβ and observed that, after BBG treatment, T cells migrated more to the cardiac muscle of mdx/mdx mice (Figure 6F). Moreover, we monitored a group of mice for 5 weeks during and after BBG treatment, measuring muscle damage markers for cardiac muscle (CK-MB) (Figure 7A) and skeletal muscle (CK-NAC) (Figure 7B). BBG-treated mdx/mdx mice exhibited higher CK-MB and CK-NAC activity until 2 weeks of treatment, compared with all other groups; after 2 weeks, CK-NAC activity decreased, reaching levels similar to those observed in vehicle-treated mdx/mdx mice.
      Figure thumbnail gr7
      Figure 7Analysis of muscle damage and motor activity with BBG treatment. A and B: mdx/mdx and C57BL/10 mice were treated with BBG (45.5 mg/kg) or vehicle from 9 to 12 weeks of age (weeks 1 to 3) and CK-MB (A) and CK-NAC (B) were measured once a week during the treatment and for 2 weeks more after the treatment (weeks 4 and 5). C: Motor spontaneous activity was evaluated on the second week of treatment, using a Noldus EthoVision XT6 video tracking system. *P ≤ 0.05 mdx/mdx versus treatment-matched C57BL/10 mice; P ≤ 0.05 treated versus untreated mdx/mdx mice.
      To determine whether the increase in muscle lesion observed after BBG treatment was due to increased physical activity, we evaluated motor spontaneous activity using an EthoVision video tracking system and observed that BBG-treated mdx/mdx mice have even lower spontaneous activity, compared with vehicle-treated mdx/mdx mice (Figure 7C).
      Because T. cruzi infection induces a myocarditis composed mostly of CD8+ T cells, we infected 6- and 12-week-old mdx/mdx mice to test whether fewer blood CD62L+ T cells would lead to nonlymphoid inflammatory infiltrates. The 6- and 12-week-old infected mdx/mdx and C57BL/10 mice had equivalent numbers of cardiac infiltrates per field, and the numbers were higher, compared with noninfected mdx/mdx mice (Figure 8A). The number of inflammatory cells per inflammatory focus in infected mdx/mdx mice was higher than in noninfected mdx/mdx and infected C57BL/10 mice (Figure 8B). H&E staining of cardiac samples from 12-week-old uninfected (Figure 8C) and infected (Figure 8D) mdx/mdx mice showed a general appearance of inflammatory infiltration. Regarding the presence of T cells, we did not observe inflammatory infiltration and CD3 labeling in noninfected C57BL/10 mice (Figure 8E). On the other hand, cardiac tissues from infected C57BL/10 mice showed high numbers of CD3+ T cells (Figure 8F), in contrast to noninfected and infected 12-week-old mdx/mdx mice, which have few CD3+ cells (Figure 8, G and H). TCR αβ staining in Western blotting of cardiac extracts from infected and noninfected 12-week-old mdx/mdx and C57BL/10 mice confirmed these data (Figure 8I). Regardless of the technique used, throughout our study we always found more cardiac T cells in control mdx/mdx mice, compared with control C57BL/10 mice.
      Figure thumbnail gr8
      Figure 8T. cruzi infection of mdx/mdx mice. A and B: C57BL/10 and mdx/mdx mice were infected with T. cruzi and hearts were collected 15 days after infection. The number of imflammatory foci per microscopic field (A) and the number of cells per inflammatory focus (B) were quantified by light microscopy in H&E-stained slices from 6- and 12-week-old mice. C and D: H&E-stained samples from 12-week-old uninfected (C) and infected (D) mdx/mdx mice were used to illustrate general characteristics of inflammatory foci. E–H: Identification of T cells was performed in heart slices using anti-CD3 monoclonal antibody in 12-week-old uninfected (E) and infected (F) C57BL/10 mice and in uninfected (G) and infected (H) mdx/mdx mice. I: Cardiac extracts from 12-week-old mice were used in Western blotting to evaluate T-cell lymphocytic infiltration; membranes were incubated with anti-αβ TCR monoclonal antibody. Arrows indicate CD3+ cells. *P ≤ 0.05. Scale bar = 50 μm.

      Discussion

      Although skeletal muscles are more studied in DMD, cardiac function and anatomy are also affected, in both patients and experimental models. Using echocardiography, Quinlan et al
      • Quinlan J.G.
      • Hahn H.S.
      • Wong B.L.
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      Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings.
      found that 8-week-old mdx/mdx mice had normal cardiac function, but 29-week-old mice exhibited minor alterations and 42-week-old mice exhibited dilated cardiomyopathy; they also found a four-fold increase in interstitial cardiac fibrosis in 17-week-old mdx/mdx mice. Moreover, Nakamura et al
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      Progression of dystrophic features and activation of mitogen-activated protein kinases and calcineurin by physical exercise, in hearts of mdx mice.
      found an increase in left ventricular wall with inflammation and fibrosis after regular treadmill exercise. Nonetheless, the precise role of the inflammatory response to the onset of cardiac pathology is currently unknown, especially the reason why T lymphocytes are rarely found in cardiac muscle. Many reports have described a great number of myeloid cells, mainly macrophages, mast cells, and some eosinophils in cardiac and skeletal muscles.
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      Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype.
      In contrast to DMD, other myopathies (including polymyositis, inclusion body myositis, dermatomyositis, and cardiomyopathies induced by the protozoan T. cruzi and coxsackie B3 virus) induce migration of T lymphocytes.
      • Dalakas M.C.
      Review An update on inflammatory and autoimmune myopathies.
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      We observed that T cells, mainly CD4+ T cells, are present in the cardiac tissue of 6-week-old mdx/mdx mice and that their frequency decreases with age, being almost undetectable in 24-week-old animals. Thus, reduced cardiac function and increased damage in older mice seem to be T cell-independent or likely delayed from early T-cell function.
      Different possibilities might account for the reduced number of T cells in muscles from DMD patients and mdx/mdx mice, such as high levels of lymphocyte death in the tissue. This finding could be due to either cytotoxic activity of other cells targeting T lymphocytes or high production of soluble factors, such as TNF. However, we (data not shown) observed that cardiac muscles have few TUNEL-positive cells, suggesting low levels of apoptosis in the muscle, including T cells. On the other hand, the paucity of TUNEL-positive cells could be due to a very efficient scavenging process of dying cells. This can be illustrated by the presence of very few TUNEL-positive cells in thymus, despite the death of approximately 95% of thymocytes not rescued by positive selection (death by neglect) and during negative selection. Therefore, at this point and regardless of our present results, we cannot exclude the possibility that apoptosis undetected by current techniques could be occurring in cardiac muscle and contributing more or less to the reduced number of T cells.
      Another possibility is inefficient migration of blood T cells to the cardiac tissue, which could be due to defective activation in lymph nodes, reduced muscle production of T cell chemotactic factors, reduced expression of chemotactic receptors by T cells, or inefficient T lymphocyte/endothelial cell interaction due to reduced expression/activation of adhesion molecules, such as selectins and integrins. Many of these alternatives remain to be tested. However, regarding the first possibility, mdx/mdx mice are not considered more susceptible to infections, for example, nor do they show any other characteristic of significant reduced immunocompetence. In accord, we observed no difference in blood parasitemia after T. cruzi infection between mdx/mdx and C57BL/10 mice (data not shown). Moreover, we observed no significant differences in total CD44high blood T cells between 6-week-old (5.38% ± 1.17) and 12-week-old (4.54% ± 0.96) mdx/mdx mice. Regarding the production of TNF, IL-6, IFN-γ, IL-10, MCP-1, and IL-12 (potential regulators for T-cell migration) in extracts of reperfused hearts, we observed no significant differences between 6- and 12-week-old mdx/mdx mice (data not shown). On the other hand, of the various adhesion molecules for endothelial interaction (CD62L, CD49d, LFA-1, ICAM, and VCAM), only CD62L was absent from blood T cells in a window of time in mdx/mdx mice (10 to 20 weeks old). The 24-week-old mdx/mdx mice recovered expression of CD62L on T lymphocytes, although these cells were still not observed in the cardiac tissue. This suggests that CD62L-independent mechanisms can still prevent T-cell migration at this age, but this alternative remains to be tested.
      Our data also showed that naïve and long-term activated T cells (CD69) retain CD62L on cell surface, and only a part of the early-activated T cells (CD69+) shed the selectin. These data further support the concept that lack of CD62L in T-cell subpopulations does not affect the acquired immune response to most antigens.
      CD62L down-regulation could be a result of reduced molecular transcription or translation, with membrane molecules targeted to lysosomal destruction or shedding. Because we observed high levels of serum CD62L in 12-week-old mdx/mdx mice, we considered proteolytic cleavage from the cell surface as the most likely alternative. Data from the literature
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      and our present results show that CD62L is required for T-cell migration to nonlymphoid tissues, although we and others have observed that LFA-1 seems to be more important.
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      Studies using CD62L−/− mice showed that leukocytes, including T cells, have significantly reduced ability to roll and enter sites of inflammation, which blunts inflammatory responses.
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      Moreover, anti-CD62L antibody administration suppressed clinical signs and T-lymphocyte infiltration in the central nervous system in an experimental autoimmune encephalomyelitis model.
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      also proposed that rapid CD62L endoproteolytic release from T-cell surface modulates lymphocyte ability to migrate and enter sites of inflammation. These data further support that CD62L is necessary for T-cell rolling along the endothelium of blood vessels and for subsequent migration.
      Our in vitro and in vivo results show that P2X7 activity led to CD62L shedding in 12-week-old mdx/mdx mice. On the other hand, although C57BL/10 and mdx/mdx mice expressed similar levels of P2X7, ATPe-induced membrane permeabilization was increased only in 6-week-old mdx/mdx mice. We expected a different result, that permeabilization would be increased also in 12-week-old mdx/mdx mice, concomitant with P2X7-induced CD62L shedding. However, this is not the first finding of receptor expression, intracellular biochemical pathways, and resultant cellular responses independently triggered by P2X7 agonistic stimuli. For example, we observed previously that both T. cruzi-infected and noninfected C57BL/6 thymocytes expressed P2X7, but agonistic stimulus did not induce a Ca2+ signal or permeabilization in control mice, which acquired these functions only after infection.
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      found increased expression of P2X7 in dystrophic skeletal muscles, concomitant with higher levels of phospho-ERK1/2 and calcium influx induced by ATPe; however, sustained activation of P2X7 by ATPe did not induce pore formation in these cells. Together, these data show that pore formation and other P2X7 signaling pathways can be dissociated.
      BBG treatment was efficient in blocking P2X7-dependent CD62L shedding and membrane permeabilization. In terms of an in vivo treatment, however, we do not know other possible activities of BBG over P2X7 function in other cell types, such as macrophages and mast cells that migrate to muscles, and even over other P2 receptor-dependent pathways. Regarding T cells, sustained expression of CD62L allowed cell migration to cardiac muscle and induced cardiac and skeletal lesions for 2 weeks after BBG treatment, as ascertained by CK-NAC and CK-MB. These data are in agreement with other studies showing that T cells increase pathology
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      and corroborate the importance of P2X7 and selectins to T cell-dependent muscle damage in DMD. However, we do not know whether the treatment also affected other cell types, inducing increased migration of other leukocyte subpopulations to the muscles, which could lead to more tissue damage. Nonetheless, after 3 weeks of treatment, when the mice were 12 weeks of age, we found no differences in CK levels between BBG-treated and untreated mdx/mdx mice, despite the migration of more T cells to the heart. This lack of difference may be due to a reduced functional effector activity of T cells, because we found that <10% of harvested cardiac T cells from 12-week-old mdx/mdx mice retain CD2 and CD44high on cell membrane, unlike 6-week-old mice, which showed higher levels of both molecules (data not shown).
      To our knowledge, mdx/mdx mice were the first experimental model of T. cruzi infection that induced a nonlymphocytic myocarditis. C57BL/10 infected mice, as well as Chagas' disease patients and other experimental models, have inflammatory foci composed mostly of CD8+ T lymphocytes.
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      • Paiva M.M.
      • Correa A.F.
      • Batista M.M.
      • Bisaggio R.C.
      • Liu C.C.
      • Cotta-De-Almeida V.
      • Coutinho C.M.
      • Persechini P.M.
      • Araujo-Jorge T.C.
      Evidence for a perforin-mediated mechanism controlling cardiac inflammation in Trypanosoma cruzi infection.
      Although it is still not completely known which cytotoxic pathways and cells induce cardiomyocyte death in the infection, it is largely accepted that T cells are mainly responsible for cell death and inflammation. In 12-week-old mdx/mdx mice, however, T cell competence to interact with endothelial cells is apparently so inefficient that, even after infection, most of these cells were still not able to reach the muscle. Even though the infection induced a new assortment of alterations in the immune system of mdx/mdx mice, it is noteworthy that T-cell migration to damaged muscles was actively prevented.
      The present findings suggest that blood T cells from 12-week-old mdx/mdx mice lack the necessary repertoire of adhesion molecules, which may be, at least in part, responsible for the paucity of T cells found in muscles of mdx/mdx mice.

      Acknowledgments

      We thank Marcelo Meuser Batista for infection of the mice and Dr. Robson Coutinho Silva for Western blotting support.

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