Interleukin-13 Protects Against Experimental Autoimmune Myocarditis by Regulating Macrophage Differentiation
2008; Elsevier BV; Volume: 172; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.070207
ISSN1525-2191
AutoresDaniela Čiháková, Jobert G. Barin, Marina Afanasyeva, Miho Kimura, DeLisa Fairweather, Michael G. Berg, Monica V. Talor, G. Christian Baldeviano, Sylvia Frisancho, Kathleen L. Gabrielson, Djahida Bedja, Noel R. Rose,
Tópico(s)Immune Cell Function and Interaction
ResumoWe report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45+ leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1β, IL-18, interferon-γ, transforming growth factor-β1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4+ T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4+CD25+Foxp3+ regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206+ and CD204+ macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1β and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function. We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45+ leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1β, IL-18, interferon-γ, transforming growth factor-β1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4+ T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4+CD25+Foxp3+ regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206+ and CD204+ macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1β and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function. Idiopathic cardiomyopathy of nonischemic origin is often preceded by myocarditis and represents an increasing public health problem.1Rose NR Afanasyeva M The inflammatory process in experimental myocarditis.in: Feuerstein GZ Libby P Mann DL Inflammation and Cardiac Diseases. Birkhauser Verlag, Basel2003: 325-333Crossref Google Scholar, 2Carniel E Sinagra G Bussani R Di Lenarda A Pinamonti B Lardieri G Silvestri F Fatal myocarditis: morphologic and clinical features.Ital Heart J. 2004; 5: 702-706PubMed Google Scholar Similar to the human disease, infection of mice with coxsackievirus B3 (CVB3) results in the development of an acute, self-limited myocarditis in a majority of mice, but a few genetically susceptible strains proceed to autoimmune myocarditis and dilated cardiomyopathy (DCM) by day 35 after infection.3Fairweather D Afanasyeva M Rose NR Cellular immunity: a role for cytokines.in: Doria A Pauletto P The Heart in Systemic Autoimmune Diseases. Elsevier, Amsterdam2004: 3-17Google Scholar, 4Fairweather D Rose NR Inflammatory heart disease: a role for cytokines.Lupus. 2005; 14: 646-651Crossref PubMed Scopus (76) Google Scholar Cardiac myosin heavy chain is a major target of autoimmune responses in CVB3-induced myocarditis.5Neu N Beisel KW Traystman MD Rose NR Craig SW Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis.J Immunol. 1987; 138: 2488-2492PubMed Google Scholar Immunization with cardiac myosin purified from murine hearts or cardiac myosin heavy chain peptide in complete Freund's adjuvant can induce experimental autoimmune myocarditis (EAM).6Neu N Craig SW Rose NR Alvarez F Beisel KW Coxsackievirus induced myocarditis in mice: cardiac myosin autoantibodies do not cross-react with the virus.Clin Exp Immunol. 1987; 69: 566-574PubMed Google Scholar, 7Neu N Rose NR Beisel KW Herskowitz A Gurri-Glass G Craig SW Cardiac myosin induces myocarditis in genetically predisposed mice.J Immunol. 1987; 139: 3630-3636PubMed Google Scholar, 8Pummerer CL Luze K Grassl G Bachmaier K Offner F Burrell SK Lenz DM Zamborelli TJ Penninger JM Neu N Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice.J Clin Invest. 1996; 97: 2057-2062Crossref PubMed Scopus (153) Google Scholar Both cell-mediated and antibody-mediated immunity contribute to the final pathological picture of chronic inflammation and DCM in EAM and CVB3-induced myocarditis.9Afanasyeva M Wang Y Kaya Z Park S Zilliox MJ Schofield BH Hill SL Rose NR Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype.Am J Pathol. 2001; 159: 193-203Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Fairweather D Rose NR Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.Methods. 2007; 41: 118-122Crossref PubMed Scopus (145) Google Scholar We previously demonstrated that EAM in A/J mice carry hallmarks of Th2-like pathology. Blockade of interleukin (IL)-4 partially suppresses the development of EAM, indicating that IL-4 is cardiopathogenic in this strain.9Afanasyeva M Wang Y Kaya Z Park S Zilliox MJ Schofield BH Hill SL Rose NR Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype.Am J Pathol. 2001; 159: 193-203Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar The importance of IL-4 in the pathogenesis of EAM suggests that other Th2 cytokines could also augment EAM pathology. Therefore, we postulated that IL-13, another Th2 cytokine, could synergize with IL-4, and that IL-13 knockout (KO) mice would develop reduced EAM and CVB3-induced myocarditis. However, our unexpected results presented in this article have led to the novel conclusion that IL-13 exerts protective effects in this autoimmune disease. IL-13 is a pleiotropic cytokine produced by T-helper-type 2 (Th2) CD4+ T cells, CD8+ T cells, mast cells, dendritic cells, and eosinophils.11de Vries JE The role of IL-13 and its receptor in allergy and inflammatory responses.J Allergy Clin Immunol. 1998; 102: 165-169Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 12Reiman RM Thompson RW Feng CG Hari D Knight R Cheever AW Rosenberg HF Wynn TA Interleukin-5 (IL-5) augments the progression of liver fibrosis by regulating IL-13 activity.Infect Immun. 2006; 74: 1471-1479Crossref PubMed Scopus (156) Google Scholar IL-13 does not use the classical receptor for IL-4 (IL-4Rα/γc), but shares use of the alternative IL-4 receptor, consisting of IL-4Rα and the IL-13 receptor α1 (IL-13Rα1) subunit.13Wills-Karp M Interleukin-13 in asthma pathogenesis.Immunol Rev. 2004; 202: 175-190Crossref PubMed Scopus (540) Google Scholar In addition to the common receptor with IL-4, IL-13 has an additional receptor: IL-13Rα2, which possesses antagonistic decoy functions, in addition to unique signaling functions.14Fichtner-Feigl S Strober W Kawakami K Puri RK Kitani A IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis.Nat Med. 2006; 12: 99-106Crossref PubMed Scopus (686) Google Scholar There is growing evidence of unique physiological functions of IL-13, not shared by IL-4, in models of helminthic parasitism,15McKenzie GJ Bancroft A Grencis RK McKenzie AN A distinct role for interleukin-13 in Th2-cell-mediated immune responses.Curr Biol. 1998; 8: 339-342Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 16Bancroft AJ McKenzie AN Grencis RK A critical role for IL-13 in resistance to intestinal nematode infection.J Immunol. 1998; 160: 3453-3461PubMed Google Scholar schistosomiasis,17Fallon PG Richardson EJ McKenzie GJ McKenzie AN Schistosome infection of transgenic mice defines distinct and contrasting pathogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent.J Immunol. 2000; 164: 2585-2591PubMed Google Scholar, 18McKenzie GJ Fallon PG Emson CL Grencis RK McKenzie AN Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses.J Exp Med. 1999; 189: 1565-1572Crossref PubMed Scopus (287) Google Scholar lung immunity and atopy,19Zhu Z Homer RJ Wang Z Chen Q Geba GP Wang J Zhang Y Elias JA Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.J Clin Invest. 1999; 103: 779-788Crossref PubMed Scopus (1470) Google Scholar, 20Whittaker L Niu N Temann UA Stoddard A Flavell RA Ray A Homer RJ Cohn L Interleukin-13 mediates a fundamental pathway for airway epithelial mucus induced by CD4 T cells and interleukin-9.Am J Respir Cell Mol Biol. 2002; 27: 593-602Crossref PubMed Scopus (169) Google Scholar and tumor immunity.21Terabe M Matsui S Noben-Trauth N Chen H Watson C Donaldson DD Carbone DP Paul WE Berzofsky JA NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway.Nat Immunol. 2000; 1: 515-520Crossref PubMed Scopus (588) Google Scholar T cells are not known to express functional IL-13Rα1 and so IL-13 is probably not directly acting on T cells. The main targets of IL-13 are monocytes/macrophages. IL-13 signaling in monocytes yielded a transcriptional profile unique and distinct from macrophages classically activated by interferon (IFN)-γ. IL-13 and IL-4 alternatively activated macrophages show distinct phenotypic changes: mannose receptor up-regulation, induction of selective chemokines, and expression of arginase. Also, IL-13 prevents lipopolysaccharide-dependent caspase-1 activity in monocytes, therefore decreasing production of IL-1 and IL-18. This role of IL-13 on macrophages contrasts with the activation of macrophages by IFN-γ: up-regulation of iNOS, as well as the proinflammatory cytokines IL-6, tumor necrosis factor (TNF)-α, and IL-1.22Scotton CJ Martinez FO Smelt MJ Sironi M Locati M Mantovani A Sozzani S Transcriptional profiling reveals complex regulation of the monocyte IL-1 beta system by IL-13.J Immunol. 2005; 174: 834-845PubMed Google Scholar, 23Gordon S Alternative activation of macrophages.Nat Rev Immunol. 2003; 3: 23-35Crossref PubMed Scopus (4595) Google Scholar We report here that IL-13 KO mice on a BALB/c background developed significantly increased myosin- and CVB3-induced myocarditis. IL-13 reduces myocarditis by regulating monocyte/macrophage populations during EAM. IL-13 KO and IL-13/IL-4 DKO mice on the BALB/c background were generated in the laboratory of Andrew McKenzie, as described15McKenzie GJ Bancroft A Grencis RK McKenzie AN A distinct role for interleukin-13 in Th2-cell-mediated immune responses.Curr Biol. 1998; 8: 339-342Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 18McKenzie GJ Fallon PG Emson CL Grencis RK McKenzie AN Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses.J Exp Med. 1999; 189: 1565-1572Crossref PubMed Scopus (287) Google Scholar and were bred and maintained in the Johns Hopkins University School of Medicine conventional animal facility. IL-4 KO and WT BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in the Johns Hopkins University School of Medicine conventional animal facility. All experiments were conducted on 6- to 8-week-old male mice. All methods and protocols involving mice were approved by the Animal Care and Use Committee of the Johns Hopkins University. For the induction of EAM, we used the myocarditogenic peptide MyHCα614–629 derived from the sequence of the murine cardiac myosin heavy chain (Ac-SLKLMATLFSTYASAD-OH)8Pummerer CL Luze K Grassl G Bachmaier K Offner F Burrell SK Lenz DM Zamborelli TJ Penninger JM Neu N Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice.J Clin Invest. 1996; 97: 2057-2062Crossref PubMed Scopus (153) Google Scholar, 24Eriksson U Kurrer MO Sonderegger I Iezzi G Tafuri A Hunziker L Suzuki S Bachmaier K Bingisser RM Penninger JM Kopf M Activation of dendritic cells through the interleukin 1 receptor 1 is critical for the induction of autoimmune myocarditis.J Exp Med. 2003; 197: 323-331Crossref PubMed Scopus (121) Google Scholar commercially synthesized by fMOC chemistry and purified by high performance liquid chromatography (Global Peptide, Fort Collins, CO). On days 0 and 7, mice received subcutaneous injections of 200 μg of MyHCα614–629 peptide emulsified in complete Freund's adjuvant (Sigma, St. Louis, MO) supplemented with 5 mg/ml of Mycobacterium tuberculosis, strain H37Ra (Difco, Detroit, MI). On day 0, mice additionally received 500 ng of pertussis toxin intraperitoneally (List Biologicals, Campbell, CA). For CVB3-induced myocarditis, IL-13 KO and WT BALB/c mice were inoculated intraperitoneally with 103 plaque-forming units of a heart-passaged stock of CVB3 (Nancy strain), originally obtained from the American Type Culture Collection (Manassas, VA), diluted in sterile phosphate-buffered saline (PBS) on day 0. Individual experiments were conducted at least three times with 7 to 10 mice per group. Mice were evaluated for the development of EAM at the peak of disease on day 21 or in chronic stage of EAM at day 30 or chronic CVB3-induced myocarditis at day 35 after infection. Heart tissues were fixed in 10% phosphate-buffered formalin. Five-μm sections were cut longitudinally and stained with hematoxylin and eosin, and Masson's trichrome for fibrosis. Myocarditis severity was evaluated by histopathological microscopic approximation of the percent area of myocardium infiltrated with mononuclear cells or fibrosis determined from five sections per heart according to the following scoring system: grade 0, no inflammation; grade 1, less than 10% of the heart section is involved; grade 2, 10 to 30%; grade 3, 30 to 50%; grade 4, 50 to 90%; grade 5, more then 90%. For CVB3-induced myocarditis the severity of the disease was assessed as the percentage of the heart section with inflammation compared with the overall size of the heart section, with the aid of a microscope eyepiece grid. Two independent researchers scored slides separately in a blinded manner. Mice were bled on days 0 and 21 from the retro-orbital venous plexus using heparinized capillary tubes. Sera were collected by heart puncture on day 35 after infection of CVB3-induced myocarditis. Serum levels of MyHCα614–629-reactive (EAM) or cardiac myosin (CVB3) antibodies were determined using microtiter plates coated with 0.5 μg of MyHCα614–629 or cardiac myosin incubated with phosphatase-conjugated isotype-specific secondary antibodies. Adjusted optical density (OD) was calculated as follows: adjusted OD = mean OD of a sample − mean OD of a negative control. The autoantibody titer is expressed as the reciprocal of the highest serum dilution having an OD greater than that of the negative control serum plus three SD. Negative control serum consisted of pooled sera from 10 uninfected BALB/c mice. Half of the heart or spleen was snap-frozen on dry ice immediately after resection and stored at −80°C until homogenized in minimal essential medium plus 2% fetal bovine serum, debris cleared by centrifugation, and stored at −80°C until used in ELISA. Cytokine levels were measured in homogenized heart and spleen supernatants using Quantikine cytokine ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. The limit of detection for the cytokine kits were as follows: IFN-γ, 2 pg/ml; transforming growth factor (TGF)-β1, 1.6 pg/ml; IL-1β, 3 pg/ml; IL-4, 2 pg/ml; IL-13, 1.5 pg/ml; IL-17, 5 pg/ml; IL-18, 25 pg/ml; histamine, 1.5 ng/ml. Heart cytokine and histamine levels were expressed as pg/g of heart tissue. Caspase-1 activity was measured from 5 × 106 splenocytes from IL-13 KO mice on day 21 of EAM. Splenocytes were snap-frozen and stored at −80°C until caspase-1 activity was measured. Caspase-1 activity was measured by the Casp-1/ICE fluorometric assay kit (BioVision, Mountain View, CA). Single cell suspension was prepared from mice spleens. After enumeration in a hemocytometer and standardization of cell densities in RPMI 1640 and 10% fetal bovine serum, 2.5 × 105 responder cells were plated with 10 μg/ml of MyHCα614–629 in triplicate in 96-well format. Control cells were cultured with media in the absence of MyHCα614–629. All cells were incubated at 37°C, 5% CO2 for 48 hours. Proliferation assay plates were pulsed 24 hours before harvest with 1 μCi/well [3H]-methyl thymidine (GE Amersham, Buckinghamshire, UK), harvested onto glass filters, and specific radio-uptake detected on a Trilux β-direct counter (Packard, Waltham, MA). The heart was perfused at a constant flow of 14 ml/minute with cold PBS (Biofluids, Carlsbad, CA) for 2 minutes, and then digested with collagenase II (100 μg/ml; Sigma-Aldrich, St. Louis, MO) and protease XIV (50 μg/ml; Sigma- Aldrich) in PBS for 7 minutes at 37°C.25Afanasyeva M Georgakopoulos D Belardi DF Ramsundar AC Barin JG Kass DA Rose NR Quantitative analysis of myocardial inflammation by flow cytometry in murine autoimmune myocarditis: correlation with cardiac function.Am J Pathol. 2004; 164: 807-815Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 26Fairweather D Frisancho-Kiss S Yusung SA Barrett MA Davis SE Steele RA Gatewood SJ Rose NR IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart.J Immunol. 2005; 174: 261-269PubMed Google Scholar After careful mincing, single cell suspensions were sequentially passed through 70-μm and 40-μm cell strainers (BD Falcon, Franklin Lakes, NJ). Splenocytes were also extracted into single cell suspension in 1× PBS, and red blood cells lysed by incubation in ACK lysis buffer (Biofluids). Cells were washed and FcγRII/III blocked with αCD16/32 (eBiosciences, San Diego, CA). Surface markers were stained with fluorochrome-conjugated mAbs to CD3ε, CD4, CD8α, CD11b (Mac1), CD11c, CD19, CD25, CD28, CD44, CD45, CD45R (B220), CD62L, CD69, CD71, CD80 (B7.1), CD86 (B7.2), CD117 (c-kit), CD152 (CTLA4), CD204 (SR-A), CD206 (MR), CD274 (PD-L1), DX5, F4/80, FcεRIα, Gr1 (Ly6G), Mac3, MHC Class II (I-A/I-E), and TCRβ (eBiosciences; BD Pharmingen, San Diego, CA; Biolegend, San Diego, CA; and AbD Serotec, Raleigh, NC). Treg cells were further stained by intracellular staining of Foxp3 with a kit according to manufacturer's instructions (eBiosciences). Samples were acquired on a four-color dual-laser FACScalibur cytometer running the CellQuest software package or the LSR II quad-laser cytometer running FACSDiva (BD Immunocytometry, San Jose, CA). Trans-thoracic echocardiography was performed using the visualsonic Vevo 660 imaging system equipped with a 40 MHz transducer (VisualSonics Inc., Toronto, Canada). Conscious, previously trained mice were gently held in a supine position in the palm of the hand, as described.25Afanasyeva M Georgakopoulos D Belardi DF Ramsundar AC Barin JG Kass DA Rose NR Quantitative analysis of myocardial inflammation by flow cytometry in murine autoimmune myocarditis: correlation with cardiac function.Am J Pathol. 2004; 164: 807-815Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Olson LE Bedja D Alvey SJ Cardounel AJ Gabrielson KL Reeves RH Protection from doxorubicin-induced cardiac toxicity in mice with a null allele of carbonyl reductase 1.Cancer Res. 2003; 63: 6602-6606PubMed Google Scholar The left hemi-thorax was shaved and ultrasonic transmission gel (Parker Laboratories, Fairfield, NJ) was applied to the thorax. The heart was imaged in the two-dimensional mode in the parasternal short axis view. From this mode, an M-mode cursor was positioned perpendicular to the interventricular septum (IVS) and the left ventricular posterior wall (LVPW) at the level of the papillary muscles. From the M-mode, the left ventricular wall thickness and chamber dimensions were measured. For each mouse, three to five values for each measurement were obtained and averaged for evaluation. The left ventricular (LV) end diastolic dimension (LVEDD), LV end systolic dimension (LVESD), LV septal wall thickness at end diastole (IVSED) and end systole (IVSES), LV posterior wall thickness at end diastole (PWTED) and end systole (PWTES) were measured from a frozen M-mode tracing. Fractional shortening (FS %) is the percent change in LV cavity dimensions. Ejection fraction (EF %) represents stroke volume as a percentage of end diastolic LV volume. The heart rate is automatically determined from the M-mode image by positioning the first and second caliper point on two systolic phases. LV mass is determined automatically by the software or by using the following standard cube function formula: LV mass (mg) = 1.055 [(IVST + LVEDD + PWT)3 − (LVEDD)3] (1.055 is the specific gravity of the cardiac muscle).28Yang XP Liu YH Rhaleb NE Kurihara N Kim HE Carretero OA Echocardiographic assessment of cardiac function in conscious and anesthetized mice.Am J Physiol. 1999; 277: H1967-H1974PubMed Google Scholar Relative wall thickness (RWT) indicates the overall thickness of the LV wall and is calculated as follows: (RWT) = 2 × PWTD/LVEDD.28Yang XP Liu YH Rhaleb NE Kurihara N Kim HE Carretero OA Echocardiographic assessment of cardiac function in conscious and anesthetized mice.Am J Physiol. 1999; 277: H1967-H1974PubMed Google Scholar, 29Basset A Blanc J Messas E Hagege A Elghozi JL Renin-angiotensin system contribution to cardiac hypertrophy in experimental hyperthyroidism: an echocardiographic study.J Cardiovasc Pharmacol. 2001; 37: 163-172Crossref PubMed Scopus (37) Google Scholar Mice were immunized with MyHCα614–629 and received 2 mg of rat anti-mouse IL-4 mAb IgG1 clone 11B.11 (American Type Culture Collection) on days −1, 3, 6, 9, 12, 15, and 18. Control mice were injected with isotype control, anti-Escherichia coli β-galactosidase mAb IgG1 clone GL113 (kindly provided by Fred Finkelman, University of Cincinnati, Cincinnati, OH). Monoclonal Abs were purified from the concentrated hybridoma supernatants using a HiTrap protein G column (Supelco, Bellefonte, PA). For blocking of IL-13, mice were immunized with MyHCα614–629 and injected with 100 μg of αIL-13 mAb rat IgG2b clone 38213 (R&D Systems) on days 0, 4, 8, 12, and 16 of EAM. Normal rat IgG2b clone 141945 (R&D Systems) was used as an isotype control. All Abs were administered in sterile 1× PBS intraperitoneally. The Mann-Whitney U-test was used to compare EAM severity scores between treatment groups. Normally distributed data on continuous parametric axes were analyzed with the two-tailed Student's t-test. Values of P < 0.05 were considered statistically significant. We found previously that blockade of IL-4 partially suppresses the development of myosin-induced myocarditis, indicating that IL-4 increases the severity of myocarditis in A/J mice.9Afanasyeva M Wang Y Kaya Z Park S Zilliox MJ Schofield BH Hill SL Rose NR Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype.Am J Pathol. 2001; 159: 193-203Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar To investigate the role of another Th2 cytokine, IL-13, in myocarditis, we infected IL-13 KO and WT BALB/c controls with CVB3 and examined hearts for inflammation and viral replication during the early (day 21 after infection) and late phase (day 35 after infection) of chronic myocarditis. We found that IL-13 deficiency significantly increased the severity of chronic CVB3-induced myocarditis at day 35 after infection (P = 0.001) (Figure 1A). IL-13 deficiency was also associated with decreased survival. Only 40% of IL-13 KO mice survived past day 21 after infection, whereas none of the WT control mice had died by that time point (Figure 1B). Total IgG autoantibodies against cardiac myosin were also significantly increased in IL-13 KO mice, compared to WT controls (P < 0.01) (Figure 1C). When we examined IL-13 KO mice and WT controls with CVB3-induced myocarditis on day 21 by echocardiography, we found significantly compromised cardiac function. IL-13 KO mice that survived to this time point had increased left ventricular end systolic dimension (LVESD) (P < 0.001), decreased fractional shortening (%FS) (P = 0.02), and decreased ejection fraction (%EF) (P = 0.05) (Figure 1, E–G). Left ventricular end diastolic dimension (LVEDD) was not significantly changed at this time point (Figure 1D) although DCM is usually not observed by histology until day 35 after infection. To exclude the possibility that increased viral replication or persistence resulted in increased myocarditis severity; we examined viral replication in IL-13 KO mice at several time points by plaque assay. We found no significant differences in viral replication in the hearts of IL-13 KO mice on day 7 (P = 0.85), on day 11 (P = 0.11), or on day 14 after infection (P = 0.15) (Figure 1, H–J). By day 14, CVB3 was already cleared from most of the WT and IL-13 KO hearts. Viral replication in heart tissue was not detectable at days 21 or 35 in either WT or IL-13 KO mice (data not shown). Thus, IL-13 reduces chronic myocarditis, autoantibody production, and heart failure after viral infection. To investigate the role of IL-13 in EAM, we immunized IL-13 KO BALB/c males with the myocarditogenic peptide MyHCα614–629 emulsified in CFA.8Pummerer CL Luze K Grassl G Bachmaier K Offner F Burrell SK Lenz DM Zamborelli TJ Penninger JM Neu N Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice.J Clin Invest. 1996; 97: 2057-2062Crossref PubMed Scopus (153) Google Scholar Consistent with the results from CVB3-induced myocarditis, IL-13 deficiency significantly increased the incidence and severity of EAM on day 21 (P < 0.0001) (Figure 2, A–C). In EAM hearts, total infiltrating CD45+ cells were significantly increased in IL-13 KO mice (Figure 2D). To test antigen-specific autoantibody responses, we examined the level of MyHCα614–629-reactive serum antibodies by ELISA at day 21 of EAM. IL-13 KO mice developed significantly higher levels of MyHCα614–629-reactive total IgG, IgG1, IgG2a, and IgG2b, compared to WT mice (Figure 2E). To additionally confirm that IL-13 is protective in EAM, we blocked IL-13 in WT BALB/c mice on days 0, 4, 8, 12, and 16 with anti-IL-13 mAb and also observed increased disease severity at day 21, compared to isotype control-treated mice (Figure 2F). Thus, IL-13 also limits myocarditis in EAM model. All further studies were done on EAM. To analyze changes in cytokine expression at day 21 of EAM, we homogenized hearts of IL-13 KO and WT control mice and analyzed levels of IFN-γ, IL-1β, IL-4, IL-17, IL-18, TGF-β1, and histamine by ELISA. Th17 cells can mediate severe cardiac pathology when Th1 signaling is disrupted30Rangachari M Mauermann N Marty RR Dirnhofer S Kurrer MO Komnenovic V Penninger JM Eriksson U T-bet negatively regulates autoimmune myocarditis by suppressing local production of interleukin 17.J Exp Med. 2006; 203: 2009-2019Crossref PubMed Scopus (212) Google Scholar, 31Marty RR Eriksson U Dendritic cells and autoimmune heart failure.Int J Cardiol. 2006; 112: 34-39Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar; blocking IL-17 with monoclonal antibody decreased myocarditis severity (our unpublished results). Therefore, we measured levels of intracardiac IL-17 to test the hypothesis that Th17 is the driving CD4+ subset mediating increased cardiac pathology in IL-13 KO EAM. However, we found that IL-17 was significantly lower in heart homogenates from IL-13 KO mice at day 21 of EAM, compared to WT BALB/c controls (Figure 3A). However, we did observe significantly increased levels of IL-1β, IL-18, IFN-γ, TGF-β1, IL-4, and also histamine in the hearts of IL-13 KO mice (Figure 3A). The most markedly increased cytokines were IL-18 and IL-1β; both were increased in hearts as well as in spleens of IL-13 KO mice (data not shown). Pro-IL-18 and pro-IL-1β are converted to their active forms by caspase-1. We found greatly increased caspase-1 activity in IL-13 KO splenocytes on day 21 of EAM (P = 0.0002) (Figure 3B). Thus, IL-13 probably protects against myocarditis by down-regulating caspase-1 activity and decreasing levels of active IL-1β and IL-18. We have previously shown that fibrosis is associated with progression to DCM and heart failure.32Fairweat
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