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Natural Killer Cells Limit Cardiac Inflammation and Fibrosis by Halting Eosinophil Infiltration

Open AccessPublished:January 23, 2015DOI:https://doi.org/10.1016/j.ajpath.2014.11.023
      Myocarditis is a leading cause of sudden cardiac failure in young adults. Natural killer (NK) cells, a subset of the innate lymphoid cell compartment, are protective in viral myocarditis. Herein, we demonstrated that these protective qualities extend to suppressing autoimmune inflammation. Experimental autoimmune myocarditis (EAM) was initiated in BALB/c mice by immunization with myocarditogenic peptide. During EAM, activated cardiac NK cells secreted interferon γ, perforin, and granzyme B, and expressed CD69, tumor necrosis factor–related apoptosis-inducing ligand treatment, and CD27 on their cell surfaces. The depletion of NK cells during EAM with anti-asialo GM1 antibody significantly increased myocarditis severity, and was accompanied by elevated fibrosis and a 10-fold increase in the percentage of cardiac-infiltrating eosinophils. The resultant influx of eosinophils to the heart was directly responsible for the increased disease severity in the absence of NK cells, because treatment with polyclonal antibody asialogangloside GM-1 did not augment myocarditis severity in eosinophil-deficient ΔdoubleGATA1 mice. We demonstrate that NK cells limit eosinophilic infiltration both indirectly, through altering eosinophil-related chemokine production by cardiac fibroblasts, and directly, by inducing eosinophil apoptosis in vitro. Altogether, we define a new pathway of eosinophilic regulation through interactions with NK cells.
      Myocarditis is a leading cause of sudden cardiac failure in individuals <40 years, with 9% to 16% of cases progressing to inflammatory dilated cardiomyopathy.
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      Correlations between eosinophil frequency and poor clinical outcomes have been reported in other chronic inflammatory disease models, including asthma, inflammatory bowel disease, and experimental autoimmune encephalomyelitis.
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      IFN-gamma determines distinct clinical outcomes in autoimmune encephalomyelitis.
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      • Riddell R.H.
      The role of eosinophils in inflammatory bowel disease.
      • Kay A.B.
      The role of eosinophils in the pathogenesis of asthma.
      Herein, we investigated the connection between eosinophils and natural killer (NK) cells, highlighting a new pathway responsible for the control of eosinophilic accumulation in sites of inflammation.
      Our group and others have reported that NK cells, an innate lymphoid cell subset, are protective in coxsackievirus B3 and murine cytomegalovirus animal models of myocarditis by limiting viral replication.
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      From infection to autoimmunity.
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      Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis.
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      • Yokoyama W.M.
      • Virgin H.W.
      Natural killer cells utilize both perforin and gamma interferon to regulate murine cytomegalovirus infection in the spleen and liver.
      Because myocarditis is also an autoimmune-mediated disease, it is unknown if NK cells can protect against disease through limiting viral replication, as well as by reducing the autoimmune response.
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      • Berg L.
      • Hall H.
      • Hoglund P.
      NK cells: elusive players in autoimmunity.
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      • Shoenfeld Y.
      NK cells in autoimmunity: a two-edg'd weapon of the immune system.
      The data regarding NK cells and autoimmunity are extensive, but conflicting. NK cells accumulate in joints during rheumatoid arthritis (RA), skin lesions during psoriasis, and brain lesions during multiple sclerosis.
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      • Kay C.
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      • Carsons S.E.
      Rheumatoid arthritis serum or synovial fluid and interleukin 2 abnormally expand natural killer-like cells that are potent stimulators of IgM rheumatoid factor.
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      • Thompson R.N.
      • Christmas S.E.
      • Moots R.J.
      Natural killer cells in the synovial fluid of rheumatoid arthritis patients exhibit a CD56bright,CD94bright,CD158negative phenotype.
      Activated NK cells from the joints of RA patients induce differentiation of monocytes, signifying an active role in the immune environment,
      • Dalbeth N.
      • Gundle R.
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      • Lee Y.C.
      • McMichael A.J.
      • Callan M.F.
      CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation.
      and indicating that NK cells play a proinflammatory role in autoimmunity.
      This directly contradicts the observations that myocarditis, RA, Sjögren syndrome, and systemic lupus erythematosus patients have decreased NK cell numbers and cytotoxicity potential.
      • Ren J.
      • Feng Z.
      • Lv Z.
      • Chen X.
      • Li J.
      Natural killer-22 cells in the synovial fluid of patients with rheumatoid arthritis are an innate source of interleukin 22 and tumor necrosis factor-alpha.
      • Dalbeth N.
      • Callan M.F.
      A subset of natural killer cells is greatly expanded within inflamed joints.
      • Shi F.D.
      • Takeda K.
      • Akira S.
      • Sarvetnick N.
      • Ljunggren H.G.
      IL-18 directs autoreactive T cells and promotes autodestruction in the central nervous system via induction of IFN-gamma by NK cells.
      • Yang Y.Z.
      • Jin P.Y.
      • Wang Q.D.
      [Natural killer cell activity and induction of alpha and gamma interferon in patients with Coxsackie B viral myocarditis].
      • Kanda T.
      • Ohshima S.
      • Yuasa K.
      • Watanabe T.
      • Suzuki T.
      • Murata K.
      Idiopathic myocarditis associated with T-cell subset changes and depressed natural killer activity.
      A limited study of biopsy specimens from myocarditis patients revealed a lack of NK cells in the cardiac tissue.
      • Chow L.H.
      • Ye Y.
      • Linder J.
      • McManus B.M.
      Phenotypic analysis of infiltrating cells in human myocarditis: an immunohistochemical study in paraffin-embedded tissue.
      Peripheral NK cells from RA patients failed to induce apoptosis in major histocompatibility complex I–deficient K562 cells versus healthy controls in vitro. Patients with multiple sclerosis in remission had higher frequencies of activated peripheral NK cells than those with active disease, supporting the notion that defects in NK cells are associated with increased risk of autoimmunity.
      • Takahashi K.
      • Aranami T.
      • Endoh M.
      • Miyake S.
      • Yamamura T.
      The regulatory role of natural killer cells in multiple sclerosis.
      Altogether, it is unclear whether autoimmune diseases are exacerbated by deficiencies or excesses of NK cells, making animal studies necessary.
      Herein, we investigated the role of NK cells in autoimmune myocarditis using a mouse model of experimental autoimmune myocarditis (EAM). EAM is induced by s.c. immunization of myocarditogenic peptide in complete Freund's adjuvant, the same antigen targeted by autoaggressive T cells in coxsackievirus group B type 3 (CB3)-induced myocarditis.
      • Afanasyeva M.
      • Georgakopoulos D.
      • Rose N.R.
      Autoimmune myocarditis: cellular mediators of cardiac dysfunction.
      • Cihakova D.
      • Sharma R.B.
      • Fairweather D.
      • Afanasyeva M.
      • Rose N.R.
      Animal models for autoimmune myocarditis and autoimmune thyroiditis.
      • Neu N.
      • Beisel K.W.
      • Traystman M.D.
      • Rose N.R.
      • Craig S.W.
      Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis.
      • Rose N.R.
      The significance of autoimmunity in myocarditis.
      Susceptible mice strains develop myocarditis, followed by inflammatory dilated cardiomyopathy.
      • Cihakova D.
      • Sharma R.B.
      • Fairweather D.
      • Afanasyeva M.
      • Rose N.R.
      Animal models for autoimmune myocarditis and autoimmune thyroiditis.
      EAM induces the immune response independent of persistent virus, allowing us to separate autoimmune- from virus-mediated disease. We report herein the ability of NK cells to control myocarditis in the absence of a viral pathogen.

      Materials and Methods

      Mice

      BALB/c, Rag1−/−, C.Cg-Gata1tm6Sho/J (ΔdoubleGATA1), CD3δ-IL5Tg NJ.1636, Ccr3−/−, interferon γ receptor 1 (IFNγR1)−/−, and IFNγ−/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were bred and maintained in the conventional housing facilities at Johns Hopkins University (Baltimore, MD). All protocols have been reviewed and approved by the Johns Hopkins Animal Care and Use Committee.

      Immunization with MHC614-629 and Assessment of EAM

      Male 6- to 8-week-old BALB/c mice were injected s.c. with 100 μg of myocarditogenic peptide of cardiac myosin heavy chain (MyHC), MyHC614-629, emulsified in an equal volume of complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO), supplemented with 4 mg/mL of H37Ra extract (Difco, Lawrence, KS) on days 0 and 7, as previously described.
      • Cihakova D.
      • Sharma R.B.
      • Fairweather D.
      • Afanasyeva M.
      • Rose N.R.
      Animal models for autoimmune myocarditis and autoimmune thyroiditis.
      Pertussis toxin (500 ng in 100 μL of phosphate-buffered saline (PBS; Sigma-Aldrich) was administrated i.p. on day 0. Mice were sacrificed on day 21; hearts were collected and sections were stained with hematoxylin and eosin, as previously described.
      • Baldeviano G.C.
      • Barin J.G.
      • Talor M.V.
      • Srinivasan S.
      • Bedja D.
      • Zheng D.
      • Gabrielson K.
      • Iwakura Y.
      • Rose N.R.
      • Cihakova D.
      Interleukin-17A is dispensable for myocarditis but essential for the progression to dilated cardiomyopathy.
      • Barin J.G.
      • Baldeviano G.C.
      • Talor M.V.
      • Wu L.
      • Ong S.
      • Quader F.
      • Chen P.
      • Zheng D.
      • Caturegli P.
      • Rose N.R.
      • Cihakova D.
      Macrophages participate in IL-17-mediated inflammation.
      • Barin J.G.
      • Talor M.V.
      • Baldeviano G.C.
      • Kimura M.
      • Rose N.R.
      • Cihakova D.
      Mechanisms of IFNgamma regulation of autoimmune myocarditis.
      • Cihakova D.
      • Barin J.G.
      • Afanasyeva M.
      • Kimura M.
      • Fairweather D.
      • Berg M.
      • Talor M.V.
      • Baldeviano G.C.
      • Frisancho S.
      • Gabrielson K.
      • Bedja D.
      • Rose N.R.
      Interleukin-13 protects against experimental autoimmune myocarditis by regulating macrophage differentiation.
      The degree of myocardial infiltration and fibrosis was determined blindly by two individuals (D.C. and S.O.), and histology was scored as follows: 0, no infiltration; 1, ≤10%; 2, 11% to 30%; 3, 31% to 50%; 4, 51% to 90%; and 5, >90%.
      • Cihakova D.
      • Sharma R.B.
      • Fairweather D.
      • Afanasyeva M.
      • Rose N.R.
      Animal models for autoimmune myocarditis and autoimmune thyroiditis.

      Assessment of Fibrosis

      Mice were sacrificed on day 21. Hearts were collected and sections were stained with Masson’s trichrome, as previously described.
      • Baldeviano G.C.
      • Barin J.G.
      • Talor M.V.
      • Srinivasan S.
      • Bedja D.
      • Zheng D.
      • Gabrielson K.
      • Iwakura Y.
      • Rose N.R.
      • Cihakova D.
      Interleukin-17A is dispensable for myocarditis but essential for the progression to dilated cardiomyopathy.
      • Barin J.G.
      • Baldeviano G.C.
      • Talor M.V.
      • Wu L.
      • Ong S.
      • Quader F.
      • Chen P.
      • Zheng D.
      • Caturegli P.
      • Rose N.R.
      • Cihakova D.
      Macrophages participate in IL-17-mediated inflammation.
      • Barin J.G.
      • Talor M.V.
      • Baldeviano G.C.
      • Kimura M.
      • Rose N.R.
      • Cihakova D.
      Mechanisms of IFNgamma regulation of autoimmune myocarditis.
      • Cihakova D.
      • Barin J.G.
      • Afanasyeva M.
      • Kimura M.
      • Fairweather D.
      • Berg M.
      • Talor M.V.
      • Baldeviano G.C.
      • Frisancho S.
      • Gabrielson K.
      • Bedja D.
      • Rose N.R.
      Interleukin-13 protects against experimental autoimmune myocarditis by regulating macrophage differentiation.
      Images of Masson's trichrome–stained cardiac sections were uploaded into ImageJ software version 1.48 (NIH, Bethesda, MD). The background space was deleted and the left ventricle, the region of interest, was selected using the freeform loop tool. Pixels within the selected area were deconstructed into red (tissue) or blue (collagen) channels, and fibrosis was calculated as a percentage of blue versus total red plus blue pixels in the region of interest.

      Echocardiography

      An Acuson Sequoia 256 high-resolution microimaging system with a 13-MHz transducer was used (Visualsonic, Toronto, ON, Canada). In conscious mice, the heart was imaged in the two-dimensional mode in the parasternal short-axis view. From the M-mode, the left ventricular (LV) wall thickness and chamber dimensions were measured. The M-mode cursor was positioned perpendicular to the intraventricular septum and the LV posterior wall, with three to five readings taken for each measurement. The LV end diastolic dimension, LV end systolic dimension, LV posterior wall thickness at end diastole, and the intraventricular septal wall thickness at end diastole were measured from a frozen M-mode tracing. Fractional shortening, ejection fraction, and relative wall thickness were calculated as previously described.
      • Baldeviano G.C.
      • Barin J.G.
      • Talor M.V.
      • Srinivasan S.
      • Bedja D.
      • Zheng D.
      • Gabrielson K.
      • Iwakura Y.
      • Rose N.R.
      • Cihakova D.
      Interleukin-17A is dispensable for myocarditis but essential for the progression to dilated cardiomyopathy.

      Intracardiac and Splenic Flow Cytometry

      The aorta was cannulated to perfuse hearts with 15 mL of cold 1× PBS for 3 minutes to remove blood. To generate cardiac single-cell suspensions, hearts were bisected, placed in C-tubes, and dissociated on the GentleMACS system (Miltenyi Biotech, Bergisch Gladbach, Germany) under program heart_01. Cells were placed in a rotating incubator with 10 mg of collagenase II and 1.5 mg of DNase I (Worthington Biochemical, Lakewood, NJ) for 30 minutes at 37°C. Cells were dissociated again and rinsed twice with 1× PBS with 0.05% bovine serum albumin (BSA; Sigma-Aldrich) and 2 mmol/L EDTA (Corning Cellgro, Corning, NY). To generate a splenic single-cell suspension, spleens were dissociated between two frosted glass slides and incubated with 2 mL of ACK Lysing Buffer for 1 minute. The cells were rinsed with 1× PBS (Mediatech, Manassas, VA) and filtered through a 40-μm mesh. Cells (1 to 3 × 106) were incubated with 1 μL of LIVE/DEAD Aqua (Invitrogen, Carlsbad, CA) for 30 minutes in 1× PBS to stain dead cells. Cells were then incubated with 2 μg of αCD16/32 at 4°C for 10 minutes before the addition of fluorescent antibodies (CD3, CD4, CD8, CD45, Ly6G, SiglecF, NKp46, DX5, CD11b, CD11c, and F4/80) (eBioscience, San Diego, CA). Samples were incubated with antibodies at 4°C for 10 to 20 minutes, washed in 1 mL of 0.5% BSA in 1× PBS, and fixed in fixation and permeabilization buffer (BD Bioscience, Franklin Lakes, NJ) for 30 minutes. For intracellular cytokine staining, suspensions were incubated for 4 to 6 hours with 20 ng/mL 4β-phorbol 12-myristate 13-acetate, 1 μg/mL ionomycin, and Golgistop (BD Bioscience). Cells were surface stained and then permeabilized with 1× permeabilization buffer (BD Bioscience) overnight at 4°C. Cells were then incubated with anti-cytokine antibodies (eBioscience) for 30 minutes at 4°C. Cells were washed in 1× permeabilization buffer and resuspended in 100 to 200 μL of buffer. Samples were acquired on the LSR II quad-laser cytometer running FACSDiva 6 (BD Immunocytometry, Franklin Lakes, NJ). Data were analyzed with FlowJo version 7.6 (Treestar Software, Ashland, OR).

      Depletion of NK Cells with Anti-Asialo GM1 Antibody

      To deplete NK cells before immunization, 6-week-old male BALB/c mice were injected i.p. with 1 mg of anti-asialo GM1 (Wako Chemicals USA, Richmond, VA) antibody every day 6 days before the first immunization (days -6 to 0).
      • Kasai M.
      • Yoneda T.
      • Habu S.
      • Maruyama Y.
      • Okumura K.
      • Tokunaga T.
      In vivo effect of anti-asialo GM1 antibody on natural killer activity.
      • Habu S.
      • Fukui H.
      • Shimamura K.
      • Kasai M.
      • Nagai Y.
      • Okumura K.
      • Tamaoki N.
      In vivo effects of anti-asialo GM1, I: reduction of NK activity and enhancement of transplanted tumor growth in nude mice.
      To maintain decreased levels of NK cells after the first immunization (day 0), 1 mg of anti-asialo GM1 antibody was administered every other day until day 20. Control mice received 1 mg of rabbit IgG (Sigma-Aldrich) by the same schedule.

      Isolation of Primary Adult Mouse Cardiac Fibroblasts

      Primary adult mouse cardiac fibroblasts were isolated with minor modifications from protocols previously described.
      • O'Connell T.D.
      • Rodrigo M.C.
      • Simpson P.C.
      Isolation and culture of adult mouse cardiac myocytes.
      Hearts dissected from 6- to 8-week-old naïve BALB/c mice were perfused through the aorta with warmed 37°C calcium-free buffer, followed by collagenase type II (Worthington Biochemical, Lakewood, NJ) for 15 minutes. Tissue was dissolved into a single-cell suspension and filtered through a 70-μm mesh. Cells were seeded, and nonadherent cells were washed off after 1 hour. Cells were either collected immediately in TRIzol reagent (Invitrogen) for ex vivo experiments or passaged twice before in vitro use in complete Dulbecco’s modified Eagle’s medium with 20% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1× penicillin/streptomycin, 25 mmol/L HEPES, 1× Anti-Anti (Gibco, Carlsbad, CA), and 1× nonessential amino acids.

      Isolation of Primary NK Cells

      NK cells were negatively isolated from Rag1−/− BALB/c spleens by manual magnetic cell sorting using the Mouse NK Isolation Kit II (Miltenyi Biotech) and cultured for 24 hours with 10 ng/mL IL-12 and 5 ng/mL IL-15.

      Isolation of Primary Eosinophils

      Eosinophils were isolated from naïve CD3δ-IL5Tg NJ.1636 peripheral blood mononuclear cells using a Percoll (GE Lifesciences, Marlborough, MA) gradient and subsequent negative fluorescence-activated cell sorting for SSChiLy6GDX5 eosinophils.

      Apoptosis Measurement

      Cells were harvested from culture and rinsed twice with 1× PBS with 0.05% BSA (Sigma-Aldrich) and 2 mmol/L EDTA (Corning Cellgro). The cells were rinsed with 1× PBS and incubated with 1 μL of LIVE/DEAD Aqua (Invitrogen) for 30 minutes in 1× PBS to stain dead cells. Cells were then incubated with 2 μg of αCD16/32 at 4°C for 10 minutes before the addition of fluorescent antibodies (Ly6G, SiglecF, and NKp46) (eBioscience). Samples were incubated with antibodies at 4°C for 10 to 20 minutes and washed in 1 mL of 0.5% BSA in 1× PBS. Cells were then resuspended in 1× Annexin Binding Buffer (eBioscience) and stained with 2 μL of annexin V. Cells were acquired after 15 minutes of incubation on ice on the LSRII flow cytometer (Becton Dickenson, Franklin Lakes, NJ).

      mRNA

      For real-time quantitative PCR (qPCR), cells or tissues were homogenized in TRIzol reagent and chloroform extracted. Samples were DNase treated, and cDNA libraries were made using iScript Reverse Transcriptase Supermix (BioRad, Hercules, CA). mRNA was amplified using SYBR Green (Applied Biosystems, Foster City, CA), and all values were calculated against hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA. Values were controlled against isotype control groups and shown as a function of fold induction using the formula 2−Δ(ΔCT).
      Murine primers were as follows (forward and reverse primers, respectively): Hprt, 5′-TCCTCCTCAGACCGCTTTT-3′ and 5′-TCTGCTGGAGTCCCCTTG-3′; collagen 1a1 (Col1a1), 5′-AGCAGGTCCTTGGAAACCTT-3′ and 5′-AAGGAGTTTCATCTGGCCCT-3′; Col1a2, 5′-GTGAACGGGGCGAAGCTGGTT-3′ and 5′-GCGGCTCCTGGAAGCCCATTTG-3′; Col1a3, 5′-AACCTGGTTTCTTCTCACCCTTC-3′ and 5′-ACTCATAGGACTGACCAAGGTGG-3′; Il4, 5′-AAGGCAACTTTCTTGATATT-3′ and 5′-GGCCTTTCAGACTAATCTT-3′; Il13, 5′-TGAGGAGCTGAGCAACATCACACA-3′ and 5′-TGCGGTTACAGAGGCCATGCAATA-3′; eosinophil peroxidase, 5′-AGATGCAACAACAAGAAGCATCC-3′ and 5′-TGATTGGAGACATCCCGGAC-3′; major basic protein 2, 5′-TGAAACTTCTGACTCCAAAAGCC-3′ and 5′-CGGCATTAGCTCTTCCCCT-3′; Il1b, 5′-CAACCAACAAGTGATATTCTCCATG-3′ and 5′-GATCCACACTCTCCAGCTGCA-3′; transforming growth factor (Tgf) b1, 5′-CTCCCGTGGCTTCTAGTGC-3′ and 5′-GCCTTAGTTTGGACAGGATCTG-3′; chemokine ligand 11 (Ccl11), 5′-GAATCACCAACAACAGATGCAC-3′ and 5′-TCCTGGACCCACTTCTTCTT-3′; Ccl24, 5′-TCTTAGGGCCCTTCTTGGTG-3′ and 5′-AATTCCAGAAAACCGAGTGG-3′; and Cxcl9, 5′-GTGGAGACCACCAGAGTTGG-3′ and 5′-TGCCACTAAGCTACAGCCAC-3′.

      Enzyme-Linked Immunosorbent Assays

      Samples were run using tissue homogenates in 1× PBS or cell culture supernatant using enzyme-linked immunosorbent assay kits for Ccl11 (RND Biosystems, Minneapolis, MN) or a multiplex LINCO kit (Millipore, Jaffery, NH).

      Statistical Analysis

      Multiple group comparisons were performed by ordinary one-way analysis of variance, followed by the Tukey-Kramer post test (if parametric) or Kruskal-Wallis, followed by Dunn's post test (if nonparametric) (GraphPad Prism 5; GraphPad, San Diego, CA). All statistics of two groups were performed by Student's t-test. P < 0.05 was considered statistically significant.

      Results

      NK Cells Suppress Cardiac Inflammation and Severe Myocarditis in EAM

      To determine whether NK cells modulate EAM, we depleted NK cells in vivo using 1 mg of anti-asialogangloside GM-1 (ASGM-1) polyclonal antibody injected i.p. from day -6 to day 21, as described in Materials and Methods (Figure 1A). The depletion caused myocarditis to increase in severity as assessed by histology and total count of infiltrating CD45+ cells in the heart at the peak of inflammation on day 21 (Figure 1, B–D). This was accompanied by increased circulating anti–MyHCα614-629-specific IgG antibodies (Figure 1E). At this time, ASGM-1 antibody reduced CD3DX5+NKp46+ NK cells, but not CD3+DX5+ NK T cells (Figure 1, F and G) in the spleen and heart during EAM. ASGM-1 had little effect on other cell types in naïve animals, because antibody treatment depleted CD3DX5+NKp46+ NK, but not CD3+DX5+NKp46+, NK T cells or Ly6GloSiglecF+ eosinophils (Supplemental Figure S1). Development of severe myocarditis after depletion of NK cells suggests that NK cells protect from severe cardiac inflammation.
      Figure thumbnail gr1
      Figure 1Depletion of natural killer (NK) cells increases cardiac inflammation and the severity of experimental autoimmune myocarditis (EAM). A: Schematic of phosphate-buffered saline (PBS), rabbit IgG, and asialogangloside GM-1 (ASGM-1) antibody treatment schedule throughout EAM. Representative histology from rabbit IgG (RaIgG) and ASGM-1–treated animals (B) and scores of hematoxylin and eosin–stained cardiac sections from PBS, rabbit IgG, and ASGM-1 antibody treated animals at day 21 of EAM (analysis of variance P < 0.01; C). D: Total CD45+ cells infiltrating the hearts of isotype control and ASGM-1–treated animals at day 21 of EAM, as assessed by flow cytometry (P < 0.001). E: Levels of anti-myocarditogenic peptide of cardiac myosin heavy chain (MyHC)614-629 total IgG antibodies in the serum of PBS, isotype control, and ASGM-1–treated animals at day 21 of EAM (analysis of variance P < 0.001). Percentage of CD3DX5+NKp46+ NK cells in the heart (P < 0.001) and spleen (P < 0.002) (F) and CD3+DX5+ NK T cells out of CD45+ cells (G) of rabbit IgG and ASGM-1 monoclonal antibody–treated wild-type (WT) mice at day 21 of EAM, as assessed by flow cytometry. ∗∗P < 0.05, ∗∗∗P < 0.001.

      Absence of NK Cells Increases Collagen Deposition in the Heart

      We explored whether NK cells inhibit fibrosis development and inflammation. To assess fibrosis quantitatively, images of cardiac histology slides stained by Masson's trichrome were processed by ImageJ software to enumerate collagen-positive (blue) versus nonfibrotic (red) pixels. NK-depleted animals had increased cardiac collagen deposition on day 21 of EAM (Figure 2, A and B). This accompanied a decline in cardiac function, as shown by decreased ejection fraction with increased LV end systolic diameter and no changes in LV end diastolic diameter or intraventricular septal thickness (Figure 2, C–F). We quantified active collagen production in NK-depleted mice at day 21 and showed that NK depletion led to increased mRNA levels of collagen 1 and collagen 3 in the heart (Figure 2, G–I). Thus, the depletion of NK cells during EAM leads to increased cardiac fibrosis and a decline in cardiac function.
      Figure thumbnail gr2
      Figure 2Depletion of natural killer cells increases collagen deposition and fibrosis during experimental autoimmune myocarditis (EAM). A: Representative histology of Masson's trichrome–stained cardiac sections from rabbit IgG (RaIgG) and asialogangloside GM-1 (ASGM-1)–treated animals at day 21 of EAM. B: Enumeration of fibrosis by calculation of red versus blue pixels on ImageJ software (P = 0.012). Statistics calculated by unpaired t-test. C: Ejection fraction (P = 0.026) calculated from fractional shortening and left ventricular (LV) diastole and systole. LV end diastolic dimension (LVEDD; D) and LV end systolic dimension (LVESD; P = 0.03; E). F: Intraventricular septum diameter (IVS), as measured by echocardiography. Statistics by ordinary one-way analysis of variance with post testing by Tukey's multiple-comparisons test. Collagen production as measured using real-time quantitative PCR for collagen 1a1 (Col1a1; P = 0.04; G), collagen 1a2 (Col1a2; P = 0.03; H), and collagen 3a1 (Col3a1P = 0.02; I). Values calculated as a function of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and compared against rabbit IgG. Statistics calculated by unpaired t-test. P < 0.05.

      Activated NK Cells Accumulate in the Heart during EAM

      To examine NK cell kinetics during EAM, we quantified cardiac NK cells by flow cytometry (Figure 3A). CD3DX5+NKp46+ NK cells increased from day 0 to day 21 in absolute counts; however, their proportion out of CD45+ cells was stable (Figure 3, B and C). To determine their phenotype, we profiled cytokine and receptor expression from NK cells by flow cytometry on day 21 of EAM to see if they represented an activated population compared to those in the periphery. Cardiac NK cells produced more Ifnγ than splenic NK cells, but equivalent levels of Il-13 (Figure 3, D and E). Greater proportions of cardiac NK cells up-regulated perforin and were positive for lysosomal-associated membrane protein 1 (LAMP-1), a correlate for granzyme B secretion (Figure 3, F and G). In addition, cardiac NK cells up-regulated activation markers CD27, CD69, and tumor necrosis factor–related apoptosis-inducing ligand treatment (Figure 3, H–J), with decreased NKG2D (Figure 3K). Thus, cardiac NK cells secrete Ifnγ and have increased expression of multiple activation receptors compared to NK cells in periphery during the course of myocarditis.
      Figure thumbnail gr3
      Figure 3Activated natural killer (NK) cells accumulate in the heart during experimental autoimmune myocarditis (EAM). A: Gating strategy for cardiac CD3DX5+NKp46+ NK cells. Absolute numbers (analysis of variance P = 0.002; B) and percentages (C) of CD3DX5+NKp46+ NK cells on days 0, 14, and 21 of EAM by flow cytometry. Significance by ordinary one-way analysis of variance with post testing by Tukey's multiple-comparisons test. To determine NK physiology, cardiac and splenic CD3DX5+NKp46+ NK cells were stained at day 21 of EAM. Percentage of interferon γ (IFNγ; P < 0.001; D), Il-13 (E), and perforin (P < 0.001; F) positive cells were based on intracellular antibody staining after 4 to 6 hours of 4β-phorbol 12-myristate 13-acetate/ionomycin and Golgistop in vitro. G: Percentage of lysosomal-associated membrane protein 1 (LAMP-1+), a marker for granzyme B release, staining (P < 0.001). Percentage of NK cells positive for activation-associated markers CD27 (P < 0.001; H), CD69 (P < 0.001; I), tumor necrosis factor–related apoptosis-inducing ligand treatment (TRAIL; P < 0.001; J), and NKG2D (P < 0.001; K). D–F: Significance calculated by unpaired t-test. ∗∗P < 0.01, ∗∗∗P < 0.001. FSC, forward scatter; SSC, side scatter.

      Activated Fibroblasts Are Not Targeted for Cytotoxic Killing by NK Cells in Vitro

      The depletion of NK cells led to accelerated fibrosis (Figure 2) in the heart; therefore, we hypothesized that NK cells lysed activated fibroblasts, as described in animal models of liver fibrosis.
      • Gonzalez A.
      • Katz J.D.
      • Mattei M.G.
      • Kikutani H.
      • Benoist C.
      • Mathis D.
      Genetic control of diabetes progression.
      • Poirot L.
      • Benoist C.
      • Mathis D.
      Natural killer cells distinguish innocuous and destructive forms of pancreatic islet autoimmunity.
      We explored this putative mechanism using a co-culture system in vitro with an outcome focused on cell death. First, adult cardiac fibroblasts were isolated from naïve BALB/c hearts and passaged twice. The presence of contaminating macrophages was ruled out by qPCR and immunofluorescence (Supplemental Figure S2). Second, NK cells were isolated from naïve or poly(I:C)-treated BALB/c spleens (Supplemental Figure S3A). Cardiac fibroblasts were activated with angiotensin II, illustrated by increased expression of vimentin (Supplemental Figure S3B). Finally, we co-cultured cardiac fibroblasts and NK cells together for 48 hours at a 1:1 ratio. Cardiac fibroblasts were stained green with calcein AM (live cells) and red with ethidium homodimer (dead cells) and examined by immunofluorescence microscopy (Supplemental Figure S3C). Although NK cells induced cell death of cardiac fibroblasts, no difference was seen between untreated and activated cardiac fibroblasts (Supplemental Figure S3, D and E), indicating that NK-mediated killing of activated fibroblasts was not a likely mechanism.

      NK Cells Prevent Eosinophils from Accumulating in the Heart during EAM

      Given the lack of direct killing seen between NK cells and activated fibroblasts, we hypothesized that NK cells altered disease severity and fibrosis in EAM through alterations in the hematopoietic populations infiltrating the heart. Therefore, we examined changes in the cardiac infiltrate during EAM after the depletion of NK cells by flow cytometry. NK-depleted animals had increased proportion of SSChiCD45+ granulocytic cells (Figure 4A). We identified that SSChiLy6GloSiglecF+ eosinophils were responsible for the increase in granulocytic cells. SSChiLy6GloSiglecF+ eosinophils increased twofold on day 14 and 10-fold on day 21 out of CD45+ cells (Figure 4B). We found no changes in SSChiLy6Ghi neutrophils on day 14 or 21 (Figure 4C).
      Figure thumbnail gr4
      Figure 4Depletion of natural killer (NK) cells increases eosinophilic infiltration in the heart. A: Representative side-scatter histograms of isotype and asialogangloside GM-1 (ASGM-1) antibody treated animals at day 21. Percentage of Ly6GloSiglecF+ eosinophils from cardiac CD45+ cells on day 14 (P = 0.043) and day 21 (P < 0.001) (B) and SiglecFLy6Ghi neutrophils at days 14 and 21 (C). Significance calculated by unpaired t-test. P < 0.05, ∗∗∗P < 0.001. SSC, side scatter.
      To determine whether the increased eosinophils in the heart seen in ASGM-1–treated mice during EAM were a cardiac-specific observation or a reflection of systemic eosinophilia in response to NK depletion, we analyzed the blood and spleens of mice depleted of NK cells during EAM at days 14 and 21. We found that, although there was a mild trend toward increased eosinophils, it was not significant (Supplemental Figure S4), underlining that the mechanism of eosinophilic control by NK cells is specific for the cardiac environment during EAM.
      We also explored whether there were shifts in any other infiltrating cardiac populations during EAM. SSCmidCD11b+ myeloid cells, as a percentage of total CD45+ cells (Supplemental Figure S5A), remained unchanged, as were CD11b+Ly6Chi and CD11b+Ly6Clo monocytes as a proportion of total SSCmidCD11b+ cells on day 21 (Supplemental Figure S5, B and C). The proportions of total infiltrating CD45+ cells of both CD11chi dendritic cells and FcεRIα+cKit+ mast cells were also unaltered (Supplemental Figure S5, D and E). Also comparable were proportions of CD11bB220+ B cells and CD3+ and CD3+CD4+ T cells (Supplemental Figure S5, F–H) of total CD45+ cells. Because EAM and eosinophil trafficking are influenced by CD4+ T-cell polarization,
      • Kay A.B.
      The role of eosinophils in the pathogenesis of asthma.
      • Afanasyeva M.
      • Georgakopoulos D.
      • Rose N.R.
      Autoimmune myocarditis: cellular mediators of cardiac dysfunction.
      • Barin J.G.
      • Cihakova D.
      Control of inflammatory heart disease by CD4+ T cells.
      we examined if NK cells affected types 1, 2, and 17 helper T cell (Th1, Th2, and Th17, respectively) populations during EAM.
      • Kay A.B.
      The role of eosinophils in the pathogenesis of asthma.
      • Afanasyeva M.
      • Georgakopoulos D.
      • Rose N.R.
      Autoimmune myocarditis: cellular mediators of cardiac dysfunction.
      • Habu S.
      • Fukui H.
      • Shimamura K.
      • Kasai M.
      • Nagai Y.
      • Okumura K.
      • Tamaoki N.
      In vivo effects of anti-asialo GM1, I: reduction of NK activity and enhancement of transplanted tumor growth in nude mice.
      No changes in IFNγ+CD3+CD4+ Th1 cells in the heart after NK depletion were found (Supplemental Figure S6A). However, Il-13+ Th2 and Il-17A+ Th17 cells, as a fraction of total CD3+CD4+ cells, increased at day 21, but not day 14 (Supplemental Figure S6, B and C). Thus, depletion of NK cells resulted in accumulation of eosinophils in the heart, reaching up to a 10-fold increase at the peak of inflammation, and was accompanied by a shift toward mixed Th2 and Th17 milieu in the heart.

      Eosinophils in the Heart of ASGM-1–Treated Mice Have an Activated Profile

      To determine whether the increased flux of eosinophils to the heart played a role in the increased disease severity, we examined the phenotype of the heart-infiltrating eosinophils for their maturation and activation status. It has been shown that mature eosinophils from the lungs of helminth-infected or the blood of asthma-induced mice have increased expression of CD11b and SiglecF protein and decreased expression of eosinophil granule–associated genes due to these granules being fully preloaded with protein.
      • Johansson M.W.
      Activation states of blood eosinophils in asthma.
      • Voehringer D.
      • van Rooijen N.
      • Locksley R.M.
      Eosinophils develop in distinct stages and are recruited to peripheral sites by alternatively activated macrophages.
      Indeed, paraffin-embedded cardiac tissue sections of ASGM-1–treated animals with EAM showed positive staining for eosinophil granule major basic protein (Figure 5A), and cardiac eosinophils from ASGM-1–treated hearts had up-regulated levels of SiglecF and CD11b protein compared to spleen cells at day 21 (Figure 5, B and C). Consistent with this activated phenotype, cardiac eosinophils down-regulated expression of eosinophil granule–associated eosinophil peroxidase and major basic protein 2 due to their granules having already been formed during the immature stages of development (Figure 5, D and E). Cardiac eosinophils also had increased expression of Il-1b, Ccl11, and Il-6, and showed no changes in Il-4 or Il-13 expression (Figure 5, F–J). In summary, eosinophils infiltrating the heart at day 21 of ASGM-1–treated mice represent a distinct and activated population.
      Figure thumbnail gr5
      Figure 5Activated eosinophils infiltrate the heart in the absence of natural killer cells. A: Immunofluorescence staining of paraffin-embedded cardiac sections from asialogangloside GM-1 (ASGM-1)–treated mice on day 21 of experimental autoimmune myocarditis (EAM). Sections stained with 1:500 rat anti-major basic protein (MBP) primary or rat IgG antibody, 1:200 anti-rat donkey phosphatidylethanolamine (PE)–Texas Red antibody, and DAPI. Relative mean fluorescence intensity of SiglecF (P < 0.001; B) and CD11b (P < 0.001; C) on Ly6GloSiglecF+ eosinophils from the heart and spleen of ASGM-1–treated animals at day 21 of EAM by flow cytometry. Levels of eosinophil peroxidase (Epx; P = 0.05; D), major basic protein 2 (Prg2; P = 0.05; E), Il1b (P = 0.04; F), chemokine ligand (Ccl) 11 (P = 0.03; G), Il6 (P = 0.02; H), Il4 (I), and Il13 (J) mRNA from fluorescence-activated cell sorted Ly6GloSiglecFhi eosinophils from ASGM-1–treated mice at day 21 of EAM. Values shown as fold induction compared to spleen and controlled against hypoxanthine-guanine phosphoribosyltransferase (HPRT). Significance calculated by Student's t-test. P < 0.05, ∗∗∗P < 0.001. MFI, mean fluorescence intensity.

      Eosinophils Are Necessary for Increased Myocarditis Severity in the Absence of NK Cells

      To establish if the greater EAM severity in NK-depleted animals depended on the influx of activated and mature eosinophils, we depleted NK cells from eosinophil-deficient ΔdblGATA1 mice.
      • Yu C.
      • Cantor A.B.
      • Yang H.
      • Browne C.
      • Wells R.A.
      • Fujiwara Y.
      • Orkin S.H.
      Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo.
      These mice have a deletion in the GATA1 promoter, leading to a specific ablation in eosinophils due to the inability of progenitors to differentiate in the bone marrow. In the absence of eosinophils, NK depletion had no effect on cardiac inflammation, as assessed from histological readings by two independent investigators using hematoxylin and eosin–stained cardiac slides at day 21 of EAM (Figure 6, A and B). In addition, we saw no increases in fibrosis evaluated by Masson's trichrome staining. In addition, cardiac function was preserved, as shown by no differences in echocardiographic parameters between NK-depleted and isotype ΔdblGATA1 groups at day 21 (Figure 6, C–G). ΔdblGATA1 mice did not respond to ASGM-1 treatment with the increased collagen 1 and 3 mRNA seen in wild-type mice (Figure 6, H–J). Therefore, myocarditis severity and cardiac fibrosis in ΔdblGATA1 mice are unaffected by NK cell depletion. This would indicate that NK cells protect the heart from eosinophilic accumulation and the subsequent development of severe myocarditis.
      Figure thumbnail gr6
      Figure 6Eosinophil-deficient mice are phenotypically unresponsive to natural killer depletion. Representative histology from rabbit IgG (RaIgG) and asialogangloside GM-1 (ASGM-1) antibody-treated (A) and scores of rabbit phosphate-buffered saline, IgG, and ASGM-1 antibody-treated ΔdblGATA1 animals (analysis of variance; B). Representative histology (C) and percentage of fibrosis (D) by Masson's trichrome staining of cardiac sections at day 21 of experimental autoimmune myocarditis (EAM) from rabbit IgG and ASGM-1–treated mice. Ejection fraction (E), left ventricular end diastolic dimension (LVEDD; F), and left ventricular end systolic dimension (LVESD; G) by echocardiography at day 21. Collagen 1 and 3 production as measured using real-time quantitative PCR for collagen 1a1 (Col1a1; H), collagen 1a2 (Col1a2; I), and collagen 3a1 (Col3a1; J) mRNA in hearts of rabbit IgG and ASGM-1–treated animals at day 21 of EAM. Values calculated as a function of hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels and compared against rabbit IgG. Statistics calculated by unpaired t-test.

      Ccl11 Is Involved But Not Required for the Control of Eosinophilic Infiltration by NK Cells

      NK cells are not major producers of eosinophil-associated chemokines.
      • Lanier L.L.
      Activating and inhibitory NK cell receptors.
      • Lanier L.L.
      NK cell recognition.
      • Vivier E.
      • Tomasello E.
      • Baratin M.
      • Walzer T.
      • Ugolini S.
      Functions of natural killer cells.
      However, we have determined that cardiac fibroblasts are an active source of cytokines and chemokines and can control the types of immune cells infiltrating the heart during myocarditis.
      • Wu L.
      • Ong S.
      • Talor M.V.
      • Barin J.G.
      • Baldeviano G.C.
      • Kass D.A.
      • Bedja D.
      • Zhang H.
      • Sheikh A.
      • Margolick J.A.
      • Iwakura Y.
      • Rose N.R.
      • Cihakova D.
      Cardiac fibroblasts mediate IL-17A-driven inflammatory dilated cardiomyopathy.
      Eotaxins are prominent modulators of eosinophil trafficking and accumulation. In EAM, the depletion of NK cells increased eotaxin expression in whole heart tissue (Figure 7A) and isolated cardiac fibroblasts on day 21 of EAM (Figure 7, A–C), as seen by qPCR. To examine if NK cells affected eotaxin production in vitro, we co-cultured naïve adult cardiac fibroblasts and NK cells for 96 hours. NK cells suppressed Il-4–mediated Ccl11 (Figure 7D) and increased IP-10 (Cxcl10), a negative eosinophil regulator, produced by cardiac fibroblasts (Supplemental Figure S7A). Down-regulation of monocyte chemoattractant protein-1 (Ccl2), macrophage inflammatory protein-1β (Ccl4), regulated on activation normal T cell expressed and secreted (Ccl5), and keratinocyte chemoattractant (Cxcl1) (Supplemental Figure S7, B–E) also occurred in the presence of NK cells. In response to Il-4, cardiac fibroblasts produced Il-5, but were unresponsive to NK cells (Supplemental Figure S7F).
      Figure thumbnail gr7
      Figure 7Chemokine ligand (Ccl) 11 is involved, but not required, for the suppression of eosinophils by natural killer (NK) cells. Ccl11 mRNA at day 21 of experimental autoimmune myocarditis (EAM) in whole heart (P = 0.004; A) and isolated cardiac fibroblasts of rabbit IgG and asialogangloside GM-1 (ASGM-1)–treated animals (B). C: CCL24 mRNA in isolated cardiac fibroblasts of rabbit IgG (RaIgG) and ASGM-1–treated animals at day 21 of EAM. D: Ccl11 in the supernatant after a 96-hour culture of wild-type cardiac fibroblasts with or without NK cells (P < 0.001). E: Percentage of Ly6GloSiglecF+ eosinophils at day 21 of EAM in rabbit IgG and ASGM-1–treated Ccr3−/− animals. Significance by Student's t-test. P < 0.05, ∗∗∗P < 0.001.
      To address whether NK suppression of eotaxins would be sufficient to prevent eosinophil accumulation, we used mice deficient in Ccr3, the sole receptor of Ccl11 and Ccl24.
      • Xanthou G.
      • Duchesnes C.E.
      • Williams T.J.
      • Pease J.E.
      CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11.
      Depletion of NK cells in Ccr3−/− mice still resulted in the influx of eosinophils into the heart during EAM (Figure 7E), indicating that NK cells do not control eosinophils through only eotaxins and that other eosinophilic chemokines are able to drive eosinophil trafficking in the absence of NK cells. Thus, Ccl11 is involved, but not required, for the control of eosinophilic infiltration in the heart by NK cells.

      Ifnγ and Cxcl9 Are Not Required by NK Cells to Control Eosinophilic Infiltration

      During EAM, IFNγ was significantly up-regulated in cardiac NK cells accumulating in the heart. Monokine induced by interferon-γ (Cxcl9), a major negative regulator of eosinophil trafficking, is directly controlled by Ifnγ. We determined that in ASGM-1–treated mice, expression of Cxcl9 was decreased compared to the isotype control groups at day 21 of EAM (Supplemental Figure S8A), and that similar to Cxcl10 (Supplemental Figure S7A), the presence of NK cells in culture with primary cardiac fibroblasts significantly increased levels of Cxcl9 in the supernatant (Supplemental Figure S8B). In addition, our data indicate that Ifnγ produced by NK cells is absolutely required for Cxcl9 production being instigated, because Ifnγ receptor–deficient (IFNγR1−/−) cardiac fibroblasts were unresponsive to the presence of NK cells (Supplemental Figure S8C). These findings were consistent in vivo, because we observed significant decreases in the protein levels of Cxcl9 in the hearts of IFNγ−/− animals at day 14 of EAM (Supplemental Figure S8D). By using IFNγ−/− mice to model the effects of diminished Cxcl9 and the absence of NK-sourced Ifnγ, we depleted NK cells in IFNγ−/− mice and examined eosinophilic accumulation in the heart on day 21. Similar to the Ccr3−/− mice, IFNγ−/− mice still continued to accumulate significantly more eosinophils in the heart (Supplemental Figure S8E). In conclusion, both Cxcl9 and IFNγ may contribute to the NK-mediated control of eosinophils in the heart, but they are not required.

      NK Cells Directly Induce Apoptosis of Eosinophils

      Finally, we examined whether NK cells could inhibit eosinophil infiltration through direct interactions between the two cell types. Recent studies showed that human NK cells induce activation and apoptosis of eosinophils.
      • Barnig C.
      • Cernadas M.
      • Dutile S.
      • Liu X.
      • Perrella M.A.
      • Kazani S.
      • Wechsler M.E.
      • Israel E.
      • Levy B.D.
      Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma.
      • Awad A.
      • Yassine H.
      • Barrier M.
      • Vorng H.
      • Marquillies P.
      • Tsicopoulos A.
      • Duez C.
      Natural killer cells induce eosinophil activation and apoptosis.
      We explored this as one avenue of NK-mediated control of eosinophil accumulation in the heart during cardiac inflammation. NK cells and eosinophils were negatively sorted from naïve Rag1−/− spleens and Cd3δ–IL-5–transgenic (NJ.1638) blood, respectively, and co-cultured for 3 hours at 5:1 and 10:1 ratios. Compared to eosinophils alone, NK cells induced apoptosis, but not degranulation, as measured by side scatter, of eosinophils in a dose-dependent manner (Figure 8A). At this 3-hour time point, early apoptosis was measurable using annexin V, but no changes were yet seen with a viability dye measured through cell permeabilization (Figure 8, B and C). NK cells did not induce eosinophil activation either, as shown by changes in levels of SiglecF and CD11b, unlike reports in human cells (Figure 8, D and E).
      • Awad A.
      • Yassine H.
      • Barrier M.
      • Vorng H.
      • Marquillies P.
      • Tsicopoulos A.
      • Duez C.
      Natural killer cells induce eosinophil activation and apoptosis.
      In summary, we show that, similarly to human NK cells, mice NK cells cause apoptosis of eosinophils.
      Figure thumbnail gr8
      Figure 8Natural killer (NK) cells induce apoptosis in eosinophils (Eos) in vitro. A: Representative side scatter versus SiglecF and LIVE/DEAD viability dye versus annexin V plots of Ly6GloSiglecF+ eosinophils after a 3-hours co-culture with primary naïve NK cells. Percentage of annexin V–positive (analysis of variance P < 0.001; B) and LIVE/DEAD stain–positive Ly6GloSiglecF+ eosinophils (C). Average mean fluorescence intensity of activation markers SiglecF (D) and CD11b (E) of Ly6GloSiglecFhi eosinophils. Statistics calculated by one-way analysis of variance with post testing by Tukey's test. P < 0.05, ∗∗∗P < 0.001. SSC, side scatter.

      Discussion

      Previous studies
      • Fairweather D.
      • Kaya Z.
      • Shellam G.R.
      • Lawson C.M.
      • Rose N.R.
      From infection to autoimmunity.
      • Godeny E.K.
      • Gauntt C.J.
      Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis.
      have demonstrated that NK cells limit disease severity in CB3- and mouse cytomegalovirus–induced myocarditis by suppressing virus replication. In addition, we showed that depletion of NK cells rendered previously resistant strains of mice susceptible to viral myocarditis.
      • Fairweather D.
      • Kaya Z.
      • Shellam G.R.
      • Lawson C.M.
      • Rose N.R.
      From infection to autoimmunity.
      Herein, we demonstrate that the protective qualities of NK cells extend beyond viral inhibition. Activated NK cells accumulated in the heart throughout EAM and actively suppressed autoimmune-mediated inflammation, because the depletion of NK cells during EAM led to increased cardiac inflammation and fibrosis. This was caused primarily by a 10-fold increase in the proportion of eosinophils in the cardiac infiltrate, because eosinophil-deficient ΔdblGATA1 animals did not display increased myocarditis severity in response to NK cell depletion. Furthermore, this increase of eosinophils was specific for the cardiac environment and was not found in the periphery during EAM. On the basis of these data, we conclude that NK cells limited eosinophil accumulation in the heart during EAM and in the absence of NK cells, increases in eosinophils drove the amplified disease severity.
      Similar to studies in RA patients, we showed that NK cells occupying local sites of autoimmune-mediated inflammation have a distinct and activated phenotype compared to those in the periphery.
      • Dalbeth N.
      • Gundle R.
      • Davies R.J.
      • Lee Y.C.
      • McMichael A.J.
      • Callan M.F.
      CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation.
      • Dalbeth N.
      • Callan M.F.
      A subset of natural killer cells is greatly expanded within inflamed joints.
      Cardiac NK cells up-regulated CD27, tumor necrosis factor–related apoptosis-inducing ligand treatment, and CD69 on their cell surfaces, but down-regulated NKG2D. The down-regulation of NKG2D is surprising because nonobese diabetes can be prevented with anti-NKG2D antibodies and NKG2D+ cells are correlated with clinical Crohn's and RA severity.
      • Sungur C.M.
      • Tang-Feldman Y.J.
      • Zamora A.E.
      • Alvarez M.
      • Pomeroy C.
      • Murphy W.J.
      Murine NK-cell licensing is reflective of donor MHC-I following allogeneic hematopoietic stem cell transplantation in murine cytomegalovirus responses.
      • Zhang B.
      • Yamamura T.
      • Kondo T.
      • Fujiwara M.
      • Tabira T.
      Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells.
      • Lassen M.G.
      • Lukens J.R.
      • Dolina J.S.
      • Brown M.G.
      • Hahn Y.S.
      Intrahepatic IL-10 maintains NKG2A+Ly49- liver NK cells in a functionally hyporesponsive state.
      However, total NKG2D in these studies was targeted, including those expressed on NK T and δγ T cells.
      • Fathman J.W.
      • Bhattacharya D.
      • Inlay M.A.
      • Seita J.
      • Karsunky H.
      • Weissman I.L.
      Identification of the earliest natural killer cell-committed progenitor in murine bone marrow.
      Therefore, the role of NKG2D on NK cells is undetermined and its down-regulation in EAM indicates it may have a minimal role in autoimmunity.
      Our finding that eosinophils promote severe cardiac inflammation is supported by other reports of eosinophils driving myocarditis severity. In CB3-induced myocarditis, infiltrating eosinophils significantly increased disease. Soluble ST2, an Il-33 receptor decoy, prevented eosinophilia and reduced myocarditis without altering viral burden.
      • Abston E.D.
      • Barin J.G.
      • Cihakova D.
      • Bucek A.
      • Coronado M.J.
      • Brandt J.E.
      • Bedja D.
      • Kim J.B.
      • Georgakopoulos D.
      • Gabrielson K.L.
      • Mitzner W.
      • Fairweather D.
      IL-33 independently induces eosinophilic pericarditis and cardiac dilation: ST2 improves cardiac function.
      Similarly, IFNγ−/−Il17A−/− mice display massively inflamed hearts and up to 50% fatality by day 21 of EAM, a phenotype that is reversed in the absence of eosinophils by crossing these animals to dblGATA1 mice.
      • Barin J.G.
      • Baldeviano G.C.
      • Talor M.V.
      • Wu L.
      • Ong S.
      • Fairweather D.
      • Bedja D.
      • Stickel N.R.
      • Fontes J.A.
      • Cardamone A.B.
      • Zheng D.
      • Gabrielson K.L.
      • Rose N.R.
      • Cihakova D.
      Fatal eosinophilic myocarditis develops in the absence of IFN-gamma and IL-17A.
      Although the numbers of eosinophils infiltrating the heart after NK cell depletion (10% to 15%) during EAM do not approach clinical or animal models of eosinophilic myocarditis (up to 50%), we clearly demonstrate that even this moderate increase has significant consequences on disease outcome.
      • Abston E.D.
      • Barin J.G.
      • Cihakova D.
      • Bucek A.
      • Coronado M.J.
      • Brandt J.E.
      • Bedja D.
      • Kim J.B.
      • Georgakopoulos D.
      • Gabrielson K.L.
      • Mitzner W.
      • Fairweather D.
      IL-33 independently induces eosinophilic pericarditis and cardiac dilation: ST2 improves cardiac function.
      • Barin J.G.
      • Baldeviano G.C.
      • Talor M.V.
      • Wu L.
      • Ong S.
      • Fairweather D.
      • Bedja D.
      • Stickel N.R.
      • Fontes J.A.
      • Cardamone A.B.
      • Zheng D.
      • Gabrielson K.L.
      • Rose N.R.
      • Cihakova D.
      Fatal eosinophilic myocarditis develops in the absence of IFN-gamma and IL-17A.
      Clinically, infiltrating eosinophils are found in severe giant cell myocarditis and these patients, along with necrotizing eosinophilic myocarditis, have poor clinical prognosis.
      • Hyogo M.
      • Kamitani T.
      • Oguni A.
      • Kawasaki S.
      • Miyanaga H.
      • Takahashi T.
      • Kunishige H.
      • Andachi H.
      Acute necrotizing eosinophilic myocarditis with giant cell infiltration after remission of idiopathic thrombocytopenic purpura.
      • Litovsky S.H.
      • Burke A.P.
      • Virmani R.
      Giant cell myocarditis: an entity distinct from sarcoidosis characterized by multiphasic myocyte destruction by cytotoxic T cells and histiocytic giant cells.
      Other clinical entities in which moderate levels of eosinophils are found infiltrating the myocardium include hypersensitivity and drug reactions, parasitic infections, vasculitis and granulomatous diseases, and idiopathic hypereosinophilic syndrome.
      • Rezaizadeh H.
      • Sanchez-Ross M.
      • Kaluski E.
      • Klapholz M.
      • Haider B.
      • Gerula C.
      Acute eosinophilic myocarditis: diagnosis and treatment.
      Little is known about the cardiac eosinophil profile; however, in all cases, cardiac necrosis, thrombosis, and fibrosis are found.
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      Therefore, the increased eosinophil numbers seen in the absence of NK cells may be the result of a deficiency in apoptotic signals during cardiac inflammation.
      These data open the possibility for NK cells or their products as a biological therapy for myocarditis. NK-related therapies are an area of avid cancer research, and the same resources could be used to treat autoimmune disorders.
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      Developments in the treatment for clinical myocarditis are constricted by the opposing needs in the viral and autoimmune components of disease. Ideally, an intervention could be designed that would target both needs simultaneously. Our laboratory has now shown that NK cells are clearly protective in both viral and autoimmune-mediated driven forms of myocarditis. In essence, NK cells or NK-derived products concurrently could serve as an antiviral therapy and also as an immunosuppressant.

      Acknowledgment

      We thank Djahida Bedja for technical assistance in performing echocardiography on mice.

      Supplemental Data

      • Supplemental Figure S1

        The polyclonal antibody asialogangloside GM-1 (ASGM-1) selectively depletes natural killer (NK) cells from naïve animals. Percentage of CD3DX5+NKp46+ NK cells (A), CD3+DX5+NKp46+ NK T cells (B), and Ly6GloSiglecF+ eosinophils (C) in the liver, heart, lung, and spleen of naïve wild-type (WT) mice after 3 days of rabbit IgG (RaIgG) or ASGM-1 treatment of 1 mg of antibody per day. All statistics calculated by unpaired t-tests. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

      • Supplemental Figure S2

        Cardiac fibroblasts can be isolated from adult mice. A: Immunofluorescence images of adult mouse cardiac fibroblasts (AMCFs) isolated and passaged twice and primary bone marrow–derived macrophages (BMMs), fixed with 4% paraformaldehyde and stained with DAPI, 1:1000 anti-α smooth muscle rabbit IgG antibody and 1:200 anti-rabbit rat fluorescein isothiocyanate–conjugated antibody, and 1:500 anti-CD11b rat antibody and 1:100 anti-rat Texas Red–conjugated antibody. B and C: Levels of CD11b Itgam mRNA as measured by real-time quantitative PCR relative to hypoxanthine-guanine phosphoribosyltransferase (Hprt) in untreated cardiac fibroblasts and BMMs (B) and untreated cardiac fibroblasts and naïve BALB/c heart homogenates (C). ND, no data.

      • Supplemental Figure S3

        Activated fibroblasts are not targeted for cytotoxic killing by natural killer (NK) cells in vitro. A: Expression of NKG2D and CD69 in splenic NK cells from naïve or poly(I:C)-treated mice after 24 hours. B: Levels of vimentin mRNA in cardiac fibroblasts after 1 μmol/L angiotensin II (AngII) treatment for 1 hour, as controlled by hypoxanthine-guanine phosphoribosyltransferase (Hprt); P = 0.02). C: Representative images and percentage of live cardiac fibroblasts after 48 hours of co-culture with naïve and activated NK cells, as determined by red versus green cells. D and E: Quantification of naïve (D) and activated (E) NK cells in C. Statistics for B calculated by unpaired t-test and for D and E by one-way analysis of variance with Tukey's post testing. P < 0.05. AMCF, adult mouse cardiac fibroblast; NT, no treatment.

      • Supplemental Figure S4

        Depletion of the polyclonal antibody asialogangloside GM-1 (ASGM-1) during experimental autoimmune myocarditis does not induce peripheral eosinophilia. Percentage of Ly6GSiglecF+ eosinophils in the blood (A) and spleen (B) in isotype control and natural killer (NK) cell–depleted animals at day 14 and the spleen at day 21 (C) as a function of total CD45+ hematopoietic cells. Statistics calculated by unpaired t-test. RaIgG, rabbit IgG.

      • Supplemental Figure S5

        Noneosinophil CD45+ populations are unchanged proportionally by natural killer cell depletion during experimental autoimmune myocarditis (EAM). A: SSCmidCD11b+ monocytes as a percentage of total CD45+ cardiac cells at day 14 of EAM in isotype and asialogangloside GM-1 (ASGM-1)–treated wild-type (WT) animals. Ly6Chi inflammatory (B) and Ly6Clomid resident (C) subsets as a percentage of total SSCmidCD11b+ cardiac monocytes. CD11c+ dendritic cells (D) and FCεR1α+cKit+ mast cells (E) as a percentage of total CD45+ cardiac cells at day 14 of EAM in isotype and ASGM-1–treated WT animals. Lymphocyte percentages of B220+ B cells (F), CD3+ T cells (G), and CD3+CD4+ helper T cells (H) as a percentage of total CD45+ cardiac cells at day 21 of EAM in isotype control and ASGM-1–treated animals. Significance determined by unpaired t-test. RaIgG, rabbit IgG.

      • Supplemental Figure S6

        Depletion of natural killer cells increases type 2 helper T cell (Th2) and Th17 proportions at later stages of experimental autoimmune myocarditis (EAM). T-cell subset examination by intracellular cytokine staining for interferon γ (IFNγ) (Th1; A), Il-13 (Th2; B), and Il-17A (Th17; C), as a proportion of cardiac CD3+CD4+ cells at days 14 and 21 of EAM after 4 to 6 hours of 4β-phorbol 12-myristate 13-acetate and ionomycin stimulation with Golgistop. Significance calculated by unpaired t-test. ∗∗P < 0.01, ∗∗∗P < 0.001. RaIgG, rabbit IgG.

      • Supplemental Figure S7

        Natural killer (NK) cells alter levels of eosinophil-associated chemokines from cardiac fibroblasts in vitro. Cardiac fibroblasts cultured with Il-4 or Il-4 and magnetically sorted NK cells (1:2 ratio) for 94 hours. Levels of Cxcl10 (P < 0.001; A), chemokine ligand (Ccl2; P < 0.001; B), Ccl4 (P < 0.001; C), Ccl5 (P = 0.002; D), Cxcl1 (P < 0.001; E), and Il-5 (P < 0.001; F) protein in supernatant were measured by enzyme-linked immunosorbent assay. Significance by ordinary one-way analysis of variance with post testing by Tukey's multiple-comparisons test. ∗∗∗P < 0.001.

      • Supplemental Figure S8

        Cxcl9 and interferon γ (IFNγ) are not required by natural killer (NK) cells for control of eosinophil trafficking. A: Levels of Cxcl9 mRNA by real-time quantitative PCR (qPCR) from rabbit IgG (RaIgG) and asialogangloside GM-1 (ASGM-1)–treated hearts at day 21 of experimental autoimmune myocarditis (EAM; P = 0.004). Cxcl9 protein from supernatants of cardiac fibroblasts co-cultured with or without naïve NK cells for 96 hours (P < 0.001; B) and naïve NK co-cultures from wild-type (WT) or IFNγ receptor 1 (IFNγR1)−/− cardiac fibroblasts for 96 hours (P < 0.001; C). D: Cxcl9 mRNA by qPCR from WT and IFNγ−/− hearts at day 14 of EAM (P = 0.050). E: Percentage of Ly6GloSiglecF+ eosinophils at day 14 of EAM in rabbit IgG and ASGM-1–treated IFNγ−/− animals. Significance by Student's t-test. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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