- Cooper L.T.
- Baughman K.L.
- Feldman A.M.
- Frustaci A.
- Jessup M.
- Kuhl U.
- Levine G.N.
- Narula J.
- Starling R.C.
- Towbin J.
- Virmani R.
Materials and Methods
Immunization with MHC614-629 and Assessment of EAM
Assessment of Fibrosis
Intracardiac and Splenic Flow Cytometry
Depletion of NK Cells with Anti-Asialo GM1 Antibody
Isolation of Primary Adult Mouse Cardiac Fibroblasts
Isolation of Primary NK Cells
Isolation of Primary Eosinophils
Enzyme-Linked Immunosorbent Assays
NK Cells Suppress Cardiac Inflammation and Severe Myocarditis in EAM
Absence of NK Cells Increases Collagen Deposition in the Heart
Activated NK Cells Accumulate in the Heart during EAM
Activated Fibroblasts Are Not Targeted for Cytotoxic Killing by NK Cells in Vitro
NK Cells Prevent Eosinophils from Accumulating in the Heart during EAM
Eosinophils in the Heart of ASGM-1–Treated Mice Have an Activated Profile
Eosinophils Are Necessary for Increased Myocarditis Severity in the Absence of NK Cells
Ccl11 Is Involved But Not Required for the Control of Eosinophilic Infiltration by NK Cells
Ifnγ and Cxcl9 Are Not Required by NK Cells to Control Eosinophilic Infiltration
NK Cells Directly Induce Apoptosis of Eosinophils
- Nielsen N.
- Pascal V.
- Fasth A.E.
- Sundstrom Y.
- Galsgaard E.D.
- Ahern D.
- Andersen M.
- Baslund B.
- Bartels E.M.
- Bliddal H.
- Feldmann M.
- Malmstrom V.
- Berg L.
- Spee P.
- Soderstrom K.
- Supplemental Figure S1
The polyclonal antibody asialogangloside GM-1 (ASGM-1) selectively depletes natural killer (NK) cells from naïve animals. Percentage of CD3−DX5+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 Ly6G−SiglecF+ 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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Supported by NIH/National Heart, Lung, and Blood Institute National Research Service Awards F31 HL112665-01A1 (S.O.), R01HL118183 (D.C.), and R01HL113008 (N.R.R.), the Michel Mirowski MD Discovery Foundation (D.C.), a W.W. Smith Charitable Trust Heart Research grant H1103 (D.C.), and the Children's Cardiomyopathy Foundation (D.C.).
Disclosures: None declared.
Current address of D.L.L., Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.
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