CTLA-4 During Innate Immunity Inflammatory Heart Disease By

Cutting Edge: T Cell Ig Mucin-3 Reduces Inflammatory Heart Disease by Increasing CTLA-4 during Innate Immunity

This information is current as Sylvia Frisancho-Kiss, Jennifer F. Nyland, Sarah E. Davis, of September 26, 2021. Masheka A. Barrett, Shannon J. L. Gatewood, Dolores B. Njoku, Daniela Cihakova, Ellen K. Silbergeld, Noel R. Rose and DeLisa Fairweather J Immunol 2006; 176:6411-6415; ;

doi: 10.4049/jimmunol.176.11.6411 Downloaded from http://www.jimmunol.org/content/176/11/6411

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JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: T Cell Ig Mucin-3 Reduces Inflammatory Heart Disease by Increasing CTLA-4 during Innate Immunity1 Sylvia Frisancho-Kiss,* Jennifer F. Nyland,*† Sarah E. Davis,*† Masheka A. Barrett,* Shannon J. L. Gatewood,*† Dolores B. Njoku,*‡ Daniela Cihakova,* Ellen K. Silbergeld,† Noel R. Rose,*§ and DeLisa Fairweather2*†

Autoimmune diseases can be reduced or even prevented if velopment of acute myocarditis from days 7 to 14 postinfection proinflammatory immune responses are appropriately (p.i.) that progresses to chronic myocarditis and dilated cardio-

down-regulated. Receptors (such as CTLA-4), cytokines myopathy (4). Downloaded from (such as TGF-␤), and specialized cells (such as The family of genes encoding T cell Ig mucin (Tim) proteins ؉ ؉ CD4 CD25 T regulatory cells) work together to keep reside in syntenic chromosomal regions (5q33.2 in human; immune responses in check. T cell Ig mucin (Tim) family 11B1.1 in mouse) that are linked to susceptibility to allergy and proteins are key regulators of inflammation, providing an autoimmune diseases (5). Tim-3 is highly expressed on differ- inhibitory signal that dampens proinflammatory re- entiated Th1 cells, with lower levels on monocytes, NK, and sponses and thereby reducing autoimmune and allergic re- NKT cells (6–8). Treatment of mice with Abs that block http://www.jimmunol.org/ sponses. We show in this study that reducing Tim-3 sig- Tim-3 (anti-Tim-3) in a mouse model of multiple sclerosis leads to hyperacute disease with increased numbers of neutro- naling during the innate immune response to viral phils and macrophages and greater demyelination (5, 7, 9, 10), infection in BALB/c mice reduces CD80 costimulatory showing that Tim-3 is important in decreasing inflammation molecule expression on mast cells and macrophages and ؉ (5). Although it is believed that Tim-3 induces tolerance by reduces innate CTLA-4 levels in CD4 T cells, resulting down-regulating inflammation, the precise mechanisms are less in decreased T regulatory cell populations and increased clear. inflammatory heart disease. These results indicate that by guest on September 26, 2021 regulation of inflammation in the heart begins during in- Materials and Methods nate immunity and that Tim-3 signaling on cells of the Myocarditis and anti-Tim-3 treatment innate immune system critically influences regulation of Male BALB/cJ (BALB/c) mice (6–8 wk old) (The Jackson Laboratory) were the adaptive immune response. The Journal of Immu- inoculated i.p. with 103 PFU of a heart-passaged stock of CVB3 (Nancy strain; nology, 2006, 176: 6411–6415. American Type Culture Collection) or control PBS on day 0 and examined at 30 min or 6 h (innate response) or at day 10 or 12 p.i. for myocarditis (acute response) (11). Mice also received one i.p. injection of 100 ␮g of anti-Fc (clone ntigen presentation during innate immunity influ- 93; eBioScience) to reduce nonspecific binding and either 100 ␮g of anti-Tim- 3-blocking Ab (clone 8B.2C12; eBioScience) or control IgG (catalog no. 16- ences the development of the adaptive immune re- 4301; eBioScience) on day 0. Mice were maintained under pathogen-free con- A sponse. The costimulatory molecules CD80 (B7-1) ditions in the animal facility at Johns Hopkins School of Medicine, and and CD86 (B7-2) on APC bind both CD28 and the regulatory approval was obtained from the Animal Care and Use Committee of the Johns molecule CTLA-4. CTLA-4 down-regulates T cells by mecha- Hopkins University for all procedures. ␤ nisms that include TGF- and transcription factor forkhead FACS analysis box p3 (Foxp3)3 expression, T regulatory cell (Treg) induction, Mast cells (MC) and macrophages were separated from heart cells, splenocytes, and reverse signaling via CD80 and CD86 on APC (1, 2). or peritoneal lavage cells using anti-CD117, anti-FITC, or anti-CD45 (eBio- CTLA-4-deficient mice spontaneously develop a lymphoprolif- Science; clone 30-F11) paramagnetic beads on a magnetic column (Miltenyi erative autoimmune response, with particularly severe pancre- Biotec), as described previously (11). MC and macrophages comprise ϳ6% of atitis and myocarditis (3). Coxsackievirus B3 (CVB3) infection the cells isolated from the heart at 6 h p.i., a number similar to the level in uninfected or PBS inoculated mice. Cells were stained with the following mAbs of susceptible BALB/c mice produces inflammatory heart dis- (eBioScience) diluted in 1% FBS in PBS: CD3 (total T cells, clone 17A2), CD4 ease that is similar to the disease observed in humans, with de- (CD4 T cells, clone GK1.5), CD117 (MC, clone ACK2), F4/80 (macrophages,

*Department of Pathology, †Department of Environmental Health Sciences, ‡Department 1 This work was supported by National Institutes of Health Grants HL67290, HL70729, of Anesthesiology and Critical Care Medicine, and §W. Harry Feinstone Department of AI51835, ES03819, and T32 ES07141 (to J.F.N.). Molecular Microbiology and Immunology, Johns Hopkins University, Baltimore, MD 2 Address correspondence and reprint requests to Dr. DeLisa Fairweather, Department of 21205 Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins Uni- Received for publication September 8, 2005. Accepted for publication March 22, 2006. versity, 615 North Wolfe Street, Room E7628, Baltimore, MD 21205. E-mail address: [email protected] The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. 3 Abbreviations used in this paper: Foxp3, forkhead box p3; Treg, T regulatory cell; CVB3, Section 1734 solely to indicate this fact. coxsackievirus B3; p.i., postinfection; Tim, T cell Ig mucin; MC, mast cell.

FIGURE 1. Anti-Tim-3 increases myocarditis. A, BALB/c mice were inoculated with anti-Tim-3 (aTIM3), isotype control IgG Ab (ISO), or PBS control at day 0 of CVB3 infection. Myocarditis was assessed on day 12 as the percentage of the heart with inflammation compared with the overall size of the heart and shown as the mean Ϯ SEM. B–C, Tissues were fixed with formalin and stained with H&E. Original magnification, ϫ20. B, Histopathology of isotype control IgG-treated .p Ͻ 0.05 ,ء .mice. C, Histopathology of anti-Tim-3-treated mice. Experiments were repeated three times with five to seven mice per group

clone BM8), CD11b (macrophages and neutrophils, clone M1/70), B220 (B Fluorescence microscopy cells, clone RA3-6B2), Tim-3 (clone 8B.2C12), CD80 (B7-1, clone 16-10A1), D Immune cells were collected by flushing the peritoneal cavity with 1% BSA in CD86 (B7-2, clone GL1), I-A (MHC class II, clone AMS-32.1; BD Pharm- Downloaded from ingen), CD152 (CTLA-4, clone UC10-4B9), CD25 (clone PC61.5), Foxp3 PBS at 30 min or 6 h after CVB3 i.p. infection and compared with PBS, un- (clone FJK-165), and CD28 (clone 37.51). For intracellular staining, cells were infected controls at 0 h. MC and macrophages from the peritoneum were sep- fixed and permeabilized using a BD Cytofix/Cytoperm or anti-mouse Foxp3 arated using paramagnetic beads and labeled using FITC CD117 or F4/80 staining kit (BD Pharmingen). Cell fluorescence was measured using a FACS- (green fluorescence). Biotinylated anti-Tim-3 mAb were labeled with Alexa- Calibur flow cytometer, and data were analyzed using CellQuest software (BD 555 (red fluorescence) (Molecular Probes), and cells were visualized using a Ni- Biosciences). kon E800 Microscope. Images were recorded with a 5-MHz Black and White http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Tim-3 expression is increased on APC6haf- ter CVB3 infection. A, Peritoneal MC (FITC-CD117) or macrophages (Mac; FITC-F4/80) (green, left column) were costained with Alexa-555 (red, middle column). Merged images (yellow, right column) show colocalization of CD117 or F4/80 with Tim-3 on the surface of cells 30 min after infection. B, Tim-3 expression (solid red line) is increased on peritoneal MC 6 h after infection by FACS analysis compared with uninfected (filled line) and isotype controls (fine blue line). C, MC were isolated from the heart, and the expression level (% cells) of Tim-3, CD80, CD86, or MHC class II (IAD) was analyzed by FACS. Expression levels of PBS inoculated, uninfected controls (un) were compared with CVB3-inoculated BALB/c mice 6 h (6h) after infection. D, Degranulation of peritoneal MC stained with CD117 (green) and Tim-3 (red) 30 min after CVB3 infection showing vesicles with Tim-3 (arrow). E, Cytokine levels (pg/g of heart) were measured by ELISA from heart homogenates at 6 h after infection and compared with uninfected controls p Ͻ ,ءء ;p Ͻ 0.05 ,ء .un) and shown as the mean Ϯ SEM) p Ͻ 0.001. Data represent one of three separate ,ءءء ;0.01 experiments using five to seven mice per group. The Journal of Immunology 6413

FIGURE 3. Anti-Tim-3 reduces CD80 and CTLA-4 levels during the innate immune response. BALB/c mice were inoculated with anti-Tim-3 (aTIM3) or isotype control IgG Ab (ISO) at day 0 of CVB3 infection. A, MC and macrophages (Mac) were isolated from the heart or spleen (Spl), and the expression level (% cells) of Tim-3, CD80, CD86, or MHC class II (MHCII or IAD) was analyzed by FACS. Expression levels of ISO (fine lines and white bars) are compared with Downloaded from anti-Tim-3-treated (aTIM3, heavy lines and black bars) BALB/c mice at 6h.B, Expression levels of CTLA-4 (intracellular) or CD28 (surface) were assessed on CD4ϩ T cells from the spleen 6 h after CVB3 infection. Data represent one of three separate experi- http://www.jimmunol.org/ ments using five to seven mice per group. by guest on September 26, 2021

CCD MicroMAX Camera (Princeton Instruments) and analyzed with Meta- carditis indicates the critical role Tim-3 plays during the innate Morph Software (Universal Imaging). immune response in regulating the progression to inflamma- Cytokine measurement tory heart disease. Tim-3 is expressed on monocytes at low levels and highly ex- Cytokines were examined using Quantikine ELISA kits (R&D Systems) ac- cording to the manufacturer’s instructions, as described previously (11). pressed on Th1 cells after three to four rounds of in vitro stim- ulation (6–8). However, Tim-3 expression on APC during the Results and Discussion innate immune response to infection has not been previously Our laboratory has recently shown that myocarditis and examined. In this study, we confirmed Tim-3 expression on chronic heart failure in mice is influenced by macrophage and MC and macrophages obtained from the peritoneum 30 min ϩ ϩ neutrophil infiltration, cardiac fibrosis, and CD4 CD25 T after CVB3 infection by observing colocalization of Tim-3 with cell populations (11–13). Because Tim-3 influences macro- cell-specific Abs using fluorescence microscopy (Fig. 2A). At 6 h phage and neutrophil populations and the development of or- p.i., Tim-3 levels were increased on MC (Fig. 2, B and C) and gan-specific autoimmune diseases, we investigated whether macrophages (data not shown) isolated from the heart (Fig. 2, B Tim-3 alters CVB3-induced myocarditis. Because tolerance to and C), spleen, or peritoneum (data not shown) compared with myocarditis can be broken by administering LPS during the in- uninfected controls by FACS analysis. To our knowledge nate immune response to CVB3 infection (14, 15), we blocked Tim-3 has not been reported to be expressed on MC. The co- Tim-3 during the innate immune response. Mice received only stimulatory molecules CD80 and CD86, and MHC class II one 100-␮g dose of anti-Tim-3-blocking or isotype control IgG were also increased on MC (Fig. 2C) and macrophages (data Ab i.p. at day 0 of CVB3 infection, and myocarditis was as- not shown) isolated from the heart at 6 h p.i., indicating that sessed on day 12 p.i. Anti-Tim-3 treatment during the innate MC can serve as APC during CVB3 infection. Similar increases immune response to CVB3 significantly increased acute myo- in the expression of Tim-3, CD80, CD86, and MHC class II carditis at day 12 p.i. (Fig. 1A). Histochemical staining revealed were observed on MC and macrophages from the spleen at 6 h an increased mixed lymphocytic infiltrate in the myocardium of p.i. (data not shown). We also observed Tim-3 on the vesicles of mice treated with anti-Tim-3 compared with controls (Fig. 1, B degranulating MC following CVB3 infection by fluorescence and C). The fact that a single treatment with anti-Tim-3 during microscopy (Fig. 2D), and a global increase in cytokines in the the innate immune response significantly increased acute myo- heart at 6 h p.i. (Fig. 2E). MC are known to release an array of 6414 CUTTING EDGE: Tim-3 REDUCES INFLAMMATORY HEART DISEASE

FIGURE 4. Anti-Tim-3 increases macrophages/neutrophils and reduces Treg populations in the heart during acute myocarditis. A, BALB/c mice were inoculated with anti-Tim-3 (aTIM3) or isotype control IgG Ab (ISO) at day 0 of CVB3 infection. B, CD45ϩ immune cells were isolated from the heart during acute myocarditis, and individual cell populations were assessed by FACS analysis. C, Percentage of CD4ϩ T cells expressing Tim-3, CD25, Foxp3, CTLA-4, or coexpressing CD25 and Foxp3 (25&FOX), or Tim-3 and CTLA-4 (T&CTLA4) by FACS analysis. Data represent one of at least three separate experiments using five to seven mice per group. Downloaded from preformed and secreted mediators after activation, including heart. We found increased inflammation in the heart in anti- cytokines, that may directly influence the function of the adap- Tim-3-treated mice by FACS analysis (Fig. 4A), similar to re- tive immune response (15–17). Our results provide evidence sults obtained by histology (Fig. 1). Examination of the inflam- that MC may be important immunoregulatory cells during the matory infiltrate of anti-Tim-3-treated mice revealed increased ϩ initiation of immune responses. numbers of CD11b cells (Fig. 4B), such as macrophages and To investigate innate immune mechanisms that lead to in- neutrophils. In contrast, other immune cells in the heart were http://www.jimmunol.org/ creased myocarditis after a single anti-Tim-3 treatment (Fig. 1), decreased in anti-Tim-3-treated mice during acute myocarditis we examined the level of CD80 and CD86 on APC from the (Fig. 4B). Reducing Tim-3 expression during the innate im- ϩ heart or spleen, and CTLA-4 (intracellular) and CD28 (surface) mune response reduced the percentage of CD4 T cells ex- ϩ levels on CD4 T cells from the spleen at 6 h p.i. There is no pressing CD25, Foxp3, or CTLA-4 (classical Treg) during inflammatory infiltrate in the heart at 6 h p.i., and so T cells are acute myocarditis (Fig. 4C), as well as reducing the percentage ϩ not present in the heart to analyze by FACS. We found that of other potentially regulatory CD4 T cells such as those ex- anti-Tim-3 administered during the innate immune response pressing Tim-3 (Fig. 4C) (6, 9, 10). Thus, reducing Tim-3 lev- partially blocked Tim-3 expression and reduced CD80 levels on els during the innate immune response to CVB3 infection re- by guest on September 26, 2021 MC and macrophages isolated from the heart or spleen at 6 h sults in increased macrophages/neutrophils and reduced Treg p.i. compared with isotype controls (Fig. 3A). CD80 levels were populations in the heart during the adaptive immune response. most profoundly reduced by anti-Tim-3 treatment on APC in Overall, these findings indicate that the regulatory effect of the heart, compared with the spleen (Fig. 3A). In contrast, anti- Tim-3 begins during the innate immune response. We demon- Tim-3 had little effect on MHC class II or CD86 levels on MC strate that Tim-3 signaling on MC and macrophages reduces or macrophages (Fig. 3A), which were expressed at levels similar inflammatory heart disease by altering CD80 and CTLA-4 lev- to those induced by CVB3 infection alone (Fig. 2C). Reducing els during the innate immune response. Understanding how Tim-3 expression on APC resulted in reduced intracellular immune responses are regulated is critical for developing effec- ϩ CTLA-4 levels in CD4 T cells from the spleen, whereas CD28 tive therapies to reduce or prevent allergy and autoimmune ϩ levels were increased on CD4 T cells at 6 h p.i. (Fig. 3B). Our diseases. results suggest an interaction of CD80 with CTLA-4, and CD86 with CD28 (Fig. 3). Although there are many conflict- Acknowledgments ing reports (18), it has been suggested that the interaction of We thank L. Meszler and L. Blosser for technical assistance. CD86 with CD28 dominates during costimulation, whereas CD80 and CTLA-4 govern negative signaling (19, 20). The in- Disclosures teraction between CD80 and CTLA-4 is one of the highest af- The authors have no financial conflict of interest. finities described for cell surface molecules (21), and CD80 transgenic mice exhibit markedly impaired immune responses– References consistent with the ability of CD80 to bind to CTLA-4 (21, 1. Chen, L. 2004. Coinhibitory molecules of the B7-CD28 family in the control of T cell ϩ immunity. Nat. Rev. Immunol. 4: 336–347. 22). Blocking CD80 results in increased CD4 T cell prolifer- 2. Nelson, B. H. 2004. IL-2, regulatory T cells, and tolerance. J. 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