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(Circulation Research. 2006;99:1109.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Coxsackievirus B3 Induces T Regulatory Cells, Which Inhibit Cardiomyopathy in Tumor Necrosis Factor- Transgenic Mice

Sally A. Huber, Arthur M. Feldman, Danielle Sartini

From the Department of Pathology (S.A.H., D.S.), University of Vermont, Colchester; and Department of Medicine (A.M.F.), Center for Translational Medicine, Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Dr Sally Huber, Department of Pathology, University of Vermont, 208 South Park Dr, Ste 2, Colchester, VT 05446. E-mail Sally.Huber{at}uvm.edu

	Abstract

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Innate immunity promotes both the generation of autoimmunity and immunoregulation of adaptive immunity. Transgenic mice expressing the tumor necrosis factor- (TNF-) gene under the cardiac myosin promoter (TNF1.6 mice) develop dilated cardiomyopathy. Transgenic mice show extensive cardiac inflammation, suggesting that immunopathogenic mechanisms may promote cardiomyopathy. Two coxsackievirus B3 (CVB3) variants infect and replicate in the heart. H3 variant is highly myocarditic, but H310A1 variant activates CD4⁺ T regulatory cells, which protect against viral myocarditis. T-cell depletion of TNF1.6 mice using monoclonal anti-CD3 or anti-CD4 antibody significantly reduced heart size and plasma troponin I concentrations compared with control TNF1.6 mice. Cardiomyopathy in TNF1.6 mice correlates to a CD4⁺Th1 response and autoimmune IgG2a antibodies. TNF1.6 mice infected with H310A1 virus reduced heart size and cardiac inflammation corresponding to the activation of CD4⁺CD25⁺FoxP3⁺ (T regulatory cells). Immunosuppression is dependent on IL-10 but not TGFß. Adoptive transfer of the CD4⁺CD25⁺ cells from H310A1-infected mice into uninfected TNF1.6 recipients abrogated cardiomyopathy. Exogenous administration of recombinant TNF- to H310A1-infected mice for 4 days abrogated immunosuppression. Cardiac enlargement in TNF1.6 mice is partly attributable to T-cell activation and humoral autoimmunity caused by cytokine expression. T regulatory cells induced by H310A1 virus abrogate autoimmunity caused by TNF- overexpression. H3 virus infection induces high levels of systemic TNF-, whereas H310A1 virus does not. The low TNF- response during H310A1 infections is likely responsible for the T regulatory cell response in these animals.

Key Words: myocarditis • inflammation • infection

	Introduction

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Coxsackievirus B3 (CVB3) induces myocarditis and dilated cardiomyopathy.^1,2 Cardiac injury results from both direct viral injury and host responses to infection.¹ Cytokines, including tumor necrosis factor- (TNF-), interleukin (IL)-2, IL-1ß, and IL-6, cause cardiac dysfunction.³ Increased plasma concentrations of TNF-, a proinflammatory cytokine induced by infection and ischemic injury, are found in patients with congestive heart failure and dilated cardiomyopathy.^4–6 Lymphoid cells, especially macrophage,^7,8 and cardiac myocytes⁷ produce TNF-. TNF- decreases myocardial contractile efficiency, reduces ejection fraction, and induces biventricular dilation.⁵ TNF- causes acute disruption of calcium transients by affecting L-type channel–induced calcium influx through increases in sphingosine.⁵ Sustained contractile dysfunction results from TNF- induced desensitization of myofilaments to intracellular calcium through upregulation of nitric oxide.⁵ Other effects of TNF- on cardiac function include death of myocytes, disruption of excitation/contraction coupling, and induction of reactive oxygen species.⁵ As might be expected, a transgenic mouse containing the TNF- gene under the cardiac myosin promoter develops cardiomyopathy.⁹ The cardiomyopathy results from TNF- because anti–TNF- antibody treatment partially reverses cardiac failure.¹⁰ Nonetheless, the transgenic mice also have extensive cardiac inflammation,¹¹ and this raises the question of whether immunopathogenic mechanisms also precipitate cardiomyopathy.

H3 and H310A1 CVB3 differ by a single nonconserved amino acid in the VP2 capsid protein.¹² H3 causes severe myocarditis and induces a strong TNF- cytokine response. H310A1 is nonmyocarditic and fails to elicit TNF-. H310A1 preferentially induces a CD4⁺ T regulatory cell population that is not induced by H3.¹³ The CD4⁺ T regulatory cell effectively prevents H3-induced myocarditis when adoptively transferred before H3 virus infection. This communication demonstrates that the T regulatory cell is highly effective in abrogating dilated cardiomyopathy in TNF- transgenic mice (TNF1.6),^10,11 showing that virus-induced T regulatory cells suppress already established heart disease as well as preventing the initiation of myocarditis.

	Materials and Methods

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Mice
Male TNF1.6 transgenic mice were used at 5 to 6 weeks of age. The TNF1.6 mice have cardiac-specific TNF- overexpression and are on the FVB/J background strain.⁹ FVB/J mice were purchased from The Jackson Laboratory (Bar Harbor, Me). The investigation conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). The animal facility at the University of Vermont is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and the experiments were approved by the University Internal Animal Care and Use Committee.

Virus and Virus Titrations
The H3 and H310A1 variants of CVB3 were used.¹² Adult mice were infected by IP injection with 10⁵ plaque-forming units (pfu) of virus in PBS and killed 7 days later. Hearts were perfused with PBS, weighed, and then homogenized in 0.9 mL of RPMI medium 1640. Debris was removed by centrifugation at 300g, and the supernatant was titered using the plaque forming assay.¹²

TNF- and Antibody Injections
Mice were injected IP with 0.5 mL of PBS containing either 300 ng of recombinant mouse TNF- (Pharmingen) or 100 µg of monoclonal anti-CD3 (clone 17A2; Pharmingen), anti-CD4 (clone GK1.5; American Type Culture Collection), or anti-CD8 (clone 2.43; American Type Culture Collection). Some H310A1-infected mice were injected IV through the tail vein with 0.25 mg of monoclonal anti–IL-10 (clone JES5–2A5; Pharmingen) or anti-TGFß1 (clone 1D11; R&D Systems) on days 2 and 5 after infection.

Histology
Hearts were fixed in 10% buffered formalin, paraffin embedded, sectioned, stained with hematoxylin/eosin, and evaluated for myocarditis using a 0 to 4 scale, where 0 represents no inflammation; 1 represents 1 to 10 inflammatory foci/section; 2 represents 11 to 20 inflammatory loci/section; 3 represents 21 to 40 inflammatory loci/section; and 4 represents >40 inflammatory loci/section as published previously.¹⁴

Flow Cytometry
Inflammatory cells in the heart were isolated by digesting finely minced hearts with 0.4% collagenase II (Sigma Chemical Co, St Louis MO) and 0.25% pancreatin (Sigma). Spleens were pressed through fine mesh screens. The cellular debris was allowed to settle and the supernatant was centrifuged on Histopaque (Sigma). Blood was collected by intracardiac puncture in EDTA and centrifuged on Histopaque. The intracellular cytokine staining protocol has been published.¹⁵ Cells (10⁵) were cultured for 4 hours with 10 µg/mL brefeldin A (BFA) (Sigma), 50 ng/mL phorbol myristate acetate (PMA) (Sigma), and 500 ng/mL ionomycin (Sigma); washed; incubated with a 1:100 dilution of Fc Block, APC-Cy7 rat–anti-mouse CD4 (clone GK1.5), and fluorescein isothiocyanate (FITC)-hamster anti-V4 (clone UC3), APC-Cy7 rat IgG1 (clone R3-34), and FITC-hamster IgG (clone G235-2356). The cells were washed, fixed in 2% paraformaldehyde; resuspended in PBS-BSA containing 0.5% saponin, Fc Block, and 1:100 dilutions of phycoerythrin (PE) anti–interferon- (anti-IFN) (clone XMG 1.2), PE anti-FoxP3, or PE-rat IgG1 (clone R3–34); washed; and resuspended in 2% paraformaldehyde for flow cytometry. Additional cells (10⁵ cells/well) were washed once with PBS containing 1% BSA and labeled with 1:100 dilutions of APC-Cy7 anti-CD11b (clone M1/70), PE anti-CD45R/B220 (clone RA3–6B2), and FITC-anti-CD3 (clone 17A2), FITC-anti-CD4 (clone GK1.5), and APC-Cy7 anti-CD25 (clone PC61). All reagents were purchased from Pharmingen/eBiosciences. Cells were evaluated on a Coulter Epics Elite flow cytometer with a single excitation wavelength (488 nm) and band filters for FITC (525 nm) and PE (575 nm). The excitation wavelength for APC-Cy7 was 595 nm and a band filter of 650 nm. Each cell population was classified for cell size (forward scatter) and complexity (side scatter). At least 10 000 cells were evaluated. Positive staining was determined relative to isotype controls.

IL-10, TNF-, and TGFß ELISAs
Splenocytes (1x10⁶) from individual mice were cultured for 4 hours at 37°C in RPMI medium 1640 containing 5% FBS, PMA, and ionomycin without BFA. The supernatants were assayed for IL-10 and TGFß using commercial ELISA kits according to the directions of the manufacturer (R&D Systems). Mice were bled by intracardiac puncture into EDTA-treated tubes, and hearts were perfused with 10 mL of PBS. Tissue was homogenized in 1 mL of PBS and centrifuged at 1000g to remove cellular debris. Blood was centrifuged at 1000g to retrieve plasma. One hundred microliters of the homogenate supernatant and plasma were assayed at undiluted, 1:10 and 1:100 dilutions for TNF- using an Endogen TNF- ELISA kit according to the directions of the manufacturer. Troponin I concentrations were determined on plasma from individual animals using the murine troponin I ELISA kit (Life Diagnostics Inc, West Chester, Pa) according to the directions of the manufacturer.

Autoantibody ELISA
Neonatal FVB myocytes were made as described previously¹⁶ and were plated (0.5x10⁵ cells/well) into 96-well tissue culture plates. Sequential 2-fold dilutions of plasma in PBS-BSA were incubated on the myocytes for 30 minutes at room temperature. The cells were washed, incubated with 1:100 dilutions of biotinylated rat anti-mouse IgG, rat anti-mouse IgG1, or rat anti-mouse IgG2a (Pharmingen) for 45 minutes, washed, incubated with streptavidin–horseradish peroxidase, washed, and incubated with R&D color substrate for 45 minutes. Stop solution was added to each well, and the plates were read at 450 nm. Titer was the inverse dilution giving >2-fold background (no plasma) absorbance.

Adoptive Transfer of CD4⁺CD25^– and CD4⁺CD25⁺ Cell Populations
Male FVB/J mice were either uninfected or infected with 10⁵ pfu H3 or H310A1 virus IP. The spleen CD4⁺ cell population was isolated 7 days after infection by negative selection using the BD Biosciences CD4⁺ cell enrichment kit according to directions of the manufacturer (>95% CD4⁺ cell purity). Cells were labeled with FITC-anti-CD4 and PE-Cy5.5-anti-CD25 in PBS-BSA, washed, sorted using BD FACS Aria (BD Biosciences, San Jose, Calif), washed with PBS, resuspended to 3x10⁵ (CD4⁺CD25⁺) or 1x10⁶ (CD4⁺CD25^–) cells per 0.1 mL and injected (0.1 mL) IV into the tail veins of uninfected TNF1.6 recipient animals. Recipients were killed 7 days after cell transfer.

Statistics
Data were analyzed for skewness and kurtosis using the SSPS for Windows program (Version 11.0, 2001; SPSS Inc, Chicago, Ill). Statistical analysis was done by Wilcoxon ranked score because variance was not normally distributed in many groups. Data represent 1 of at least 2 experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heart-Specific Antibody and CD4⁺Th1 Cells in TNF1.6 Mice
Hearts, plasma, and spleen of uninfected TNF1.6 transgenic mice were assayed for TNF- (Figure 1A). TNF- expression in uninfected TNF1.6 transgenic mice is restricted to the heart (P<0.001 compared with hearts of FVB mice). Next, TNF1.6 mice were uninfected or infected 7 days earlier with 10⁵ pfu H3 or H310A1 virus (Figure 1B). TNF- concentrations in plasma from H3-infected mice were significantly increased compared with uninfected or H310A1-infected animals. TNF1.6 mice develop dilated cardiomyopathy accompanied with extensive myocardial inflammation.⁹ To determine whether inflammation contributes to cardiomyopathy, TNF1.6 mice were treated with 100 µg of monoclonal anti-CD4 or anti-CD8 antibodies twice 3 days apart and then killed 6 days after the last antibody treatment (Figure 1C to 1E). Heart weight was significantly reduced in only mice treated with anti-CD4 antibody. Analysis of CD4⁺IFN⁺ (Th1) and CD4⁺IL-4⁺ (Th2) cells showed that Th1 cells predominate. Because previous studies showed IgG deposits in the myocardium of TNF1.6 mice,¹¹ plasma was evaluated for cardiac myocyte-reactive antibody and showed high titers of IgG2a anti-myocyte antibodies but little IgG1 antibody.

View larger version (18K): [in this window] [in a new window]   Figure 1. A, Heart, spleen, and plasma were isolated from uninfected FVB and TNF1.6 mice. Heart and spleen were homogenized in 1 mL of medium. Homogenate supernatant and plasma were assayed for TNF- by ELISA using the Endogen Inc kit according to the directions of the manufacturer. Results are mean±SEM of 6 or more mice per group. B, Plasma from uninfected TNF1.6 mice and mice infected 7 days earlier with 105 pfu of either H3 or H310A1 virus was evaluated for TNF-. Each dot represents an individual animal. In A, *significantly different from FVB at P<0.01 (A). In B, *indicated groups are significantly different at P<0.05. C, TNF1.6 mice were injected IP with 100 µg of rat IgG (isotype), monoclonal anti-CD4, or anti-CD8 antibody twice and killed 6 days after the last injection. Hearts were perfused and weighed. D, Peripheral blood mononuclear cells (PBMCs) were cultured with PMA, ionomycin, and BFA for 4 hours, and the cells were stained with antibodies to CD4 and intracellularly with antibodies to IFN and IL-4. E, Plasma was titered for antibody to cardiac myocytes as described in Materials and Methods. #Results are significantly different from isotype control at P<0.05. Groups consisted of 3 mice.

H310A1 Virus Infection Abrogates Cardiomyopathy
TNF1.6 transgenic mice were uninfected or infected for 7 days with 10⁵ pfu of either H3 or H310A1 CVB3 (Figure 2). H310A1 virus does not induce myocarditis¹⁷ but infects and replicates in the myocardium to levels similar to those of the pathogenic H3 virus. Some uninfected TNF1.6 mice received 2 IP injections of 100 µg of monoclonal anti-CD3 antibody 7 days before euthanasia. No significant differences were observed in body weights (Figure 2B). Uninfected TNF1.6 hearts were significantly enlarged compared with FVB animals (Figure 2A). H3 infection caused a slight, but not significant, increase in heart weight compared with uninfected TNF1.6 mice. H310A1 infection reduced heart weights to FVB levels, suggesting that this virus protects against cardiomyopathy induced by TNF-. Anti-CD3 antibody treatment also reduced heart weights. Troponin I concentrations were determined in plasma from individual mice (Figure 2C). Anti-CD3–treated uninfected TNF1.6 mice and H310A1-infected TNF1.6 mice had significantly less troponin I than uninfected TNF1.6 animals. No significant difference in troponin I levels occurred between uninfected and H3-infected TNF1.6 mice. Uninfected and H3-infected hearts had a globular morphology (Figure 2E) compared with normal morphology for H310A1 infected and anti-CD3–treated hearts. Uninfected TNF1.6 mice have a diffuse inflammatory infiltrate but minimal myocyte necrosis (Figure 2F). H3-infected TNF1.6 mice have substantial myocyte necrosis and increased inflammatory cell infiltrates. H310A1 infected and anti-CD3–treated hearts show reduced inflammation and no necrosis. Cardiac virus titers showed no significant difference between infected groups (5.6±1.0 log10 pfu/heart for H3-infected animals and 4.9±1.3 log 10 pfu/heart for H310A1-infected mice).

View larger version (78K): [in this window] [in a new window]   Figure 2. A, Hearts were obtained from 6-week-old male uninfected FVB mice and TNF1.6 mice, which were either uninfected or infected for 7 days with 105 pfu H3 or H310A1 virus, were weighed in milligrams. Uninfected TNF1.6 mice were treated on 2 consecutive days with 100 µg of monoclonal anti-CD3 and killed 7 days after the last antibody treatment. H310A1-infected mice were treated on days 2 and 5 relative to infection with 0.25 mg of anti-IL-10 or anti-TGFß. B, Animals were weighed in grams. C, Plasma murine troponin I concentrations in TNF1.6 mice. D, Splenocytes from TNF1.6 mice were stimulated with PMA and ionomycin for 4 hours in vitro, and the supernatants of the cultures were evaluated by ELISA for IL-10 and TGFß. E, Photographs of hearts from representative mice. F, Histology of hearts from representative mice. Results represent mean±SEM of 6 mice per group. *Significantly different from uninfected FVB at P<0.05. #Mice are significantly different from uninfected and H3-infected TNF1.6 mice at P<0.05.

H310A1 infection actively protected TNF1.6 mice from cardiomyopathy, raising the question of immunoregulation. Two likely mediators of immunoregulation would be IL-10 or TGFß. Splenocytes from uninfected, H3-infected, and H310A1-infected TNF1.6 mice were activated with PMA/ionomycin. The supernatants from H310A1 immune cells had increased IL-10 and TGFß concentrations (Figure 2D). H310A1-infected TNF1.6 mice were injected IV with either 250 µg of monoclonal anti–IL-10 or anti-TGFß on days 2 and 5 days then killed 7 days after infection (Figure 2A through 2C). Anti–IL-10 treatment abrogated protection as determined by heart weight and troponin I levels, whereas anti-TGFß was minimally effective. These results demonstrate that IL-10 is the major immunoregulatory factor during H310A1 infection.

Next, inflammatory cells were isolated from the heart. Total numbers of recovered cells/heart (n=6/group) are given in Figure 3A. Inflammatory cells in uninfected TNF1.6 animals consisted mostly of CD11b⁺, CD3⁺, CD4⁺, and B220⁺ (B lymphocyte) cells (Figure 3B). H3 infection greatly increased numbers of all of these cells. H310A1 infection decreased CD11b⁺, CD3⁺, and CD4⁺ populations over uninfected mice but increased in CD4⁺CD25⁺ cells. All CD4⁺CD25⁺ cells in the heart are also FoxP3⁺. As expected, anti-CD3 treatment substantially reduced T cells in the heart, although numbers of CD11b⁺ cells were equivalent to uninfected TNF1.6 mice.

View larger version (27K): [in this window] [in a new window]   Figure 3. TNF1.6 mice were either uninfected or infected 7 days earlier with 105 pfu H3 or H310A1 virus. Additional TNF1.6 mice were injected IP with 100 µg of anti-CD3 on 2 sequential days, then killed 7 days after the second injection. Individual hearts were minced and digested with collagenase to release inflammatory cells. The inflammatory cells were counted by trypan blue exclusion, and total cells recovered are given in A. The inflammatory cells were labeled with fluorochrome-conjugated antibodies to the molecules indicated in graph B, and the percentage of cells positive was determined by flow cytometry. The number of cells per heart represents the percentage of cells labeling with an antibody times the number of cells isolated per heart. Results are mean±SEM. *Significantly different from uninfected at P<0.05.

Peripheral blood mononuclear cells were isolated from H310A1-infected mice and infected mice treated with anti–IL-10 or anti-TGFß (Figure 4). All CD4⁺CD25⁺ cells were FoxP3⁺ in H310A1-infected mice. Anti–IL-10 treatment significantly reduced the number of Treg cells, whereas anti-TGFß treatment had no significant effect.

View larger version (21K): [in this window] [in a new window]   Figure 4. PBMCs were isolated from TNF1.6 mice 7 days after infection with 105 pfu H310A1 virus. Mice were either treated with 0.25 mg/mouse with anti–IL-10 or anti-TGFß on days 2 and 5 relative to infection. Cells were labeled with antibodies to CD4 and CD25 and intracellularly with antibody to FoxP3. Gating on the CD4+CD25+ cells, the graphs show FoxP3 staining. Number indicates percentage of total PBMCs, which are CD4+CD25+FoxP3+.

To confirm immunoregulation, CD4⁺CD25⁺ and CD4⁺CD25^– cell populations were isolated from the spleens of uninfected, H3-infected, and H310A1-infected TNF1.6 mice and adoptively transferred into uninfected TNF1.6 recipients (Table 1). The transferred cells were negative for virus as shown by titering homogenized cells on Hela cell monolayers in the plaque forming assay. Figure 5 shows representative purity of the transferred cell populations. Recipient TNF1.6 mice were killed 7 days after cell transfer. Animals were evaluated for heart and body weight, plasma troponin I levels, and CD4⁺CD25⁺ cells in the spleen. CD4⁺CD25⁺ cells from H310A1-infected mice inhibited cardiomyopathy (reduced heart weight; decreased plasma troponin I concentrations) in recipient mice. Neither CD4⁺CD25⁺ cells from uninfected nor H3-infected donors were inhibitory. Giving CD4⁺CD25⁺ cells from H310A1-infected mice also increased splenic CD4⁺CD25⁺ cells in recipients. Plasma troponin I levels were increased in recipients given CD4⁺CD25^– cells from H310A1-infected donors, suggesting that a cardiopathic T cell is activated during H310A1 infection but inhibited by the CD4⁺CD25⁺ cells in the same animal.

View this table: [in this window] [in a new window]   Table 1. Adoptive Transfer of CD4+CD25+ and CD4+CD25– Cell Populations Into TNF 1.6 Mice

View larger version (16K): [in this window] [in a new window]   Figure 5. Representative original unsorted CD4+ (A) and sorted CD4+CD25+ (B) and CD4+CD25– (C) cell populations isolated from donor mice for adoptive transfer into uninfected TNF1.6 recipients. Top number is percentage of CD4+CD25+, and bottom number is percentage of CD4+CD25–.

TNF- Inhibits T Regulatory Cell Response
H3 virus induces TNF- expression and does not induce T regulatory cells, and exogenous TNF- treatment of CVB3 resistant mice restores myocarditis susceptibility.¹⁸ The following study evaluated whether TNF- modulates the T regulatory cell response during viral infection. Uninfected and H310A1-infected TNF1.6 mice were treated with 300 ng of recombinant TNF- daily from days 0 to 3 relative to infection. Additional mice were infected with H3 virus. All mice were killed 7 days after infection (Table 2). TNF1.6 mice infected with H310A1 virus showed reduced cardiomyopathy compared with uninfected and H3-infected mice, but TNF- treatment resulted in increased heart weight. Cytokine treatment of uninfected TNF1.6 mice had no effect on heart weight. Cardiac virus titers were not significantly different between any of the infected groups. However, H310A1-infected mice had fewer CD4⁺IFN⁺ and increased numbers of CD4⁺CD25⁺FoxP3⁺ cells compared with H3-infected or uninfected groups. These H310A1-infected mice also had fewer activated V4⁺IFN⁺ cells, a cell population known to be important in myocarditis induction.¹⁵

View this table: [in this window] [in a new window]   Table 2. Exogenous TNF- Treatment of H310A1-Infected Mice Abrogates Protection

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The immune system is divided into innate and adaptive immunity, in which the former involves rapid and broad reactivity to a wide range of infectious agents, whereas the latter is highly specific to each inducing microbe. The innate system help control infections until the more effective and specific adaptive immune response develops and clears the microbes. However, the initial innate immune system also has a profound effect on the strength and type of adaptive immune response.^19,20 Transgenic expression of TNF-, a proinflammatory cytokine usually induced by infection, initiates the development of cardiomyopathy.⁹ Depletion of CD4⁺ T cells in TNF1.6 mice is partially protective. The presence of IgG2a autoimmune anti-myocyte antibodies, substantial IgG deposits in the myocardium, and widespread myocardial inflammation in TNF1.6 mice¹¹ is consistent with a role for humoral autoimmunity in cardiomyopathy. The ability of T regulatory cells to suppress existing dilated cardiomyopathy in the TNF- transgenic mice is also consistent with immunopathogenicity. Adoptive transfer of H310A1 immune CD4⁺CD25⁺ cells substantially reduces cardiomyopathy in TNF1.6 recipients, whereas the same populations from uninfected or H3 virus–infected mice have no inhibitory effects.

TNF- is a potent proinflammatory cytokine known to promote induction or progression of autoimmunity.²¹ The high levels of TNF- likely affect dendritic cells in the heart, resulting in self antigen processing and migration of the maturing dendritic cells to peripheral lymphoid tissues for autostimulation. High peripheral TNF- levels are clearly not necessary for autoimmunity induction because TNF1.6 mice have minimal plasma levels of this cytokine despite extremely high levels in the myocardium. The most surprising observation is that systemic TNF- is effective in abrogating T regulatory cell activity. T regulatory cells are important modulators of the adaptive immune response.²² There are three known types of T regulatory cells. T regulatory 1 (Tr1) cells produce high levels of IL-10 and IL-5 but either no or only moderate amounts of TGFß.²³ Tr1 cells express both CCR5 and T1-ST2. Because Th1 cells preferentially express CCR5 (CD195) over T1/ST2, whereas Th2 cells preferentially express T1-ST2 over CCR5, the expression of both markers on Tr1 cells implies that these cells represent a distinct subset of CD4⁺ T cells.^24,25 IL-10 is required for Tr1 induction and neutralization of IL-10 abrogates Tr1-mediated suppression.²⁵ Other regulatory populations include Th3 cells, which express high levels of TGFß,²⁴ and natural T regulatory cells, which require direct cell/cell contact rather than cytokine secretion for suppression.²⁶ The T regulatory cell induced during H310A1 infection probably is a Tr1 cell because IL-10 suppresses cardiomyopathy and Tr1 cells usually inhibit Th1 cell responses,^24,27 the dominant CD4⁺ cells in TNF1.6 mice. IL-10 produced by dendritic cells promote the differentiation of Tr1 cells,²⁵ possibly by upregulating costimulatory factors CD40, CD80, and/or CD86.^28,29 Although TNF- can promote the induction of IL-10–producing Tr1 cells,³⁰ in the CVB3 model, TNF- is highly effective in abrogating immunosuppression because exogenous administration of this cytokine substantially enhanced dilated cardiomyopathy and reduced CD4⁺CD25⁺FoxP3⁺ cells.

The reason(s) why TNF- promotes Tr1 cells in another system but abrogates Tr1 cells in H310A1 infection is probably complex. Although most studies find Tr1 cells suppress Th1 cells, some reports indicate they suppress Th2 responses.^24,27 This means either that different Tr1 cell types exist or that Tr1 cell function varies depending on environmental and immunological conditions. Tr1 cells have been induced in response to both noninfectious (ovalbumin, nickel, or allograft immunity),^31–33 and infectious (helminth, bacterial and viral)^25,34,35 stimuli. Both CD4⁺Th1 and Tr1 cells may respond to the same antigenic epitopes, indicating that antigen specificity is not unique for Tr1 cells but the conditions (high IL-10) prevalent during activation cause Tr1 rather than Th1 responses.³⁵ Coxsackievirus infection has many direct effects on cells through activation of nuclear factor B, p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase signal pathways.^36–38 TNF- might suppress Tr1 cell activation in CVB3 infections through effects on virus signaling combined with the effects of TNF- itself. Toll-like receptors (TLRs) activated during infections can modulate T regulatory responses. TLR4 activated by Bordetella pertussis initiates IL-10–dependent Tr1 responses.³⁹ CVB3 activates TLR4⁴⁰ and may induce Tr1 cells through this mechanism.

	Acknowledgments

 
Sources of Funding

The work was supported by NIH grants HL58583 and P01 AI45666.

Disclosures

None.

	Footnotes

 
Original received May 12, 2006; revision received August 30, 2006; accepted September 28, 2006.

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