Role of CD1d in coxsackievirus B3-induced myocarditis

The myocarditic (H3) variant of Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice and BALB/c mice lacking the invariant J alpha 281 gene, but minimal disease in BALB/c CD1d(-/-) animals. This indicates that CD1d expression is important in this disease but does not involve the invariant NKT cell often associated with CD1d-restricted immunity. The H3 variant of the virus increases CD1d expression in vitro in neonatal cardiac myocytes whereas a nonmyocarditic (H310A1) variant does not. V gamma 4(+) T cells show increased activation in both H3-infected BALB/c and J alpha 281(-/-) mice compared with CD1d(-/-) animals. The activated BALB/c V gamma 4(+) T cells from H3-infected mice kill H3-infected BALB/c myocytes and cytotoxicity is blocked with anti-CD1d but not with anti-MHC class I (K(d)/D(d)) or class II (IA/IE) mAbs. In contrast, H3 virus-infected CD1d(-/-) myocytes are not killed. These studies demonstrate that CD1d expression is essential for pathogenicity of CVB3-induced myocarditis, that CD1d expression is increased early after infection in vivo in CD1d(+) mice infected with the myocarditic but not with the nonmyocarditic CVB3 variant, and that V gamma 4(+) T cells, which are known to promote myocarditis susceptibility, appear to recognize CD1d expressed by CVB3-infected myocytes.     

An attenuated variant of Coxsackievirus B3 preferentially induces immunoregulatory T cells in vivo.

BALB/c mice infected with the Woodruff variant of coxsackievirus group B type 3 (CVB3W) develop myocarditis mediated by autoimmune cytolytic T lymphocytes. A variant of CVB3W (designated H3-10A1) which infects the myocardium but induces minimal mortality of myocarditis compared to the parental virus was selected. Although H3-10A1 infections stimulate normal CTL responses to CVB3-infected myocytes, the autoimmune response to myocardial antigens is absent. Treatment of H3-10A1-infected mice with 50 mg of cyclophosphamide per kg of body weight, a treatment which preferentially eliminates suppressor cells, allows both the development of the autoimmune cytotoxic T-lymphocyte response and the expression of myocarditis. Similar treatment of CVB3W-infected mice had no effect on the disease. The presence of the immunoregulatory cells was confirmed by adoptive transfer of T lymphocytes from either H3-10A1 or CVB3W-infected donor mice into syngeneic CVB3W-infected recipients. Animals given H3-10A1-immune cells had minimal myocardial inflammation, while animals given CVB3W-immune lymphocytes developed enhanced cardiac disease. Elimination of the T-lymphocyte population from the donor cells prior to transfer abrogated suppression with the H3-10A1-immune population, showing that immunoregulation depended upon T lymphocytes. Both H3-10A1 and CVB3W have cross-reactive epitopes between the adenine translocator protein and the virion which are indicative of antigenic mimicry and may be the basis for the autoimmunity to cardiac antigens. These results suggest that immunoregulatory T cells may be primarily responsible for the nonpathogenicity of the H3-10A1 variant.     

Modulation of cytokine expression by CD4+ T cells during coxsackievirus B3 infections of BALB/c mice initiated by cells expressing the gamma delta + T-cell receptor.

Two variants of coxsackievirus B3 have been used to investigate the pathogenesis of myocarditis in BALB/c mice. H3 virus induces moderate myocarditis and H310A1 virus induces minimal myocarditis, although both viruses infect and replicate in the heart. Cells expressing the gamma delta+ T-cell receptor composed 5 to 13% of the lymphocytes infiltrating the hearts of H3 virus-infected mice and belonged to either the CD4- CD8+ gamma delta+- or CD4- CD8- gamma delta+-cell population. Giving 5,000 gamma delta+ cells isolated from the hearts of H3 virus-infected mice to H310A1 virus-infected recipients restored myocarditis susceptibility in the recipient animals and shifted the pattern of cytokine production in the virus-immune CD4+-cell population from being predominantly interleukin-4 producing to being predominantly gamma interferon producing in the H310A1 virus-infected mice. Apoptosis was evident in the infiltrating lymphocyte population in the myocardia of H3 virus-infected mice by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay and in splenic lymphocytes by DNA fragmentation in agarose gel electrophoresis and was confined to the CD4+ population. No apoptosis was observed in H310A1 virus-infected mice, but apoptosis was induced subsequent to gamma delta +-T-cell transfer. These results are consistent with the hypothesis that gamma delta+ T cells may help modulate cytokine responses during virus infections in vivo and that apoptosis might be involved in this modulation.     

Cytokine production by Vgamma(+)-T-cell subsets is an important factor determining CD4(+)-Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis

Two coxsackievirus B3 (CVB3) variants (H3 and H310A1) differ by a single amino acid mutation in the VP2 capsid protein. H3 induces severe myocarditis in BALB/c mice, but H310A1 is amyocarditic. Infection with H3, but not H310A1, preferentially activates Vgamma4 Vdelta4 cells, which are strongly positive for gamma interferon (IFN-gamma), whereas Vgamma1 Vdelta4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vgamma1(+) cells using monoclonal anti-Vgamma1 antibody enhanced myocarditis and CD4(+)-, IFN-gamma(+)-cell responses in both H3- and H310A1-infected mice yet decreased the CD4(+)-, IL-4(+)-cell response. Depleting Vgamma4(+) cells suppressed myocarditis and reduced CD4(+) IFN-gamma(+) cells but increased CD4(+) IL-4(+) T cells. The role of cytokine production by Vgamma1(+) and Vgamma4(+) T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-gamma expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vgamma4 and Vgamma1(+) T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-gamma, whereas these cell populations derived from Stat6ko mice expressed IFN-gamma but no IL-4. Stat4ko Vgamma1(+) cells (IL-4(+)) suppress myocarditis. Stat6ko Vgamma1(+) cells (IFN-gamma(+)) were not inhibitory. Stat6ko Vgamma4(+) cells (IFN-gamma(+)) significantly enhanced myocarditis. Stat4ko Vgamma4(+) cells (IL-4(+)) neither inhibited nor enhanced disease. These results show that distinct gammadelta-T-cell subsets control myocarditis susceptibility and bias the CD4(+)-Th-cell response. The cytokines produced by the Vgamma subpopulation have a significant influence on the CD4(+)-Th-cell phenotype.     

Vgamma4(+) T cells promote autoimmune CD8(+) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(+) Th1 cells

T cells expressing the Vgamma4 T-cell receptor (TCR) promote myocarditis in coxsackievirus B3 (CVB3)-infected BALB/c mice. CD1, a major histocompatibility complex (MHC) class I-like molecule, is required for activation of Vgamma4(+) cells. Once activated, Vgamma4(+) cells initiate myocarditis through gamma interferon (IFN-gamma)-mediated induction of CD4(+) T helper type 1 (Th1) cells in the infected animal. These CD4(+) Th1 cells are required for activation of an autoimmune CD8(+) alphabeta TCR(+) effector, which is the predominant pathogenic agent in this model of CVB3-induced myocarditis. Activated Vgamma4(+) cells can adoptively transfer myocarditis into BALB/c mice infected with a nonmyocarditic variant of CVB3 (H310A1) but cannot transfer myocarditis into either uninfected or CD1(-/-) recipients, demonstrating the need for both infection and CD1 expression for Vgamma4(+) cell function. In contrast, CD8(+) alphabeta TCR(+) cells transfer myocarditis into either infected CD1(-/-) or uninfected recipients, showing that once activated, the CD8(+) alphabeta TCR(+) effectors function independently of both virus and CD1. Vgamma4(+) cells given to mice lacking CD4(+) T cells minimally activate the CD8(+) alphabeta TCR(+) cells. These studies show that Vgamma4(+) cells determine CVB3 pathogenicity by their ability to influence both the CD4(+) and CD8(+) adaptive immune response. Vgamma4(+) cells enhance CD4(+) Th1 (IFN-gamma(+)) cell activation through IFN-gamma- and CD1-dependent mechanisms. CD4(+) Th1 cells promote activation of the autoimmune CD8(+) alphabeta TCR(+) effectors.     

Selection of an attenuated Coxsackievirus B3 variant, using a monoclonal antibody reactive to myocyte antigen.

Previously, we described a heart-reactive monoclonal antibody (MAb), 10A1, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectively inhibits infection of HeLa cells and myocytes with the myocarditic virus variant (CVB3W). A plaque-purified variant (H3) of CVB3W was isolated from the heart of an infected animal, and a second virus (H3-10A1) was obtained by growing H3 in HeLa cells in the presence of MAb 10A1. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb 10A1, but the antibody-selected H3-10A1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 10(6) PFU of CVB3W, H3, or H3-10A1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10A1, suggesting that the escape mutant is less pathogenic. Additionally, hearts from animals infected with H3-10A1 demonstrated only half the amount of myocarditis observed in either CVB3W- or H3-infected mice. Cardiac virus titers were also reduced approximately 200-fold in H3-10A1-infected animals compared with those in mice given the pathogenic variants. In vitro studies indicate that H3-10A1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.     

Differential Th1 and Th2 cell responses in male and female BALB/c mice infected with coxsackievirus group B type 3.

Male and female BALB/c mice differ dramatically in susceptibility to myocarditis subsequent to coxsackievirus B3 (CVB3) infection. CVB3 infection of male mice results in substantial inflammatory cell infiltration of the myocardium, and virus-immune lymphocytes from these animals give predominantly a Th1 cell phenotypic response, as determined by predominant immunoglobulin G2a isotypic antibody production and elevated numbers of gamma interferon and interleukin-2 (IL-2)-producing CD4+ T lymphocytes. Females infected with the same virus give predominantly a Th2 cell phenotypic response, as determined by preferential immunoglobulin G1 antibody isotypic responses and increased precursor frequencies of IL-4- and IL-5-producing CD4+ T cells. Treatment of females with testosterone or males with estradiol prior to infection alters subsequent Th subset differentiation, suggesting that the sex-associated hormones have either a direct or indirect effect on CD4+ lymphocyte responses in this model. Treatment of females with 0.1 mg of monoclonal antibody to IL-4 reduces precursor frequencies of IL-4-producing CD4+ T cells and increases frequencies of gamma interferon-producing cells. This treatment also enhances myocardial inflammation, indicating a correlation between Th1-like cell responses and pathogenicity in CVB3 infection. The Th2-like cell may regulate Th1 cell activation. Adoptive transfer of T lymphocytes from CVB3-infected female mice into male animals suppresses the development of myocarditis in the recipients. Treatment of the female donors with monoclonal antibodies to either CD3, CD4, or IL-4 molecules abrogates suppression.     

Augmentation of pathogenesis of coxsackievirus B3 infections in mice by exogenous administration of interleukin-1 and interleukin-2.

Two variants of coxsackievirus B3 (CVB3) which differ dramatically in the ability to induce myocarditis in BALB/c mice were studied. H3 virus infection of murine monocytes in vitro resulted in release of concentrations of interleukin 1 (IL-1) and alpha/beta interferon that were high compared with those of cells infected with the H310A1 virus variant. In vivo, H3 virus infection caused substantial inflammatory cell infiltration of the myocardium, and lymphocytes from these animals gave predominantly Th1-cell responses to either whole H3 virus or overlapping peptides of the CVB3 vp1 capsid protein, as determined by IL-2 production. In contrast, H310A1 virus infection produced minimal myocarditis and Th1-cell responses, but Th2-cell activation was more pronounced than in H3 virus-infected mice (as determined by IL-4 concentrations). Exogenous treatment of H310A1 virus-infected mice with either IL-1 or IL-2 restored both myocarditis susceptibility and Th1-cell responses to whole virus and vp1 peptides. Furthermore, H310A1 virus-infected mice given exogenous IL-1 showed substantial in situ IL-2 deposition in the myocardium. These results indicate that CVB3-induced myocarditis may depend upon release of specific cytokines during infection and that activation of Th1 cells may be an important factor in pathogenesis.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Cardiac injury in myocarditis induced by Coxsackievirus group B, type 3 in Balb/c mice is mediated by Lyt 2 + cytolytic lymphocytes

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (CVB3) develop severe myocarditis 7 days later. The lesions are characterized by mononuclear cell inflammation and myocyte necrosis. Infected T-lymphocyte-deficient mice show either minimal or no cardiac injury, although virus concentrations in the hearts of T-cell-deficient and -sufficient animals are similar. Adoptive transfer of 2 X 10(6) CVB3 immune Thy 1+ cells into CVB3-infected T-cell-deficient mice effectively restored myocarditis to levels observed in intact animals. Similar reconstitution with immune Ig+ cells or serum resulted in only a minimal increase in cardiac injury. To determine whether T-lymphocyte-dependent humoral or cellular immunity was responsible for myocarditis. T lymphocytes were obtained from Balb/c mice 6 days after infection with CVB3, separated into Lyt 1+2- (helper) and Lyt 1-2+ (cytolytic/suppressor) cell populations, and 2 X 10(6) of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt2+ cells developed severe myocarditis, had cytolytic T lymphocytes to both CVB3-infected and uninfected myocytes, but lacked a detectable IgG antibody response. Recipients of the Lyt 1+ cells failed to develop either myocarditis or cytolytic T cells but had normal serum IgG antibody titers to the virus. These results demonstrate that cardiac myocarditis is the product of cellular immune mechanisms.     

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.     

gamma delta+ T cells regulate major histocompatibility complex class II(IA and IE)-dependent susceptibility to coxsackievirus B3-induced autoimmune myocarditis.

Coxsackievirus B3 (CVB3) infection induces myocardial inflammation and myocyte necrosis in some, but not all, strains of mice. C57BL/6 mice, which inherently lack major histocompatibility complex (MHC) class II IE antigen, develop minimal cardiac lesions despite high levels of virus in the heart. The present experiments evaluate the relative roles of class II IA and IE expression on myocarditis susceptibility in four transgenic C57BL/6 mouse strains differing in MHC class II antigen expression. Animals lacking MHC class II IE antigen (C57BL/6 [IA+ IE-] and ABo [IA- IE-]) developed minimal cardiac lesions subsequent to infection despite high concentrations of virus in the heart. In contrast, strains expressing IE (ABo Ealpha [IA- IE+] and Bl.Tg.Ealpha [IA+ IE+]) had substantial cardiac injury. Myocarditis susceptibility correlated to a Th1 (gamma interferon-positive) cell response in the spleen, while disease resistance correlated to a preferential Th2 (interleukin-4-positive) phenotype. Vgamma/Vdelta analysis indicates that distinct subpopulations of gamma delta+ T cells are activated after CVB3 infection of C57BL/6 and Bl.Tg.Ealpha mice. Depletion of gamma delta+ T cells abrogated myocarditis susceptibility in IE+ animals and resulted in a Th1-->Th2 phenotype shift. These studies indicate that the MHC class II antigen haplotype controls myocarditis susceptibility, that this control is most likely mediated through the type of gamma delta T cells activated during CVB3 infection, and finally that different subpopulations of gamma delta+ T cells may either promote or inhibit Th1 cell responses.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis

Coxsackievirus group B type 3 (CVB3) induces myocarditis in male Balb/c mice but produces little cardiac injury in females. Males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury observed in the disease. Infected female mice lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation. Four lines of evidence demonstrate suppressor cells in females. First, females develop myocarditis when treated with low-dose cyclophosphamide under conditions known to preferentially eliminate suppressor cells but not other immune cells. Second, lymphocytes obtained from females at various times after infection prevent myocarditis when adoptively transferred into CVB3-infected males. Virus concentrations in the hearts of males receiving immune female cells and control males were equivalent. Thus protection did not result from accelerated virus elimination in recipient males. Third, CTL from CVB3 infected male mice could induce myocarditis in infected T-lymphocyte depleted but not in intact females suggesting the presence of an inhibitory T cell in the intact animals. Finally, male lymphocytes cultured on heart cell monolayers for 5 days generate significant cytolytic activity to myocyte targets. CTL generation could be inhibited by co-culture of the male cells with immune female lymphocytes. Nonimmune female cells were not inhibitory.     

Histamine H(1) receptor signaling regulates effector T cell responses and susceptibility to coxsackievirus B3-induced myocarditis

Susceptibility to autoimmune myocarditis has been associated with histamine release by mast cells during the innate immune response to coxsackievirus B3 (CVB3) infection. To investigate the contribution of histamine H(1) receptor (H(1)R) signaling to CVB3-induced myocarditis, we assessed susceptibility to the disease in C57BL/6J (B6) H(1)R(-/-) mice. No difference was observed in mortality between CVB3-infected B6 and H(1)R(-/-) mice. However, analysis of their hearts revealed a significant increase in myocarditis in H(1)R(-/-) mice that is not attributed to increased virus replication. Enhanced myocarditis susceptibility correlated with a significant expansion in pathogenic Th1 and Vγ4(+) γδ T cells in the periphery of these animals. Furthermore, an increase in regulatory T cells was observed, yet these cells were incapable of controlling myocarditis in H(1)R(-/-) mice. These data establish a critical role for histamine and H(1)R signaling in regulating T cell responses and susceptibility to CVB3-induced myocarditis in B6 mice.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

紫外灭活CVB3 病毒诱导BALB/c小鼠产生免疫保护作用的研究

目的:探讨紫外灭活型CVB3病毒诱导BALB/c小鼠产生特异性免疫应答及保护作用的评估。方法:采用紫外灭活的方法处理野生型CVB3m株,按照0.1 LD50(10~4 PFU)的剂量免疫小鼠,设置PBS免疫组作为对照,免疫后第3,5,7天收集小鼠血清,检测细胞因子含量;在第3,5,7,14天分离小鼠脾脏,流式分析T细胞亚群的分布比例;在免疫后一个月分离小鼠血清,检测中和抗体的滴度;同时间给予小鼠100LD 50野生型CVB3感染,观察小鼠的死亡率。结果:与对照组相比,在检测日期内紫外灭活型CVB3组小鼠血清中细胞因子IL-1α,TNF-α,IL-6的表达量明显增高(P0.05),IL-4的表达量没有明显差异;免疫后第14天CD3~+CD4~+T细胞的分布较对照组明显升高(P0.05);在免疫后一个月,紫外灭活型CVB3免疫组可以诱导机体产生高滴度中和抗体,同时,小鼠应对高致死量CVB3感染时有较高的存活率。结论:紫外灭活型CVB3感染能诱导机体产生特异性免疫应答,同时,产生的中和抗体可以提高小鼠应对致死剂量CVB3感染时的生存率,对机体有明显的保护作用。     

Proteasome inhibition attenuates coxsackievirus-induced myocardial damage in mice

Coxsackievirus B3 (CVB3) is one of the most prevalent pathogens of viral myocarditis, which may persist chronically and progress to dilated cardiomyopathy. We previously demonstrated a critical role of the ubiquitin-proteasome system (UPS) in the regulation of coxsackievirus replication in mouse cardiomyocytes. In the present study, we extend our interest to an in vivo animal model to examine the regulation and role of the UPS in CVB3-induced murine myocarditis. Male myocarditis-susceptible A/J mice at age 4-5 wk were randomized to four groups: sham infection + vehicle (n = 10), sham infection + proteasome inhibitor (n = 10), virus + vehicle (n = 20), and virus + proteasome inhibitor (n = 20). Proteasome inhibitor was administered subcutaneously once a day for 3 days. Mice were killed on day 9 after infection, and infected hearts were harvested for Western blot analysis, plaque assay, immunostaining, and histological examination. We showed that CVB3 infection led to an accumulation of ubiquitin conjugates at 9 days after infection. Protein levels of ubiquitin-activating enzyme E1A/E1B, ubiquitin-conjugating enzyme UBCH7, as well as deubiquitinating enzyme UCHL1 were markedly increased in CVB3-infected mice compared with sham infection. However, there was no significant alteration in proteasome activities at 9 days after infection. Immunohistochemical staining revealed that increased expression of E1A/E1B was mainly localized to virus-damaged cells. Finally, we showed that application of a proteasome inhibitor significantly reduced CVB3-induced myocardial damage. This observation reveals a novel mechanism of coxsackieviral pathogenesis, and suggests that the UPS may be an attractive therapeutic target against coxsackievirus-induced myocarditis.     

Direct gene transfer with IP-10 mutant ameliorates mouse CVB3-induced myocarditis by blunting Th1 immune responses

Background

Myocarditis is an inflammation of the myocardium that often follows the enterovirus infections, with coxsackievirus B3 (CVB3) being the most dominant etiologic agent. We and other groups previously reported that chemokine IP-10 was significantly induced in the heart tissue of CVB3-infected mice and contributed to the migration of massive inflammatory cells into the myocardium, which represents one of the most important mechanisms of viral myocarditis. To evaluate the direct effect of IP-10 on the inflammatory responses in CVB3 myocarditis, herein an IP-10 mutant deprived of chemo-attractant function was introduced into mice to antagonize the endogenous IP-10 activity, and its therapeutic effect on CVB3-induced myocarditis was evaluated.

Methodology/Principal Findings

The depletion mutant pIP-10-AT, with an additional methionine after removal of the 5 N-terminal amino acids, was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection. Compared with vector or no treatment, pIP-10-AT treatment had significantly reduced heart/body weight ratio and serum CK-MB level, increased survival rate and improved heart histopathology, suggesting an ameliorated myocarditis. This therapeutic effect was not attributable to an enhanced viral clearance, but to a blunted Th1 immune response, as evidenced by significantly decreased splenic CD4⁺/CD8⁺IFN-γ⁺ T cell percentages and reduced myocardial Th1 cytokine levels.

Conclusion/Significance

Our findings constitute the first preclinical data indicating that interfering in vivo IP-10 activity could ameliorate CVB3 induced myocarditis. This strategy may represent as a new therapeutic approach in treating viral myocarditis.     

Expression of immunoregulatory cytokines by recombinant coxsackievirus B3 variants confers protection against virus-caused myocarditis

Clinical and laboratory investigations have demonstrated the involvement of viruses and bacteria as potential causative agents in cardiovascular disease and have specifically found coxsackievirus B3 (CVB3) to be a leading cause. Experimental data indicate that cytokines are involved in controlling CVB3 replication. Therefore, recombinant CVB3 (CVB3rec) variants expressing the T-helper-1 (T(H)1)-specific gamma interferon (IFN-gamma) or the T(H)2-specific interleukin-10 (IL-10) as well as the control virus CVB3(muIL-10), which produce only biologically inactive IL-10, were established. Coding regions of murine cytokines were cloned into the 5' end of the CVB3 wild type (CVB3wt) open reading frame and were supplied with an artificial viral 3Cpro-specific Q-G cleavage site. Correct processing releases active cytokines, and the concentration of IFN-gamma and IL-10 was analyzed by enzyme-linked immunosorbent assay and bioassays. In mice, CVB3wt was detectable in pancreas and heart tissue, causing massive destruction of the exocrine pancreas as well as myocardial inflammation and heart cell lysis. Most of the CVB3wt-infected mice revealed virus-associated symptoms, and some died within 28 days postinfection. In contrast, CVB3rec variants were present only in the pancreas of infected mice, causing local inflammation with subsequent healing. Four weeks after the first infection, surviving mice were challenged with the lethal CVB3H3 variant, causing casualties in the CVB3wt- and CVB3(muIL-10)-infected groups, whereas almost none of the CVB3(IFN-gamma)- and CVB3(IL-10)-infected mice died and no pathological disorders were detectable. This study demonstrates that expression of immunoregulatory cytokines during CVB3 replication simultaneously protects mice against a lethal disease and prevents virus-caused tissue destruction.