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1.  Relevance of Molecular Mimicry in the Mediation of Infectious Myocarditis 
Heart disease, the leading cause of death in humans, is estimated to affect one in four American adults in some form. One predominant cause of heart failure in young adults is myocarditis, which can lead to the development of dilated cardiomyopathy, a major indication for heart transplantation. Environmental microbes, including viruses, bacteria, and fungi that are otherwise innocuous, have the potential to induce inflammatory heart disease. As the list is growing, it is critical to determine the mechanisms by which microbes can trigger heart autoimmunity and, importantly, to identify their target antigens. This is especially true as microbes showing structural similarities with the cardiac antigens can predispose to heart autoimmunity by generating cross-reactive immune responses. In this review, we discuss the relevance of molecular mimicry in the mediation of infectious myocarditis.
PMCID: PMC3943813  PMID: 24263348
Heart, infectious myocarditis; Autoimmunity; Molecular mimicry; Coxsackievirus; Microbial mimics; Mimicry epitopes
2.  The role of sex differences in autophagy in the heart during coxsackievirus B3 induced myocarditis 
Under normal conditions, autophagy maintains cardiomyocyte health and integrity through turnover of organelles. During stress, oxygen and nutrient deprivation or microbial infection, autophagy prolongs cardiomyocyte survival. Sex differences in induction of cell death may to some extent explain the disparity between the sexes in many human diseases. However, sex differences in gene expression, which regulate cell death and autophagy were so far not taken in consideration to explain the sex bias of viral myocarditis. Coxsackievirus B3 (CVB3) induced myocarditis is a sex-biased disease, with females being substantially less susceptible than males and sex hormones largely determine this bias. CVB3 was shown to induce and subvert the autophagosome for its optimal viral RNA replication. Gene expression analysis on mouse and human, healthy and CVB3 infected, cardiac samples of both sexes, suggests sex differences in autophagy related gene expression. This review discusses the aspects of sex bias in autophagy induction in cardiomyocytes.
PMCID: PMC4115281  PMID: 24323874
coxsackievirus B3 (CVB3); autophagy; sex bias; myocarditis
3.  T Helper Cell Polarization in Healthy People: Implications for Cardiovascular Disease 
Atherosclerosis is a chronic inflammatory disease characterized by T lymphocyte infiltration into the atherosclerotic plaque. Assessments of T cell subtypes have demonstrated a predominance of CD4+ T helper (Th) cells, implicated Th1 and Th17 immunity in both human and mouse atherogenesis, and provided some evidence suggesting protective roles of Th2 and T regulatory cells. Observations that certain inbred mouse strains have an inherent T helper bias suggests a genetic predisposition toward developing a particular T helper phenotype. This review summarizes our current understanding of mechanisms of antigen processing for major histocompatibility complex (MHC) molecules, describes the different T helper cell subsets and their roles in atherosclerosis, and discusses mechanisms of genetic predisposition toward Th1/Th2 bias in mice. We also present data from our laboratory demonstrating inherent Th1/Th2 phenotypes in apparently healthy human volunteers that are stable over time, and discuss the potential implications for cardiovascular disease.
PMCID: PMC3806306  PMID: 23921946
Atherosclerosis; cardiovascular disease; CD4+ lymphocyte; T helper cell; immunology; inflammation; immunoglobulin
4.  Coxsackievirus B detection in cases of myocarditis, myopericarditis, pericarditis and dilated cardiomyopathy in hospitalized patients 
Molecular Medicine Reports  2014;10(6):2811-2818.
Coxsackieviruses B (CV-B) are known as the most common viral cause of human heart infections. The aim of the present study was to assess the potential role of CV-B in the etiology of infectious heart disease in hospitalized patients. The present study is based on blood, pericardial fluid and heart biopsies from 102 patients and 100 control subjects. All of the samples were examined for the detection of specific enteroviral genome using the reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis. Immunohistochemical investigations for the detection of the enteroviral capsid protein, VP1, from the biopsies were performed. The samples were cultured on confluent KB monolayer cell line for possible virus isolation. The epidemiological data were also collected. CV-B was detected in 28 of the 102 patients. The sequence analysis demonstrated that 27 strains were identical to CV-B3 and only one strain was identical to CV-B1. Furthermore, VP1 in the heart biopsies was detected in enterovirus-positive cases, as revealed by RT-PCR. Pericarditis infection was more frequent than myocarditis (P<0.05) or myopericarditis (P=0.05). The epidemiological data demonstrate that CV-B heart infections occur mainly during autumn and winter, and young male adults are more susceptible than adolescents or adults (P<0.5). The present findings demonstrate a higher prevalence of viral heart infections, suggesting that CV-B may significantly contribute to heart infections.
PMCID: PMC4227425  PMID: 25241846
coxsackievirus B; human heart infections; molecular diagnosis; immunohistochemical investigations; epidemiology
5.  c-FLIP-Short Reduces Type I Interferon Production and Increases Viremia with Coxsackievirus B3 
PLoS ONE  2014;9(5):e96156.
Cellular FLIP (c-FLIP) is an enzymatically inactive paralogue of caspase-8 and as such can block death receptor-induced apoptosis. However, independent of death receptors, c-FLIP-Long (c-FLIPL) can heterodimerize with and activate caspase-8. This is critical for promoting the growth and survival of T lymphocytes as well as the regulation of the RIG-I helicase pathway for type I interferon production in response to viral infections. Truncated forms of FLIP also exist in mammalian cells (c-FLIPS) and certain viruses (v-FLIP), which lack the C-terminal domain that activates caspase-8. Thus, the ratio of c-FLIPL to these short forms of FLIP may greatly influence the outcome of an immune response. We examined this model in mice transgenically expressing c-FLIPS in T cells during infection with Coxsackievirus B3 (CVB3). In contrast to our earlier findings of reduced myocarditis and mortality with CVB3 infection of c-FLIPL-transgenic mice, c-FLIPS-transgenic mice were highly sensitive to CVB3 infection as manifested by increased cardiac virus titers, myocarditis score, and mortality compared to wild-type C57BL/6 mice. This observation was paralleled by a reduction in serum levels of IL-10 and IFN-α in CVB3-infected c-FLIPS mice. In vitro infection of c-FLIPS T cells with CVB3 confirmed these results. Furthermore, molecular studies revealed that following infection of cells with CVB3, c-FLIPL associates with mitochondrial antiviral signaling protein (MAVS), increases caspase-8 activity and type I IFN production, and reduces viral replication, whereas c-FLIPS promotes the opposite phenotype.
PMCID: PMC4015977  PMID: 24816846
6.  Autoimmune Myocarditis, Valvulitis, and Cardiomyopathy 
Cardiac myosin-induced autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by CD4+ T cells (Smith and Allen 1991; Li, Heuser et al. 2004). It is a paradigm of the immune-mediated cardiac damage believed to play a role in the pathogenesis of a subset of postinfectious human cardiomyopathies (Rose, Herskowitz et al. 1993). Myocarditis is induced in susceptible mice by immunization with purified cardiac myosin (Neu, Rose et al. 1987) or specific peptides derived from cardiac myosin (Donermeyer, Beisel et al. 1995; Pummerer, Luze et al. 1996) (see Basic Protocol 1), or by adoptive transfer of myosin-reactive T cells (Smith and Allen 1991) (see Alternate Protocol). Myocarditis has been induced in Lewis rats by immunization with purified rat or porcine cardiac myosin (Kodama, Matsumoto et al. 1990; Li, Heuser et al. 2004) (see Basic Protocol 2) or S2-16 peptide (Li, Heuser et al. 2004), or by adoptive transfer of T cells stimulated by specific peptides derived from cardiac myosin (Wegmann, Zhao et al. 1994). Myocarditis begins 12 to 14 days after the first immunization, and is maximal after 21 days.
Other animal models commonly used to study myocarditis development include the pathogen-induced models in which disease is initiated by viral infection. The first murine model of acute viral myocarditis causes sudden death via viral damage to cardiomyocytes (Huber, Gauntt et al. 1998; Horwitz, La Cava et al. 2000; Fong 2003; Fuse, Chan et al. 2005; Fairweather and Rose 2007; Cihakova and Rose 2008) whereas the second model is based on inoculation with heart-passaged coxsackievirus B3 (CVB3) that includes damaged heart proteins (Fairweather, Frisancho-Kiss et al. 2004; Fairweather D 2004; Fairweather and Rose 2007; Cihakova and Rose 2008)
In addition to the protocols used to induce EAM in mice and rats, support protocols are included for preparing purified cardiac myosin using mouse or rat heart tissue (see Support Protocol 1), preparing purified cardiac myosin for injection (see Support Protocol 2), and collecting and assessing hearts by histopathological means (see Support Protocol 3).
PMCID: PMC3672855  PMID: 23564686
7.  Sex differences in TLR2 and TLR4 expression and their effect on coxsackievirus-induced autoimmune myocarditis 
Coxsackievirus B3 (CVB3) infection of C57Bl/6 mice shows a sex bias with males developing more severe cardiac inflammation than females because males develop a Th1 inflammatory response, whereas females develop a Th2 response. Since their discovery, Toll-like receptors have been shown to play an important role in the development of the immune response against harmful pathogens. To assess the role of TLRs in coxsackievirus-induced myocarditis wild type and Toll-like Receptor 2 −/− male and female mice were infected and assessed for viral replication, myocarditis, helper T-cell generation, and regulatory T-cell generation. TLR2−/− mice show reduced Th1 expression compared to controls. Treatment of wild type mice with either Pam3CSK4 (TLR2) or LPS (TLR4) specific TLR agonists resulted in increased Th1 expression in male and female mice and a decrease in FoxP3+ regulatory T-cells in male mice. The suppression of Tregulatory cells by TLR signaling in males but not females correlates with the increased myocarditis susceptibility of the males.
PMCID: PMC3485413  PMID: 22750431
Coxsackievirus B3; Toll-like receptors; myocarditis
8.  Caspase-8 and FLIP regulate RIG-I/MDA5-induced innate immune host responses to picornaviruses 
Future virology  2012;7(12):1221-1236.
Picornaviruses are small, nonenveloped, positive-stranded RNA viruses, which cause a wide range of animal and human diseases, based on their distinct tissue and cell type tropisms. Myocarditis, poliomyelitis, hepatitis and the common cold are the most significant human illnesses caused by picornaviruses. The host response to picornaviruses is complex, and the damage to tissues occurs not only from direct viral replication within infected cells. Picornaviruses exhibit an exceptional ability to evade the early innate immune response, resulting in chronic infection and autoimmunity. This review discusses the detailed aspects of the early innate host response to picornaviruses infection mediated by RIG-I-like helicases, their adaptor, mitochondrial ant iviral signaling protein, innate immune-induced apoptosis, and the role of caspase-8 and its regulatory paralog, FLIP, in these processes.
PMCID: PMC3595017  PMID: 23503762
apoptosis; caspase-8; FLIP; innate immunity; MAVS; MDA5; mitochondria; picornavirus; RIG-I
9.  Decreased Naive and Increased Memory CD4+ T Cells Are Associated with Subclinical Atherosclerosis: The Multi-Ethnic Study of Atherosclerosis 
PLoS ONE  2013;8(8):e71498.
Adaptive immunity has been implicated in atherosclerosis in animal models and small clinical studies. Whether chronic immune activation is associated with atherosclerosis in otherwise healthy individuals remains underexplored. We hypothesized that activation of adaptive immune responses, as reflected by higher proportions of circulating CD4+ memory cells and lower proportions of naive cells, would be associated with subclinical atherosclerosis.
Methods and Findings
We examined cross-sectional relationships of circulating CD4+ naive and memory T cells with biomarkers of inflammation, serologies, and subclinical atherosclerosis in 912 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Circulating CD4+ naive cells were higher in women than men and decreased with age (all p-values <0.0001). European-Americans had higher levels of naive cells and lower levels of memory cells compared with African-Americans and Hispanic-Americans (all p-values ≤0.0005). Lower naive/higher memory cells were associated with interleukin-6 levels. In multivariate models, cytomegalovirus (CMV) and H. Pylori titers were strongly associated with higher memory and lower naive cells (all p-values <0.05). Higher memory cells were associated with coronary artery calcification (CAC) level in the overall population [β-Coefficient (95% confidence interval (CI))  = 0.20 (0.03, 0.37)]. Memory and naive (inversely) cells were associated with common carotid artery intimal media thickness (CC IMT) in European-Americans [memory: β =  0.02 (0.006, 0.04); naive: β = −0.02 (−0.004, −0.03)].
These results demonstrate that the degree of chronic adaptive immune activation is associated with both CAC and CC IMT in otherwise healthy individuals, consistent with the known role of CD4+ T cells, and with innate immunity (inflammation), in atherosclerosis. These data are also consistent with the hypothesis that immunosenescence accelerates chronic diseases by putting a greater burden on the innate immune system, and suggest the importance of prospective studies and research into strategies to modulate adaptive immune activation in chronic disease states such as atherosclerosis.
PMCID: PMC3751895  PMID: 24009662
10.  IL-21R Expression on CD8+T Cells Promotes CD8+ T cell Activation in Coxsackievirus B3 Induced Myocarditis 
IL-21 is a multi-functional cytokine which can promote survival, proliferation and activation of T and B lymphocytes including CD8 T cells. Previous studies have shown that autoimmune CD8+ T cells are the primary pathogenic effector cell in coxsackievirus B3 (CVB3) induced myocarditis in C57Bl/6 mice. To evaluate the role of IL-21 in promoting CD8+ T cell mediated cardiac injury in myocarditis, C57Bl/6 and IL-21RKO mice were infected with CVB3. IL-21RKO mice developed significantly less myocarditis than C57Bl/6 animals although cardiac virus titers were equivalent between the mouse strains. Numbers of CD8+IFNγ+ cells were decreased in IL-21RKO mice but numbers of either CD4+IFNγ+ or CD4+IL-4+ cells were not significantly different from C57Bl/6 animals indicating a selective effect of IL-21 signaling on the CD8+ T cell response. To confirm that IL-21 signaling exclusively functions at the level of the CD8+ T cell in CVB3 induced myocarditis, purified CD8+ cells were isolated from either C57Bl/6 or IL-21RKO donors and adoptively transferred into CD8KO recipients prior to CVB3 infection. CD8KO recipients given either C57Bl/6 or IL-21RKO CD8+ cells showed equivalent reconstitution of the CD8+ cells in the spleen but the recipients given C57Bl/6 CD8+ cells showed significantly greater myocarditis than recipients of IL-21RKO CD8+ cells. These data demonstrate that IL-21 signaling directly in the CD8+ cell population is required for CVB3-induced myocarditis.
PMCID: PMC3354021  PMID: 22465422
coxsackievirus B3; myocarditis; IL-21R; CD8 T cells; autoimmunity
11.  T‐Helper Type 1 Bias in Healthy People Is Associated With Cytomegalovirus Serology and Atherosclerosis: The Multi‐Ethnic Study of Atherosclerosis 
Although T‐helper type 1 (Th1) cells are considered important in atherosclerosis, the relationships between Th1 and Th2 cells and atherosclerosis have not been examined in population‐based studies.
Methods and Results
We measured Th cells as a percentage of lymphocytes by flow cytometry using CD4 staining (%CD4) in 917 participants of the Multi‐Ethnic Study of Atherosclerosis. We also measured interferon gamma–positive and interleukin‐4‐positive CD4+ cells, representing Th1 and Th2 subpopulations (%Th1 and %Th2), respectively. We found that %CD4 was 1.5% lower per 10 years of age (P<0.0001). Whites had higher %CD4 and lower %Th1 and %Th2 values than other race/ethnic groups. Body mass index (BMI) and blood pressure were associated with %CD4, but no traditional cardiovascular disease (CVD) risk factors were associated with %Th1 or %Th2. In multivariable models, the major independent variable associated with %Th1 was cytomegalovirus (CMV) antibody titer, with minor contributions from age, sex, seasonality, and interleukin‐6. In models with coronary artery calcification level as the outcome, significant independent variables included age, sex, smoking status, and %Th1 (β=0.25; P≤0.01). Both %Th1 and %Th2 were associated with common carotid intimal media thickness (β=0.02 and −0.02, respectively; both P<0.05), as were age, sex, race/ethnicity, blood pressure, and BMI.
Th1 bias is associated with subclinical atherosclerosis in a multiethnic population. The main Th1 correlate was CMV infectious burden. These findings are consistent with a role of Th1 cells in atherosclerosis and suggest the importance of prospective studies of T‐helper cell biasing in CVD.
PMCID: PMC3698770  PMID: 23688675
atherosclerosis; epidemiology; immunology; inflammation; T‐helper cell
12.  Histamine H1 receptor signaling regulates effector T cell responses and susceptibility to coxsackievirus B3-induced myocarditis 
Cellular immunology  2011;272(2):269-274.
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 H1 receptor (H1R) signaling to CVB3-induced myocarditis, we assessed susceptibility to the disease in C57BL/6J (B6) H1R−/− mice. No difference was observed in mortality between CVB3-infected B6 and H1R−/− mice. However, analysis of their hearts revealed a significant increase in myocarditis in H1R−/− 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 H1R−/− mice. These data establish a critical role for histamine and H1R signaling in regulating T cell responses and susceptibility to CVB3-induced myocarditis in B6 mice.
PMCID: PMC3244571  PMID: 22078271
CVB3; autoimmune; myocarditis; histamine receptor; Th1; γδ T cells; Tregs; mice
13.  Sex-specific signaling through Toll-Like Receptors 2 and 4 contributes to survival outcome of Coxsackievirus B3 infection in C57Bl/6 mice 
Coxsackievirus B3 (CVB3) induces myocarditis, an inflammatory heart disease, which affects men more than women. Toll-like receptor (TLR) signaling has been shown to determine the severity of CVB3-induced myocarditis. No direct role for signaling through TLR2 had been shown in myocarditis although published studies show that cardiac myosin is an endogenous TLR2 ligand and stimulates pro-inflammatory cytokine expression by dendritic cells in vitro. The goal of this study is to determine which TLRs show differential expression in CVB3 infected mice corresponding to male susceptibility and female resistance in this disease.
Male and female C57Bl/6 mice were infected with 102 PFU CVB3 and killed on day 3 or 6 post infection. Hearts were evaluated for virus titer, myocardial inflammation, and TLR mRNA expression by PCR array and microarray analysis. Splenic lymphocytes only were evaluated by flow cytometry for the number of TLR+/CD3+, TLR+/CD4+, TLR+F4/80+ and TLR+/CD11c+ subpopulations and the mean fluorescence intensity to assess upregulation of TLR expression on these cells. Mice were additionally treated with PAM3CSK4 (TLR2 agonist) or ultrapure LPS (TLR4 agonist) on the same day as CVB3 infection or 3 days post infection to confirm their role in myocarditis susceptibility.
Despite equivalent viral titers, male C57Bl/6 mice develop more severe myocarditis than females by day 6 after infection. Microarray analysis shows a differential expression of TLR2 at day 3 with female mice having higher levels of TLR2 gene expression compared to males. Disease severity correlates to greater TLR4 protein expression on splenic lymphocytes in male mice 3 days after infection while resistance in females correlates to preferential TLR2 expression, especially in spleen lymphocytes. Treating male mice with PAM reduced mortality from 55% in control CVB3 infected animals to 10%. Treating female mice with LPS increased mortality from 0% in control infected animals to 60%.
CVB3 infection causes an up-regulation of TLR2 in female and of TLR4 in male mice and this differential expression between the sexes contributes to disease resistance of females and susceptibility of males. While previous reports demonstrated a pathogenic role for TLR4 this is the first report that TLR2 is preferentially up-regulated in CVB3 infected female mice or that signaling through this TLR directly causes myocarditis resistance.
PMCID: PMC3586360  PMID: 23241283
14.  Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem 
BMC Infectious Diseases  2012;12:212.
Viral myocarditis is a major cause of sudden unexpected death in children and young adults. Until recently, coxsackievirus B3 (CVB3) has been the most commonly implicated virus in myocarditis. At present, no standard diagnosis is generally accepted due to the insensitivity of traditional diagnostic tests. This has prompted health professionals to seek new diagnostic approaches, which resulted in the emergence of new molecular pathological tests and a more detailed immunohistochemical and histopathological analysis. When supplemented with immunohistochemistry and molecular pathology, conventional histopathology may provide important clues regarding myocarditis underlying etiology.
This study is based on post-mortem samples from sudden unexpected death victims and controls who were investigated prospectively. Immunohistochemical investigations for the detection of the enteroviral capsid protein VP1 and the characterization and quantification of myocardial inflammatory reactions as well as molecular pathological methods for enteroviral genome detection were performed.
Overall, 48 sudden unexpected death victims were enrolled. As for controls, 37 cases of unnatural traffic accident victims were studied. Enterovirus was detected in 6 sudden unexpected death cases (12.5 %). The control samples were completely enterovirus negative. Furthermore, the enteroviral capsid protein VP1 in the myocardium was detected in enterovirus-positive cases revealed by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike control samples, immunohistochemical investigations showed a significant presence of T and B lymphocytes in sudden unexpected death victims.
Our findings demonstrate clearly a higher prevalence of viral myocarditis in cases of sudden unexpected death compared to control subjects, suggesting that coxsackie B enterovirus may contribute to myocarditis pathogenesis significantly.
PMCID: PMC3462138  PMID: 22966951
15.  Chromosome Y Regulates Survival Following Murine Coxsackievirus B3 Infection 
G3: Genes|Genomes|Genetics  2012;2(1):115-121.
Coxsackievirus B3 (CVB3) contributes to the development of myocarditis, an inflammatory heart disease that predominates in males, and infection is a cause of unexpected death in young individuals. Although gonadal hormones contribute significantly to sex differences, sex chromosomes may also influence disease. Increasing evidence indicates that Chromosome Y (ChrY) genetic variants can impact biological functions unrelated to sexual differentiation. Using C57BL/6J (B6)-ChrY consomic mice, we show that genetic variation in ChrY has a direct effect on the survival of CVB3-infected animals. This effect is not due to potential Sry-mediated differences in prenatal testosterone exposure or to differences in adult testosterone levels. Furthermore, we show that ChrY polymorphism influences the percentage of natural killer T cells in B6-ChrY consomic strains but does not underlie CVB3-induced mortality. These data underscore the importance of investigating not only the hormonal regulation but also ChrY genetic regulation of cardiovascular disease and other male-dominant, sexually dimorphic diseases and phenotypes.
PMCID: PMC3276194  PMID: 22384388
heart disease; sexual dimorphism; heterochromatin
16.  Sex chromosome complement contributes to sex differences in coxsackievirus B3 but not influenza A virus pathogenesis 
Both coxsackievirus B3 (CVB3) and influenza A virus (IAV; H1N1) produce sexually dimorphic infections in C57BL/6 mice. Gonadal steroids can modulate sex differences in response to both viruses. Here, the effect of sex chromosomal complement in response to viral infection was evaluated using four core genotypes (FCG) mice, where the Sry gene is deleted from the Y chromosome, and in some mice is inserted into an autosomal chromosome. This results in four genotypes: XX or XY gonadal females (XXF and XYF), and XX or XY gonadal males (XXM and XYM). The FCG model permits evaluation of the impact of the sex chromosome complement independent of the gonadal phenotype.
Wild-type (WT) male and female C57BL/6 mice were assigned to remain intact or be gonadectomized (Gdx) and all FCG mice on a C57BL/6 background were Gdx. Mice were infected with either CVB3 or mouse-adapted IAV, A/Puerto Rico/8/1934 (PR8), and monitored for changes in immunity, virus titers, morbidity, or mortality.
In CVB3 infection, mortality was increased in WT males compared to females and males developed more severe cardiac inflammation. Gonadectomy suppressed male, but increased female, susceptibility to CVB3. Infection with IAV resulted in greater morbidity and mortality in WT females compared with males and this sex difference was significantly reduced by gonadectomy of male and female mice. In Gdx FCG mice infected with CVB3, XY mice were less susceptible than XX mice. Protection correlated with increased CD4+ forkhead box P3 (FoxP3)+ T regulatory (Treg) cell activation in these animals. Neither CD4+ interferon (IFN)γ (T helper 1 (Th1)) nor CD4+ interleukin (IL)-4+ (Th2) responses differed among the FCG mice during CVB3 infection. Infection of Gdx FCG mice revealed no effect of sex chromosome complement on morbidity or mortality following IAV infection.
These studies indicate that sex chromosome complement can influence pathogenicity of some, but not all, viruses.
PMCID: PMC3162877  PMID: 21806829
17.  Tumor Necrosis Factor-α Promotes Myocarditis in Female Mice Infected with Coxsackievirus B3 Through Upregulation of CD1d on Hematopoietic Cells 
Viral Immunology  2010;23(1):79-86.
Coxsackievirus B3 (CVB3) induces cardiac inflammation (myocarditis) in male but not female C57BL/6 mice. Protection of females correlates with reduced expression of TNF-α and IL-1β at both the mRNA and protein levels in the heart. Treatment of females with 300 ng/mouse of recombinant TNF-α on days +1 and +3 relative to infection restores myocarditis susceptibility to levels approximating those of infected male mice, showing that TNF-α deficiency is central to disease resistance. Female mice express little CD1d on spleen lymphocytes or cardiac myocytes, while females treated with TNF-α show increased CD1d expression in both cell populations. TNF-α treatment of male or female CD1d knockout (CD1dKO) mice failed to restore myocarditis susceptibility, demonstrating that of the multiple potential TNF-α activities, its ability to upregulate this non-classical major histocompatibility complex antigen is its dominant function in myocarditis susceptibility. Bone marrow chimeric mice were produced between female C57BL/6 and C57BL/6 CD1dKO mice so that either hematopoietic or non-hematopoietic cells were CD1d deficient. TNF-α treatment of chimeric mice having wild-type (CD1d+) hematopoietic cells restored myocarditis susceptibility, while TNF-α treatment of chimeric mice having CD1dKO hematopoietic cells, but CD1d+ myocytes, failed to develop myocarditis, showing that CD1d expression in lymphoid cells controls disease susceptibility.
PMCID: PMC2883509  PMID: 20121405
18.  Cross-talk between cd1d-restricted nkt cells and γδ cells in t regulatory cell response 
Virology Journal  2011;8:32.
CD1d is a non-classical major histocompatibility class 1-like molecule which primarily presents either microbial or endogenous glycolipid antigens to T cells involved in innate immunity. Natural killer T (NKT) cells and a subpopulation of γδ T cells expressing the Vγ4 T cell receptor (TCR) recognize CD1d. NKT and Vγ4 T cells function in the innate immune response via rapid activation subsequent to infection and secrete large quantities of cytokines that both help control infection and modulate the developing adaptive immune response. T regulatory cells represent one cell population impacted by both NKT and Vγ4 T cells. This review discusses the evidence that NKT cells promote T regulatory cell activation both through direct interaction of NKT cell and dendritic cells and through NKT cell secretion of large amounts of TGFβ, IL-10 and IL-2. Recent studies have shown that CD1d-restricted Vγ4 T cells, in contrast to NKT cells, selectively kill T regulatory cells through a caspase-dependent mechanism. Vγ4 T cell elimination of the T regulatory cell population allows activation of autoimmune CD8+ effector cells leading to severe cardiac injury in a coxsackievirus B3 (CVB3) myocarditis model in mice. CD1d-restricted immunity can therefore lead to either immunosuppression or autoimmunity depending upon the type of innate effector dominating during the infection.
PMCID: PMC3033358  PMID: 21255407
19.  Inhibition of arterial lesion progression in CD16-deficient mice: evidence for altered immunity and the role of IL-10 
Cardiovascular Research  2009;85(1):224-231.
Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR−/−) and FcgRIII−/−×LDLR−/− double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet.
Methods and results
Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR−/− controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR−/− controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-γ, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII −/−×LDLR−/− double knockouts relative to LDLR−/− controls, while total IgG levels were similar.
These results reveal altered immunity in FcgRIII−/−×LDLR−/− mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.
PMCID: PMC2791057  PMID: 19720605
Fc receptors; CD16; Murine atherosclerosis; T-cells; Oxidized LDL; Lesions; IL-10; Interferon; Lymphoid follicle
20.  Local Production of Interferon Gamma by Invariant Natural Killer T cells Modulates Acute Lyme Carditis 
The Lyme disease spirochete, Borrelia burgdorferi, is the only known human pathogen that directly activates invariant natural killer T (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. We now report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57Bl/6 mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFNγ, which we demonstrate ameliorates the severity of murine Lyme carditis by at least two mechanisms. First, IFNγ enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Secondly, IFNγ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate the severity of murine Lyme carditis through the action of IFNγ, which appears to self-renew through a positive feedback loop during infection.
PMCID: PMC2679988  PMID: 19265151
Invariant NKT cells; Lyme carditis; Borrelia burgdorferi; Interferon gamma
21.  Inhaled Asbestos Exacerbates Atherosclerosis in Apolipoprotein E–Deficient Mice via CD4+ T Cells 
Environmental Health Perspectives  2008;116(9):1218-1225.
Associations between air pollution and morbidity/mortality from cardiovascular disease are recognized in epidemiologic and clinical studies, but the mechanisms by which inhaled fibers or particles mediate the exacerbation of atherosclerosis are unclear.
Objective and methods
To determine whether lung inflammation after inhalation of a well-characterized pathogenic particulate, chrysotile asbestos, is directly linked to exacerbation of atherosclerosis and the mechanisms involved, we exposed apolipoprotein E–deficient (ApoE−/−) mice and ApoE−/− mice crossed with CD4−/− mice to ambient air, NIEHS (National Institute of Environmental Health Sciences) reference sample of chrysotile asbestos, or fine titanium dioxide (TiO2), a nonpathogenic control particle, for 3, 9, or 30 days.
ApoE−/− mice exposed to inhaled asbestos fibers had approximately 3-fold larger atherosclerotic lesions than did TiO2-exposed ApoE−/− mice or asbestos-exposed ApoE−/−/CD4−/− double-knockout (DKO) mice. Lung inflammation and the magnitude of lung fibrosis assessed histologically were similar in asbestos-exposed ApoE−/− and DKO mice. Monocyte chemoattractant protein-1 (MCP-1) levels were increased in bronchoalveolar lavage fluid and plasma, and plasma concentrations correlated with lesion size (p < 0.04) in asbestos-exposed ApoE−/− mice. At 9 days, activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), transcription factors linked to inflammation and found in the promoter region of the MCP-1 gene, were increased in aortas of asbestos-exposed ApoE−/− but not DKO mice.
Our findings show that the degree of lung inflammation and fibrosis does not correlate directly with cardiovascular effects of inhaled asbestos fibers and support a critical role of CD4+ T cells in linking fiber-induced pulmonary signaling to consequent activation of AP-1– and NF-κB–regulated genes in atherogenesis.
PMCID: PMC2535625  PMID: 18795166
AP-1; atherosclerosis; CD4+ T-cells; chrysotile asbestos; fibrosis; inflammation; knockout mice; lung; MCP-1; NF-κB
22.  γδ T Cells Promote a Th1 Response during Coxsackievirus B3 Infection In Vivo: Role of Fas and Fas Ligand 
Journal of Virology  2002;76(13):6487-6494.
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-γ+]) response to the virus, and a predominance of γδ 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 γδ 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 γδ 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 γδ Τ cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.
PMCID: PMC136276  PMID: 12050361
23.  Cytokine Production by Vγ+-T-Cell Subsets Is an Important Factor Determining CD4+-Th-Cell Phenotype and Susceptibility of BALB/c Mice to Coxsackievirus B3-Induced Myocarditis 
Journal of Virology  2001;75(13):5860-5869.
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 Vγ4 Vδ4 cells, which are strongly positive for gamma interferon (IFN-γ), whereas Vγ1 Vδ4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vγ1+ cells using monoclonal anti-Vγ1 antibody enhanced myocarditis and CD4+-, IFN-γ+-cell responses in both H3- and H310A1-infected mice yet decreased the CD4+-, IL-4+-cell response. Depleting Vγ4+ cells suppressed myocarditis and reduced CD4+ IFN-γ+ cells but increased CD4+ IL-4+ T cells. The role of cytokine production by Vγ1+ and Vγ4+ T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-γ expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vγ4 and Vγ1+ T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-γ, whereas these cell populations derived from Stat6ko mice expressed IFN-γ but no IL-4. Stat4ko Vγ1+ cells (IL-4+) suppress myocarditis. Stat6ko Vγ1+ cells (IFN-γ+) were not inhibitory. Stat6ko Vγ4+ cells (IFN-γ+) significantly enhanced myocarditis. Stat4ko Vγ4+ cells (IL-4+) neither inhibited nor enhanced disease. These results show that distinct γδ-T-cell subsets control myocarditis susceptibility and bias the CD4+-Th-cell response. The cytokines produced by the Vγ subpopulation have a significant influence on the CD4+-Th-cell phenotype.
PMCID: PMC114301  PMID: 11390587

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