Differential Macrophage Polarization in Male and Female BALB/c Mice Infected With Coxsackievirus B3 Defines Susceptibility to Viral Myocarditis
Rational: Myocardial infiltrating macrophages play an important role in the pathogenesis of viral myocarditis in male BALB/c mice following coxsackievirus B3 (CVB3) infection. Interestingly, comparable macrophage numbers were observed in the myocardium of female mice during acute myocarditis.
Objective: Given CVB3 infection causes severe myocarditis in male but not female mice, we postulated that macrophages infiltrating the myocardium of female mice may display distinct functional properties that contribute to differential susceptibility to CVB3 myocarditis.
Methods and Results: Here, we found that myocardial infiltrating macrophages from CVB3-infected male mice expressed high levels of classically activated macrophages (M1) markers, including inducible nitric oxide synthase, interleukin-12, tumor necrosis factor-α, and CD16/32, whereas those of females showed enhanced expression of arginase 1, interleukin-10, macrophage mannose receptor (MMR) and macrophage galactose type C-type lectin (MGL) that were associated with alternatively activated macrophage (M2) phenotype. Moreover, distinct myocardial-derived cytokines were found to play a critical role in differential macrophage polarization between sexes after CVB3 infection. Adoptive transfer of ex vivo programmed M1 macrophages, as expectedly, significantly increased myocarditis in both male and female mice. Strikingly, transfer of M2 macrophages into susceptible male mice remarkably alleviated myocardial inflammation by modulating local cytokine profile and promoting peripheral regulatory T cell (Treg) differentiation.
Conclusions: Taken together, this study may facilitate the understanding of the mechanism underlying gender bias in susceptibility to CVB3 myocarditis and the development of therapeutic strategies based on macrophage polarization for inflammatory heart disease.
Viral myocarditis is a principal cause of heart failure in young adults and often progresses to chronic myocarditis, dilated cardiomyopathy, and congestive heart failure.1 Coxsackievirus (CVB3) is believed to be the most common causative agent in human myocarditis,2 and the same virus strain induced similar inflammatory heart disease in genetically susceptible strains of mice. Previously, using the male BALB/c model of viral myocarditis, which develops acute myocarditis from days 7 to 14 postinfection,3 we reported that myocardial MCP-1 expression after CVB3 infection led to migration of mononuclear cells into the myocardium.4 Although CVB3-induced myocarditis in the males had been considered to be T lymphocyte–mediated inflammatory heart disease,2,5 accumulating data demonstrated that F4/80+ macrophages represented the major inflammatory infiltrates and played a pathogenic role in the development of acute viral myocarditis, because the severity of myocardial inflammation correlated with the intensity of the macrophage infiltration6 and macrophage-depleted mice failed to develop viral myocarditis.7 However, substantial macrophage infiltration is not always indicative of severe myocarditis. Huber et al5 and Frisancho-Kiss et al3 observed an obvious macrophage infiltrate after CVB3 infection in female BALB/c mice which are less susceptible to viral myocarditis. This highlights the heterogeneity of macrophages.
Macrophages are highly versatile, as local environment factors shape their phenotypic and functional properties.8 Operationally, 2 distinct activation states have been defined for macrophages: the M1 (or classically activated) and M2 (or alternatively activated) macrophages, each being associated with particular phenotypes and functions.8–10 M1 macrophages, induced by lipopolysaccharide (LPS) and interferon (IFN)-γ, typically produce copious amounts of proinflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-12) and generate reactive oxygen species. M1 macrophages participate as inducers and effectors in T helper-1 (Th1) immunity. M2 macrophages, polarized by Th2 cytokines (IL-4, IL-13), secrete high levels of antiinflammatory cytokines (IL-10) and are characterized by increased arginase 1 (Arg-1) activity and surface expression of macrophage mannose receptor (MMR, CD206) and macrophage galactose type C-type lectin (MGL, CD301). Functionally, M2 macrophages are considered to tune inflammatory responses and promote tissue repair.11,12
The M1/M2 dichotomy provides a useful tool for studying macrophage heterogeneity.13,14 In CVB3-infected male BALB/c mice, because infiltration of mononuclear cells into the myocardium is correlated with MCP-1 expression4 which preferentially directs recruitment of proinflammatory macrophages to sites of tissue damage,15 it could be speculated that macrophages infiltrating the myocardium of males in the acute phase of viral myocarditis develop a classically activated phenotype. This is supported by previous reports that macrophages express high levels of inducible nitric oxide synthase,16 IL-12, IL-1β, and TNF-α,17 which promote the development of viral myocarditis. However, the phenotype and function of local macrophages in female BALB/c mice have been scarcely documented.
Given macrophages display remarkable plasticity, the present study was undertaken to characterize the phenotype of myocardial infiltrating macrophages derived from male and female BALB/c mice after CVB3 infection and define their role in the development of viral myocarditis.
Male and female BALB/c (H-2d) mice, 6 weeks of age, were purchased from Shanghai Experimental Animal Centre of Chinese Academy of Sciences. Green fluorescent protein (GFP) transgenic mice of BALB/c background were obtained from The Jackson Laboratory. All animal experiments were performed according to the guidelines for the Care and Use of Laboratory Animals (Ministry of Health, PR China, 1998) and the guidelines of the Laboratory Animal Ethical Commission of Fudan University.
Virus Infection and Virus Titration
The original stock of CVB3 (Nancy strain) was maintained by passage through Hela cells (ATCC number: CCL-2). Mice were inoculated IP with 103 50% tissue culture infectious dose (TCID50) of the virus, and heart from individual mice was removed aseptically, weighed, and homogenized in DMEM containing 2% FBS. Cellular debris was removed by centrifugation, and viral titers were determined on HeLa cell monolayers using TCID50 assay as previously described.18
Cardiac myocytes from neonatal mice within 72 hour of birth were prepared as previously reported.4 Primary cardiac myocytes (2×105) were infected with 10 TCID50 of the virus. At the appropriate intervals, the cells and supernatant were frozen and thawed thrice. Virus titer was determined as described above.
Purification and Counting of Myocardial Infiltrating Macrophages
The mononuclear cells from inflamed hearts were separated as described.19 To determine the number of infiltrating macrophages per heart and to further analyze the phenotype of macrophages, the mononuclear cells were pooled from the hearts of 7 to 10 equivalently affected mice and F4/80+ macrophages were isolated using purified antimouse F4/80 (BM8; eBioscience) in conjunction with goat anti-rat microbeads on a magnetic column (Miltenyi Biotec) as described previously.20 This yields a population of >90% macrophages, assessed by fluorescence-activated cell-sorting (FACS) analysis using a FITC anti-F4/80 monoclonal antibody staining. And the number of macrophages per heart is calculated by dividing purified macrophage counts with the number of hearts pooled.
Bone marrow–derived macrophages (BMDMs) were prepared according to the protocol of Lake et al.21 M1 and M2 macrophages were generated as previously described.22 BMDMs were cultured under IFN-γ, IL-4, IL-13, IFN-γ plus IL-13, IL-4 plus IL-13 at the following concentrations: 7 ng/mL (IFN-γ), 25 ng/mL (IL-4), and 3 ng/mL (IL-13) for 24 hours.
Real-Time Polymerase Chain Reaction
Real-time polymerase chain reaction (PCR) was performed as previously described.15
Arginase activity was analyzed as described.15
Enzyme-Linked Immunosorbent Assay
Cell culture supernatant and heart homogenate were examined for IFN-γ (R&D Systems), IL-4 (eBioscience), IL-13 (eBioscience), TNF-α (R&D Systems), IL-10 (R&D Systems), and IL-12 (eBioscience) levels according to the instructions of the manufacturer.
Myocardial infiltrating macrophages and BMDMs were stained with the following monoclonal antibodies diluted in 1% FBS in PBS:FITC anti-F4/80 (eBioscience), PE anti-F4/80 (eBioscience), purified anti–inducible nitric oxide synthase (iNOS) (Santa Cruz), PE anti-CD16/32 (BD Biosciences), PE anti-CD206 (BD Biosciences), and anti-MGL (a kind gift from Dr Pieter J.M. Leenen, Erasmus Medical Center, Rotterdam, The Netherlands). FACS analysis of iNOS was performed as previously described.23 Regulatory T cells (Tregs) were measured using phycoerythrin (PE) anti-Foxp3 staining set (eBioscience) according to the protocols of the manufacturer.
In Vivo Neutralization of Cytokines
The rat monoclonal antibodies anti-mIFN-γ, anti-mIL-4, anti-mIL-13, anti-mIL-4 plus anti-mIL-13, or rat IgG (R&D Systems) were injected IP (2.5 mg/kg) on days 0, 2, 4, and 6 relative to CVB3 inoculation. Mice were euthanized on day 7 postinfection.
Adoptive Transfer of Macrophages
Mice were injected IV through the tail vein with M0, M1, or M2 macrophages (2.5×106) 3 days after recipients were infected with the virus. In other experiments, M2 macrophages (2.5×106) were mixed with M1 macrophages (2.5×106, 5×106, 1×107, and 2×107) to achieve ratios of M2 to M1 (M2:M1) of 1:1, 1:2, 1:4, or 1:8.
Hearts were cut longitudinally, fixed in 10% phosphate-buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin/eosin. Stained sections were observed using a Nikon Eclipse TE2000-S microscope (Nikon, Japan) and true color digital images were captured. The myocarditis was determined by identifying both infiltrating mononuclear cells and myocyte necrosis. The severity of myocarditis was judged by percentage area of the myocardium with inflammation as described previously.24,25 Five fields of view (×100 magnification) of each slide were chosen randomly, and three different sections per mouse were examined. The entire areas of the fields and of regions affected by myocarditis were determined with the use of ImageJ software (NIH) and the percent area of the myocardium undergoing inflammation (affected/entire area in percent) was calculated and averaged. All data were analyzed in a blind fashion by 2 independent investigators and averaged.
The presence of F4/80-positive macrophages in the heart tissue was analyzed by immunohistology as previously described.25
Data are presented as means±SEM. Differences between experimental groups were analyzed for statistical significance by unpaired Student’s t test or by 1-way ANOVA followed by Bonferroni test for the selected pairs using GraphPad Prism version 4.01 (GraphPad Software Incorporated). A value of P<0.05 was considered significant.
Female BALB/c Mice Showed Less Susceptibility to CVB3-Induced Myocarditis Than Males
Age-matched male and female BALB/c mice were infected IP with CVB3. On day 7 postinfection, the mice were euthanized and the incidence and severity of myocarditis were compared. Histopathologic examination showed an obvious inflammatory infiltrate in the myocardial tissue of male mice, whereas only a few inflammatory cells diffuse in the myocardium of females (Figure 1A). Consistently, quantitative analysis revealed that smaller myocardial areas were affected in female mice compared to males (P<0.01) (Figure 1B). Survival rate of male and female BALB/c mice after CVB3 inoculation was also monitored. During the 10-day infection period, mortality occurred in 50% of male mice. In contrast, only 7% of female mice died (Figure 1C). These results indicated that female BALB/c mice developed significantly less CVB3 myocarditis than males.
Male and Female BALB/c Mice Displayed Comparable Cardiac Viral Loads and Macrophage Infiltration During CVB3-Induced Myocarditis
To determine whether differential efficiency of CVB3 infection in male and female BALB/c mice contributed to the gender bias in susceptibility, we examined the levels of viral titer in vivo and in vitro during the early phase (day 3 postinfection) and acute phase (day 7 postinfection) of viral myocarditis by TCID50 assay. There was no significant difference in viral titers in the hearts (P>0.05) and cardiac myocytes (P>0.05) between the sexes at day 3 (Figure 2A and 2B). Furthermore, there were even slightly higher myocardial virus titers in female mice as compared to males (P<0.05) at day 7 (Figure 2A), although females showed milder myocarditis than male mice. In vitro studies, however, showed that there was no sex difference in viral titer in the cardiac myocytes at day 7 (P>0.05) (Figure 2B). Together, decreased inflammation in the myocardium of female mice with viral myocarditis was not attributable to less susceptibility to CVB3 infection.
Accumulating data indicated that abundant macrophages were recruited into the myocardium in male mice after CVB3 infection, which played an indispensable role in the development of viral myocarditis.6,7,26 Given female mice are less prone to acute myocarditis, we speculated that quantitatively and/or functionally distinct macrophages may exist in female mice. Age-matched male and female BALB/c mice were infected with CVB3 and compared for macrophage infiltration by immunohistological analysis. Macrophage-specific F4/80 immunostaining revealed an obvious macrophage infiltration in the myocardium both in males and females (Figure 3A). Interestingly, macrophages in male mice were more frequently clustered, shown as an intense accumulation at the inflamed lesions. As for female mice, however, macrophages tended to be diffusely distributed within the myocardium (Figure 3A). We next determined the absolute numbers of macrophages accumulating in the myocardium using cell yield analyses of F4/80+ cells. No significant differences were observed in the cell counts of infiltrating macrophages per heart between the 2 sexes (male, 7.9±0.4×105 versus female, 7.5±0.6×105; P>0.05) (Figure 3B). Comparative analysis demonstrated that sex difference in susceptibility to viral myocarditis was not attributable to quantitative distinction in macrophages.
Myocardial Infiltrating Macrophages Exhibited a Predominant M2 Phenotype in Females During CVB3-Induced Myocarditis
The above data suggested that there were no significant quantitative differences in infiltrating macrophages between male and female BALB/c mice following CVB3 infection, we then determined whether sex differences in macrophage polarization existed by analyzing markers associated with M1 and M2 polarization. It was shown that infiltrating macrophages from male mice significantly overexpressed the M1-asssocated inflammatory genes Nos2 (iNOS) and Tnfa (TNF-α) compared with female mice (Figure 4A). Strikingly, macrophages derived from females showed increased expression of M2-specific genes Arg1 (Arg-1), Il10 (IL-10), Mrc (MMR), and Mgl (MGL) compared with the macrophages isolated from male mice (Figure 4A). Consistently, FACS analysis revealed significantly increased iNOS and CD16/32 expression in male-derived macrophages, which were markers of M1 phenotype, and dramatically upregulated MMR and MGL expression in females (Figure 4B). As shown in Figure 4C, higher arginase activity, a typical characteristic of M2 macrophages, was observed in macrophages from female than in male mice. Taken together, we revealed phenotypic differences between myocardial infiltrating macrophages derived from male and female BALB/c mice during acute phase of viral myocarditis, with males being polarized to M1 phenotype, females being of predominantly M2 phenotype.
Myocardial Th2 Cytokine Primed Macrophages Into M2 Phenotype in Female Mice After CVB3 Infection
Given that there was a preferential M1 and M2 polarization under Th1 (IFN-γ) and Th2 cytokines (IL-4, IL-13), respectively, we determined whether distinct cytokine milieu contributed to sex difference in macrophage polarization in BALB/c mice. We first confirmed that male mice had significantly increased levels of myocardial IFN-γ expression compared to females (P<0.01), whereas females produced a larger amount of IL-4 in the hearts (P<0.01) (Figure 5A). Interestingly, another prototypical Th2 cytokine, IL-13, did not show significant difference between sexes (P>0.05) (Figure 5A). To assess the role of these cytokines in macrophage polarization in vivo, male and female BALB/c mice were injected with neutralizing antibody on days 0, 2, 4, and 6 relative to CVB3 infection. After cytokine blockade, macrophage phenotypes were examined by quantitative analysis of Nos2 and Arg1 expression, which were widely viewed as markers of M1 and M2 polarization, respectively. In susceptible male mice, as compared to isotype antibody treatment, macrophages showed decreased Nos2 (P<0.05) and increased Arg1 expression (P<0.05) after anti–IFN-γ neutralization (Figure 5B). No significant differences were observed after anti–IL-4 and/or anti-IL-13 treatment (Figure 5B). In contrast, Arg1 expression was strongly suppressed in nonsusceptible female mice after treatment with anti-IL-4 (P<0.01) or a combination of anti-IL-4 and anti-IL-13 neutralizing antibody (P<0.01), suggesting an essential role of IL-4 in M2 polarization in female BALB/c mice (Figure 5B). IL-13 neutralization alone also slightly reduced Arg1 expression in female mice, indicating less dominant role of IL-13 (Figure 5B). In vitro studies showed that BMDMs cultured at the presence of IFN-γ exhibited M1 phenotype, whereas IL-4, IL-13, or IL-4 plus IL-13 polarized BMDMs toward an M2 phenotype (Figure 5D), consistent with the in vivo data. Interestingly, although IL-13 alone primed macrophages along M2 program, when it was used in combination with high dose of IFN-γ, M2 polarization was markedly impaired, as shown by decreased Arg1 and enhanced Nos2 expression (Figure 5D). Because hypoxia-inducible transcription factor (HIF)-1α and peroxisome proliferator-activated receptor (PPAR)-γ are essential transcription factors for M1 and M2 differentiation, respectively,22,27,28 we further determined whether differential cytokine milieu between sexes regulated macrophage polarization at the transcription level. As shown in Figure 5C, higher mRNA levels of HIF-1α was observed in male BALB/c mice (P<0.01), whereas PPAR-γ was significantly overexpressed in females on day 7 after CVB3 infection (P<0.01).
Collectively, these data suggest that myocardial Th2 cytokines, notably IL-4, play a bona fide critical role in M2 polarization in female BALB/c mice.
Adoptive Transfer of Ex Vivo–Programmed M2 Macrophages Significantly Ameliorated Myocarditis in Susceptible Male Mice After CVB3 Infection
To explore the role of macrophage polarization in the development of viral myocarditis, we first generated M1 and M2 macrophages in vitro. It was shown that M1 macrophages induced by IFN-γ and LPS produced more proinflammatory cytokines IL-12 (P<0.01) and TNF-α (P<0.01) (Figure 6A), M2 macrophages polarized by IL-4 showed increased IL-10 production (P<0.05) (Figure 6A) and high levels of arginase activity (P<0.01) (Figure 6B). FACS analysis revealed high levels of iNOS and CD16/32 expression in M1 macrophages and increased MMR and MGL expression by M2 macrophages (Figure 6C). To exclude the possibility of induction of Foxp3+ Tregs in the programmed cells, FACS analysis was performed and showed that no CD4+ Foxp3+ cells could be detected in ex vivo–programmed macrophages (Figure 6D).
Then the programmed M1 or M2 macrophages were adoptively transferred into male and female recipient BALB/c mice at day 3 following CVB3 infection when substantial macrophages started invading the myocardium in the model.5 Histological analysis showed that M1 transfer into nonsusceptible female mice led to severe myocarditis on day 7 postinfection, with obvious inflammatory infiltration (Figure 7A), whereas M0 and M2 transfer did not increase myocarditis. In male mice, significantly more intense infiltrates (Figure 7C) and larger areas of inflammation (P<0.05) (Figure 7D) were detected in male mice given M1 macrophages compared to that of mice receiving no macrophages. Importantly, only M2 macrophages transfer led to alleviation of myocardial inflammation, reflected by restricted inflammation foci (Figure 7C) and decreased inflamed area (P<0.01) (Figure 7D), indicating significant therapeutic effect. These data confirmed the pathogenic role of M1 macrophages and indicated that M2 could effectively rescue mice from severe myocarditis. To further explore the lowest M2 percentage facilitating the protection, M2 macrophages were mixed with M1 at various ratios of M2:M1 (1:1, 1:2, 1:4 or 1:8) and transferred into female BALB/c mice 3 days after CVB3 infection. Only when the M2:M1 ratio reached to 1:4, the protective effect of M2 against myocarditis was abrogated (Figure 7E), indicating the high potency of M2 macrophages in rescuing severe myocarditis.
M2 Macrophage Transfer Led to Elevated IL-10 Production and Treg Frequency
Using GFP-positive (GFP+) donor macrophages, we examined the migration pattern of ex vivo–polarized macrophages in recipient mice. Comparable percentages of GFP+ M0, M1, or M2 macrophages were detected in the spleens on day 2 (Figure 8A) and within myocardium at day 4 after transfer (Figure 8B), indicated as the percentage of GFP+ F4/80+ among all F4/80+ cells, suggesting no preferential myocardial recruitment of either M1 or M2 macrophages into the heart tissue.
Cytokine profiles in the heart homogenate reflect the local immune environment. To better understand the mechanisms responsible for decreased inflammation after M2 transfer, the cytokine profiles of infected male mice that received M0, M1, or M2 macrophages were detected by ELISA at day 4 after adoptive transfer. We found that mice transfused with M1 macrophages showed increased level of proinflammatory cytokine TNF-α (P<0.01), whereas M2 transfusion resulted in significantly less TNF-α (P<0.01) but more antiinflammatory IL-10 production (P<0.01) (Figure 8C) in the heart. We next evaluated the in vivo effect of the donor macrophages on the T cell response. FACS analysis showed that CD4+ Foxp3+ Treg populations were slightly decreased in the spleen 4 days after M1 transfer, whereas M2 treatment resulted in an increased frequency of CD4+ Foxp3+ Tregs (Figure 8D), indicating the protective effect of M2 transfer may exert through promoting Treg differentiation.
We report here that there were not quantitative but phenotypic differences in the myocardial infiltrating macrophages between males and females following CVB3 infection. To the best of our knowledge, this study is the first to reveal that macrophages of female BALB/c mice are polarized toward an M2 phenotype after CVB3 infection which could effectively suppress the development of CVB3-induced myocarditis.
Several lines of evidence supported the notion that tissue-derived cytokines were responsible for M2 polarization in female BALB/c mice after CVB3 infection. First, our observations that similar percentage of donor polarized macrophages was detected within the myocardium after adoptive transfer suggested that there was no preferential M1 and M2 recruitment into the heart tissue. Secondly, high myocardial IL-4 and IL-13 levels were detected in females, although IL-13 expression was also found in male mice. Thirdly, conditioned medium containing IL-4 and/or IL-13 increased the expression of M2 marker, whereas the medium containing high levels of IFN-γ could potently suppressed the expression of IL-13–induced M2 marker, suggesting high levels of IFN-γ may have an overwhelming effect over IL-13 on macrophage polarization in male mice after CVB3 infection. Most importantly, in female mice, M2 polarization was inhibited by IL-4 and/or IL-13 neutralizing antibody and in male mice treated with anti-IFN-γ neutralizing antibody, macrophages increased M2 marker expression. Furthermore, at the transcriptional level, high expression levels of HIF-1α and PPAR-γ were observed in male and female BALB/c mice on day 7 after CVB3 infection, respectively. These data together indicated that differential myocardial cytokine expression between male and female BALB/c mice after CVB3 infection played a critical role in gender specific macrophage polarization.
Adoptive-transfer experiments using ex vivo programmed macrophages help elucidate the role of macrophage polarization in CVB3 myocarditis. The excessive presence of M1 macrophages may cause damage to the host after CVB3 infection. This is supported by the fact that mice transferred with M1 macrophages developed a more severe inflammation in the hearts of both sexes indicating excessive M1 residence caused myocardial damage. The most striking observation is that exogenous M2 macrophages transfer protected against CVB3-induced myocarditis in male mice, suggesting a potentially beneficial function of M2 macrophages in myocarditis. Because M2 macrophages have been found to be protective in many inflammatory conditions, such as obesity,15 diabetes,22 and experimental autoimmune encephalomyelitis,29 etc, adoptive transfer of ex vivo programmed M2 macrophages might be a useful strategy for treating these diseases.
It has been shown that proinflammatory cytokines have an important role in the development of inflammatory myocardial diseases.19,30 Suppression of inflammatory cytokines has beneficial effects on ameliorating myocarditis.31,32 Our findings that M2 macrophages transfer reduced myocardial TNF-α expression and enhanced IL-10 production in recipient mice may explain, at least partly, the mechanisms underlying M2 macrophages mediated protection. The secretion of IL-10 may help protect against the deleterious effects of TNF-α and related cytokines in myocarditis.32 The most interesting part of the study was the observation that transferred M2 were capable of migrating into spleen and increasing peripheral Treg cell populations. In the CVB3 myocarditis model, infection with pathogenic variants induces an autoimmune Th1 and CTL response that is directly responsible for cardiac injury in BALB/c mice.33 In this regard, donor M2 macrophage transfusion may facilitate development of Treg cells and prevent immunopathology. Our study highlights the critical role of M2 macrophages in induction of Treg cells in vivo and supports the beneficial role of Treg cells in CVB3 myocarditis.
In conclusion, our findings suggest that macrophages abundant in myocardium of female BALB/c mice predominantly exhibit alternative activation phenotype, which may confer protection against CVB3-induced myocarditis by limiting the generation of proinflammatory responses and promoting Treg cell differentiation. These findings suggested the possible role of macrophage polarization in defining the sex-related susceptibility to viral myocarditis and emphasized the potential of macrophage polarization in therapeutic intervention of inflammatory heart disease.
We thank Dr Pieter J.M. Leenen (Erasmus Medical Center, Rotterdam, The Netherlands) for kindly providing the anti-MGL antibody.
Sources of Funding
This work was funded in part by National Natural Science Foundation of China (30890141, 30772020, and 30872355), Major State Basic Research Development Program of China (2007CB512401), and a grant from Science and Technology Commission of Shanghai Municipality (07JC14004, LJ06011).
Original received July 4, 2008; resubmission received March 10, 2009; revised resubmission received July 5, 2009; accepted July 6, 2009.
Shen Y, Xu W, Chu YW, Wang Y, Liu QS, Xiong SD. Coxsackievirus group B type 3 infection upregulates expression of monocyte chemoattractant protein 1 in cardiac myocytes, which leads to enhanced migration of mononuclear cells in viral myocarditis. J Virol. 2004; 78: 12548–12556.
Fairweather D, Yusung S, Frisancho S, Barrett M, Gatewood S, Steele R, Rose NR. IL-12 receptor beta 1 and Toll-like receptor 4 increase IL-1 beta- and IL-18-associated myocarditis and coxsackievirus replication. J Immunol. 2003; 170: 4731–4737.
Hirasawa K, Tsutsui S, Takeda M, Mizutani M, Itagaki S, Doi K. Depletion of Mac1-positive macrophages protects DBA/2 mice from encephalomyocarditis virus-induced myocarditis and diabetes. J Gen Virol. 1996; 77: 737–741.
Shioi T, Matsumori A, Sasayama S. Persistent expression of cytokine in the chronic stage of viral myocarditis in mice. Circulation. 1996; 94: 2930–2937.
Gay RT, Belisle S, Beck MA, Meydani SN. An aged host promotes the evolution of avirulent coxsackievirus into a virulent strain. Proc Natl Acad Sci U S A. 2006; 103: 13825–13830.
Huber SA, Sartini D. Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis. J Virol. 2005; 79: 2659–2665.
Shibata Y, Henriksen RA, Honda I, Nakamura RM, Myrvik QN. Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG. J Leukoc Biol. 2005; 78: 1281–1290.
Futamatsu H, Suzuki J, Mizuno S, Koga N, Adachi S, Kosuge H, Maejima Y, Hirao K, Nakamura T, Isobe M. Hepatocyte growth factor ameliorates the progression of experimental autoimmune myocarditis: a potential role for induction of T helper 2 cytokines. Circ Res. 2005; 96: 823–830.
Cihakova D, Barin JG, Afanasyeva M, Kimura M, Fairweather D, Berg M, Talor MV, Baldeviano GC, Frisancho S, Gabrielson K, Bedja D, Rose NR. Interleukin-13 protects against experimental autoimmune myocarditis by regulating macrophage differentiation. Am J Pathol. 2008; 172: 1195–1208.
Timmer AM, Nizet V. IKKbeta/NF-kappaB and the miscreant macrophage. J Exp Med. 2008; 205: 1255–1259.
Ponomarev ED, Maresz K, Tan Y, Dittel BN. CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci. 2007; 27: 10714–10721.
Huber SA, Feldman AM, Sartini D. Coxsackievirus B3 induces T regulatory cells, which inhibit cardiomyopathy in tumor necrosis factor-alpha transgenic mice. Circ Res. 2006; 99: 1109–1116.
Watanabe K, Nakazawa M, Fuse K, Hanawa H, Kodama M, Aizawa Y, Ohnuki T, Gejyo F, Maruyama H, Miyazaki J. Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation. Circulation. 2001; 104: 1098–1100.