REVIEW doi: 10.1111/sji.12298 ..................................................................................................................................................................

Immunopathogenesis of Myocarditis: The Interplay Between Cardiac Fibroblast Cells, Dendritic Cells, Macrophages and CD4+T Cells B. Prince Amoah*†, H. Yang*, P. Zhang*, Z. Su*‡ & H. Xu*

Abstract *Department of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, China; †Department of Biomedical and Forensic Sciences, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana; and ‡The Central Laboratory, The Fourth Affiliated Hospital of Jiangsu University, Zhenjiang, China

Received 26 January 2014; Revised 21 February 2015; Accepted in revised form 14 March 2015 Correspondence to: Z. Su and H. Xu, Department of Immunology, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China. Emails: [email protected] and [email protected]

The myocardium responds to aetiologically different pathological injuries through a common multistep process involving highly co-ordinated interactions between cardiac and immune cells. Cardiac fibroblast cells which constitute the prevalent cell type in the heart to have their functional effects that express contractile proteins and exhibit increased migratory, proliferative and secretory properties. During the pathogenesis of myocarditis, cardiac fibroblast, dendritic cells, macrophages, CD4+ T cells and other immune cells are known to play variable roles. It is becoming increasingly clear that cardiac fibroblasts are not passive players in immune responses, and several evidences show this through the release of soluble signals and/or direct interactions with these immune cells. Typically, fibroblasts are involved in synthesizing factors such as cytokines, chemokines, prostanoids, matrix components and matrix-degrading enzymes to influence dendritic cells, CD4+ T cells and macrophage functions and vice versa in the pathogenesis of myocarditis. Again, evidence proves a crosstalk between cardiac fibroblasts and immune cells recruited into the myocardium during myocarditis in the microenvironments. This piece reviews the properties and roles of cardiac fibroblast cells, dendritic cells, macrophages and CD4+ T cells in the pathogenesis of myocarditis, and how these cells interplay on each other in the microenvironment.

Introduction Myocarditis, which describes inflammatory disorders of the heart muscle of varied infectious and non-infectious origins, can lead to dilated cardiomyopathy (DCM) in young patients. Clinical presentation of myocarditis ranges from non-specific systemic symptoms (fever, myalgias, palpitations or exertional dyspnea) to fulminant hemodynamic collapse and sudden death. The extreme diversity of clinical manifestations has made the true incidence of myocarditis difficult to be determined. It can be caused by any kind of infection, drugs, toxic substances or be associated with autoimmune conditions. DCM is the most common form of cardiomyopathy responsible for heart failure [1–3]. Coxsackie virus B3 (CVB3) is a major cause of myocarditis and DCM in humans, [2] and infection of mice with heartpassaged CVB3 (containing infectious virus and cardiac tissue) and induced autoimmune myocarditis leading to DCM [3]. A mouse model for this CD4+ Th cell-mediated post-infectious myocarditis is characterized by the infiltration of inflammatory cells into the myocardium, cardio-

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myocytes necrosis and deposition of collagen by cardiac fibroblasts/myofibroblasts [4]. It is known that many inflammatory cells comprising resident inflammatory cells as well as other immune cells which are recruited into the heart play important roles in the pathogenesis of myocarditis. These immune cells are known to have diverse and multiple functions. Some are known to play pro-inflammatory roles while others are anti-inflammatory. With their various functions, cardiac fibroblasts are at the interface of multiple interactions, playing an integral part in maintaining homoeostasis in the heart [5]. Under physiological conditions, cardiac fibroblasts contribute to the structural, mechanical, biochemical and electric properties of the heart [6]. They maintain ECM homoeostasis [7]. Cytokines and growth factors, secreted into the extracellular and interstitial space by cardiac cells, promote, as well as sustain cardiac inflammasome activation, fibrosis and hypertrophy [8]. A number of excellent reviews have discussed the cellular components of the heart and the contributions of immune cells like T lymphocytes and monocyte/macrophage lineage cells to cardiovascular

1

2 Mycarditis, Cardiac Fibroblast, Inflammation, Dendritic Cells, Macrophages B. Prince Amoah et al. .................................................................................................................................................................. diseases. In this review, we will provide an overview of literature on cardiac fibroblast cells, macrophages, dendritic cells and CD4 T cells and how they interplay to influence each other during the pathogenesis of myocarditis.

Cardiac fibroblast cells Origin and organization of cardiac fibroblast

There is little knowledge concerning the structure and function of cardiac fibroblast cells compared to the other cellular components of the heart. Traditionally, cardiac fibroblasts were defined by their morphological appearance by their branched cytoplasm surrounding an elliptical, speckled nucleus that typically has 1 or 2 nucleoli [9]. Cardiac fibroblast (CF) cells are reported to originate from two main sources. Firstly, it has been proven that CF originates from a spatiotemporal locus in the developing embryo and is derived from mesenchymal cells originating in the proepicardial organ [10]. These cells migrate over the surface of the heart to form the epicardium which in turn give rise to epicardial-derived cells (EPDCs) that later in development invade the myocardial walls and establish a compact myocardium through interaction with cardiomyocytes [11]. A number of growth factors, including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), are known to regulate the differentiation of these epicardiac-derived cells into cardiac fibroblast cells [12, 13]. During embryonic development, these EPDCs are reported to have the ability to alternate between the spindle-shaped fibroblast and the a-SMAexpressing myofibroblast phenotypes. Ultimately, most EPDCs assume the fibroblast phenotype which in adult life can be reactivated to a myofibroblast during events that trigger pathological remodelling [14]. Cardiac fibroblasts can also originate from endothelial cells or they can be recruited from the bone marrow into the heart during heart disease occurrence [7, 15], and these are termed bone marrow derived cardiac fibroblast. It is however not yet known whether different cardiac fibroblast origins which assemble in the heart contribute to phenotypic and functional cardiac fibroblast heterogeneity. Fibroblast (FB) cells are classified as either inactive or activated based on their morphological appearance. The activated fibroblasts are considered to be larger with a prominent Golgi apparatus [16], are believed to produce more collagen and, hence, are considered the main culprits in fibrotic diseases [16].

Figure 1 Cardiac fibroblast cells produce extracellular proteins (collagens, elastins, fibronectins) ECM-regulatory proteins, Matrix metalloproteinases (MMP’s), Tissue Inhibitors of Matrix metalloproteins (TIMP’s), Cytokines, Growth factors (VEGF, Vascular Endothelial Growth factor) and Natriuretic peptides (Ang II, Angiotensin II; ET-1, Endothelin 1; A-type Natriuretic Peptide, BNP, B-type Natriuretic Peptide).

identified for FBs and CFBs, but over time their specificity to these cells has been challenged. Vimentin, for example, has been extensively used to label cardiac fibroblasts [15]. However, although antibodies to Vimentin label fibroblasts with great sensitivity, they also label various other cell types, including endothelial cells [17, 18]. It is however considered the best of all the other proteins to be used to select fibroblast cells (Figs 1–3). FSP1 Fibroblast-specific protein (FSP)1, a filament associated calcium-binding protein in FBs [9], is another fibroblast marker, which was identified in a differential expression screen comparing kidney fibroblasts and kidney epithelial cells [19]. These proteins are known to be expressed by metastatic cancer cells, and existing evidence suggests that it is specific for cardiac fibroblasts in the heart [19]. However, FSP1 antibodies detect only a subset of cardiac fibroblasts, and FSP1 fibroblasts are rare in the normal heart [19]. a-SMA

Proteins expressed on cardiac fibroblast cell

Cardiac fibroblast cells like all other fibroblast cells lack distinct markers. Their known markers either are not specific for fibroblasts or detect only subpopulations of fibroblasts. A number of cell surface markers have been

It is known that valvular fibroblasts (also termed valvular interstitial cells) express a smooth muscle actin (a -SMA), but not interstitial fibroblasts of the myocardium [20]. In cardiac fibrosis, myocardial fibroblasts become a-SMA and are then termed myofibroblasts.

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Figure 2 Schematic diagram of interplay among cardiac fibroblast cells and adaptive immune cells IFN, interferon; IL, interleukin; MMP, matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of matrix metalloproteinase; TNF, tumour necrosis factor.

DDR1 and DDR2 Discoidin domain receptors (DDR) 1 and 2 are collagen receptors [21, 22]. They belong to the family of protein tyrosine kinases involved in a variety of cellular functions such as growth, migration and differentiation [23]. DDR1 is expressed mainly in epithelial cells, whereas DDR2 expressed in mesenchymal cells and lymphocytic lineages identifies only subsets of fibroblasts. (DDR)-2 has been used to identify and sort cardiac fibroblasts [8, 17]. Periostin Periostin, a matricellular protein, is specifically expressed by fibroblasts that originate developmentally from epicardial-derived progenitor cells [16]. However, because it is an extracellular protein, it has limited utility to detect fibroblasts by means of antibody labelling [16]. The issue of identifying fibroblasts is becoming further complicated because evidence is emerging that cardiac fibroblasts are not a homogenous cell type but a highly heterogeneous population [19, 24].

and secretion of bioactive regulatory molecules, cardiac fibroblasts are tightly associated with other cardiac cells via cell–cell contacts. Thus, cardiac fibroblasts are in a key position to serve as sentinel cells in the myocardium [27]. They maintain the homoeostasis of adjacent cells and orchestrate the maintenance of inflammatory infiltrates, indicating their importance in tissue development, differentiation, remodelling and repair [8, 25]. Besides directly modulating ECM equilibrium, cardiac fibroblasts also sense and integrate various environmental stimuli and response by secreting bioactive molecules such as cytokines, vasoactive peptides and growth factors [27]. Fibroblasts have the ability to participate in the maintenance of an inflammatory response via the expression of chemokines [25].

Innate and adaptive Immune responses in the pathogenesis of myocarditis Various innate and adaptive immune cells have been reported to be involved in the pathogenesis of myocarditis including inflammatory monocytes, neutrophils, macrophages and CD4+ Th cells [28, 29].

Functions of the cardiac fibroblast cells

In the heart, fibroblasts account for 60–70% of the cells [8, 25]. Typically, CFs are key components of the myocardial extracellular matrix (ECM) due to their ability to synthesize and secrete fibrillar collagen types I and III [26]. In addition to their role in production of the ECM network

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Innate immune responses

As the first-line defence, innate immune system contributes to the activation of cardiac fibroblasts in the pathogenesis of myocarditis through pattern recognition receptors [8]. Cardiac fibroblasts have been shown to

4 Mycarditis, Cardiac Fibroblast, Inflammation, Dendritic Cells, Macrophages B. Prince Amoah et al. ..................................................................................................................................................................

Figure 3 Schematic diagram of interplay among cardiac fibroblast cells and adaptive immune cells. IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; MBP, major basic protein; MMP, matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of matrix metalloproteinase; TNF, tumour necrosis factor.

express variety of pattern recognition receptors, including Toll-like receptors (TLRs), and the subsequent ligand activation of those receptors can directly activate fibroblasts and promote their differentiation into collagenproducing myofibroblasts [30]. Excessive collagen deposition leads to cardiac fibrosis which is characterized by overproduction of ECM, predominantly collagen types I and III, into the interstitial and perivascular space [31]. This eventually causes myocardial stiffening, impaired cardiac relaxation and filling (diastolic dysfunction), and overload of the heart, perhaps as a consequence of transformation of quiescent fibroblasts, responsible for basal ECM homoeostasis, to activated myofibroblasts [32]. Again, clinical data by Querejeta et al. [33] have confirmed excess cardiac collagen type I synthesis and deposition in heart failure patients which may be involved in the enhancement of myocardial fibrosis that accompanies the development of heart failure. It therefore implies that any therapeutic agent which is able to inhibit fibroblast-to-myofibroblast transformation may provide a means to inhibit maladaptive tissue remodelling in response to profibrotic stimuli and the excessive production of collagens.

Dendritic cells in the heart

Dendritic cells (DCs) were initially identified as potent antigen-presenting cells that play a key role in induction of the innate immune response [34]. DCs are known now play a critical role by bridging the innate and adaptive immune responses [35, 36], and they have been confirmed to be involved in many different disease conditions, such as rheumatoid arthritis, pulmonary allergic disease, rhinitis and other autoimmune diseases [37–39]. The type of dendritic cells in the heart is the conventional, lymphoid tissue resident dendritic phenotype. They are known to express markers such as CD1c, CD11c, CD33 and CD209 via TLR2, TLR4 and TLR7. They can produce different cytokines such as the tumour necrosis factor (TNF) – a, interleukin (IL)-12 and IL-23 [40, 41], which have specific functions to play. Interactions between cardiac fibroblast and dendritic cells in myocarditis

DCs interact with a microenvironment composed of the extracellular matrix and stromal cells such as fibroblasts,

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macrophages and endothelial cells. It is not surprising to find them therefore recruited into the heart during myocarditis. Fibroblasts are thought to be involved in the regulation of DC functions because they synthesize factors known to influence DC function, such as cytokines, chemokines, prostanoids, matrix components and matrixdegrading enzymes [42, 43]. Indeed, blocking of prostaglandins (PG)E2 secretion by indomethacin completely blocked IL-23 release in the co-culture of activated DC and fibroblasts to IL-23 levels produced by activated DC alone. Little is known about the interaction between DC and cardiac fibroblasts. Saalbach et al. [44] demonstrated in their study for the first time that fibroblasts actively participate in the regulation of DC function, resulting in the maturation of DC and the subsequent T cell activation. It can therefore be hypothesized that the interaction between cardiac fibroblast and DC in the heart could influence the pathogenesis of myocarditis. Interactions between cardiac fibroblast and macrophages

Macrophages are indispensable effector cells known to be greatly involved in the pathogenesis of myocarditis. Human models have increasingly implicated an autoimmune pathogenesis, as supported by the isolation of macrophage antigens from giant cells along with prominent T cell lymphocytic proliferation in the surrounding myocardium [45, 46]. Macrophages are of two types; those stimulated by TLR ligands and interferon (IFN)-c undergo classical M1 activation, while others stimulated by IL-4 and -13 become M2 macrophages [47]. M2 macrophages are involved in wound healing, tissue remodelling, fibrosis and inflammatory responses [48], they contribute to cardiac fibrosis [49] and are shown to directly promote collagen I expression in cardiac fibroblasts [50]. M2 macrophages release Arginase 1 capable of controlling L-proline production essential for the collagen synthesis of activated myofibroblasts. Macrophages are the main source of the several types of matrix metalloproteinases (MMPs) such as MMP-1, -7, -8, -9 and -12, as well as their endogenous suppressors, tissue inhibitors of MMPs (TIMPs) [51]. The MMPs released by macrophages mediate not only signalling through proteolysis of both ECM and non-matrix substrates as well as amplifying the inflammatory response. MMP-2 degrades the matrix proteins, cleaves monocyte chemotactic protein (MCP)-3, thereby lowering its chemotactic activity and diminishing the invasion of inflammatory cells and subsequently the inflammatory response and fibrosis in viral-induced myocarditis [52]. Macrophages are a leading producer of TGF-b, considered the most significant pro-fibrotic agent involved in the progression of chronic cardiac fibrosis [53]. TGF-b induces the expression of ECM genes and suppresses the activity of genes encoding MMPs, which are capable of degrading ECM [54]. Inhibition of TGF-b Ó 2015 John Wiley & Sons Ltd

via neutralizing antibodies has been shown to be well tolerated and to successfully reduce the development of fibrosis in different experimental models [55, 56]. Studies have demonstrated the direct effect of TGF-b on fibroblast differentiation to myofibroblasts [57, 58]. Early inhibition of TGF-b is associated with increased mortality, leucocyte infiltration and chemokine expression [59], while the later elimination of TGF-b results in improved survival and reduced tissue fibrosis [60]. In addition, macrophage depletion in the early phase of post-myocardial injury remarkably impairs the wound healing and increases remodelling and mortality, proving that macrophages are a key player in myocardial wound healing [61]. The activated macrophages produce TGF-b, IL-4, IL-10, IL-13 [52], tumour necrosis factor (TNF)-a and IL-1, which have been shown to activate fibroblasts and overproduce proteins of the ECM. TNF-a has also been found to activate the extracellular regulated kinasespecific pathway in fibroblasts resulting in increased expression of TGF-b [62] and support the excessive production of pro-inflammatory cytokines via the nuclear factor-kappa b (NF-kB) pathway [63]. High concentrations of TNF-a have been detected in patients with myocardial infarctions dilated cardiomyopathy and chronic heart failures [64, 65]. Once activated, macrophages not only secrete pro-fibrotic factors, but also recruit myofibroblasts and exacerbate inflammatory cell infiltration to sites of tissue injury, leading to profound production of a variety of chemokines, cytokines and growth factors, which endpoint of repair turns to excessive and poorly ordered matrix deposition and fibrosis [66, 67]. Adaptive immune responses influence myocarditis disease progression

CD4+ T helper cells regulate appropriate cellular and humoral immune responses in many diseases progressions including myocarditis. Emerging evidence has demonstrated that several Th subsets, such as Th1, Treg, Th17 but not Th9 cells, are involved in the pathogenesis of myocarditis [68–70]. Effect of Th1 and Th2 cells

The balance of the earliest determined CD4+ T helper cell subsets, Th1 and Th2, play an important role in allergy and autoimmune diseases. Th1 cells are the primary source for the inflammatory cytokines IFN-c, IL2 and TNFb. Many controversies surround the role of the Th1 cytokine IFN-c in inflammation and fibrosis, with numerous reports showing pro-fibrotic and antifibrotic effects. In an Angiotensin II-treated IFN-cR knockout mice study, the mice exhibited reduced cardiac hypertrophy, decreased infiltration of cardiac macrophages and

6 Mycarditis, Cardiac Fibroblast, Inflammation, Dendritic Cells, Macrophages B. Prince Amoah et al. .................................................................................................................................................................. T cells, and less cardiac fibrosis. IFN-c mediated its profibrotic activities via intensifying the production of proinflammatory and pro-fibrotic mediators such as TNF-a [71, 72]. IFN-c-producing T cells are known to control the differentiation, migration and activation of macrophages as well as their MCP-1 expression, which eventually leads to inflammation and fibrosis [66, 73]. No data currently are available on the role of IFN-c signalling in the differentiation of resident fibroblasts to myofibroblasts. IFN-a and IFN-c potently inhibit the collagen production of human fibroblasts, regulating the normal and pathological fibrogenesis [74]. IFN-c inhibits the IL-4- and IL-13-promoted differentiation from fibrocytes into myofibroblasts [75]. In a model of a chronic viral myocarditis, IFN-c reduced TGF-b1-, IL-1b- and IL-4-associated inflammation and fibrosis. T helper type 2 (Th2) cells are characterized by the secretion of the cytokines IL-4, IL-5 and IL-13 [66]. IL-13 in combination with IL-4 is proven to be capable of inducing the phenotypic transition of human fibroblasts to myofibroblasts in a c-Jun NH2-terminal kinase-dependent manner [76]. IL-13 has again been found to inhibit fibroblast MMP synthesis and subsequently downregulate the matrix degradation, which results in excessive collagen deposition [77]. Th 17 cells influence other cells involved in myocarditis

Th17 cells expressing IL-17A and IL-22 are another essential player in the development and progression of fibrotic disease in the heart [78]. IL-17A is characterized by its ability to induce the expression of a variety of proinflammatory mediators, such as IL-1, IL-6, TNF-a, CXC chemokine ligand 8 (CXCL8), granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor by fibroblast cells, which ultimately results in the recruitment and activation of immune cells [79]. IL-17 is known to promote MMP-1 expression in cardiac fibroblasts via NF-kB, activating protein-1 and CytidineCytidine-Adenosine-Adenosine-Thymidine (CCAAT)enhancer-binding protein (C/EBP)-b activation 131 and induces cardiac fibrosis via activation of the protein kinase C (PKC)b/Erk1/2/NF-kB pathway [80]. During the course of viral myocarditis, IL-17 causes the proliferation of cardiac fibroblasts and, in parallel, induces the degradation of collagen Type I and III via upregulation of MMP-2 [81]. CD4+ Th17 cells are important for the development of myocarditis by enhancing inflammatory injury and autoantibody production [2]. In addition, Th17 cells and their characteristic cytokine IL-17 can activate cardiac fibroblast cells by cell contacts [82]. Again work by Yu et al. suggested that cardiac fibroblast cells induce the production of C–C chemokine ligand 20 (CCL20). In their study, it was shown that the resident cardiac fibroblast cells were involved in the

recruitment of Th17 cells infiltrating into the myocardium by secreting CCL20. Recent studies have revealed that C–C chemokine receptor 6 (CCR6), the specific receptor for CCL20 mainly secreted by cardiac fibroblast [83] is dominantly expressed on Th17 cells and mediates Th17 cells migration. CCL20 produced by the cardiac fibroblast cells had no effect on the Th17 differentiation and IL-17 production [84]. In the EAM model, our laboratory previously has found that active secretion of High Mobility Group Box 1 (HMGB1) by cardiac fibroblast/myofibroblasts leads to cardiac fibrosis via PKCb activation by autocrine means [84]. Moreover, HMGB1 increases the proliferation of Th17 cells and subsequently increases the severity of EAM, and blockade of HMGB1 production will decrease Th17 cell proliferation [85]. Th22 cells may influence cardiac fibroblast cells in myocarditis

Recently, the Th22 cell has been recognized as a novel Th cell subset, which is characterized by abundant production of IL-22 but not IL-17 or IFN-c [86–88]. This newly identified CD4+ T cells clones have low or undetectable expression of transcription factor T-bet and RORct (Th1 and Th17). IL-22–IL-22R interactions can modulate the expressions of considerable genes such as IL-17, IFN-c, IL-1b, TNF-a and IL-6 and highlighted the fact that IL-22–IL-22R interactions are an integral pathway through which cells of the innate and adaptive immune responses regulate immunity and inflammation [89–91]. The pathogenic effect of Anti-IL-22 Ab in a AVMC model and protective role of the IL-22-Ig fusion gene in experimental autoimmune myocarditis (EAM) model [92] suggest that this cytokine has an important myocardium-protective role regardless of the cell source. Emerging literature indicates that IL-22 participates in immunity and tissue inflammation through the IL-22– IL-22R pathway [20, 22]. IL-22 neutralizations are known to significantly promote cardiac IL-17, IL-6, TNF-a, which have been identified as important proinflammatory cytokines and exacerbating myocarditis by inflammation and immunity [68, 93]. Kong et al. [88] speculated that IL-22 may play a myocardium-protective role through the IL-22–IL-22R pathway in acute viral myocarditis (AVMC). Further studies focusing on the differentiation, regulation, downstream pathways of IL22-producing Th22 cells, especially cardiac myofibroblast cells in human systems are required to comprehensively explore the therapeutic potential of Th22 cells in AVMC. We also speculate that IL22 from Th22 could activate cardiac fibroblast cells and drive the later to create a specific local microenvironment that will promote a Th17 immune response in myocarditis. Even though no work has been done on how cardiac fibroblasts cells interplay on Th22 cells, there could be an indirect crosstalk between these cells.

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B. Prince Amoah et al. Mycarditis, Cardiac Fibroblast, Inflammation, Dendritic Cells, Macrophages 7 .................................................................................................................................................................. T regulatory cells in myocarditis

Regulatory T cells (Tregs) are a subset of CD4+ lymphocytes expressing Fork head box protein 3 (Foxp3). Tregs can either suppress or promote cardiac fibrosis. They are known to release important immunosuppressive cytokines including IL-10 and TGF-b that have control over the inflammatory response and contribute to the maintenance of self-tolerance and host immune defence. Tregs play a considerable role during the inflammatory process and the subsequent progression of fibrosis contributing [94] or suppressing [95] the development of fibrosis. One important molecular mechanism of Tregs to regulate immune response has been demonstrated to be mediated through production of immune-suppressive cytokines, IL-10 [96, 97]. IL-10 is most commonly recognized as an immunosuppressive cytokine to control inflammation thought to be driving fibroblast proliferation. Intravenous injection of IL10-overexpressing monocytes/macrophages greatly reduced myocarditis and cardiac fibrosis in a murine model of autoimmune myocarditis [98]. Adoptive transfer of Tregs has been shown to have a protective role in the fibrotic process of CVB3-induced cardiac fibrosis via secreting IL10 [99]. IL-10 produced by Tregs in vivo and in vitro significantly inhibits the collagen synthesis by cardiac fibroblasts [99]. However, the molecular mechanism of Treg-mediated resolution of CVB3-induced cardiac fibrosis needs further exploration. Interactions between Cardiac fibroblast cells, dendritic cells, macrophages and CD4+ T cells influence the pathogenesis of myocarditis

Fibroblasts not only modulate the recruitment of immune cells, but also regulate their behaviour, retention and survival in damaged tissue. In general, the crosstalk between fibroblasts and leucocytes depends on the interaction between the leucocyte surface antigen CD40 on fibroblasts and its ligand, CD40L, which is expressed on immune cells. Fibroblasts also express the co-stimulatory molecule B7 [100], suggesting that similar to interactions between lymphocytes and antigen-presenting cells, CD40-CD40L and co-stimulatory Cytotoxic T-Lymphocyte Antigen 4, CTLA4-B7 interactions play an important role in the fibroblast–immune cells crosstalk. Van Linthout et al. [53] suggest that the interaction between fibroblasts and macrophages may be an important early event in the recruitment of monocytes and may facilitate a cytokine network that maintains the activation of tissue inflammation. T cells infiltration in mouse hearts is known to be a major source of IFN-c production, and contact-mediated effect between IFN-c-producing T cells and macrophage stimulates macrophages to produce MCP-1, which recruits more macrophages and fuels an inflammatory positive feedback loop [73]. This means that contact-mediated effect between

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macrophages and T cells stimulates MCP-1 production and macrophage migration in WT mice during myocarditis. Fairweather et al. [101] reported that IFN-c deficiency increased chronic myocarditis in CB3 virus infection.

Conclusion During the course of myocarditis, it is evidently clear that the complex interactions between cardiac fibroblast cells and immune cells are very vital and contribute to the myocardium inflammation and remodelling in myocarditis.

Acknowledgment This work was supported by National Natural Science Foundation of China (Grant No. 81370084, 81001319, 81101677, 31270947), Postdoctoral Foundation of China (2012M511705, 2013T60508 and Postdoctoral Foundation of Jiangsu Province (1102129C).

Conflict of interest None to declare.

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