Autoimmune attack on the heart is linked to host immune responses against cardiac myosin, the most abundant protein in the heart. Although adaptive immunity is required for disease, little is known about innate immune mechanisms. In this study we report that human cardiac myosin (HCM) acted as an endogenous ligand to directly stimulate human TLRs 2 and 8 and to activate human monocytes to release proinflammatory cytokines. In addition, pathogenic epitopes of human cardiac myosin, the S2 fragment peptides S2-16 and S2-28, stimulated TLRs directly and activated human monocytes. Our data suggest that cardiac myosin and its pathogenic T cell epitopes may link innate and adaptive immunity in a novel mechanism that could promote chronic inflammation in the myocardium.

Myocarditis is an acute inflammatory disease of the heart that may lead to chronic inflammation of the myocardium and to dilated cardiomyopathy (DCM)5 (1). In myocarditis and DCM, autoantibodies react with cardiac myosin (2). In animal models of cardiac myosin-induced myocarditis, pathogenic roles for CD4+ T cells (3, 4) and autoantibodies (5) have been described. Although s virus is important in acute myocarditis (3), the development of chronic myocarditis and cardiomyopathy may require exposure and release of cardiac myosin from damaged myocytes (6). However, little is known how an intracellular protein autoantigen like cardiac myosin activates inflammatory cells.

Although studies of myocarditis over the past 20 years focused on adaptive immunity, the activation of innate immunity in the induction of myocarditis and progression to DCM has only recently been appreciated (7, 8). Innate immune cells, including macrophages, neutrophils, and dendritic cells (9, 10, 11), and several cytokines, including IL-6, IL-1, and TNF-α, are present in an inflamed myocardium (9, 12, 13). In addition, IL-6 knockout mice are resistant to disease induction (14), and overexpression of TNF-α resulted in severe myocarditis in susceptible mice (15).

Although TLRs were initially discovered to play essential roles in innate immune recognition of pathogen-associated molecular patterns (PAMPs), increasing evidence indicates that TLR activation by endogenous ligands stimulates acute and chronic inflammatory responses (16). Endogenous ligands released from damaged tissue may activate TLRs, leading to chronic inflammation.

The involvement of TLRs has been reported in myocarditis (7, 9, 17). Activation of TLRs in dendritic cells through MyD88 signaling is critical, because MyD88 deficiency rendered susceptible mice resistant to myocarditis (8). It was shown that several TLR ligands replaced complete Freund’s adjuvant in a mouse model of myocarditis where dendritic cells loaded with α-myosin H chain-derived peptide caused disease (7). Activation of innate immunity through TLRs appears to be essential for prolonged adaptive responses to cardiac myosin. We propose that cardiac myosin released from damaged cardiomyocytes may act as an endogenous ligand for TLRs and stimulate chronic inflammation, leading to cardiomyopathy.

Our study shows that human cardiac myosin (HCM) and its pathogenic peptides directly stimulated human TLRs 2 and 8 and activated human monocytes to secrete proinflammatory cytokines in a TLR-dependent manner. Thus, cardiac myosin is not only a major target of the adaptive immune response, but it may directly stimulate innate immune responses that promote chronic inflammation in the myocardium.

HCM was purified as previously described (18). S2 peptides were previously described (13) and analyzed for purity by mass spectrometry. Rabbit skeletal myosin (SKM) and chloroquine were from Sigma-Aldrich. The mAb to human TLR2 (TL2.1) was from Stressgen Biotechnologies. Lipoteichoic acid, LPS, and CL075 were from InvivoGen.

PBMCs were prepared on Histopaque-1077 (Sigma-Aldrich) from human peripheral blood of healthy volunteers and monocytes enriched by negative selection using a monocyte isolation kit (Dynal MPC-L). PBMCs and monocytes were plated in 24-well plates and cultured in RPMI 1640 medium supplemented with either 5 or 10% heat-inactivated FBS or human sera from AB blood group or monocyte donors, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. In certain cases, monocytes were preincubated with gammaglobulin to block Fc receptors followed by incubation with TL2.1 mAb (50 ng/ml) for 1 h before treatment with HCM. Monocytes were treated with HCM in the presence of chloroquine (50 nM).

The human monocytic cell line THP-1 was obtained from the American Type Culture Collection and maintained according to the supplier’s instructions.

Cells were stained with the following: PE-anti-human TLR2 (eBioscience), FITC-anti-human TLR8 (Imgenex), allophycocyanin-anti-human CD14 (monocyte marker) (BD Biosciences), and appropriate isotype controls. Intracellular staining with anti-TLR8 was done using an IC-Flow kit (Imgenex). Samples were analyzed on a FACSCalibur flow cytometer.

Human PBMC, monocytes, and THP-1 cells were treated with TLR2 stimulants for 24 h. Supernatants were collected and assayed using ELISA kits (human IL-8 was from BD Pharmingen and human IL-6 and TNF-α were from Mabtech).

HEK293 cells stably transfected with human TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, and control untransfected HEK293 cells were from InvivoGen. Expression of TLRs was verified by Western blotting (data not shown). HEK293-TLR transfectants were maintained in DMEM, 4.5 g/L glucose, 10% heat-inactivated FBS or human AB serum, 10 μg/ml blasticidin, and 100 μg/ml Normocin. Cells were treated with HCM, S2-16 peptide, SKM, or known TLR ligands. Supernatants were harvested after 72 h and assayed for IL-8 as an indicator of TLR activation.

psiRNA-hTLR2 and psiRNA-TLR8 plasmids (InvivoGen) were used to silence TLR2 and TLR8 in human monocytes using a Nucleofector kit (Amaxa) according to the manufacturer’s instruction. Cells (8 × 106) were subjected to Nucleofection using the Y001 program. Control cells were transfected with a control small interfering RNA (siRNA) (psiRNA-LucGL3). Transfected cells were prewarmed in RPMI 1640 medium with 10% FBS. After 24 h, transfection efficiency was monitored by GFP-positive cells by immunofluorescence microscopy. Cells were treated with HCM and supernatants were harvested 24 h later and assayed for IL-8. Silencing efficiency was ∼100% as determined by Western blotting.

Data are expressed as the mean ± SEM of triplicate wells in cytokine ELISA. The effect of multiple treatment groups was evaluated by one-way ANOVA using GraphPad Prism4 software. Difference of the means was evaluated by Tukey’s test. For HCM S2-16 effects on HEK293-hTLRs, mAb and chloroquine inhibition on monocyte IL-8, and siRNA data, comparison between treatment and media was performed by Student’s unpaired two-tailed t test.

To determine TLR activation by HCM, we used HEK293 cells transfected with hTLR2, hTLR3, hTLR4, hTLR6, hTLR7, hTLR8, or hTLR9. HCM-treated cells demonstrated a 2- to 5-fold increase in IL-8 secretion in HEK293 cells transfected with TLR2 (Fig. 1,A) and TLR8 (Fig. 1,B). No increase of IL-8 secretion was observed over control in HEK293 cells transfected with TLR3, TLR4, TLR6, or TLR9 when treated with HCM (data not shown). The data show that HCM activates TLR2 and TLR8, but not other TLRs. Although not shown or studied further, TLR7 was activated by HCM but not by SKM. Interestingly, SKM, which does not induce myocarditis in animal models (18, 19), did not significantly activate TLR2 and TLR8 (Fig. 1, A and B). The HCM peptides S2-16, a cryptic pathogenic peptide of HCM, and S2-28, a dominant epitope in experimental autoimmune myocarditis (13), activated TLR2 and TLR8, but not TLR4 (Fig. 1 C) or other TLRs (not shown). Other HCM peptides such as S2-2 did not have the same stimulatory effect (not shown). FACS analysis of transfectants (HEK293-hTLR2 and HEK293-hTLR8) confirmed surface expression of TLR2 and intracellular expression of TLR8 (data not shown). Untransfected HEK293 cells did not express TLR2 or TLR8.

FIGURE 1.

HCM activates TLR2 and TLR8. A, HEK293-hTLR2 cells were stimulated with HCM or SKM. Supernatants harvested at 24 h were assayed for IL-8. LTA, Lipoteichoic acid (TLR2 ligand). B, HCM stimulated IL-8 secretion from HEK293-hTLR8 cells; p < 0.001. C, HCM S2-16 and S2-28 peptides activated TLR2 (p < 0.0001) and TLR8 (p < 0.0001). S2-16 is a cryptic pathogenic HCM peptide and S2-28 is the dominant epitope of cardiac myosin in experimental autoimmune myocarditis (13 ). CL075 is a TLR7/8 ligand and LPS is a TLR4 ligand. Values are means ± SEM. Representative results from three to six independent experiments are shown. The p value was obtained by Tukey’s test using one-way ANOVA.

FIGURE 1.

HCM activates TLR2 and TLR8. A, HEK293-hTLR2 cells were stimulated with HCM or SKM. Supernatants harvested at 24 h were assayed for IL-8. LTA, Lipoteichoic acid (TLR2 ligand). B, HCM stimulated IL-8 secretion from HEK293-hTLR8 cells; p < 0.001. C, HCM S2-16 and S2-28 peptides activated TLR2 (p < 0.0001) and TLR8 (p < 0.0001). S2-16 is a cryptic pathogenic HCM peptide and S2-28 is the dominant epitope of cardiac myosin in experimental autoimmune myocarditis (13 ). CL075 is a TLR7/8 ligand and LPS is a TLR4 ligand. Values are means ± SEM. Representative results from three to six independent experiments are shown. The p value was obtained by Tukey’s test using one-way ANOVA.

Close modal

Our discovery that HCM activated TLRs led us to study human monocytes that express TLR2 and TLR8. Treatment of PBMCs with HCM resulted in a dose-dependent 2.5-fold increase in IL-8 secretion (Fig. 2,A). We isolated human monocytes and found that HCM stimulated IL-8 secretion from human monocytes in a dose-dependent manner up to a 4.3-fold increase (Fig. 2,B). Importantly, monocytes cultured with the HCM peptide S2-16 led to a dose-dependent increase of 2.5-fold in IL-8 secretion. Stimulation of IL-8 by HCM and the S2-16 peptide was also observed in human monocytic THP-1 cells. No stimulation was observed with SKM (Fig. 2,C). The HCM S2 peptides 16 and 28 stimulated IL-8 secretion from human monocytes, with S2-16 being moderately and S2-28 highly stimulatory (Fig. 2 D). The S2-30 peptide and SKM were not stimulatory. In further studies of human monocytes, S2-28 (10 μg/ml) consistently stimulated IL-8 in PBMCs to high levels (55,000 pg/ml) above that seen for the HCM peptide S2-16 (20,000 pg/ml). Our findings suggest that pathogenic epitopes for T cells may link adaptive and innate immunity.

FIGURE 2.

A–C, HCM activates human monocytes and induces IL-8 from PBMCs (A; p < 0.001); monocytes (B; p < 0.001); and human monocytic THP-1 cells (C; p < 0.001) treated with HCM, S2-16 HCM peptide, or SKM for 24h. Supernatants were assayed for IL-8. D, The HCM peptides S2-16 and S2-28 stimulated IL-8 secretion from human monocytes. Values are means ± SEM. The p value was obtained by Tukey’s test using one-way ANOVA.

FIGURE 2.

A–C, HCM activates human monocytes and induces IL-8 from PBMCs (A; p < 0.001); monocytes (B; p < 0.001); and human monocytic THP-1 cells (C; p < 0.001) treated with HCM, S2-16 HCM peptide, or SKM for 24h. Supernatants were assayed for IL-8. D, The HCM peptides S2-16 and S2-28 stimulated IL-8 secretion from human monocytes. Values are means ± SEM. The p value was obtained by Tukey’s test using one-way ANOVA.

Close modal

To further confirm that HCM activates human monocytes, we analyzed several proinflammatory cytokines besides IL-8 (Fig. 3,A). We found that HCM also stimulated IL-6 (Fig. 3,B) and TNF-α (Fig. 3 C). SKM did not have a stimulatory effect on the secretion of proinflammatory cytokines from human monocytes. In further studies, we found that S2-28 (10 μg/ml) stimulated the production of more IL-6 (1500 pg/ml) from PBMCs than that seen with S2-16 (500 pg/ml).

FIGURE 3.

HCM and peptide S2-16 stimulated the proinflammatory cytokines IL-8 (A), IL-6 (B), and TNF-α (C) from human monocytes as measured by ELISA, whereas the cytokines in monocytes treated with SKM were not stimulated. Values are means ± SEM. LTA, lipoteichoic acid. The p value obtained by Tukey’s test using one-way ANOVA.

FIGURE 3.

HCM and peptide S2-16 stimulated the proinflammatory cytokines IL-8 (A), IL-6 (B), and TNF-α (C) from human monocytes as measured by ELISA, whereas the cytokines in monocytes treated with SKM were not stimulated. Values are means ± SEM. LTA, lipoteichoic acid. The p value obtained by Tukey’s test using one-way ANOVA.

Close modal

Human monocytes are known to express several TLRs, with TLR2 and TLR8 the most dominant (20). We hypothesize that HCM activates human monocytes in a TLR-dependent manner. To demonstrate that the activation of human monocytes by HCM was in part dependent on TLR2, we preincubated monocytes with a mAb to TLR2 for 1 h before HCM treatment. The mAb inhibited HCM stimulated-IL-8 secretion by 40% (Fig. 4 A).

FIGURE 4.

Anti-TLR2, chloroquine, and siRNA inhibit HCM stimulation of TLR2 or TLR8. A, Human monocytes were preincubated with mAb to human TLR2 and treated with HCM for 24 h. Supernatants were assayed for IL-8. Anti-TLR2 inhibited HCM stimulation (p = 0.0002). B, Chloroquine inhibited HCM stimulation of IL-8 secretion from human monocytes (p < 0.0001). C, Silencing of TLR2 and TLR8 RNA inhibited HCM-stimulated human monocyte activation. Human monocytes were transfected with siRNA against TLR2 or TLR8 or both and then stimulated with HCM. Bar graphs show IL-8 (pg/ml) above medium control for human monocytes containing control plasmid (psiRNA-LucGL3) or TLR siRNA plasmids after HCM activation of human monocytes. Representative results from three independent experiments shown. Values are means ± SEM.

FIGURE 4.

Anti-TLR2, chloroquine, and siRNA inhibit HCM stimulation of TLR2 or TLR8. A, Human monocytes were preincubated with mAb to human TLR2 and treated with HCM for 24 h. Supernatants were assayed for IL-8. Anti-TLR2 inhibited HCM stimulation (p = 0.0002). B, Chloroquine inhibited HCM stimulation of IL-8 secretion from human monocytes (p < 0.0001). C, Silencing of TLR2 and TLR8 RNA inhibited HCM-stimulated human monocyte activation. Human monocytes were transfected with siRNA against TLR2 or TLR8 or both and then stimulated with HCM. Bar graphs show IL-8 (pg/ml) above medium control for human monocytes containing control plasmid (psiRNA-LucGL3) or TLR siRNA plasmids after HCM activation of human monocytes. Representative results from three independent experiments shown. Values are means ± SEM.

Close modal

To determine whether HCM activated monocytes in part through TLR8, we cultured monocytes with HCM in the presence of chloroquine, a chemical inhibitor of endosome acidification that interferes with the activation of TLR8. We found that chloroquine inhibited HCM-stimulated IL-8 production by 60% (Fig. 4 B).

FACS analysis of PBMCs and human monocytes from three different individuals showed little difference in TLR expression before and after treatment with HCM and S2 peptides. Percent difference ranged from 1 to 4% (TLR2) and 1–9% (TLR8).

To further confirm that stimulation of monocyte IL-8 by HCM was dependent upon activation of TLR2 and TLR8, we transfected human monocytes with siRNA against TLR2, TLR8, or both. Control siRNA did not affect HCM stimulation of monocyte IL-8 (Fig. 4,C). Knocking down TLR2 in monocytes resulted in the complete inhibition of stimulation of IL-8 by HCM (Fig. 4,C). Knocking down TLR8 resulted in almost complete inhibition of HCM-stimulated increase of IL-8 (Fig. 4,C). However, when knocking down both TLR2 and TLR8, the stimulation of IL-8 by HCM was again strongly inhibited (Fig. 4 C), suggesting cooperation between TLR2 and TLR8 in the activation of human monocytes by HCM.

Our results show for the first time that cardiac myosin, the major autoantigen in myocarditis, activated TLR2 and TLR8 while skeletal myosin did not, and HCM directly activated human monocytes to release proinflammatory cytokines. It is currently well accepted that endogenous TLR ligands may play an important role in the pathogenesis of some autoimmune diseases (16).

The specificity of TLRs for cardiac myosin is novel and intriguing. The structural similarity between cardiac myosin and virulence factors of pathogens may be important in its overall ability to activate innate immunity. Most known endogenous TLR ligands have been shown to activate TLR2 and/or TLR4 (16, 21). Only nucleic acid self-Ags have been reported to activate TLR7, TLR8, and TLR9 (21). Cardiac myosin may function as a TLR ligand by mimicking epitopes on pathogens or PAMPs. To support this hypothesis, immunological mimicry has been shown between streptococcal M protein, coxsackievirus, and cardiac myosin (22, 23) indicating that cardiac myosin shares structural similarities with pathogens. Evidence has demonstrated mimicry between α-helical molecules such as streptococcal M protein and myosin and DNA (24). Recently, it was shown that the α-helical coiled-coil streptococcal M protein activated TLR2 on monocytes and was a potent inducer of inflammatory cytokines (25). The strong similarity of cardiac myosin and streptococcal M protein supports the recognition of TLR2 by both molecules (25). The crystal structure of streptococcal M protein reveals that nonidealities of the α-helices may interact with the immune system (26). This may be the case for cardiac myosin.

Microbial PAMPS did not appear to be in our purified HCM preparations and peptides, because they did not activate TLR4 and several other TLRs that are activated by bacterial LPS or other microbial products. ssRNA, which activates TLR7 and TLR8, would be rapidly degraded. SKM was negative with all TLRs, and analysis of HCM preparations and peptides for 260 nm/280 nm ratio of <0.57 indicated that nucleic acids were not present. All peptides were tested for purity by mass spectrometry.

Stimulation of TLRs on monocytes was observed not only with intact HCM but also with peptides from the S2 region of cardiac myosin, in particular the peptide S2-16 and the dominant HCM epitope S2-28. Our findings suggest that the two dominant T cell epitopes of HCM have the ability to directly activate innate immune cells, thus linking adaptive and innate immunity.

It is well known that patients with myocarditis or dilated cardiomyopathy have elevated levels of autoantibodies against cardiac myosin (2) and that Abs against cardiac myosin deposit in the hearts of animals with myocarditis (5). Immune complexes containing HCM or its peptides may target TLR7 or TLR8 on endosomes after internalization through Fc receptors on immune cells. Cooperation of Fc receptors and TLRs might be important in tissue-specific autoimmune diseases that display elevated levels of autoantibodies to tissue-specific Ags (27, 28).

Although the classic view of immune recognition was based on discrimination of self-nonself, more and more evidence suggests that self-molecules with hydrophobic regions exposed to the immune system are perceived as a “danger signal” and have the ability to activate the immune response in a similar way as nonself molecules (29). Our hypothesis is supported by our data, which suggest that cardiac myosin is not only a major target of the adaptive immune response but may serve as a “signal” from within the heart to crosstalk with the immune system.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant R01 HL56267. M.W.C. is the recipient of a NHLBI merit award HL35280.

5

Abbreviations used in this paper: DCM, dilated cardiomyopathy; HCM, human cardiac myosin; hTLR, human TLR; PAMP, pathogen-associated molecular pattern; siRNA, small interfering RNA; SKM, skeletal myosin.

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