The protective effect of interfering TLR9-IRF5 signaling pathway on the development of CVB3-induced myocarditis

Highlights

• The TLR9-IRF5 signaling is activated in development of CVB3-inducecd myocarditis.

• TLR9-IRF5 signaling aggravates CVB3-induced cardiac damage in mice.

• Interfering TLR9-IRF5 signaling could alleviate myocarditis in CVB3-infected mice.

• Targeting TLR9-IRF5 signaling pathway could be an approach for treating VMC.

Abstract

Since toll-like receptor 9 (TLR9) or interferon regulatory factor 5 (IRF5) was reported to be associated with the development of myocarditis, we wondered if the TLR9-IRF5 pathway could contribute to the development of coxsackievirus B3 (CVB3)-induced myocarditis. We detected signaling molecules of TLR9-IRF5 pathway in CVB3-infected patients and mice. The results showed that TLR9, IRF5 and its downstream molecules such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly increased, and the increase was correlated with the severity of heart injury during CVB3 infection. In addition, we demonstrated that an AAAG ODN with IRF5 interfering activities significantly decreased the levels of the TLR9-IRF5 pathway molecules in hearts, spleens as well as white blood cells, and alleviated the myocarditis in CVB3-infected mice. The data suggest that interfering TLR9-IRF5 pathway could be an approach to treat CVB3-induced myocarditis.

Introduction

Viral myocarditis (VMC), caused by viruses, is inflammation of the heart muscle [1]. Coxsackievirus B3 (CVB3), a type of enteroviruses, is the most common pathogen for the VMC, implicated in 20–40% of VMC patients [2]. Being treated with ribavirin, interferon-beta, immunoglobulins and corticosteroids, CVB3-induced myocarditis remains one of the most challenging clinical problems in cardiology, and still accounts for up to 20–30% of dilated cardiomyopathy in the VMC patients, and 2% to 12% human sudden deaths [1,3]. Thus, novel medications are required to be developed for treating the CVB3-induced myocarditis.

In recent years, the innate immune response induced by CVB3 has been realized to be both beneficial and detrimental to hearts. During the infection, the innate immune cells are activated through recognizing pathogen-associated molecular patterns (PAMPs), such as viral RNAs, by a broad panel of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), such as TLR9 [4]. TLR9 is activated by the endogenous molecules released from the heart cells damaged by CVB3 replication in the development of the CVB3-induced myocarditis [5,6]. The released molecules, such as high-mobility group box 1 (HMGB1) and mitochondrial DNA (mtDNA), act as damage associated molecular patterns (DAMPs) to initiate sterile inflammatory responses, aggregating the heart damage. The DAMPs activate TLR9 and consequently trigger the recruitment of myeloid differentiation primary response gene 88 (MyD88), interleukin-1 associated kinase (IRAK4) and TNF receptor-associated factor 6 (TRAF6). The TRAF6 in turn activates transforming growth factor activated kinase-1 (TAK1), and subsequently mitogen-activate protein kinase (MAPK) and the IκB kinase (IKK) complex, to activate nuclear factor kappa B (NF-κB). The NF-κB induces the production of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) [7]. The TLR9/MyD88/NF-κB/TNF-α axis, if overly activated, could aggravate CVB3-induced myocarditis, exemplified by the evidences that the VMC mice with deficiency of TLR9 or MyD88 displayed improved left ventricular (LV) function and reduced cardiac inflammation [5,8], and that blocking NF-κB lessens cardiac injury in CVB3-infected mice [9].

In addition to NF-κB, interferon regulatory factor 5 (IRF5) is another downstream transcription factor in TLR9/MyD88 signaling pathways [10]. TLR9 activation induces the formation of MyD88-IRF5-TRAF6 complexes, which in turn activates IRF5 [11]. The activated IRF5 moves from cytoplasm into the nucleus where it binds cis-regulatory elements to promote the transcription of TNF-α and IL-6 [12]. TNF-α is significantly increased in hearts of the CVB3-infected mice. TLR9 deficient mice infected with CVB3 show significantly lower levels of TNF-α in the heart [5]. The increased levels of IL-6 are associated with prognosis of the VMC patients, and injecting an anti-IL-6 receptor antibody reduces CVB3 replication and improves LV functions in the VMC mice [13]. These evidences suggest that TLR9-IRF5 signaling pathway could be involved in the development of CVB3-induced myocarditis, and that IRF5 could be targeted for the treatment of VMC.

To develop IRF5 inhibitors for treating autoimmune/inflammatory diseases, various efforts have been made. Trichostatin A (TSA) capable of depressing promoter activities of IRF5 and reducing productions of TNF-α and IL-6 was tested as a potential drug for the treatment of childhood onset systemic lupus erythematosus (SLE) [14]. Subcutaneously applied sMiR-146b, a microRNA that drives degradation of IRF5 mRNA, ameliorates colitis in mice [15]. An IRF5 decoy peptide binds to IRF5 and reduces IRF5 translocation to the nucleus, therefore reducing myocardial inflammation and improving LV function in mice with systemic sclerosis (SSc) [16]. Interestingly, in our previous work, an AAAG-rich oligodeoxynucleotide (AAAG ODN) was demonstrated to interfere IRF5 activities [17]. The AAAG ODN was designed based on the sequence of human micro-satellite DNA. Coincidentally, the AAAG ODN (5′-AAAGAAAGAAAGAAAGAAAGAAAG-3′) is consensus to the IRF5 binding motif (G/C)(A)AAA(N)_2-3AAA(G/C)(T/C) in the regulatory element of TNF-α and IL-6 genes [11]. The AAAG ODN was demonstrated to rescue mice from bacterial septic peritonitis [18] and attenuate burn-induced systemic inflammatory responses in mice [19]. Moreover, the AAAG ODN was found to lessen acute lung inflammatory injury by reducing TNF-α production in the mice infected by influenza virus [17]. Together with these findings, we could propose that the AAAG ODN may alleviate the CVB3-induced myocarditis through interfering TLR9-IRF5 signaling pathway.

To confirm this, in this study, we assessed the levels of TLR9, IRF5, TNF-α and IL-6 in pericardial fluids of the VMC patients, and tested whether the AAAG ODN could lessen myocarditis by interfering TLR9-IRF5 signaling pathway in the mice. The data obtained suggest that the activation of TLR9-IRF5 pathway is involved in the pathogenesis of CVB3-induced myocarditis and inhibiting the pathway could be a novel treatment for VMC.