A Role for Toll-like Receptor 3 Variants in Host Susceptibility to Enteroviral Myocarditis and Dilated Cardiomyopathy

Abstract

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3␤ (MAP1LC3␤). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3␤ fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.Virus-induced myocarditis is an important cause of morbidity and mortality (1, 2). The enteroviruses have been considered the most common etiologic agents; however, other viruses, such as the adenoviruses, have also been implicated (3). The advent of molecular hybridization and PCR techniques has directly demonstrated infection of the myocardium with these viruses and has provided evidence of a viral etiology for dilated cardiomyopathy (DCM) 5 (1, 3). These findings further support the hypothesis that DCM is in some cases a long term sequela of acute or chronic myocarditis (4, 5), either due to the pathogenic effect of persistent viral replication or to an ongoing autoimmune process secondary to the viral infection.The recognition of viruses or viral particles by the host triggers the activation of an innate immune response that is characterized by the production of mediators such as tumor necrosis factor-␣, interleukins, interferons, and nitric oxide, all of which are toxic to replicating viruses (6). This initial antiviral response by the host is now known to be mediated at least in part by Toll-like receptors (TLRs) (5, 6). It has been shown that double-stranded (ds) RNA is the primary ligand for TLR3 (7). Although most TLR signaling pathways utilize the adaptor molecule MyD88 (myeloid differentiation primary response gene 88), TLR3 signals through the adaptor molecule called Toll-like receptor adaptor molecule 1 (Ticam1) or Toll/Interleukin 1 receptor domain-containing adapter-inducing interferon ␤ (TRIF) (8, 9). Activation of this pathway triggers produ...

A Common Polymorphism in TLR3 Confers Natural Resistance to HIV-1 Infection

“…As mentioned earlier, this latter phenotype has also been shown to occur with increased frequency in subjects homozygous for the 412Phe allele. Consistently, in an in vitro system, cells expressing TLR3 412Phe molecules allow increased replication of coxsackievirus B3 compared with 412Leu receptors (5). Similarly, our data are consistent in showing a role for the Leu412Phe variant in HIV-1 infection susceptibility, but the allelic effect is exactly the opposite of that described in enteroviral infection.…”

“…Thus, the huge amount of immunological factors released in response to HIV-induced TLR3 stimulation may be able to overwhelm the virus and inhibit the infection process. Again, this latter finding contrasts with previous data indicating that ectopic expression of TLR3 412Phe molecules results in blunted immune responses after agonist stimulation (4,5). A major difference between the results we report in this study and previous functional characterization of TLR3 variants (4, 5) resides in our using an ex vivo approach rather than relying on cell line transfection experiments.…”

“…A major difference between the results we report in this study and previous functional characterization of TLR3 variants (4, 5) resides in our using an ex vivo approach rather than relying on cell line transfection experiments. Indeed, previous data on TLR3 activity have been obtained using HEK293 or COS7 cells that were driven to express the receptor from an exogenous promoter (4,5). Thus, some tiers of physiological regulation might be lost in cells that overexpress the two TLR3 variants.…”

“…This variant was shown to be functional and to be associated with different human phenotypes, including viral infection susceptibility (5,6). Thus, we wished to verify whether rs3775291 modulates the predisposition to HIV-1 infection.…”

“…The minor 412Phe allele occurs with a frequency of ∼30% in populations with European and Asian ancestry, whereas it is virtually absent in Africans (3). In vitro analyses have suggested that TLR3 molecules carrying the 412Phe allele display reduced activation after poly(I:C) stimulation and allow increased coxsackievirus replication compared with 412Leu TLR3 receptors (4,5). Genetic association studies have provided mixed evidence concerning the role of Leu412Phe in viral infection susceptibility.…”