Cells Stimulated with More Than One Toll-Like Receptor-Ligand in the Presence of a MyD88 Inhibitor Augmented Interferon-beta via MyD88-Independent Signaling Pathway

Host exposure to pathogens engage multiple pathogen recognition receptors (PRRs) including toll-like receptors (TLRs); recruit intracellular signaling adaptor proteins primarily myeloid differentiation primary response protein 88 (MyD88) for activating downstream signaling cascades, which culminate in the production of type I interferons (IFNs), proinflammatory cytokines, and chemokines; and impede pathogen replication and dissemination. However, recent studies highlight that absence of MyD88 increased antiviral type I IFN induction, and MyD88(-/-) mice showed a higher survival rate compared with the low survival rate of the MyD88(+/+) mice, implicating MyD88 limits antiviral type I IFN response. As a single infectious agent may harbor multiple PRR agonists, which trigger different sets of TLR-initiated immune signaling, we examined whether MyD88 inhibition during stimulation of cells with more than one TLR-ligand would augment type I IFN. We stimulated human U87- and TLR3-transfected HEK293-TLR7 cells with TLR-ligands, such as lipopolysaccharides (LPS) (TLR4-ligand) plus poly I:C (TLR3-ligand) or imiquimod (R837, TLR7-ligand) plus poly I:C, in the presence of compound 4210, a previously reported MyD88 inhibitor, and measured IFN-beta response using an enzyme-linked immunosorbent assay. Our results showed that when U87- or TLR3-transfected HEK293-TLR7 cells were stimulated with TLR-ligands, such as poly I:C plus LPS or poly I:C plus R837, IFN-beta production was significantly increased with MyD88 inhibition in a dose-dependent manner. Collectively, these results indicate that during more than one TLR-ligand-induced immune signaling event, impairment of antiviral type I IFN response was restored by inhibition of MyD88 through MyD88-independent pathway of type I IFN signaling, thus, offer a MyD88-targeted approach for type I IFN induction.