Innate immune pathways are early responses important for pathogen control, and are activated by pattern recognition receptors (PRRs) that bind ligands containing pathogen- or danger-associated molecular patterns, such as modified carbohydrate or nucleic acid structures (1
). For antiviral innate immune responses, ligation of these receptors induces a signal transduction cascade that results in the production of type I IFNs, other proinflammatory cytokines, and cell-intrinsic factors important for the generation of an antiviral cellular microenvironment (2
). In addition, antiviral PRR signaling is important for activating an appropriate adaptive immune response, which is required for the eventual clearance of many viral infections. Thus, PRR-mediated innate immune pathway signaling serves a pivotal role in stimulating rapid yet nonspecific antiviral activity while also providing activation signals for the production of more specialized adaptive immune responses.
There are three general steps in innate antiviral immune responses: activation, amplification, and effector production. Antiviral PRR signaling is initiated by a variety of receptors, including the transmembrane TLR proteins 2, 3, 4, 7/8, and 9, and the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) RIG-I and melanoma differentiated-associated gene 5 (MDA5) (2
). TLR3, TLR7/8, and TLR9 recognize the non-self nucleic acid moieties dsRNA, ssRNA, and hypomethylated CpG DNA, respectively, whereas TLR4 recognizes viral glycoproteins and the viral ligand for TLR2 remains to be identified. In the cytoplasm, RIG-I binds double-stranded 5′ triphosphorylated RNAs, homopolymeric RNA motifs, and short dsRNAs less than 2 kb in length (3
), whereas MDA5 recognizes complex dsRNAs greater than 2 kb in length (5
) that can be mimicked by the synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly(I-C)) (6
). Due in part to this ligand specificity, PRRs differentially recognize and respond to distinct viral infections (1
). After PRR ligation, signal transduction is mediated by several distinct adaptor proteins, including MyD88, TIR-domain-containing adapter-inducing IFN-β (TRIF), and IFN-β promoter stimulator protein 1 (IPS-1; also referred to as Cardif, MAVS, and VISA) (1
). These adaptor protein complexes activate the transcription factors NFκB and IFN regulatory factor 3 (IRF3) via multiple downstream kinases. Activated NFκB and IRF3 subsequently upregulate the expression of many genes important for mounting a robust antiviral response, including type I IFNs (7
), which function in either a paracrine or autocrine manner to induce IFN-stimulated genes that contain IFN-stimulated response elements (ISREs) within their promoters. There are several IFN-stimulated genes that act directly as antiviral effectors, but many are also components of antiviral PRR pathways, which provides a mechanism for positive feedback regulation and amplification (1
The molecular mechanisms of antiviral PRR signaling have been defined primarily using a limited number of cell lines and primary cell types, many of which are derived from small rodent models and are professional immune cells such as dendritic cells or macrophages. These studies have revealed important cell type-specific differences in antiviral PRR pathways. For example, dendritic cells express relatively high basal levels of TLR7 and TLR9, and as a result vigorously respond to ligands for these receptors (8
). In contrast, “non-professional” immune cells such as fibroblasts use primarily cytoplasmic RLRs for innate antiviral pathway activation (9
), although some cell types such as keratinocytes (10
) and respiratory epithelial cells (11
) can also mount vigorous TLR-mediated antiviral responses. Plasmacytoid dendritic cells also constitutively express the transcription factor IRF7, which is thought to contribute to their ability to produce IFNα rapidly after PRR-mediated stimulation (8
), whereas IFNα production in other cell types occurs later if at all, and is linked to IFNβ-mediated induction of IRF7 (14
). Additional examples of cell type-specific differences in innate antiviral immunity include a lower basal activity of PRR pathways in cardiac fibroblasts compared cardiac myocytes (17
), differential responses of specific human hepatocyte cell lines to poly(I-C) and Sendai virus (SeV) stimulation (18
), and cell type-specific roles for IRF3 and IRF7 in response to West Nile virus infection (19
). Furthermore, species-specific differences also exist with respect to TLR expression, regulation, and function (21
). These observations suggest that caution should be exercised in extrapolating results on innate antiviral pathway activity between species and cell types.
Viruses from several families preferentially infect CNS neurons, and the extent of neurotropic virus-mediated cell death can be an important determinant in the severity and clinical outcome of infection (25
). Thus, an effective neuronal innate antiviral response that controls virus replication until an adaptive immune response can be generated may be crucial to prevent the essentially irreversible loss of these critical cells. However, we have limited knowledge regarding the PRR antiviral pathways that are active in CNS neurons. TLR3 expression has been reported in human neurons (26
), West Nile virus replication is enhanced in cortical neurons isolated from TLR3−/−
), and neural progenitor cells respond to poly(I-C) stimulation by reducing proliferation and neurosphere formation in a TLR3-dependent manner (32
). Furthermore, studies have demonstrated virus-mediated induction of type I IFNs in CNS neurons both in vitro (19
) and in vivo (33
). In addition to putative antiviral functions, PRR pathways have been implicated in neuronal development (34
), neuronal regeneration (28
), and neuroinflammatory diseases (36
). Altogether, these reports suggest that CNS neurons possess active PRRs that may have multiple physiologic functions, but the full extent of their activity and the downstream components that mediate their activation remain to be determined.
In this report, we use both global and targeted approaches to examine PRR expression and pathway activity in response to RLR and TLR ligands. We found that human neuronal cells show differentiation-dependent selective responses to TLR3-, TLR4-, MDA5-, and RIG-I-mediated stimulation. Furthermore, detailed genetic and pharmacologic studies revealed that select neuronal innate immune pathways were dependent on PI3K activity. These results demonstrate that human neuronal cells are immunologically active and possess specific and non-redundant functional PRR pathways that may play a protective role in neurotropic virus pathogenesis.