In this study, we have shown that in addition to TLR2, 4 and 9 (11
), activation of TLR3 or TLR7 promoted the expression of functional mFPR2 in murine microglial cells through MAPK and NF-κB dependent signaling pathways. The induction of mFPR2 enabled microglial cells to migrate in response to various peptide agonists, including Aβ42
associated with AD (16
). Activated microglial cells also increased their capacity to endocytose Aβ42
in an mFPR2 dependent manner. More importantly, we showed that suboptimal concentrations of TLR3 and TLR7 agonists synergistically cooperated to activate microglial cells through both MyD88- and TRIF-signaling cascades which converge by activation of the key transcription factor NF-κB.
Cooperation between TLRs in cellular responses is common in mammalian cells. Combination of Poly(I:C) and CPG DNA synergistically induced TNF, IL-6 and IL-12p40 in mouse macrophages (21
), and combination of TLR9 with TLR4 agonists promotes IL-12 release by murine DCs at levels higher than the activity of each agonist used alone (32
). Other reports showed that gene expression and protein secretion of TNF, IL-1β, IL-6, IL-10, IL-12, IL-23 and cyclooxygenase-2 (COX2) were several-fold higher in DCs stimulated with combinations of TLR ligands than in cells stimulated with a single agonist (17
). In general, stimulation of TLRs by agonists activates two downstream MyD88-dependent and -independent signaling pathways. MyD88 is the immediate adaptor molecule that is common to all TLRs, with the exception of TLR3. MyD88 recruits IL-1R-associated kinase and TNFR-associated factor 6 (TRAF6) leading to activation of the canonical IκB kinase (IKK)αβγ complex. IKKβ phosphorylates IκB-α resulting in its degradation and the release of NF-κB for nuclear translocation. LPS and Poly(I:C) also activates Toll/IL-1R domain-containing adaptor (TRIF; TICAM-1). This pathway is independent of MyD88, leading to delayed activation of NF-κB. TRIF also activates the transcriptional regulator, IFN regulatory factor (IRF)3 and the expression of IFN-β and IFN-inducible genes through the activation of TANK-binding kinase (TBK)1 and IKKε. TLR3 primarily uses the TRIF pathway, whereas LPS interaction with TLR4 activates both MyD88- and TRIF-dependent pathways. It has been demonstrated that all MyD88-dependent TLR agonists synergize with Poly(I:C) in vitro in inducing TNF and IL-6 production by mouse bone marrow-derived macrophages (18
). Our previous studies showed the cooperation between TLR2 and NOD2 as well as IFNγ and CD40 ligand in promoting mFPR2 expression by microglial cells (13
). The present study extended the scope of interaction between proinflammatory stimulants by showing a marked synergistic effect of combination of suboptimal concentrations of TLR3 and TLR7 agonists in the induction of mFPR2 in microglial cells.
The cooperation between TLR7 and TLR3 in activating microglial cells may have important pathophysiological consequences. Many encephalitic retroviruses such as West Nile virus and Japanese encephalitis virus produce dsRNA during replication in the CNS (35
). Microglia recognize dsRNA through TLR3 and therefore are a key sensor of dsRNA-producing viruses invading the CNS. Murine TLR7 and human TLR8 interact with guanosine (G)- and uridine (U)-rich ssRNA derived from human immunodeficiency virus-1 (HIV-1) (37
). As one of the consequences of such interaction, our present study reveals the induction of mFPR2 by TLR3 and TLR7 engagement with dsRNA and ssRNA, bacterial RNA, and endogenous ligands such as mitochondrial RNA and mRNA released from necrotic cells. Thus, TLRs on microglial cells interact with proinflammatory signals and orchestrate host responses in the CNS in the presence of multiple proinflammatory signals. It should be noted that recently other molecules such as RIG1 have been reported to sense dsRNA and mediate cell activation originally attributed to TLR3 (39
). However, structure/function analysis have confirmed the capacity of TLR3 to sense dsRNA that are composed of more than 40 nucleotides. In contrast, RIG1 mainly interacts with dsRNA of shorter sequences (40
). Thus viral dsRNA was able to interact multiple sensors in the host, which may favor the host anti-viral response.
Upon activation by proinflammatory or injurious stimulants, microglial cells assume a typical macrophage phenotype and secrete a variety of cytokines and chemokines involved in host defense against microbial infection and inflammatory responses. Microglial cells express a plethora of TLRs and are ready to sense PAMPs specific for these receptors. In fact, results from our previous and present studies indicate that PAMPs, including molecules from Gram-positive and –negative bacteria, CPG-containing DNA from bacteria and virues, as well as viral dsRNA and viral ssRNA, all are capable of inducing functional mFPR2 in microglial cells, suggesting that mFPR2 or the human homologue FPRL1 is one of the major target genes, whose transcription and translation are subject to rapid up-regulation in pathological processes where multiple TLR agonists may be present. A growing body of evidence shows that TLR-activated microglia that express mFPR2/FPRL1 actively participate in the uptake and processing of Aβ42
, which is a chemotactic agonist for mFPR2 and has direct neurotoxic effects and also stimulates microglia to release neurotoxic mediators in the AD brain (16
). While microglial endocytosis of Aβ42
has been shown to be important for the “lay-down” of fibrillary Aβ aggregates to form the cores of senile plaques seen in the AD brain, recent studies using TLR gene deletion approach demonstrated that TLR-activation is crucial for mouse microglia to be able to endocytose and degrade Aβ42
peptides in a manner depended on the induction of a G protein-coupled receptor (15
) and mFPR2 is likely to be the responsible receptor. Thus, based on its promiscuous nature of ligand recognition, mFPR2 in activated microglia may play central roles in innate host defense in the brain and actively participate in the course of AD pathogenesis.