Abstract
The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.
Highlights
Neuroinflammation, including cytokine/chemokine production by resident glial cells, is a common response to various types of central nervous system (CNS) insults, including viral infections [1,2,3,4,5]
Analysis of purified astrocytes or microglia indicated toll-like receptor 7 (TLR7) and TLR8 expression by both cell types, on microglia TLR8 was expressed at higher levels than TLR7 (Fig. 1B, C)
Time course analysis of mixed cortical cells stimulated with the TLR7/8 agonist CL075 showed four primary profiles of gene expression. mRNA expression of G-protein coupled receptor 84 (Gpr84) and type I interferon beta (Ifnb1) peaked within 3 hours post stimulation (Fig. 2A, B), while mRNA expression of proinflammatory cytokines such as Ccl2 and Tnf peaked within 6– 12 hps (Fig. 2C, D). mRNA for the microglia activation marker F4/ 80 was upregulated at later time points with peak expression at or after 48 hps (Fig. 2F)
Summary
Neuroinflammation, including cytokine/chemokine production by resident glial cells, is a common response to various types of central nervous system (CNS) insults, including viral infections [1,2,3,4,5]. The host recognizes viral infections through the detection of pathogen-associated molecular patterns (PAMPs), repeated structural motifs generated by microbes that are not normally found in the host [6,7]. These PAMPs are recognized by pattern recognition receptors (PRRs) expressed by multiple cell types including dendritic cells and macrophages. Two PRRs that are important for the recognition of viruses are toll-like receptor 7 (TLR7) and TLR8 These two receptors are closely related endosomal TLRs that recognize guanosine and uridine-rich viral ssRNA, including RNA from virus families that are known to infect the CNS and induce neurological disease [8,9,10,11]. TLR7 and TLR8 can be stimulated by synthetic molecules like imidazoquinoline compounds and guanosine analogs, which are currently used as anti-viral therapeutics [12,13,14]
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