Abstract
BackgroundToll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant expression in microglia, the major immune cells of the brain. Although individual TLRs have been shown to contribute to CNS disorders, the consequences of multiple activated TLRs on the brain are unclear. We therefore systematically investigated and compared the impact of sole and pairwise TLR activation on CNS inflammation and injury.MethodsSelected TLRs expressed in microglia and neurons were stimulated with their specific TLR ligands in varying combinations. Cell cultures were then analyzed by immunocytochemistry, FlowCytomix, and ELISA. To determine neuronal injury and neuroinflammation in vivo, C57BL/6J mice were injected intrathecally with TLR agonists. Subsequently, brain sections were analyzed by quantitative real-time PCR and immunohistochemistry.ResultsSimultaneous stimulation of TLR4 plus TLR2, TLR4 plus TLR9, and TLR2 plus TLR9 in microglia by their respective specific ligands results in an increased inflammatory response compared to activation of the respective single TLR in vitro. In contrast, additional activation of TLR7 suppresses the inflammatory response mediated by the respective ligands for TLR2, TLR4, or TLR9 up to 24 h, indicating that specific combinations of activated TLRs individually modulate the inflammatory response. Accordingly, the composition of the inflammatory response pattern generated by microglia varies depending on the identity and combination of the activated TLRs engaged. Likewise, neuronal injury occurs in response to activation of only selected TLRs and TLR combinations in vitro. Activation of TLR2, TLR4, TLR7, and TLR9 in the brain by intrathecal injection of the respective TLR ligand into C57BL/6J mice leads to specific expression patterns of distinct TLR mRNAs in the brain and causes influx of leukocytes and inflammatory mediators into the cerebrospinal fluid to a variable extent. Also, the intensity of the inflammatory response and neurodegenerative effects differs according to the respective activated TLR and TLR combinations used in vivo.ConclusionsSole and pairwise activation of TLRs modifies the pattern and extent of inflammation and neurodegeneration in the CNS, thereby enabling innate immunity to take account of the CNS diseases’ diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0166-7) contains supplementary material, which is available to authorized users.
Highlights
Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules
Pairwise stimulation of TLRs induces an inflammatory response in microglia that differs from sole TLR activation in vitro In immune cells, stimulation of TLRs with their respective specific ligands initiates the canonical signaling pathway, which results in activation of transcription factors, including NF-κB, and leads to the secretion of proinflammatory molecules [1]
Microglia isolated from C57BL/6J mice were incubated with LPS as a highly specific ligand for TLR4, Pam3CysSK4 as a specific TLR2 agonist acting mainly through TLR2/1 heterodimeric receptors, loxoribine as a ligand for TLR7, or CpG cytosine-phosphate-guanine motifs (ODN) as a TLR9-specific agonist solely or in pairwise combination, as indicated, for up to 72 h
Summary
Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant expression in microglia, the major immune cells of the brain. The family of Toll-like receptors (TLRs) comprises germline encoded pathogen recognition receptors that allow the innate immune system to differentiate among microorganisms by sensing their conserved motifs called pathogen-associated molecular patterns [1]. Engagement of TLRs by their cognate ligands leads to the recruitment of one or more of five intracellular adaptor proteins, including myeloid differentiation primary response gene 88 (MyD88) and TIR-domaincontaining adaptor inducing IFN-β (TRIF). One major TLR-induced set of responses is the activation of transcription factors, such as NF-κB, leading to the induction of proinflammatory mediators and type I interferons [1]
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