Role Of TLR3/TRIF Signaling Pathway On Immune Inflammation In MPTP Mice

Parkinson’s disease(PD) is a chronic neurodegenerative diseases characterized by the progressive degeneration and Lewy’s body formation of dopaminergic(DA) neurons in the Substantia nigra pars compacta(SNpc) of the midbrain, leading to dopamine deficiency in striatum.A large number of studies in vivo and in vitro have shown that inflammatory response plays an important role in Parkinson’s disease and is a key pathological hallmark of PD. Autopsy, genetics investigation, retrospective studies and molecular imaging all point to a fact that inflammation plays a key role in the onset and development of PD. Moreover, high expression of inflammatory cytokines such as IL-1β and TNF-α was found in both autopsy and MPTP induced animal models.Microglia are recognized as the most important resident brain immune cells in the central nervous system(CNS) and are key players in immune surveillance, immune regulation and phagocytic recognition. Microglia activation is heterogeneous, which can be divided into M1(proinflammatory phenotype) and M2(anti-inflammatory phenotype). The classical pro-inflammatory microglia M1 reactive phenotype(M1phenotype) may exacerbate the damages to dopaminergic neurons by releasing large amounts of inflammatory cytokines and reactive oxygen species. Whereas, the alternative microglia M2 reactive phenotype(M2 phenotype) somehow is intended to antagonize the pro-inflammatory functions of M1 microglia, thus inhibiting the inflammatory responses and alleviate the dopaminergic neuron damage.Toll-like receptors(TLRs) are important conservative receptors. Activation of TLR3/TRIF signaling pathway is involved in neuroinflammatory response. In the central nervous system, Toll-like receptors are expressed mainly in the glial cells exerting immune function. Among all TLRs, TLR3 is merely TRIF-dependent and TLR3/TRIF signaling pathway activation may contribute to PD pathogenesis via potential regulation of polarization of microglia M1/M2 phenotypes.At present, the mechanism underlying PD pathogenesis remain unclear.Undoubtedly,polarization of microglia M1/M2 phenotypes are involved in the onset and progression of PD. However, the end initiating factor and definite mechanismunderlying polarization of microglia M1/M2 phenotypes are not clear and rarely addressed. Therefore, reorganization of polarization of microglia M1/M2 phenotypes are of significant importance for understanding PD pathogenesis and further providing novel anti-inflammatory strategies for PD treatment. In conclusion, exploration on polarization of microglia M1/M2 phenotypes are expected to be a issue that need to be urgently addressed.ObjectivesIn this study, PD animal model was established in C57BL/6 mice, which were then intervened with TLR3 agonists Poly(I:C) and resveratrol. This study aims to explore the role of TLR3/TRIF signaling pathway in the inflammatory response involved in PD and effect of resveratrol on TLR3/TRIF signaling pathway and inflammatory microenvironment in SNpc in MPTP-induced PD animal model.MethodsIn the first part of the experiment, sex and age matched wild-type male adult C57BL/6 mice(20 to 25 g) underwent intraperitoneal injection of MPTP(35mg/kg) to build subacute PD animal model, to which brain stereotaxic injection of TLR3 agonists Poly(I:C) to the striatum was performed. Immunohistochemical technique was applied to observe the morphology and numbers of microglia, astrocytes as well as dopaminergic neurons. In addition, the expression levels of TLR3 and TRIF in SNpc were also detected using immunohistochemistry. The mRNA expression levels of proteins of TLR3/TRIF signaling pathway and markers of microglia M1/M2 phenotypes in SNpc were measured using fluorescence quantitative PCR. This part of the experiment sought to explore the potential role of TLR3/TRIF signaling pathway in inflammatory response in MPTP-induced PD mice. In the second part of the experiment, subacute PD animal model was established by intraperitoneal injection of MPTP(35mg/kg) in sex and age matched wild-type male adult C57BL/6 mice(20 to 25 g), followed by resveratrol treatment. Subsequently, microglia and astrocytes activation states in SNpc were visualized using immunohistochemistry. In addition, inflammatory cytokines in SNpc were detected using fluorescence quantitative PCR. technique. This attempt aimed to explore the effect of resveratrol on TLR3/TRIF signaling pathway and inflammatory microenvironment underlying PD pathogenesis in MPTP-induced PD mice.ResultsA. Effect of TLR3/TRIF signaling pathway on inflammatory response inMPTP-induced PD micea. Immunohistochemical results showed that the numbers of DA neurons were significantly reduced and synapses were decreased or even disappeared following MPTP treatment. The activated microglia presenting hypertrophy of cell body in circular or elliptic shape, enlarged but shorter processes were seen under microscope.Additionally, the activated astrocytes showing hypertrophy in a spindle, linear, oval or round shape, increased and enlarged processes occurred after MPTP treatment.Meanwhile, TLR3 and TRIF expression levels were elevated significantly(P < 0.05).b. qPCR results demonstrated that the mRNA expression levels of TLR3/TRIF signaling pathways related proteins(TLR3, TRIF and IFN-?) were significantly increased 7 days after Poly(I:C) intervention. Meanwhile, the mRNA expression levels of M1(TNF-α, IL-6, CD86 and iNOS) microglia markers showed a significant increasing tendency. Nevertheless, dominant elevation of M2( IL-10, CD206, Arg1 and YM1) occurred 14 days after Poly(I:C) treatment.B. Impact of resveratrol on TLR3/TRIF signaling pathway and inflammatorymicroenvironment underlying PD pathogenesis in MPTP-induced PD micea. Immunohistochemical results revealed,as compared with the MPTP group, no obvious changes in morphology and numbers of DA neurons. At the same time,relatively smaller cell bodies of microglia presenting round or oval shape as well as thinner and longer processes were seen in the resveratrol group as compared with the MPTP group. After resveratrol treatment, the astrocyte exhibited smaller cell body size in circular shape, shorter processes in SNpc as compared with the MPTP group.b. qPCR results unveiled that the mRNA expression levels of TLR3 and IFN-βwere significantly increased following resveratrol administration as compared with the MPTP group,but the mRNA expression of TRIF no significant change. Moreover,mRNA expression levels of proinflammatory cytokines including iNOS and TNF-α was significantly reduced(P<0.05), whereas, the mRNA expression levels of anti-inflammatory cytokines Arg-1 was distinctly increased.Conclusions1. TLR3/TRIF signaling pathway can activate both the microglia and astrocytes in MPTP-induced PD mice. In addition, at the late stage of neuroinflammatory process,activation of TLR3/TRIF signaling pathway can increase expression of anti-inflammatory cytokines, delivering a neuroprotective effect.2. Resveratrol might suppress proinflammatory cytokines secretion in SNpc via inhibiting activation of microglia and astrocytes as well as activating the TLR3/TRIF signaling pathway. reduce the midbrain black the release of proinflammatory factor,thus improving inflammatory microenvironment in MPTP- induced PD model of mice.