MGluR5-A Preliminary Study Of The New Target Of LPS’s Action On Microglia

The observations that immune responses are blunted in the central nervous system(CNS) and that the blood-brain barrier (BBB) prevents infiltration of immune cells andmolecules into the CNS originally established the belief that the nervous and immunesystems were isolated with little interaction except during disease and/or trauma.Recently,however, a true paradigm shift in our understanding of neuralimmune interactions hasoccurred due to the convergence of unexpected results from diverse fields showing clearand convincing evidence of bidirectional communication between these systems.Neuronal pathways are always involved in the control of the humoral and cellularimmune responses.The immune system, in turn, influences the central nervous systemprimarily through cytokines. So far, the communication between neuronal system andimmune system has been believed through the neuronal transmitters or immune cytokines,however since both immune receptors and nerve receptors can be expressed in neuron orinnate immune cell, such as microglia, whether the two kinds of receptor could interactionby protein-protein interaction directly has not been reported till now. Therefore, we try toexplore the possibility that the interaction between the immune receptor and never receptorfocus on the typical innate immune cell in brain.Microglial cells are the resident macrophages of the brain that perform surveillancefunctions to maintain the integrity of central nervous system (CNS). In response to a widerange of invading pathogens, microglia mount innate immune responses that can becharacterized by the production of cytokines and chemokines, up-regulation of cell surfacemolecules, and expansion of local immune responses. During the early onset of an infection,microglia rapidly become activated and robustly produce proinflammatory cytokines andchemokines. Production of these immune mediators may result in the infiltration of variousleukocytes across the blood-brain barrier to sites of infection. The effective functioning ofmicroglial cells is critical for controlling neuroinflammation and alleviatingneuropathogenesis. Neuron-microglia interactions play a pivotal role in controlling microglial functionsand restraining their activation. Neurons are able to influence microglial through the releaseof soluble factors as well as through direct cell-to-cell interactions mediated by membranebound antigens and their cognate receptors. Among the neuronal signals that may play anactive role in controlling microglial activation are neurotransmitters. Similar to neurons,microglial express a number of neurotransmitter receptors that enable microglial to respondto the same signals acting on neurons and, through such signals, influence neuronalresponses.Microglia endogenously express both neurotransmitter receptors and immunereceptors. The interactions between neurotransmitter receptor pathways and immunereceptor pathways in microglia are explored in this dissertation. Three types of microgliawere used:1Microglia cell line N9, with normal TLR4(Toll-like receptor4);2Microgliacell line EOC20, where TLR4carries a point mutation;3Primary cultures of microgliafrom TLR4deficient C57BL/10ScNJ mice.First, we studied the interaction between TLR4and NMDAR1(N-methyl-D-aspartatereceptor1),TLR4and D2DR(Dopamine D2receptor). Right after the interaction of TLR4and NMDAR1had been proved, we found that the LPS can trigger calcium oscillation inmicroglia. As we all know, calcium oscillation closely related with the mGluR5(metabotropic glutamate receptor5). The following experiments focused on themGluR5.Co-immunoprecipitation, fluorescence immuno-colocalization, FRET(fluorescence resonance energy transfer), NF-κB (Nuclear factor-κB) reporter gene andELISA (enzyme-linked immunosorbent assay) were used to examine the function of themGluR5receptor and the interaction among LPS(lipopolysaccharides), mGluR5, NMDAR1and TLR4in response to LPS stimulation.MAIN RESULTS AND CONCLUSIONS1D2DR receptors were expressed in N9and EOC20microglia cell lines normally.Upon LPS stimulation, the expression level of D2DR has been increased. However,TLR4/D2DR interactions were not detected by co-immunoprecipitation.2Both N9and EOC20cell lines could express TLR4receptors. The co-expression ofmGluR5and NMDAR1have been found in culture N9, EOC20, as well as TLR4-deficientprimary microglia. 3Upon LPS stimulation, in N9and EOC20cells, LPS could interact directly withmGluR5, which indicated that other signal transduction pathway(s) for LPS in addition tothe TLR4pathway except the classic LPS-TLR4pathway.4In N9, EOC20and TLR4-deficient primary microglia cells, LPS could induceoscillation of intracellular free calcium, which can be blocked by mGluR5antagonists. Theresults suggest activation of mGluR5as a direct mechanism for LPS to induce intracellularcalcium oscillation.5Upon LPS stimulation in N9, EOC20and TLR4-deficient primary microglia cells,phosphorylation of Ser896(Serine896) was detected in NMDAR1. Such phosphorylationwas eliminated by administration of mGluR5antagonists. These results suggested that LPSinduced NMDAR1phosphorylation by directly activating mGluR5.6LPS stimulation on N9and EOC20cells led to protein-protein interactions betweenTLR4and NMDAR1, which was abolished by mGluR5antagonists.7NF-κB activity was increased in LPS stimulated N9and EOC20cells, suggestingthe presence of other pathways leading from LPS to NF-κB, beside the TLR4mechanism.8Activation of mGluR5was able to attenuate TNF-α(tumor necrosis factor-α)secretion in response to LPS, suggesting involvement of mGluR5in anti-inflammatoryreactions, which can serve as a novel target for treating inflammatory complications.To summarize, we conclude that in microglia, LPS directly activates mGluR5inaddition to its classic activation of the TLR4pathway. mGluR5activation by LPS led tooscillation of intracellular free calcium and phosphorylation of NMDAR1at Ser896, whichresulted in NMDAR1translocation from the ER (endoplasmic reticulum) to the plasmamembrane, where NMDAR1and TLR4directly interact.