The Effects And Mechanisms Of Adenosine 2A Receptor (A2AR) And Metabotropic Glutamate Receptor 5 (mGluR5) In Inflammatory Response

Inflammation is a defense mechanism toward exogenous or endogenous stimulations,which could be activated through pathogen-associated molecular pattern(PAMP)or damage-associated molecular pattern(DAMP).However,excessive inflammatory response will not only aggravate the original damage,but also cause systemic inflammatory reactions and post-traumatic degenerations.Neutrophils are the main participants in acute inflammatory reaction and their functions are closely related to inflammatory modulation and development.Previous studies have shown that the enrichment of adenosine and glutamate in tissues can closely modulate inflammation by binding to adenosine A2 A receptor(A2AR)and metabotropic glutamate receptor 5(mGluR5).While it is still delusive to understand the functions and mechanisms on neutrophil mediated by these two receptors since the expressions and functions might be altered depends on different time points and disease models.In this paper,lipopolysaccharide(LPS)administration(PAMP model)and controlled cortical impact(DAMP model)were used as acute inflammatory disease models to investigate the effects of A2 AR and mGluR5 on neutrophil’s functions and related inflammatory outcomes.Section 1: PAMP inflammation modelA.In whole body systemic inflammatory response induced by LPS in mice,we found that the mortality of A2 AR knockout mice was significantly higher than that of wild-type mice,accompanied with enhanced release of pro-inflammatory factors and excessive apoptosis of neutrophils.Flow cytometry and WB analysis confirmed that the level of autophagy in neutrophils in A2 AR knockout group was significantly increased,resulting in excessive apoptosis of neutrophils mediated by autophagy.Activation of A2 AR suppressed LPS-induced autophagy by inhibiting the ROS-JNK pathway as well as promoting PI3 K β? subunit–AKT signaling in neutrophils.The A2AR-inhibited autophagy suppressed apoptosis of neutrophils,preventing inflammatory storms caused by excessively apoptotic neutrophils.B.In LPS-induced depression model,we found that depressive-like behaviors of mGluR5 knockout mice was significantly improved with a decreased level of inflammatory mediators and decreased activation of brain endothelial cells.We used the bone marrow transplantation method to selectively knock out and reconstitute mGluR5 expression of bone marrow-derived cells.It was confirmed that transplantation of mGluR5-deficient bone marrow cells to wild-type mice can significantly inhibit the release of pro-inflammatory mediators and the activation of brain endothelial cells,improving depressive-like behaviors as well.Gene chip screening further showed that the mRNA levels of inflammatory-related cytokines and chemokines from neutrophils in mGluR5 KO mice were significantly lower than those in wild-type mice.Activation of mGluR5 on neutrophil promotes cytokine release by enhancing the PLC-PKC pathway,thereby promoting the release of neutrophil inflammatory cytokines.Furthermore,neutrophil-released inflammatory mediators could promote activation of brain endothelial cells in ERK-dependent manner,enhancing neutrophils infiltration and aggravating intracranial inflammatory response simultaneously.This part indicates that in the PAMP related response,either activating A2 AR or blocking mGluR5 on neutrophils presented anti-inflammatory and protective effects.Section 2: DAMP inflammation modelA.In traumatic brain injury(TBI),we found an elevated formation of A2AR-mGluR5 complex in brain tissue after injury.Our previous study confirmed that high concentrations of glutamate in brain tissues can promote the formation of A2AR-mGluR5 complex on neutrophils.We further confirmed that the formation can be detected in neuron as well,and the functions of A2AR-mGluR5 complex were cell-specific.High concentration of glutamate can promote the formation of A2ARmGluR5 complex on neutrophils and promote the excessive release of inflammatory mediators from neutrophils.However,high concentrations of glutamate inhibit the formation of A2AR-mGluR5 complex on neurons,aggravating neuronal damage subsequently.By constructing A2 AR protein mutant plasmid and performing co-immunoprecipitation(co-IP)experiments,we showed that the A2 AR intracellular third peptide(A2AR C3)is an important fragment for A2AR-mGluR5 complex formation on neurons.By transfecting with primary isolated neurons,we proved that A2AR-mGluR5 complex formation was significantly reduced in A2ARC3 mutant group,which was accompanied with increased level of lactate dehydrogenase(LDH).Meanwhile,over-expression of wild type A2 AR plasmid leaded to increased A2AR-mGluR5 complex formation and decreased level of LDH,further confirming the protective effect of A2AR-mGluR5 complex in neurons.B.We further confirmed that the A2AR-mGluR5 complex can be formed between xenogeneic cells.Based on the results of Z-dock(protein molecular docking software),we found that there were possible amino acid binding sites of A2 AR and mGluR5 in the extracellular regions.In glutamate-induced co-culture system(A2AR-deficient neurons and mGluR5-deficient neutrophils),the formation of A2AR-mGluR5 complex between heterologous cells was still detective in vitro.By constructing traumatic brain injury of chimeric mice(brain tissue A2 AR knockout combined with bone marrow cell mGluR5 knockout),we showed the formation of the A2AR-mGluR5 complex between heterologous cells in vivo.In the above-mentioned co-culture system,the differences in nerve repair-related genes mediated by heterologous A2AR-mGluR5 complex were analyzed by gene chips.Combined with Realtime-PCR in vivo and in vitro,we found that the mRNA levels of neuropilin-1(NRP1)and protein phosphatase regulatory subunit 12A(PPP1R12)might be the target genes mediated by heterologous A2AR-mGluR5 complex,leading to neuron damage caused by neutrophils.This part indicates that in acute traumatic brain injury(DAMP related response),A2AR-mGluR5 dimer(homologous complex)can be formed on neutrophils and neurons respectively.For neutrophils,the complex formation promotes the release of inflammatory cytokines.For neurons,the complex prevents excessive damage.Meanwhile,heterologous A2AR-mGluR5 complex between neutrophils and neurons can be formed as well under high concentration of glutamate,inhibiting neuronal repair by down-regulating NRP1 and PPP1R12.