Dopamine Signaling Inhibits S.aureus Infection-induced Inflammation In Macrophages Via DRD5 Receptor

Obejectives: DA(Dopamine)is an important catecholamine neurotransmitter and its deficiency is closely related to Parkinson's disease.In addition,DA plays a critical role in regulating the immune system.Previous studies indicate that patients with Parkinson's disease are prone to be infected by nosocomial bacterial,such as Staphylococcus aureus(S.A)and severe patients with Parkinson's disease even have the symptoms of sepsis.However,the functional relevance and mechanistic basis to the effects of the neurotransmitter DA on inflammation remains unclear.This study aims to explore the important role and mechanism that DA may play in the inflammatory responses caused by S.A infection,and provide new strategies for clinical treatment of inflammation-related diseases such as sepsis and meningitis.Methods:1)We treated bone marrow-derived macrophages(BMDMs)with Pam3CSK4(Pam3),a TLR2 receptor ligand that mimics the pathogenic molecule of S.A,or S.A and analyzed the Pam3 or S.A-induced expression of cytokines such as IL-6 and TNF-a at both m RNA and protein levels after stimulation of DA.2)We performed RNA sequencing(RNAseq)to analyze the transcriptional profiles of Pam3-stimulated BMDMs treated in the absence or presence of DA,and verified the changes of related pathways by Western blot.3)We screened the potential role of each of the 5 DRD subtypes by using DRDs knockout BMDMs and DRDs selective agonists.4)Co-immunoprecipitation experiments,mutagenesis assays and pull down assays were further used to determine the domains of dopamine receptors that inhibit inflammations.The domains that inhibit inflammations were confirmed in BMDMs by rescue experiments.5)The role of dopamine in the inflammatory response was verified in vivo through mouse models such as sepsis and meningitis.Results:1)We observed that DA inhibited Pam3 or S.A-induced the expression of proinflammatory cytokines.2)RNAseq analysis and Immunoblot analysis showed that DA specifically inhibits TLR2-mediated NF-k B signaling.3)DA inhibits TLR2-induced inflammation specifically via DRD5 receptor in macrophages.4)We found that DRD5 receptor,via EFD and IYX(X)I/L motifs in its C-tail and IC3 loop respectively,can directly recruit TRAF6 and its negative regulator ARRB2 to form a multi-protein complex also containing downstream signalling proteins such as TAK1,IKKs and PP2 A,that impairs TRAF6-mediated activation of NF-kB and expression of pro-inflammatory genes.5)We found that DA could decrease the production of inflammatory cytokines in vivo to protect against S.A-induced septic shock and evaluated mice survival and DRD5 agonist SKF-38393 treatment could control neuroinflammation after S.A-induced meningitis.Conclusion: In summary,after DRD5 is activated by DA,the negative regulatory protein ARRB2 / PP2 A is recruited into the TRAF6-IKK complex in the TLR2 signaling through the DRD5 receptor EFD and IYX(X)I/L domain,thereby controlling the NFkB inflammation and preventing Sepsis and meningitis caused by Staphylococcus aureus.These findings identify DA-DRD5 axis might be a potential target for future therapeutics of inflammation-associated diseases.