GABAergic Synapses Suppress Intestinal Innate Immunity Via Muscular Insulin Signaling

Innate immunity is the first line of host defense against pathogen infection in metazoans,which is an evolutionarily conserved defense mechanism.Increasing evidence indicates that the immune response extensively rely on the regulation of nervous system.GABAergic neurotransmission is a major inhibitory signalling and plays crucial roles for physiology in central nervous system,immunomodulation in immune cell and is implicated in autoimmune diseases.However,its roles in innate immunity remain unclear.In this study,by using the host-pathogen infectious model of Caenorhabditis elegans to the human conditioned pathogen-Pseudomonas aeruginosa,the mechanisms of GABAergic nervous system mediating innate immune regulation were systematically studied from the following aspects :Firstly,we used the killing assay to screen the loss-function mutants of presynaptic and postsynaptic genes involved in GABAergic neurotransmission,and found that the deficiency in GABAergic neuromuscular junctions such as loss function of GABAA receptor UNC-49(GABAAR/UNC-49)enhances the resistance to PA14 infection.Then,CFU experiment and imaging experiment after PA14-GFP feeding were used to confirm that GABA signal regulates the immune response by affecting the host’s ability to clear intestinal pathogens.Since nematodes contain 26 GABA neurons and form synaptic connections with multiple tissues,we further used tissue-specific and GABA neuron-specific rescue assay and the expression of Tetx toxic protein to inhibit synaptic transmission to confirm that D-type motor neurons in GABA system mediate immune regulation through body wall muscle(BWM).To explore whether pathogen infection can affect the function of GABAergic neurotransmission,we conducted fluorescence imaging analysis of transgenic worms labeled with synaptic component proteins before and after PA14 infection,and found that pathogen infection can increase the expression of transcription factor UNC-30 that controls neurogenesis of GABAergic D-type motor neurons.Moreover,the synaptic clustering of presynaptic marker SNB-1 and postsynaptic receptor GABAAR/UNC-49 at GABAergic synaptic sites was upregulated.Electrophysiological data demonstrated that pathogen PA14 infection enhanced the release frequency of GABA neurotransmitter,but not the function of GABAAR/UNC-49.These results suggest that pathogen infection promotes the expression of key synaptic structural and functional components at GABAergic neuromuscular junction,and enhances the strength of GABAergic synaptic neurotransmission.Since GABAergic neuromuscular junction is formed between GABAergic D-type motor neurons and body wall muscles while the innate immunity is induced in the C.elegans intestines upon PA14 infection,we investigated the relationship between GABAergic synaptic signal and the classical p38 MAPK or DAF-2/Daf-16 innate immune pathways.The results showed that blocking the DAF-16/FOXO pathway could completely inhibit the enhanced resistance to PA14 infection by loss of GABAAR/UNC-49.Meanwhile,q RT-PCR showed that muscular GABAAR/UNC-49 significantly inhibited the expression of DAF-16 immune-related target genes,and the intestinal nuclear entry level of DAF-16::GFP was significantly enhanced in unc-30 and unc-49 mutants.Further imaging experiments verified that GABA ergic synaptic signal specifically inhibited the ROS level of oxidative stress response in intestinal cells,and these processes were directly related to the scavenging ability of pathogens.Therefore,GABAergic synaptic neurotransmission inhibits the expression of downstream stress-related genes through the DAF-16/FOXO pathway in intestine,thus inhibiting host’s immune response to pathogen infection.Transcriptome sequencing was performed on WT worms and unc-49 mutants to explore the molecular mechanism of GABA signaling in the cell-non-autonomous regulation of intestinal immunity.Because neuropeptides are important secretory small molecules that mediate trans-tissue signaling,we focused on INS-31,a member of insulin-like INS signaling peptides.The expression of INS-31 was positively regulated by GABAAR/UNC-49,which expressed in muscle and inhibited the immunity of worm to pathogen.Further killing assay analysis showed that INS-31 played an immune role in downstream of GABAAR/UNC-49.Meanwhile,INS-31 interacted with DAF-2,and its immune regulation function could be blocked by DAF-2 or DAF-16 mutation.Furthermore,INS-31 mutation significantly induced the nuclear translocation of DAF-16::GFP.These results suggest that the muscleexpressed insulin-like peptide INS-31 is a messenger molecule linking GABAergic neuromuscular junction signal to intestinal innate immune response through DAF-2/DAF-16 pathway.When screening the survival of key mutants in GABA pathway,we found that the mutants completely lacking GABA signal such as unc-25 or unc-47 exhibit enhanced sensitive phenotype to PA14 infection,while the mutants only lacking GABAergic Dtype motor neuron synaptic transmission such as unc-30 or unc-49 exhibited enhanced resistant phenotype to PA14 infection.These results suggest that GABA signal of nonD type motor neurons may mediate innate immune response.We used specific GABAergic neuron promoters to rescue the expression of UNC-25 in the context of unc-25 mutants,and preliminarily detected the immune phenotype of GABA-deficient worms in these different neurons by using killing assay.The data showed that GABAergic AVL and DVB neurons may mediate intestinal innate immunity through their postsynaptic receptor EXP-1 of enteric muscle.In summary,this study used the host-pathogen infection model of C.elegansPA14 to demonstrate that pathogen infection can induce the expression of key component and synaptic molecules of GABAergic pre-and post-synapses formed between GABAergic D-type motor neurons and body wall muscles,and enhance the strength of GABAergic synaptic neurotransmission.GABAergic synapses inhibit the DAF-2/DAF-16 signaling pathway in intestinal tissues by inhibiting the expression of INS-31,an insulin-like signaling peptide expressed and act in muscle,thus weakening the host’s ability to clear pathogens and thus inhibiting innate immunity.This study reveals a new signaling axis in the synapse-muscular insulin signaling-intestinal immune response in living organisms,and broadens our understanding of the molecular and cellular mechanisms in which synapse in the nervous system regulates intestinal innate immune response.Given that most components of this signaling axis are evolutionarily conserved,it is possible that sim ilar neuro-immune interactions exist in mammals.