Research On The Mechanism Of Neuropeptide Y Regulating Splenic Immune Response In Response To LPS Infection

Various diseases caused by pathogenic microorganism infection are one of the major challenges for animal survival.Exploring the regulatory mechanism of the immune system during bacterial infection is an essential way to resist bacteria and improve survival rate.Endotoxin lipopolysaccharide(LPS)is one of the most commonly used natural inducers for systemic inflammation models.LPS can induce a pathological response similar to the invasion of Gram-negative bacteria.The immune system will be rapidly activated and secrete cytokines when encountering bacterial invasion,but how to regulate the immune balance under pathological conditions is still unknown.The nervous system and the immune system are two relatively independent systems with their own structures and functions,but at the same time there is a close interaction between them.Some neurotransmitters and neuropeptides can mediate the immune response in vitro and in vivo and the surface of some immune cells also express a variety of neurotransmitters and receptors for neuropeptides.Neurons are also regulated by a variety of cytokines.The interaction between the two plays a key role in maintaining immune homeostasis.The study of the interaction mechanism of these two systems may provide new therapeutic ideas and diagnostic methods to resist pathogenic infection and improve the survival rate.The purpose of this study is to utilize various techniques of neurobiology and molecular biology to explore the neuroimmune circuit of the suprarenal-celiac ganglia(Sr G-CG)innervating the spleen,and study the changes that occur in the Sr G-CG during bacterial invasion and the potential neurotransmitters that specifically modulates the splenic immune response.Meantime,the regulatory mechanism of specific types of neurons in the Sr G-CG to the splenic immune response was analyzed,and the immune cells and molecular cell pathways that could respond to the signals of the Sr G-CG neurons were discovered.This research will provide a new theoretical basis for exploring the precise and rapid regulation of the immune response by the peripheral nervous system(PNS)during pathogenic invasion.The main contents of this research are divided into the following parts:1.Suprarenal-celiac ganglia respond to LPS and upregulate NPY expressionIn this study,the neuroimmune circuit from the Sr G-CG to the spleen was validated by retrograde tracing of pseudorabies virus.The RNA-seq data analysis of the Sr G-CG found that the rat Sr G-CG was rich in NPY~+neurons,and NPY was the most significant gene in the ganglia neurotransmitter in response to LPS treatment.Subsequently,we found that LPS could directly activate the Sr G-CG neurons by calcium imaging experiments,and immunofluorescence staining also revealed that the expression of TLR4 and NPY co-localized in the rat Sr G-CG neurons.These results suggested that LPS can directly activate the Sr G-CG neurons and upregulate the expression level of NPY,which may play an important role in regulating the splenic immune response.2.The immunoregulatory effect of Sr G-CG NPY on the spleen under the physiological stateWe constructed an adeno-associated virus with knockdown of NPY to further explore the specific regulatory effect of NPY secreted by the Sr G-CG neurons on the spleen.The functions of T cells,B cells,and macrophages all changed in the spleen after NPY knockdown.The results of sc RNA-seq analysis and immunohistochemistry showed that the NPY receptor NPY1R is expressed on T cells and macrophages,which can directly communicate with NPY,while the receptor is almost absent on B cells.Notley,NPY can inhibit the TNF signaling pathway of macrophages and reduce the expression of cytokines such as IL-1βand TNF-αin splenic macrophages.3.Sr G-CG NPY inhibits spleen inflammatory response induced by LPS and bacterial infectionTo further analyze the function of NPY~+neurons in the Sr G-CG when encountering systemic inflammation,we performed experiments of exogenous NPY treatment on splenocytes,neurons-splenocytes co-culture experiment with neuronal NPY knockdown,knockdown and overexpression of NPY in Sr G-CG,these results showed that Sr G-CG NPY mainly exhibited the function of inhibiting splenic inflammatory storm in the LPS-induced rat inflammatory model.4.Drosophila NPF regulates the inflammatory response to bacterial infectionA neuron NPF knockdown Drosophila strain was bred and utilized for bacterial infection experiments,the result showed that Drosophila neuron NPF(NPY homologue)has similar functions to rat NPY,and can significantly reduce the levels of antimicrobial peptides and cytokines induced by bacterial infection.This revealed that the regulation of NPY/F on the immune system is evolutionarily conserved.The communication between the nervous system and the immune system is one of the common features of lower animals to higher animals,while neuropeptides(such as NPY,etc.)are an"ancient language"that sustains this communication.Taken together,this study reveals that neuron-derived NPY/F is an ancient language for communication between the nervous system and the immune system,and its regulatory role is relatively conserved from Drosophila to rats.In rats,the nervous system can sense pathogenic infection and massively up-regulate the Sr G-CG NPY to suppress the expression of cytokines in the spleen,thereby exerting a fine regulation of immune response.Our work may provide new drug targets and therapeutic strategies for alleviating the inflammatory storm and modulating immune balance during bacterial infection.