Functional Exploration Of Circumventricular Organs Responding Systemic Inflammation Induced By LPS

During the infection caused by pathogenic invasion from bacteria,viruses and other pathogens,the immune system of animal body will generate the local inflammatory response and even systemic inflammation in severe cases through releasing abundant inflammatory mediators including cytokines and chemokines.Meanwhile,the nervous system will be affected by the activation of immune system to regulate the physiological activities(immunity,endocrine,body temperature,etc.)and behaviors(feeding,sleep,movement,social activity,etc.),so that the body present a series of sickness behaviors such as fever,anorexia,lethargy,and less activity for better resisting pathogen infection and restoring homeostasis.Such neuro-immune interaction plays a key role in the regulation of homeostasis under physiological and pathological condition.The Sensation of inflammatory reaction of the immune system by central nervous system is essential to the following stress regulation,while the sensation and subsequent regulation mechanisms still remain unclear.It is currently believed that the central nervous system senses the inflammation of immune system through the peripheral afferent nerve,transmembrane transport,and sensory circumventricular organs(sensory CVOs).Due to the blood-brain barrier,central nervous system cannot directly sense inflammation cytokines of the immune system.However,the sensory circumventricular organs(SFO,OVLT,and AP),which are located in the center of nervous system,closed to the ventricle,and lack the complete blood-brain barrier,are the junction sites of the nervous,immune,and endocrine systems.Under systemic inflammatory,sensory CVOs can up-regulate specific cytokines expression.It has been shown that disrupting sensory CVOs could attenuate the response of the brain nucleus to peripheral inflammation.Now the sensory CVOs are considered to be "the window of the brain".It is speculated that sensory CVOs may play a key role in the perception of peripheral humoral changes,especially inflammation.the neurons of sensory CVOs may play an important role during the regulation of central nervous system under systematic inflammation.However,the mechanism remains to be further investigated.In this study,a classic model mimicking systemic inflammation induced by Gramnegative bacterial infection was used,in which bacterial lipopolysaccharide(LPS)was intraperitoneally injected to cause systemic inflammation.Through the combination of tissue RNA-seq,high sensitivity fluorescence in situ hybridization(HCR-FISH),immunofluorescence staining and virus-based neural circuit tracing technologies,we explored the transcriptomic changes and property of the sensory CVOs under systemic inflammation,and initially analyzed the important projection circuits derived from sensory CVOs.Our research will provide the significant evidences and basis for the functional study of sensory CVOs.The main results are as follows:(1)By immunostaining of activated neuron marker c-Fos,we detected c-Fos positive neurons in sensory CVOs(SFO,OVLT,and AP),the paraventricular hypothalamic nucleus(PVN),the supraoptic nucleus(SON),the medial septal nucleus(MS),the anteromedial thalamic nucleus(AM),and the nucleus of the solitary tract medial part(Solm),indicating that these nuclei are activated under LPS induced systemic inflammation.(2)The transcriptome changes of SFO,OVLT,and AP under LPS-induced systemic inflammatory were detected by tissue RNA-seq analysis.The significantly changed genes included inflammatory cytokines and chemokines.And differentially expressed genes were mainly enriched in inflammatory pathway.Meanwhile,some differentially expressed receptor genes were only detected in certain sensory CVOs,such as the interleukin receptor IL2 rg in SFO,the cholinergic receptor Chrm1 in AP,and the chemokines receptor Cxcr6 in OVLT.In addition,under physiological condition,different sensory CVOs displayed certain receptor expression,like the cholinergic receptor(Chrna10)in SFO,the arginine receptor(Avpr1b)in AP,and the interleukin receptor(IL20ra)in OVLT.(3)Based on our improved high sensitivity fluorescence in situ hybridization(HCR-FISH)method,we verified the expression change of partial genes according to RNA-seq results.In order to reduce the cost for HCR-FISH,we attempted three approaches including digestion-denaturation,unsymmetrical amplification,and bridging ligation,to generate single-strand hybridization probes,but failed to obtain the ideal probes for HCR-FISH in tissue.We further established a quickHCR-FISH(qHCRFISH)method through optimizing the conditions of HCR-FISH(the reagents for permeabilization,the temperature of the hybridization,and the concentration of the hybridization probes).Via this method,the expression changes of IL-1?,IL-6,TNF? in sensory CVOs under systemic inflammation were verified.(4)We tracked the projection from the neurons of sensory circumventricular organs SFO and OVLT to paraventricular hypothalamic nucleus(PVN),the higher center of autonomic regulation,by using retrograde AAV-mCherry tracing virus.By cFos immunostaining,we observed that the SFO-PVN and OVLT-PVN circuits were partially activated under LPS-induced systemic inflammatory.Our results demonstrated that three sensory circumventricular organs can respond to systemic inflammation,and the differential expression genes among the three nuclei suggested their difference in function.Their efferent neural circuits activation suggested that the sensory CVOs may transmit the perceived changes to the downstream brain regions.This study will provide key clues for the functional study of the sensory CVOs in physiological and pathological conditions,provide the basis for understanding the mechanism of the nervous system in responding to immune system changes and coordinating the homeostasis,also contribute to the further investigation of the neuro-immune interaction mechanism.