Neuromedin U Induces The Activation Of ILC2s Through The Erk Pathway In The Pathogenesis Of Allergic Rhinitis

Background: The pathogenesis of allergic rhinitis(AR)is not fully understood,and there is no effective method or approach to completely eradicate the pathogenesis and development of AR.It is well known that AR is a group of immunoglobulin E(Ig E)-mediated type I allergic diseases and that the pathogenesis of AR is influenced by a variety of factors,including immune cells,inflammatory mediators and neuropeptides.It was found that in the mucosal barrier tissue,immune cells are present around dense populations of neurons and their activation is regulated by neuronal signalling,providing a bridge between the immune system and the nervous system.Studies have shown that neuropeptides such as Substance P(SP),Calcitonin Gene-Related Peptide(CGRP),Vasoactive intestinal peptide(VIP)and Neurokinin A(NKA)are released by neurons in the nasal mucosa in response to external stimuli,suggesting that neuropeptides play an important role in the pathogenesis of AR.Previous studies by our team have shown that neurons in the nasal mucosa of AR rats have a morphological construct with immune cells;neuropeptide P substances are involved in AR pathogenesis by inducing mast cell degranulation through a dual neuromodulatory and immunomodulatory pathway.Therefore,we hypothesize that the pathogenesis of AR is under dual neuroimmune regulation.Group 2 innate lymphoid cells(ILC2s)are a newly identified type of immune effector cells that are key regulators of the immune response at the airway barrier surface.Studies have demonstrated that the proportion of ILC2 s is elevated in the peripheral blood and nasal mucosa of AR patients and that ILC2 s are released in large amounts following stimulation by epithelial-derived cytokines(IL-33,IL-25,TSLP),type 2 inflammatory factors(e.g.IL-4,IL-5,IL-13),thereby exacerbating allergic inflammation.NMU is a multifunctional neuropeptide secreted by cholinergic neurons.In an airway model of asthmatic mice,NMU was found to activate ILC2 s in vitro and to co-activate ILC2 s with IL-25 in vivo,enhancing allergic inflammation.In addition,in a mouse model of gastrointestinal inflammation,NMU was found to be released from intestinal cholinergic neurons upon antigen stimulation,and the latter was able to co-localize with and activate ILC2 s,prompting the release of large amounts of Th2-like inflammatory factors from ILC2 s and subsequently stimulating a type 2 inflammatory response.However,the neuroimmune regulatory relationship between ILC2 s and NMU in AR and the specific mechanisms involved are not yet clear.Combining these findings with the neuroimmune mechanisms that our team has been working on for many years,we hypothesize that NMU induces an inflammatory response through activation of ILC2 s during nasal allergy,establishing a neuro-immune link.Therefore,the group focused on exploring the possible mechanisms of NMU involvement in the nasal allergic inflammatory response in the following three parts:(1)understanding the past life of NMU through bibliometric methods,comprehensively grasping the various functions of NMU and predicting its future trends;(2)observing the effects of exogenous NMU stimulation on the expression of ILC2 s and their functions in the peripheral circulation and exploring their possible mechanisms;(3)exploring the expression of local NMU in the nasal cavity during AR and its functions,and observing the effects of NMU on ILC2 s through animal models,with the intention of providing positive assistance for the prevention and treatment of AR.PartⅠ: Insights into the research status of neuromedin U: a bibliometric and visual analysis from 1987 to 2021Objectives: Neuromedin U(NMU)is a regulatory peptide that is widely distributed throughout the body and performs a variety of physiological functions through its corresponding receptors.In recent years,NMU has become the focus of attention in various fields of research as its diverse and essential functions have gradually been elucidated.However,there have been no bibliometrics studies on the development trend and knowledge structure of NMU research.Methods: In this study,the Web of Science(Wo S)database was used to search the papers published in all years containing the theme of "Neuromedin U",and the online "Wo S Literature Analysis Report" was generated to evaluate the publication characteristics,including countries/regions,institutions,journals,authors and their annual publication number,citation frequency and H-index,etc.Then,we used VOSviewer software to statistically analyze scientific data from articles related to NMU to track the developmental footprint of this research field,including relevant countries,institutions,authors,and keywords.Results: We retrieved a total of 338 papers related to NMU,written by 1661 authors from 438 organizations of 41 countries that were published in 332 journals.The first study on NMU was reported by a group in Japan in 1985.Subsequently,nine articles on NMU were published from 1987 to 2006.A small leap in this field could be detected in 2009,with 30 articles published worldwide.Among the various countries in which this research has been performed,Japan and the United States have made the most outstanding contributions.The highest collaborating institutions are National Cerebral and Cardiovascular Center Research Institute(Japan),Miyazaki University(Japan)and Texas Branch University School of Medicine(USA).Miyazato M,Kangawa K,and Mori K from the Department of Biochemistry,National Retrain and Cardiovascular Center Research Institute in Japan were the most productive authors who have the highest number of citations.Keyword analysis showed six clusters: central-nervous-system,homeostasis,energy metabolism,cancer,immune inflammation,and food intake.The keywords associated with each system are both different and interrelated.The three most highly cited articles were associated with inflammation.Conclusions: Overall,this study demonstrates the research trends and future directions of NMU,providing an objective description of the contributions in this field along with reference value for future research.NMU plays a variety of roles in the human body,and its future role in metabolism,obesity,inflammation and,above all,cancer will continue to attract scholarly attention;NMU deserves further study as a therapeutic target.PartⅡ:Neuromedin U induces activation of peripheral group 2 innate lymphoid cells through the ERK pathway in allergic rhinitis patientsObjectives: In allergic diseases,group 2 innate lymphoid cells(ILC2s)play critical roles.It was found that the proportion of ILC2 s in the peripheral circulation was significantly higher in AR patients and decreased after appropriate desensitization therapy,suggesting that ILC2 s are an important indicator of AR.Neuromedin U(NMU),a highly conserved multifunctional neuropeptide,is secreted by cholinergic neurons and involved in asthma pathogenesis by amplifying lung inflammation driven by ILC2 s.However,the precise effects of NMU on ILC2 s in allergic rhinitis(AR)patients remain unclear.Methods: A total of fifteen patients with persistent AR and eight healthy controls(HCs)were enrolled in the study.The percentages of ILC2 s in peripheral blood mononuclear cells(PBMCs)were enumerated using flow cytometry.The soluble or intracellular cytokines(IL-5 and IL-13)in PBMCs or sorted ILC2 s were assessed in response to various stimuli with IL-33 or NMU combined with ERK inhibitor in the presence of IL-2.Results: Our results confirmed that the proportion of peripheral circulating ILC2 s was significantly higher in AR patients(5.86±1.01)than in the HC group(1.55±0.32),with a statistically significant difference(P<0.0001).The levels of ILC2 s in AR patients were positively correlated with VAS scores(R2=0.5888,P=0.0008).We also found that NMU or IL-33 stimulated the release of IL-5 and IL-13 in PBMC of AR patients significantly higher than in the HC group,with a statistically significant difference.In AR patients,NMU stimulated PBMC or purified ILC2 s produced more type 2 inflammatory factors IL-5 and IL-13 compared to IL-33.In addition,we observed that when the ERK pathway was inhibited,the activation and proliferation function of ILC2 s promoted by NMU was significantly restricted,whereas the IL-33 activated ILC2 s capacity was not significantly affected.Conclusion: NMU effectively activated ILC2 s in AR patients to produce Th2 type cytokines,and this activation can be prevented by ERK pathway inhibitors.Our findings shed new light on the neuroimmune mechanism of AR and offer new insights into its prevention and treatment.Part Ⅲ: NMU activates type 2 innate lymphocytes to aggravate local inflammation of the nasal cavity in allergic rhinitisObjectives: In the nasal mucosal barrier tissue,immune cells are present around a dense population of neurons,and previous studies by our team have shown that the neuropeptide substance P is involved in the pathogenesis of AR by inducing mast cell degranulation through a combination of neuromodulation and immunomodulation.It has been shown that the nasal mucosa releases Substance P(SP),Calcitonin Gene-Related Peptide(CGRP),Vasoactive intestinal peptide(VIP),Neurokinin A(NAP),and other peptides in response to external stimuli.NMU is a multifunctional neuropeptide secreted by cholinergic neurons and is involved in the pathogenesis of asthma and intestinal inflammation,however,it is not clear whether NMU is involved in the pathogenesis of AR.The present study is a preliminary investigation of the effect of the local nasal neuropeptide NMU on AR pathogenesis during nasal allergy,thus unraveling the neuroimmune mechanisms of AR.Methods: In this study,we searched for differential genes between AR and healthy controls by GSE46171 gene microarray,and performed functional enrichment to locate the target genes.The expression and location of NMU and NMUR1 in the nasal mucosa of AR and controls were observed by RT-PCR and immunofluorescence.The expression of ILC2 s in the nasal mucosa was detected by flow cytometry,and the correlation between ILC2 s and NMU and NMUR1 was analysed to initially evaluate the effect of NMU on AR patients.The animal model of AR was used to further verify the local expression of NMU in the nasal cavity and the possible mechanism of inducing inflammation,and to detect the changes of OVA-s Ig E,IL-5,IL-13,NMUR1 and ILC2 s related indexes in AR mice after NMU stimulation.Results: In this study,we analyzed the differential genes between AR patients and healthy controls in the GSE46171 gene chip through R language with the help of a big data platform,and screened a total of 1137 differential genes,of which,313 were up-regulated and 824 were down-regulated.In conjunction with the direction of our group,we focused on the neuropeptide NMU,which was significantly elevated in the AR group as an up-regulated differential gene.As a result of GO analysis we focused on the regulation of the intrinsic immune response as well as ligand receptor activation.Afterwards,the expression of NMU and NMUR1 was verified at the gene and protein levels.RT-PCR results revealed that the expression of NMU m RNA in the nasal mucosa of AR patients(17.98±3.28)was significantly higher than that of controls(1.01±0.11),with a statistically significant difference between the two(P=0.0019);the relative expression of NMUR1 m RNA(3.15±0.04)was significantly higher than that of the control group(1.0±0.09),and the difference was statistically significant(P<0.05).Immunofluorescence results revealed that NMU was widely distributed in the mucosal muscle layer of the nasal mucosa in AR patients,whereas NMUR1 was mainly highly expressed in the lamina propria,the mucosal muscle layer,and the intra-and basal glandular epithelium of the glands in AR patients.local ILC2 s were significantly elevated in the nasal mucosa of AR patients(4.5±0.25)and were positively correlated with NMU(R2=0.9945,P=0.047)and NMUR1(R2=0.9986,P=0.024)were positively correlated.It was further observed that NMU significantly enhanced the local and systemic inflammatory responses in the nasal cavity of OVA-sensitized mice and induced the activation of ILC2 s to release more type 2 inflammatory cytokines,but this effect of NMU could be blocked by ERK pathway inhibitors,suggesting that NMU was involved in the pathogenesis of AR through the activation of ILC2 s in the ERK pathway.Conclusion: The local nasal neuropeptide NMU is significantly elevated in nasal allergy,and it is involved in or exacerbates the development of AR through the activation of ILC2 s in the ERK pathway to release type 2 cytokines.This lays the foundation for the exploration of multiple factors of AR from a single immune mechanism to a neuro-immune mechanism,and also provides a new idea for the prevention and treatment of AR,i.e.breaking the limits of immunotherapy.