Sphingosine-1-Phosphate Receptor 4 Attenuates Airway Inflammation In Asthma Via Repressing Proinflammatory Macrophage Activation

Objective: Asthma is a heterogeneous disease characterized by chronic inflammation in the airway,and treatment response and prognosis of asthmatic patients with different phenotypes vary greatly.Sphingosine-1-phosphate receptor 4(S1PR4),a vital receptor of bioactive sphingosine metabolite sphingosine-1-phosphate(S1P),is particularly abundant in immune and hematopoietic tissues.It was reported that S1PR4 participated in the activation,differentiation and chemotaxis of a variety of immune cells including neutrophils,macrophages and T cells which were closely related to the pathogenesis of different phenotypes of asthma.However,the role and specific mechanism of S1PR4 in the pathogenesis of different phenotypes of asthma have not been thoroughly studied.Methods: The contents of sphingosine,dihydrosphingosine and their active metabolites in the plasma and the supernatant of induced sputum of asthma patients of different phenotypes and healthy control population were detected by targeted lipidomics,and the correlation between the concentration of these sphingolipid metabolites and the clinical indicators of asthma were analyzed.Then,the differential expression and cell localization of S1PR4 were investigated in asthmatic population and the differential expression of S1PR4 was verified in asthmatic mice with different phenotypes and macrophages with different polarization states.S1pr4 knockout mice(S1pr4-KO)and overexpressed mice(S1pr4-OE)were constructed to detect the effects of S1PR4 on airway inflammation and airway reactivity in eosinophilic asthma as well as neutrophilic asthma model,respectively,while the effect of S1PR4 on macrophage activation was further explored.In terms of mechanism,the effects of S1PR4 on lipoxin metabolism and related metabolic enzymes expression were detected.Then bioinformatics were applied to identify S1PR4-related asthma phenotype gene,and the specific mechanism of S1PR4 on macrophage activation was explored in bone marrow derived macrophages(BMDMs)extracted from S1pr4-KO and wild-type(WT)mice.Finally,S1PR4 agonist CYM50308 was administrated to asthmatic mice to explore the possibility of clinical transformation of the above experimental results.Results: Sphingosine and its metabolites were abnormal in patients with asthma,but there was no significant difference between asthma patients with different phenotypes.As a vital receptor for bioactive sphingosine metabolites,S1PR4 was mainly expressed on macrophages,and it was significantly reduced in asthma patients and mice with neutrophilic airway inflammation,as well as in proinflammatory macrophages.Knockout of S1pr4 significantly increased neutrophilic airway inflammation in asthmatic mice,accompanied by activation of proinflammatory macrophages,but no significant effect was found in eosinophilic asthma.On the contrary,overexpression of S1pr4 could relieve the neutrophilic inflammatory infiltration in the lungs of asthmatic mice while the number of proinflammatory macrophages did not change significantly.Mechanistic studies showed that S1PR4 was strongly connected to bioactive oxylipins concurrent with bounding to formyl peptide receptor 2(FPR2)to influence the phosphorylation of c-Jun NH2-terminal kinase(JNK)and contributed to the macrophage M1 program.Besides,the treatment of S1PR4 agonist CYM50308 in asthmatic mice with neutrophilic airway inflammation significantly reduced pulmonary inflammatory infiltration and airway reactivity,but this therapeutic effect was not evident in asthmatic mice with eosinophilic airway inflammation.Conclusions: S1PR4 participated in the pathogenesis of neutrophilic asthma by inhibiting the activation of proinflammatory macrophages and S1PR4 agonist CYM50308 might be important clinical therapy for the treatment of noneosinophilic asthma.Part Ⅰ: Expression of sphingosine metabolites and its receptor S1PR4 in different phenotypes of asthmaObjective: The expression and cell localization of S1PR4 were investigated.Methods: Targeted lipidomics were applicated to measure the concentration of sphingosine and its metabolites,and correlation analysis was further adopted.The expression of S1 PRs were detected and S1PR4 expression and cell localization were subsequently detected.Eosinophilic asthma and neutrophilic asthma model were constructed.The expression of S1PR4 in mice with different phenotypes were detected.Lung macrophages and BMDMs of WT mice were extracted in vitro and polarized to different subtypes,to detect the expression of S1PR4 in macrophages with different polarization states.Results: The concentrations of sphingosine and its metabolites in asthma patients were abnormal,but there was no significant difference among asthma patients with different phenotypes.Correlation analysis showed that the concentrations of sphingosine and its metabolites were significantly correlated with clinical indicators of asthma.The expression of active sphingosine metabolites receptor S1PR4,localized in macrophages,was significantly decreased in the noneosinophilic asthma patients,neutrophilic asthma mice model and proinflammatory macrophages.Conclusions: Sphingosine and its metabolites were closely related to the pathogenesis of asthma,but the evidence as markers of different phenotypes of asthma was insufficient.Active sphingosine metabolites receptor S1PR4 may be involved in the occurrence and development of neutrophilic airway inflammation.Part Ⅱ: Role of S1PR4 in mice models of asthmaObjective: To explore S1PR4’s effects on pulmonary inflammation and airway reactivity.Methods: S1pr4-KO,S1pr4-OE and control mice were compared in neutrophilic asthma and eosinophilic asthma models.The indexes observed were pulmonary inflammatory infiltration scores,the total number and classification of bronchoalveolar lavage fluid cells,the content of inflammatory cytokines as well as the related parameters of lung function.Results: Neutrophilic airway inflammation was significantly increased in S1pr4-KO mice compared with WT mice,which was reflected in increased lung tissue inflammation scores,elevated number of bronchoalveolar lavage fluid cells(mainly macrophages and neutrophils),increased IL-12 b and IL-6 concentrations,and raised airway reactivity.There was no significant difference in eosinophilic airway inflammation between S1pr4-KO and WT mice.Compared with control mice,S1pr4-OE mice had attenuated neutrophilic airway inflammation,but airway reactivity had no significant difference.Conclusions: S1pr4 deficiency aggravated neutrophilic airway inflammation and airway reactivity,but has little effect on eosinophilic airway inflammation.Overexpression of S1pr4 reduced neutrophilic airway inflammation but had no impact on airway reactivity.Part III: S1PR4 inhibited proinflammatory macrophage activation through the FPR2-JNK pathwayObjective: To explore the possible mechanism of S1PR4 involved in neutrophilic airway inflammation and search for the downstream pathway.Methods: The proportions of activated macrophages in S1pr4-KO and S1pr4-OE mice were determined by flow cytometry.The BMDMs of WT and S1pr4-KO mice were extracted and the expressions of polarization-related proteins and possible pathway proteins were detected.BMDMs from WT mice were treated with S1PR4 agonist CYM50308 and inhibitor CYM50358.The expression of marker molecules of polarization and inflammatory cytokines in macrophages were further detected.Subsequently,the effect of CYM50308 on the lipoxin metabolic pathway and the expression difference of key metabolic enzymes were analysed.Finally,bioinformatics was applied to identify the S1PR4-related asthmatic endotype gene FPR2,and its differential expression in S1pr4-KO and S1pr4-OE models were determined.The reversion experiments were completed with FPR2 inhibitor WRW4.Results: M1 macrophages were significantly increased in S1pr4-KO mice after induction,and the polarization of S1pr4-KO mice-derived BMDMs increased significantly after stimulation,which may attribute to the activation of JNK pathway.Further,the expression of polarization markers and inflammatory cytokines decreased significantly after treatment with CYM50308,while CYM50358 had no significant effect.Further,the contents of active lipoxin molecules in the supernatant of BMDMs after CYM50308 intervention changed,but there was no significant difference in the expression of its key metabolic enzymes in the BMDMs from WT and S1pr4-KO mice.Next,we confirmed FPR2 as S1PR4-related asthmatic endotype gene and both had co-localized expression on cell membranes.FPR2 and phosphorylated JNK were significantly increased in the lung tissue of S1pr4-KO mice.Finally,FPR2 inhibitor WRW4 was applied to pretreat WT and S1pr4-KO BMDMs and results showed that WRW4 could significantly reverse S1pr4-KO induced proinflammatory macrophage activation,JNK phosphorylation and inflammatory cytokines release.Conclusions: S1PR4 was involved in neutrophilic airway inflammation by restraining the activation of proinflammatory macrophages through the FPR2-JNK pathway.Part IV: Therapeutic effect of S1PR4 agonist on asthmatic modelsObjective: To investigate the therapeutic effect of CYM50308 on asthmatic mice models.Methods: In neutrophilic and eosinophilic asthma model,intraperitoneal injection of CYM50308 or equivalent solvent was performed during the challenge stage to detect pulmonary inflammation and airway reactivity,the proportion of activated macrophages in lung tissue and the expression of related pathway molecules.Results: CYM50308 treatment significantly reduced neutrophilic airway inflammatory infiltration,which was reflected by decreased lung tissue inflammation scores,declined bronchoalveolar lavage fluid total cells and neutrophil counts,reduced IL-12 b and IL-6 concentrations,as well as decreased airway resistance.In addition,CYM50308 intervention significantly decreased the proportion of M1 macrophages and the expression of M1 marker molecules in lung tissue,as well as the expression of FPR2 and the phosphorylation level of JNK pathway.However,in OVA/Alum-induced eosinophilic asthma model,there were no significant changes in eosinophilic airway inflammation after CYM50308 treatment.Conclusions: S1PR4 agonist CYM50308 significantly alleviated neutrophilic airway inflammation and airway reactivity,which may be a new treatment direction for noneosinophilic asthma patients.