The Mechanism Of Allergic Rhinitis Induction By CD169~+ Macrophages Via Dendritic Cell Migration Regulation And The Development Of Targeted Drugs

BackgroundAllergic rhinitis(AR)is a widespread respiratory disorder that impacts physical health and quality of life by causing sleep disorders,reducing work and learning efficiency,and leading to a chain reaction of negative consequences.The underlying cause of AR is an imbalance in the helper T cell immune network,wherein dendritic cells play a crucial role in antigen presentation to T cells.Uncontrolled regulation of dendritic cells can cause excessive activation of Th2 cells,leading to allergic diseases.Since T cells can only recognize antigenic peptides processed by antigen-presenting cells,such as dendritic cells,these cells are essential in the pathogenesis of AR.At present,the drugs used to treat AR primarily consist of H1 antihistamines,intranasal corticosteroids,mast cell stabilizers,and decongestants.However,due to their long treatment cycles and high drug resistance rates,there remains an urgent need for the development of novel therapies to improve AR management.Macrophages are a diverse and adaptable group of immune cells that play critical roles in human physiological processes such as inflammation and tumor immunity.Unlike conventional M1 and M2 macrophages,CD 169+macrophages are involved in regulating lymphocyte proliferation,antigen presentation,immune tolerance,and induction of inflammatory responses.Our previous study found that CD169+ macrophages were increased in the nasal mucosal epithelium of patients with AR,and knocking out CD169+ macrophages in an AR mouse model inhibited the activation of dendritic cells.Therefore,this study focuses on determining whether CD169+ macrophages induce AR by regulating dendritic cell migration.Our goal is to clarify the CD 169+ macrophages’ phenotype in nasal mucosa and their mechanism of action in AR pathogenesis,and to explore the physiological changes in various immune cells and cytokines in the AR pathway,which will lead to novel drug targets for the prevention and treatment of AR.Shikonin and polyphyllin Ⅱ,derived from shikonin and Paris polyphylla plants,possess significant pharmaceutical value.However,their clinical applications are limited by their low bioavailability,short retention time,and poor solubility.As a result,more efficient drug delivery systems are required to enhance the anti-inflammatory effects of shikonin and polyphyllin Ⅱ in treating allergic diseases.In the process of studying AR,we found that dendritic cells and neutrophils accumulate in the nasal mucosa of AR animals and play a crucial role in the allergic reaction.Therefore,the second focus of this study aims to explore the potential of modified nanoparticles,loaded with traditional Chinese medicine monomers,to target dendritic cells or neutrophils and enhance their anti-inflammatory effects.This will provide new insights into the management of allergic rhinitis.ObjectivesTo construct the AR model,we used diphtheria toxin receptor transgenic mice.We employed molecular biology experiments,flow cytometry,and metabolomics detection to investigate the expression and phenotype of CD169+macrophages in the nasal mucosa and to explore the mechanism of CD169+ macrophages in AR.Moreover,we studied the relationship between CD169+ macrophages and dendritic cells as well as other immune cells and cytokines associated with allergic reactions.Our primary objective was to identify novel therapeutic targets associated with AR and to develop targeted nanodrugs that can selectively act on these targets.We aim to verify the therapeutic effect and mechanism of these nanodrugs and provide new ideas for the prevention and treatment of allergic rhinitis.MethodsThis study explored the phenotypic changes of CD169+macrophages in AR,as well as immune network regulation and metabolic state alterations at the animal,cellular,and molecular levels.Immunofluorescence staining was used to detect the expression of CD 169+macrophages in patients with AR,and flow cytometry was employed to detect the expression of CD169+macrophages and dendritic cells in the nasal lavage fluid of AR mice models.The expression of CD169+macrophages,dendritic cells,Th1,Th2,Th17,and Treg cells in nasal lavage fluid,spleen,and lymph nodes of mice was also investigated.The concentrations of IL-4,IL-17,IL-23,TGF-β,and IFN-γ in nasal lavage fluid and peripheral blood serum were measured using ELISA.HE staining was used to evaluate the infiltration of eosinophils and neutrophils.Metabolomics technology was employed to assess the differences in metabolites in nasal lavage fluid of CD169+ macrophages knockout mice.To investigate the impact of the alanine pathway on dendritic cell migration,a cell co-culture system,wound healing assay,and transwell assay were employed.Furthermore,flow cytometry was utilized to determine the optimal approach for inhibiting AR-related inflammatory cells and factors by evaluating the effectiveness of CD169+ macrophage knockout,either before or after sensitization.The duration of CD169+macrophage recovery following knockout was also examined.Given the crucial role of dendritic cells and neutrophils in AR,nanoparticles were synthesized using nanotechnology to target dendritic cells(NGR-SHI-PM)and neutrophils(PLGA-5HT-PSII-Ce6).To investigate the therapeutic mechanism of the drug on AR,molecular biological techniques,such as ELISA,immunofluorescence,immunohistochemistry,Western blot,in vitro co-culture technology,and MTT assays,were employed.ResultsOur study of CD169+ macrophages revealed the following findings:(1)Depletion of CD169+macrophages inhibited dendritic cell maturation and migration in AR mice;(2)knockout of CD 169+macrophages reduced the expression of Th2 and Th 17 cells and their corresponding immune factors;(3)depletion of CD169+ macrophages increased the expression of Treg cells;(4)the alanine pathway mediated the regulatory role of CD169+ macrophages in AR induced by dendritic cells;(5)the recovery of CD169+macrophages was observed approximately three months after depletion in the animal model;and(6)knockout of CD169+ macrophages prior to sensitization was found to be more effective in inhibiting the occurrence and development of AR.In our investigation of the mechanism of NGR-modified shikonin nanoparticles targeting dendritic cells for the treatment of AR,our findings showed that NGR modification(1)enhanced the endocytosis of the drug by dendritic cells,(2)increased the inhibitory effect of shikonin on dendritic cells,Th2 cells,and related inflammatory cytokines in AR mice,and(3)downregulated the NF-κB pathway through the targeting of PARP,ultimately inhibiting the occurrence and development of AR.In our study examining the therapeutic effect of bis-5HT modified polyphyllin Ⅱ on AR through regulation of the alanine pathway,we found that(Ⅰ)bis-5HT modified polyphyllin Ⅱenhanced the inhibitory effect of polyphyllin Ⅱ on dendritic cells and Th2 cells in AR mice,(2)bis-5HT augmented the inhibitory effect of polyphyllin Ⅱ on allergy-related inflammatory factors in AR mice,and(3)the alanine pathway played a critical role in the treatment of AR with neutrophil infiltration using PLGA-5HT-PSII-Ce6.ConclusionIn summary,this study highlights the crucial role of CD169+macrophages in regulating dendritic cell migration via the alanine pathway,thereby contributing to the onset and progression of AR.The use of NGR-SHI-PM in AR treatment demonstrated significant inhibition of the allergic response by targeting and downregulating the NF-κB pathway.Moreover,the application of PLGA-5HT-PSII-Ce6 exhibited promising results in the treatment of AR by regulating the alanine pathway.These findings present exciting opportunities for the development of novel therapies for AR treatment,as they demonstrate the potential of targeting specific pathways and molecular targets in the prevention and management of this widespread respiratory disorder.