| Objective:The development of allergic rhinitis is the result of the influence of environmental exposure on the congenital genetic background.The interconversion of Innate lymphoid cells(ILCs)may be a key cellular basis for the presence of minimal persistent inflammation and the recurrence of allergic rhinitis,particularly in the presence of changes in ILC2 plasticity.Based on the theory of"Nei Jing"and the therapeutic principle of"harmonizing Yang Qi",the hospital preparation Bimin Kang Mixture which is clinically effective but the mechanism of action for controlling the minimal persistent inflammation is not yet exhaustive.We analyzed the regulation of the minimal persistent inflammation phase inflammatory state and the mechanism of action of Bimin Kang Mixture,clarify the regulatory role of transcription factor B-cell leukemia/lymphoma11B(BCL11B)on ILC2 plasticity,to explore the changes in the minimal persistent inflammation state in which Bimin Kang Mixture interferes with BCL11B to regulate ILC2 plasticity,and to reveal the specific mechanism of Bimin Kang Mixture to control the inflammatory state and reduce the chance of relapse is the main purpose of this study.Method:1.Regulation and mechanism of action of ILCs signature inflammatory factors and transcription factors in the minimal persistent inflammation state of Yang-Qi deficiency type allergic rhinitis patients by Bimin Kang Mixture.The cells of nasal mucosa tissue were examined by high-throughput transcriptome mRNA sequencing technology in clinical healthy volunteers,during the exacerbation of Yang Qi deficiency type allergic rhinitis, during the minimal persistent inflammation phase,and in patients after treatment with Bimin Kang Mixture.By analyzing differentially expressed genes and clarifying the pathological mechanism of Yang-Qi deficiency-type allergic rhinitis in different pathogenic periods.According to the quantitative analysis of gene expression levels of representative inflammatory and transcription factors of ILCs during the exacerbation phase of allergic rhinitis,the minimal persistent inflammation phase,and after the intervention of Bimin Kang Mixture,to clarify the regulation and mechanism of action of Bimin Kang Mixture on the inflammation of airway mucosa in patients with the minimal persistent inflammation period.2.The effect of Bimin Kang Mixture on the level of inflammation in mice with the minimal persistent inflammation model.We established a mice model of allergic rhinitis and minimal persistent inflammation,analyzed the histopathological changes of airways and the expression of serum inflammatory factors in mice at different periods of pathogenesis,and clarified the effect of Bimin Kang Mixture on the mucosal inflammation changes in the model mice.3.The effect of Bimin Kang Mixture intervention with BCL11B on subpopulations of ILCs in MPI model mice.RT-qPCR was used to detect the expression levels of BCL11B and ILC2and ILC3 subsets related genes in the lungs of mice with allergic rhinitis,the minimal persistent inflammation model,and FACS was used to detect the changes of BCL11B~+ILC and ILC2 and ILC3 subsets ratios in the nasal mucosa and lungs of model mice,to clarify the correlation between the expression of BCL11B and ILC2 and ILC3 subsets.RT-qPCR was used to detect the effects of Bimin Kang Mixture on the expression levels of BCL11B and ILC2 and ILC3 subsets in the lungs of the minimal persistent inflammation model mice,and FACS was used to detect the effects of Bimin Kang Mixture on the ratios of BCL11B~+ILC and ILC2 and ILC3 subsets in the nasal mucosa and lungs of model mice,to clarify the effect of Bimin Kang Mixture on ILC2 and ILC3 subsets by interfering with BCL11B.RT-qPCR was used to detect the effect of Bimin Kang Mixture on the expression level of ILC1 related factor gene in the lung of mice with the minimal persistent inflammation,FACS was used to detect the effect of BCL11B on the proportion of ILC1 and ILC1/ILC2 double positive subsets in the nasal mucosa and lung of model mice after Bimin Kang Mixture intervention,and to explore the possibility of Bimin Kang Mixture intervention in BCL11B regulating the transformation of ILC2 to ILC1.RT-qPCR and ELISA were used to detect the changes of IL-12 and IL-18gene and protein expression levels in the lungs of the minimal persistent inflammation model mice after Bimin Kang Mixture,and the ratios of IL-18Rα~+IL-13~+ILC and 12Rβ2~+IL-13~+ILC in the nasal mucosa and lungs of the model mice after Bimin Kang Mixture,to clarify that Bimin Kang Mixture regulates the conversion of ILC2 plasticity to ILC1 by BCL11B.Result:1.Mechanistic changes and difference of gene expression of inflammatory factors and transcription factors of ILCs during the exacerbation phase of allergic rhinitis,the minimal persistent inflammation phase,and the minimal persistent inflammation phase of Bimin Kang Mixture intervention.Transcriptome sequencing revealed that differentially expressed genes in the exacerbation phase of allergic rhinitis,the minimal persistent inflammation phase and the minimal persistent inflammation phase of Bimin Kang Mixture intervention mainly have molecular functions such as regulating transcription factors and catalytic receptors,using cytokine or receptor interactions,regulating the expression of chemokines and cell adhesion molecules,affecting cell migration ability,and participating in the regulation of the systemic immune system through Th1,Th2 and Th17 cell differentiation,IL-17,Toll-like receptors,NF-κB and JAK-STAT pathway signaling.Further analysis of the gene expression level of the key inflammatory factors and transcription factors of ILCs in the nasal mucosa tissue cells of patients with the exacerbation phase of allergic rhinitis,the minimal persistent inflammation and after the intervention of Bimin Kang Mixture,it was found that the Th2inflammatory cytokines IL-5,IL-13 secreted by ILC2 and the secretion of Th17 inflammatory factor IL-17A by ILC3 during the onset of allergic rhinitis showed an up-regulation trend,while the representative inflammatory factor IL-17A and transcriptional factor RORC of ILC3 during the minimal persistent inflammation showed an up-regulation trend.After the intervention of Bimin Kang Mixture,the Th17 inflammatory factor is down-regulated.On the contrary,the Th2 inflammatory factor IL-5 secreted by ILC2 and the Th1 inflammatory factor IFNG secreted by ILC1 are significantly up-regulated.2.The effect of Bimin Kang Mixture on the level of inflammation in the minimal persistent inflammation mice model.By airway tissue staining,it was found that compared with the Control group,mice in the AR group had complex and disorganized of nasal mucosa and lung tissues,exhibiting mucosal remodeling features,with heavy hypertrophy and hyperplasia of Goblet cells(P<0.001),and a large number of Eosinophils were seen in the nasal cavity and submucosa(P<0.001),with significant Mast cells infiltration(P<0.001).The nasal mucosa and lungs of the mice in the MPI group still had a large number of inflammatory infiltrates,Goblet cells were enlarged(P<0.01)and Eosinophils(P<0.001)and Mast cells(P<0.01)were still seen in the nasal cavity and lamina propria.The nasal mucosa and lungs of the mice in the BMK and Loratadine groups had basically regained their structural integrity,cilia defects and detachment were repaired compared with the MPI period,and the volume of Goblet cells were normalized(P<0.05),the submucosal glands were reduced,vascular congestion was not obvious,only a very small amount of inflammatory cell infiltration was seen,and the number of Eosinophils and Mast cells were significantly reduced(P<0.05).Serologically,the expression levels of IgE,IL-13 and IL-5 were significantly higher in the serum of AR and MPI mice compared with Control group(P<0.001),and the expression level of IgE and IL-13in AR group was significantly higher than that in MPI group(P<0.001).The expression level of IL-17A in the serum of AR group mice tended to increase(P<0.05),the expression level of IL-17A in MPI group was significantly higher(P<0.001)and significantly higher than AR group(P<0.01).The expression level of IFN-γin the serum of mice in the AR and MPI groups was significantly downregulated(P<0.01),but the expression level of IFN-γin the MPI group was higher than that in the AR group(P<0.05).Compared with the MPI model group,the expression levels of inflammatory factors IL-13,IL-5,IL-17A and IgE in the serum of mice in the BMK and Loratadine groups were significantly lower(P<0.001),and the expression level of IFN-γtended to increase(P<0.05),and the differences between the two groups were not statistically significant(P>0.05).3.Correlation of BCL11B expression with ILC2 and ILC3 in airway tissues of mice with allergic rhinitis and the minimal persistent inflammation model.According to the results of RT-qPCR,compared with the Control group,the gene expression level of BCL11B and IL-13 in AR group was significantly higher(P<0.001),and the gene expression level of GATA3 was significantly higher(P<0.01),IL-17A and IL-5 gene expression level also increased(P<0.05).Compared with the Control group,the gene expression level of BCL11B and IL-13 in MPI group also increased significantly(P<0.01),and IL-13 was significantly lower than that in AR group(P<0.001).The gene expression level of GATA3 in MPI group also increased(P<0.05),but it was significantly lower than AR group(P<0.05).The expression level of RORC and IL-17A in MPI group was significantly higher in MPI group(P<0.05),and MPI group was significantly higher than AR group(P<0.05).According to the results of FACS,the proportion of BCL11B~+ILC in the nasal mucosa and lung of the AR model group was significantly higher than that of the Control group(P<0.001),while the proportion of BCL11B~+ILC in the MPI model group was significantly higher than that of the AR group(P<0.01).The proportion of GATA3~+ILC2 in the lung and nasal mucosa of AR group mice increased significantly(P<0.01),while the proportion of GATA3~+ILC2 in the nasal mucosa of MPI group mice increased significantly(P<0.01),but was significantly lower than that of AR group(P<0.05).In the AR and MPI model groups,the proportion of IL-5~+ILC2 in the nasal mucosa and lung of mice increased significantly(P<0.01);Compared with AR group,the proportion of IL-5~+ILC2 in MPI group had no significant difference(P>0.05).The proportion of IL-13~+ILC2 in the nasal mucosa and lung of AR group was significantly increased(P<0.01),while the proportion of IL-13~+ILC2 in MPI group had no statistical difference(P>0.05),and was significantly lower than that in AR group(P<0.05).The ratios of RORγt~+ILC3 and IL-17A~+ILC3 in the nasal mucosa and lungs of mice in the AR group did not change significantly(P>0.05),and the ratios were significantly higher in the MPI model group(P<0.01)and significantly higher than those in the AR group(P<0.05).Correlation studies revealed a significant positive correlation between BCL11B and ILC2(P<0.05)and a significant negative correlation with ILC3(P<0.05).4.The effect of Bimin Kang Mixture on the intervention of BCL11B and the subpopulations of ILC2 and ILC3 in the minimal persistent inflammation mice model.RT-qPCR revealed that compared with the MPI group,the gene expression levels of BCL11B,GATA3,RORC and IL-5 in the lung of BMK group were significantly decreased(P<0.05),and the gene expression level of IL-17A in the lung of BMK group and Loradadine group was significantly decreased(P<0.001),while the increasing trend of IL-13 gene expression level in BMK group and Loradadine group was not obvious(P>0.05).FACS test found that compared with MPI group,BMK group and Loratadine group,the proportions of BCL11B~+ILC,IL-5~+ILC2,IL-17A~+ILC3 in nasal mucosa and lung were significantly downregulated(P<0.01),and the proportions of GATA3~+ILC2 and RORγt~+ILC3 were significantly lower(P<0.05).The ratio of IL-13~+ILC2 in the nasal mucosa and lungs of mice in the BMK and Loratadine groups increased significantly(P<0.05).Compared with the Loratadine group,the ratio of IL-13~+ILC2 in the nasal mucosa of mice in the BMK group was significantly upregulated(P<0.05).FACS assay revealed that the ratio of total ILC subpopulation did not change significantly after Bimin Kang Mixture intervention compared with the MPI group(P>0.05);the ratio of IL-17A~+IL-13~+ILC2/3 subpopulation in the nasal mucosa and lungs of mice in the BMK and Loratadine groups was significantly downregulated(P<0.05),especially in the BMK group.The proportion of GATA3~+RORγt~+ILC2/3 subpopulation in the lungs of mice in the BMK and Loratadine groups was also significantly downregulated(P<0.05).5.The intervention of BCL11B with Bimin Kang Mixture regulates ILC2 plasticity in the minimal persistent inflammation mice model.RT-qPCR revealed that the gene expression levels of TBX21 were significantly increased in the lungs of mice in the BMK and Loratadine groups compared with the MPI model group(P<0.05),and the gene expression levels of IFN-γwere significantly increased(P<0.05),with a particularly significant trend of increasing gene expression levels of TBX21 and IFN-γin the BMK group(P<0.01).FACS assay revealed that IFN-γ~+ILC1 and T-bet~+ILC1 subpopulations were significantly(P<0.01) upregulated in the nasal mucosa and lungs of mice after Bimin Kang Mixture and Loratadine interventions compared with the MPI group,with T-bet~+ILC1 being particularly(P<0.01) upregulated in the BMK group.Double-positive subgroup T-bet~+GATA3~+ILC2/1 ratio significantly increased(P<0.05),and the proportion of IFN-γ~+IL-13~+ILC2/1 subsets was significantly upregulated(P<0.01),especially in the mice nasal mucosa(P<0.001).RT-qPCR and ELISA were used to detect the expression of genes and proteins related to ILC2 plasticity.It was found that the gene expression level of IL-12 and IL-18 in the lung of mice in BMK group was significantly increased(P<0.05),and the protein expression level of IL-12 and IL-18 in BMK and Loratadine group was significantly increased(P<0.01).There was no significant difference between the two groups(P>0.05).FACS assay revealed that by detecting ILC2 plasticity-related receptors in the lungs of mice in the MPI model group after Bimin Kang Mixture intervention,the 12Rβ2~+ILC ratio was significantly higher in the Loratadine group(P<0.05)and 12Rβ2~+IL-13~+ILC ratio was also significantly higher (P<0.01),with no statistically significant difference between the two groups(P>0.05).The ratio of IL-18Rα~+ILC was significantly upregulated in the BMK and Loratadine groups compared with the MPI group,and was significantly higher in the BMK group compared with the Loratadine group(P<0.01).The proportion of IL-18Rα~+IL-13~+ILC was also significantly higher in the BMK group compared with the MPI group(P<0.001),and the difference between the two groups was not statistically significant(P>0.05).Conclusion:1.mRNA sequencing of nasal mucosal tissue cells in patients with Yang Qi deficiency allergic rhinitis during the exacerbation phase,the minimal persistent inflammation phase,and after Bimin Kang Mixture intervention revealed that Th17-like inflammatory factors were predominantly expressed during the minimal persistent inflammation phase,and Bimin Kang Mixture inhibited IL-17A expression while interfering with Th1/Th2 cell differentiation.2.Mice in the minimal persistent inflammation phase model still showed high levels of inflammation in serum and airway mucosa.The Bimin Kang Mixture was able to reduce the expression level of inflammatory factors in the serum of mice,repair the damage of airway epithelial barrier,improve mucosal remodeling,and significantly reduce the infiltration of inflammatory cells such as Eosinophils,Goblet cells and Mast cells.3.BCL11B was positively correlated with ILC2 expression and negatively correlated with ILC3 expression.4.Bimin Kang Mixture reduces the proportion of ILC2 subpopulation by decreasing BCL11B expression in airway tissues,while reducing the proportion of ILC3 and ILC2/ILC3 dual-positive subpopulations.5.Bimin Kang Mixture is mainly used for harmonizing Yang Qi and correcting the balance of ILCs subsets in striae to play the mucosal barrier immune function.Bimin Kang regulates ILC2 plasticity by interfering with BCL11B,and upregulating IL-12Rβ2 and IL-18Rαin response to local microenvironmental IL-12 and IL-18 change stimuli,and increasing the expression of T-bet transcription factor in ILC2.So that ILC2 can secrete more IFN-γ through IL-12-IL-12R and IL-18-IL-18R signals under the regulation of T-bet.Thus,it can promote the transformation of ILC2 into ILC1-like cells to play an anti-inflammatory role,which may be the key mechanism to Control the minimal persistent inflammation state and inhibit the recurrent attacks of allergic rhinitis. |
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