New insights into allergic rhinitis treatment: MSC nanovesicles targeting dendritic cells

Abstract

Allergic rhinitis (AR) is a condition with limited treatment options. This study investigates the potential use of mesenchymal stem cell (MSC) nanovesicles as a novel therapy for AR. Specifically, the study explores the underlying mechanisms of MSC nanovesicle therapy by targeting dendritic cells (DCs). The researchers fabricated DC-targeted P-D2-EVs nanovesicles and characterized their properties. Transcriptomic sequencing and single-cell sequencing analyses were performed to study the impact of P-D2-EVs on AR mice, identifying core genes involved in the treatment. In vitro cell experiments were conducted to validate the effects of P-D2-EVs on DC metabolism, Th2 differentiation, and ILC2 activation. The results showed that P-D2-EVs efficiently targeted DCs. Transcriptomic sequencing analysis revealed differential expression of 948 genes in nasal tissue DCs of mice treated with P-D2-EVs. Single-cell sequencing further revealed that P-D2-EVs had inhibitory effects on DC activation, Th2 differentiation, and ILC2 activation, with Fut1 identified as the core gene. Validation experiments demonstrated that P-D2-EVs improved IL10 metabolism in DCs by downregulating Fut1 expression, thereby suppressing Th2 differentiation and ILC2 activation. Animal experiments confirmed the inhibitory effects of P-D2-EVs and their ability to ameliorate AR symptoms in mice. The study suggests that P-D2-EVs reshape DC metabolism and suppress Th2 differentiation and ILC2 activation through the inhibition of the Fut1/ICAM1/P38 MAPK signaling pathway, providing a potential therapeutic approach for AR.

Keywords: Allergic rhinitis; Dendritic cells; Mesenchymal stem cells; Single-cell sequencing; T helper 2 cells; Type 2 innate lymphoid cells.

Fig. 1

Therapeutic effects of EVs and P-D2-EVs on AR mice. A Flowchart of EVs and P-D2-EVs treatment on AR mice. B Number of nose rubbing (itching) and sneezing events of each group of mice (n = 6) within 2 h after the last intranasal OVA administration. C Total cell count in NALF of each group of mice (n = 6). D OVA-specific serum IgE levels of each group of mice (n = 6). E Representative PAS staining images of nasal tissue (scale bar = 100 μm) and quantification of percentage of PAS-positive goblet cells. F Representative H&E staining images of nasal tissue (scale bar = 100 μm or 25 μm) and quantification of eosinophils in nasal mucosa and nasal mucosal thickness, red arrows indicate nasal mucosa. G Representative images of neutrophil infiltration in nasal mucosa (scale bar = 25 μm) and quantification in each group of mice (n = 6), black arrows indicate positive cells; NALF nasal lavage fluid, * represents statistical significance (P < 0.05)

Fig. 2

Transcriptome sequencing and biological information analysis of DEGs. A Workflow of transcriptome sequencing for mouse DCs in Model group and Model + P-D2-EVs group. B Volcano plot of DEGs between mouse DC samples from Model group (n = 3) and Model + P-D2-EVs group (n = 3). C Heatmap of DEGs between mouse DC samples from Model group (n = 3) and Model + P-D2-EVs group (n = 3). D GO enrichment analysis bubble plot of DEGs. E KEGG enrichment analysis bar plot of DEGs. F Single-cell sequencing workflow diagram of Model group (n = 3) and Model + P-D2-EVs group (n = 3) mouse nasal tissue samples. G Batch correction process diagram of Harmony, with the number of interactions on the x-axis. H Distribution of cells in PC_1 and PC_2 after Harmony batch correction, each point represents a cell, different colors represent different samples. I UMAP clustering result visualization, two-dimensionally showing the clustering and distribution of cells from Model group (n = 3) and Model + P-D2-EVs group (n = 3) mouse nasal tissue samples, where blue represents samples from the Model group, and red represents samples from the Model + P-D2-EVs group. J UMAP clustering result visualization, displaying the clustering and distribution of cells from different sources (n = 3), with each color representing a cluster. K Expression levels of known cell lineage-specific marker genes in different clusters, where darker red indicates higher average expression levels and larger circles indicate more cells expressing that gene. L Three-dimensional visualization of cell annotations based on UMAP clustering, with each color representing a cell subtype. M Visualization of cell annotation results based on UMAP clustering of Model group (n = 3) and Model + P-D2-EVs group (n = 3), where each color represents a cell subtype

Fig. 3

Pseudotime analysis of DCs and T cell subpopulations. A Proportions of different cell subpopulations in different groups (n = 3), represented by different colors. B Cell trajectory differentiation diagram for DCs, where different colors represent different states. C Pseudotime visualization of DC cell trajectories, with darker colors indicating earlier time points. D Cell trajectory visualization of DCs in different groups (n = 3), with different colors representing different groups. E Pseudo-temporal expression curves for DC cell marker genes Igtax, Cd80, and Cd86, with time on the x-axis and gene expression levels on the y-axis. F Cell trajectory differentiation diagram for T cells, where different colors represent different states. G Pseudotime visualization of T cell trajectories, with darker colors indicating earlier time points. H Cell trajectory visualization of T cells in different groups (n = 3), with different colors representing different groups. I Pseudo-temporal expression curves for marker genes associated with different T cell differentiation directions, with time on the x-axis and gene expression levels on the y-axis. J Cell trajectory differentiation map of ILC2, where different colors represent different states. K Visualizing the cell trajectory map of ILC2 in pseudo-time, with darker colors indicating earlier time points. L Visualization of ILC2 cell trajectories using different grouped samples (n = 3), where different colors represent different groups. M Pseudo-time curve of gene expression of Gata3, a marker gene for ILC2 cells, with time on the x-axis and gene expression level on the y-axis. N Interaction relationships between cells in the Model group samples (n = 3), with line thickness representing the strength of the interactions. O Interaction relationships between cells in the Model + P-D2-EVs group samples (n = 3), with line thickness representing the strength of the interactions

Fig. 4

The impact of P-D2-EVs on DCs activation and metabolism through the regulation of Fut1. A Flow cytometric analysis of the percentage of DCs in nasal tissue of each mouse group (n = 6). B Flow cytometric analysis of the percentage of Ki67-positive cells in DCs of each mouse group (n = 6). C RT-qPCR analysis of Ki67 mRNA expression in DCs of different mouse groups (n = 6). D RT-qPCR analysis of Fut1 mRNA expression in DCs of different mouse groups (n = 6). E Western blot analysis of Fut1 protein expression in DCs of different mouse groups (n = 6). F Flow cytometric analysis of the percentage of IL10-positive cells in DCs of each mouse group (n = 6). G RT-qPCR analysis of IL10 mRNA expression in DCs of different mouse groups (n = 6). H RT-qPCR analysis of IL10 mRNA expression in different treated mDCs. I ELISA analysis of IL10 levels in the supernatant of different treated mDCs. J Flow cytometric analysis of intracellular IL-10 levels in different treated mDCs. K The schematic diagram illustrates the treatment methods of NC-OE-mDCs, Fut1-OE-mDCs, NC-OE-mDCs + P-D2-EVs, and Fut1-OE-mDCs + P-D2-EVs, as well as sh-NC-mDCs, sh-Fut1-mDCs, sh-NC-mDCs + P-D2-EVs, and sh-Fut1-mDCs + P-D2-EVs. L Detection of Fut1 overexpression efficiency in iDCs using RT-qPCR. M Detection of IL10 mRNA expression in mDCs from different treatment groups using RT-qPCR. N Analysis of IL10 levels in the supernatant of mDCs from different treatment groups through ELISA. O Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry

Fig. 5

Influence of P-D2-EVs on Fut1-mediated fucosylation of ICAM1. A Western blot analysis of UEA-1-enriched mDCs under different treatments. B Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. C IP-based detection and quantification of ICAM1 binding to UEA1 in each group. D Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm. E Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. F IP-based detection and quantification of ICAM1 binding to UEA1 in each group. G Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm. H Schematic representation of Fut1-OE-iDCs treated with 2DGal; (I) Western blot analysis and quantification of α-(1,2)-fucosylation status of ICAM1 protein in each group after UEA-1 enrichment. J IP-based detection and quantification of ICAM1 binding to UEA1 in each group. K Immunofluorescence co-localization (yellow) and quantification of UEA1 and ICAM1, with DAP(I) staining the cell nucleus in blue, scale bar = 25 μm; UEA1: Ulex europaeus agglutinin 1, 2DGal: 2-deoxy-d-galactose. * indicates statistical significance compared to the control group or between two groups, with P < 0.05; all experiments were repeated three times

Fig. 6

Influence of P-D2-EVs on DC metabolism via the Fut1/ICAM1/P38 MAPK pathway. A, B Quantification of p-P38 and P38 expression, as well as the p-P38/P38 ratio, in mDCs from different treatment groups using Western blot. C mRNA expression of IL10 in mDCs from different treatment groups measured by RT-qPCR. D Levels of IL10 in the supernatant of mDCs from different treatment groups analyzed using ELISA. E Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry. F Schematic representation of the culture conditions for mDCs, mDCs + 2DGal, and m + 2DGal + Anisomycin groups. G Quantification of p-P38 and P38 expression, as well as the p-P38/P38 ratio, in mDCs from different treatment groups using Western blot. H mRNA expression of IL10 in mDCs from different treatment groups measured by RT-qPCR. I Levels of IL10 in the supernatant of mDCs from different treatment groups analyzed using ELISA. J Analysis of intracellular IL-10 levels in mDCs from different treatment groups using flow cytometry; 2DGal: 2-deoxy-d-galactose; *indicates statistical significance compared to the control group or between two groups, with P < 0.05; all experiments were repeated three times

Fig. 7

The impact of P-D2-EVs on regulating the Fut1/ICAM1/P38 MAPK pathway and IL10 metabolism in DCs on Th2 differentiation. A Schematic representation of co-culturing mDCs with CD4⁺ T cells. B Flow cytometric analysis of the percentage of IL4 and GATA3 in mDCs co-cultured with CD4⁺ T cells under different treatments. C RT-qPCR analysis of mRNA expression levels of IL4 and GATA3 in mDCs co-cultured with CD4⁺ T cells under different treatments. D ELISA analysis of IL4 levels in the supernatant of mDCs co-cultured with CD4⁺ T cells under different treatments. E Flow cytometric analysis of the percentage of IL4 and GATA3 in mDCs co-cultured with CD4⁺ T cells under different treatments. F RT-qPCR analysis of mRNA expression levels of IL4 and GATA3 in mDCs co-cultured with CD4⁺ T cells under different treatments. G ELISA analysis of IL4 levels in the supernatant of mDCs co-cultured with CD4⁺ T cells under different treatments; * indicates statistical significance compared to the control group or between two groups (P < 0.05); all experiments repeated three times. H Schematic diagram of co-culture of CD4 + T cells with mDCs after IL10 receptor blockade. I Flow cytometry analysis of the percentage of intracellular IL4 and GATA3 in CD4 + T cells co-cultured with mDCs under different treatments. J RT-qPCR detection of mRNA expression levels of IL4 and GATA3 in CD4 + T cells co-cultured with mDCs under different treatments. K ELISA analysis of the level of IL4 in the supernatant of co-cultures of mDCs and CD4 + T cells under different treatments. L Schematic diagram of co-culture of CD4 + T cells and mDCs with IL10 antibody. M Flow cytometry analysis of the percentage of intracellular IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 antibody under different treatments; (N) RT-qPCR detection of mRNA expression levels of IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 antibody under different treatments. O ELISA analysis of the level of IL4 in the supernatant of co-cultures of mDCs and CD4 + T cells with IL10 antibody. P Schematic diagram of co-culture of CD4 + T cells and mDCs with IL10. Q Flow cytometry analysis of the percentage of intracellular IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 under different treatments. R RT-qPCR detection of mRNA expression levels of IL4 and GATA3 in CD4 + T cells co-cultured with mDCs and IL10 under different treatments. S ELISA analysis of the level of IL4 in the supernatant of co-cultures of mDCs and CD4 + T cells with IL10; *indicates a significant difference between two groups with P < 0.05, all experiments were repeated three times

Fig. 8

Influence of P-D2-EVs-mediated DCs metabolic regulation on Th2 differentiation and activation of ILC2 cells. A Schematic diagram of ILC2 cell extraction and co-culture. B, C Schematic diagram of IL4, IL4 antibody, and IL4Rα antibody treatment. D Flow cytometry analysis of the percentage of IL5, IL13 and GATA3 in ILC2 cells treated with supernatant from co-culture of different treated mDCs and CD4⁺ T cells. E RT-qPCR analysis of the effects of supernatant from co-culture of different treated mDCs and CD4⁺ T cells on mRNA expression of IL5, IL13 and GATA3 in ILC2 cells. F ELISA analysis of the impact of supernatant from co-culture of different treated mDCs and CD4⁺ T cells on the level of IL5, IL13 in the supernatant of ILC2 cells. G Flow cytometry analysis of the percentage of IL5, IL13 and GATA3 in ILC2 cells treated with supernatant from co-culture of different treated mDCs and CD4⁺ T cells. H RT-qPCR analysis of the effects of supernatant from co-culture of different treated mDCs and CD4⁺ T cells on mRNA expression of IL5, IL13 and GATA3 in ILC2 cells. I ELISA analysis of the impact of supernatant from co-culture of different treated mDCs and CD4⁺ T cells on the level of IL5, IL13 in the supernatant of ILC2 cells; *denotes P < 0.05 when compared between two groups, all experiments were repeated three times

Fig. 9

In vivo validation of the regulatory mechanism of P-D2-EVs. A Number of nasal itching and sneezing events within 2 h after the final intranasal OVA administration in each group of mice (n = 6). B Total cell count in NALF of each group of mice (n = 6). C OVA-specific serum IgE levels in each group of mice (n = 6). D Representative PAS staining images (scale bar = 100 μm) and quantification of PAS-positive goblet cells in nasal tissue of each group of mice (n = 6). E Representative H&E staining images (scale bar = 100 μm or 25 μm) of nasal tissue, as well as quantification of eosinophils and nasal mucosal thickness, with the red arrow indicating nasal mucosa (n = 6). F Representative images (scale bar = 25 μm) and quantification of neutrophil infiltration in nasal mucosa of each group of mice, with the black arrow indicating positive cells (n = 6). G Protein expression and quantification of Fut1, p-P38, and P3J8 in nasal tissue of each group of mice as detected by Western blotting (n = 6). H α-(1,2)-Fucose glycosylation status and quantification of ICAM1 protein in nasal tissue of each group of mice as detected by Western blotting following UEA-1 enrichment (n = 6). I Binding and quantification of ICAM1 and UEA1 in nasal tissue of each group of mice as detected by co-immunoprecipitation (n = 6). J Expression and quantification of IL10, IL4, IL13, and GATA3 in nasal tissue of each group of mice as detected by RT-qPCR (n = 6). K Levels of IL10, IL4, and IL13 in serum of each group of mice as detected by ELISA (n = 6). L Protein expression and quantification of Fut1, p-P38, and P3J8 in nasal tissue of each group of mice as detected by Western blotting (n = 6). M α-(1,2)-Fucose glycosylation status and quantification of ICAM1 protein in nasal tissue of each group of mice as detected by Western blotting following UEA-1 enrichment (n = 6). N Binding and quantification of ICAM1 and UEA1 in nasal tissue of each group of mice as detected by co-immunoprecipitation (n = 6). O Expression and quantification of IL10, IL4, IL13, and GATA3 in nasal tissue of each group of mice as detected by RT-qPCR (n = 6). P Levels of IL10, IL4, and IL13 in serum of each group of mice as detected by ELISA (n = 6); NALF nasal lavage fluid; *indicates significant difference between two groups with P < 0.05

Fig. 10

Mechanism of P-D2-EVs regulation of DCs metabolism, Th2 differentiation, and ILC2 activation in vivo. A Percentage of DCs in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6). B Percentage of IL10-positive DCs in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6). C Percentage of Th2 cells among CD4⁺ T cells in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6). D Percentage of GATA3-positive cells among Th2 cells in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6). E Percentage of ILC2 cells in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6). F Percentage of IL13-positive cells among ILC2 cells in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6). G Percentage of GATA3-positive cells among ILC2 cells in nasal tissue of each group of mice as analyzed by flow cytometry (n = 6); *indicates significant difference between two groups with P < 0.05. H Illustration of the molecular mechanism underlying the improvement of AR by P-D2-EVs