April 2023
Volume 64, Issue 4
Open Access
Immunology and Microbiology  |   April 2023
Effects of Plasma-Derived Exosomal miRNA-19b-3p on Treg/T Helper 17 Cell Imbalance in Behçet's Uveitis
Author Affiliations & Notes
  • Qingyan Jiang
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Qingfeng Wang
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Shiyao Tan
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Jinyu Cai
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Xingsheng Ye
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Guannan Su
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Peizeng Yang
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People's Republic of China
  • Correspondence: Peizeng Yang, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Youyi Road 1, Chongqing 400016, P.R. China; peizengycmu@126.com
Investigative Ophthalmology & Visual Science April 2023, Vol.64, 28. doi:https://doi.org/10.1167/iovs.64.4.28
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Qingyan Jiang, Qingfeng Wang, Shiyao Tan, Jinyu Cai, Xingsheng Ye, Guannan Su, Peizeng Yang; Effects of Plasma-Derived Exosomal miRNA-19b-3p on Treg/T Helper 17 Cell Imbalance in Behçet's Uveitis. Invest. Ophthalmol. Vis. Sci. 2023;64(4):28. https://doi.org/10.1167/iovs.64.4.28.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: To explore the potential role of plasma-derived exosomal microRNAs (miRNAs) in the development of regulatory T cell (Treg)/T helper 17 (Th17) cell imbalances in Behçet's uveitis (BU).

Methods: The exosome treatment was conducted to evaluate the effects of plasma exosomes from patients with active BU and healthy controls on the Treg/Th17 cell balance. miRNA sequencing analysis of plasma exosomes was conducted to identify differentially expressed miRNAs between patients with active BU and healthy controls. miRTarBase analysis and dual-luciferase reporter assays were conducted to identify the target genes of miR-19b-3p. CD4+T cells were transfected with miR-19b-3p mimic or inhibitor to evaluate its regulation of the Treg/Th17 cell balance. The Treg/Th17 cell balance in CD4+T cells was evaluated by flow cytometry and enzyme-linked immunosorbent assay.

Results: Exosomes from patients with active BU promoted Th17 cell differentiation and inhibited Treg cell differentiation. MiRNA sequencing analysis revealed 177 upregulated and 274 downregulated miRNAs in plasma exosomes of patients with active BU. Among them, miR-19b-3p was significantly elevated, and its target genes were identified as being involved in T-cell differentiation. miR-19b-3p overexpression downregulated CD46 expression and the Treg/Th17 cell ratio in CD4+T cells from healthy controls, whereas miR-19b-3p inhibition reversed these regulatory effects and restored the Treg/Th17 cell balance of CD4+T cells from patients with active BU.

Conclusions: Plasma-derived exosomes from patients with active BU showed a markedly differential miRNA expression in comparison to healthy controls. Highly expressed miRNA-19b-3p could induce a Treg/Th17 cell imbalance, probably by downregulating CD46 expression.

Behçet’s disease (BD) is an autoinflammation disease caused by multiple factors1,2 and is characterized by recurrent oral and genital ulcers, multiple skin lesions, and uveitis.35 Behçet’s uveitis (BU) is one of the most common manifestations of BD and leads to blindness if not well controlled. Increasing evidence has demonstrated that the pathogenesis of BU involves aberrant regulatory T cell (Treg)/T helper 17 (Th17) cell ratios.6,7 However, the exact etiology of this change is still unclear. Recent studies have reported that exosome dysfunction could contribute to the pathogenesis of several immune diseases.8,9 Exosomes are small molecular vesicles with a lipid bilayer that measure 30 to 100 nm in diameter and are discovered in numerous bodily secretions, including breast milk, plasma, urine, and saliva.10 They are produced by various cells and mediate intercellular communication by transporting information cargoes such as microRNAs (miRNAs), messenger RNAs (mRNAs), proteins, and lipids.11 Due to the transportation of these cargoes, exosomes play key roles in regulating immune responses. Exosomal miRNAs are able to target and regulate genes involved in immune-related pathways.12 Studies on the pathogenesis of immune diseases have suggested that aberrant exosomal miRNA expression has a crucial impact on activating immune responses in systemic lupus erythematosus, multiple sclerosis, and Sjögren's syndrome.1317 However, the effects of plasma-derived exosomal miRNAs on BU development and their mechanisms, if any, are not clear. The results of this study provide evidence that the expression of BU exosomal miRNAs could contribute to development of the Treg/Th17 cell imbalance by downregulating CD46 in CD4+T cells. 
Materials and Methods
Patients and Samples
A total of 23 patients with active BU and 24 sex- and age-matched healthy controls were involved in this study. Ten active BU patient samples (one BU sample with unqualified RNA was excluded) and 10 healthy control samples were used for miRNA sequencing analysis, eight active BU patient samples and eight healthy control samples for exosomal miR-19b-3p validation, and six active BU patient samples and six healthy control samples for flow cytometry (FCM), enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and the exosome treatment experiment. Information regarding the patients with active BU is provided in Supplementary Tables S1 and S2; basic information regarding the healthy controls is shown in Supplementary Table S3. All patients with active BU had not used immunosuppressive drugs for at least 1 week or used less than 20 mg/d prednisone less than 1 week before blood sampling. The BD diagnostic guidelines proposed by the International Study Group were used for the diagnosis of BD.18 All of the patients with BU had active intraocular inflammations, as indicated by anterior chamber cells, anterior chamber flare, vitreous haze, or retinal lesions.19 Each participant provided an institutionally approved written informed consent. The Chongqing Medical University Ethics Committee (2018-048) approved this study. All of the procedures adhered to the tenets of the Declaration of Helsinki. 
Exosome and Exosomal RNA Isolation
Supernatant was obtained after centrifuging plasma samples for 30 minutes at 10,000g at 4°C after being centrifuged for 10 minutes at 1500g. After washing with phosphate-buffered saline (PBS), 500 µL of a supernatant sample was applied to the top of a 70-nm qEVoriginal column (Izon Science, Cambridge, MA, USA) and eluted with PBS. The three extracellular vesicle–rich fractions were collected and centrifuged at 7500g for 30 minutes using a Millipore Amicon Ultra-4 ultrafiltration device (pore size, 100 kDa MWCO; Sigma-Aldrich, St. Louis, MO, USA). Exosomes were used immediately or stored at −80°C and re-thawed on ice for subsequent experiments. Before RNA extraction, exosomes were treated with RNase A to ensure that extra-exosomal RNA was removed. Exosomal RNA was extracted utilizing the exoRNase Midi Kit (QIAGEN, Hilden, Germany). 
Exosome Characterization
Exosomes isolated from the plasma samples of patients with active BU and healthy controls were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and WB.20,21 Exosomes were resuspended in PBS and then placed in an electron microscope grid stained with 2% uranyl acetate solution for 10 minutes at 25°C as described previously.22 Exosomes were then identified using TEM (Tecnai G2 Spirit BioTWIN; Thermo Fisher Scientific, Waltham, MA, USA) (Fig. 1A). The light-scattering and Brownian motion properties were utilized to calculate size distributions and exosome concentrations using NTA (Fig. 1B). ZetaVIEW S/N 17-310 was used for the NTA (Particle Metrix, Meerbusch, Germany). Statistical analysis was carried out using the corresponding software (ZetaVIEW 8.04.02). Exosome markers including CD9, CD63, TSG101, and the calnexin protein were detected using WB analysis (described later) (Fig. 1C). 
Figure 1.
 
Exosomes from patients with active BU induced a Treg/Th17 cell imbalance. (A) Exosomes were isolated from plasma of healthy controls and patients with active BU, and their morphology and size were validated by TEM. Scale bars: 200 nm and 100 nm. (B) NTA was used to evaluate the size and concentration of plasma exosomes. (C) WB analysis was used to detect positive exosome markers (CD9, CD63, and TSG101) and a negative exosome marker (calnexin). (D) Representative fluorescence microscopy images showing the internalization of PKH-26-labeled exosomes (red) by PKH-67-labeled CD4+T cells (green). Scale bars: 10 µm and 1 µm. (E) The proportion of Treg and Th17 in CD4+T cells was detected by FCM following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). (F) The protein levels of IL-10 and IL-17 were measured by ELISA following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). All experiments were repeated three times. The paired ANOVA test was used for statistical analysis (*P < 0.05, **P < 0.01); NS, no significance.
Figure 1.
 
Exosomes from patients with active BU induced a Treg/Th17 cell imbalance. (A) Exosomes were isolated from plasma of healthy controls and patients with active BU, and their morphology and size were validated by TEM. Scale bars: 200 nm and 100 nm. (B) NTA was used to evaluate the size and concentration of plasma exosomes. (C) WB analysis was used to detect positive exosome markers (CD9, CD63, and TSG101) and a negative exosome marker (calnexin). (D) Representative fluorescence microscopy images showing the internalization of PKH-26-labeled exosomes (red) by PKH-67-labeled CD4+T cells (green). Scale bars: 10 µm and 1 µm. (E) The proportion of Treg and Th17 in CD4+T cells was detected by FCM following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). (F) The protein levels of IL-10 and IL-17 were measured by ELISA following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). All experiments were repeated three times. The paired ANOVA test was used for statistical analysis (*P < 0.05, **P < 0.01); NS, no significance.
Exosome Tracing
A PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) was utilized to label CD4+T cells, and a PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) was utilized to label exosomes. CD4+T cells (5 × 105/well) were seeded in 12-well plate(4.5 cm2/well), cultured in 500 µL exosome-free medium (UmiBio, Shanghai, China), then activated with 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, they were treated with 20 µL exosomes (1 × 1010 particles/20 µL/well) for 24 hours. The uptake of labeled exosomes was detected by an inverted fluorescence microscope (LAS X3.4.7; Leica Camera, Wetzlar, Germany) and by 520-nm and 580-nm lasers. To compare the uptake of exosomes by CD4+T cells between patients with BU and the healthy controls, a PKH26 Red Fluorescent Cell Linker Kit was utilized to label exosomes and anti-human CD4-APC antibody (clone RPA-T4; BioLegend, San Diego, CA, USA) was used to stain CD4+T cells. The relative quantification of exosome uptake by CD4+T cells was evaluated by FCM analysis. 
Sequencing of MicroRNAs
The QIAseq miRNA Library Kit (QIAGEN) was used to create miRNA libraries, which were sequenced using the NextSeq 550 System (Illumina, San Diego, CA, USA). Prior to amplification, each strand of the complementary DNA (cDNA) synthesis was specifically labeled by unique molecular indices, which more accurately reflected the level of endogenous miRNA. miRNA counts captured using miRBase were also obtained. The final list of miRNAs was refined and constructed using statistical, bioinformatic, and computational biology approaches. 
Isolation and Treatment of Human CD4+T Cells
Human Ficoll–Hypaque density gradient centrifugation was employed to isolate peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample. To extracted CD4+T cells from PBMCs, CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were utilized using a procedure described previously in our laboratory.23 Briefly, PBMCs (1 × 107) were incubated with 20 µL of CD4 microbeads and 80 µL of buffer solution at 4°C for 15 minutes. The cells were subjected to a mass spectrometry column (Miltenyi Biotec) filled with 500 µL of buffer solution. The cells were then collected following a buffer solution wash. CD4+T cells were cultured in exosome-free medium or Roswell Park Memorial Institute (RPMI) 1640 medium containing 100 U/mL penicillin/streptomycin and 10% fetal bovine serum and were then activated using 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28 antibodies. Finally, they were treated with exosomes, miRNA mimic, miRNA inhibitor, or PBS. The samples were incubated for 24 hours or 72 hours at 37°C under 5% CO2 conditions, and cells or supernatants were finally collected for subsequent experiments. 
Cell Transfection With miRNA Mimics and Inhibitors
miR-19b-3p mimic (Sangon Biotech, Shanghai, China) or negative control (NC) mimic and miR-19b-3p inhibitor (Sangon Biotech) or NC inhibitor was transfected into CD4+T cells using LIPO8000 transfection reagent (Beyotime Biotech, Jiangsu, China). 
Luciferase Reporter Assay
The LIPO8000 transfection reagent was used to transfect 293T cells with miRNA (miR-19b-3p mimic, miR-19b-3p inhibitor, and miR-19b-3p-NC), 3′ UTR luciferase reporter constructs (3′ UTR-NC, 3′ UTR-CD46-WT, and 3′ UTR-CD46-MUT), and Renilla luciferase as described previously.12 To observe luciferase activity, we employed the Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) after 48 hours of transfection. The firefly luciferase activity was normalized by Renilla luciferase. 
Real-Time Quantitative PCR and WB
Invitrogen TRIzol Reagent (Thermo Fisher Scientific) was utilized to extract total RNA from human CD4+T cells. Using the PrimeScript RT Reagent Kit (Takara, Kusatsu, Japan) or miRNA 1st-Strand cDNA Synthesis Kit (stem-loop method; Sangon Biotech), mRNA was reverse transcribed into cDNA. SYBR Premix (Bio-Rad Laboratories, Hercules, CA, USA) and an Applied Biosystems ABI Prism 7500 System (Thermo Fisher Scientific) were utilized for real-time quantitative PCR (RT-qPCR). For normalization, U6 snRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were utilized as internal controls. The data were analyzed utilizing the 2–∆∆Ct method. Table 1 provides the various primer sequences employed in this study. The WB assay was conducted according to the method described previously24 and used the following antibodies: anti-CD9 (1:1000, EXOAB-CD9A-1; System Biosciences, Palo Alto, CA, USA), anti-CD63 (1:1000, EXOAB-CD63-1; System Biosciences), anti-TSG101 (1:1000, EXOAB-STG101-1; System Biosciences), anti-Calnexin (1:1000, YT0613; ImmunoWay Biotechnology, Plano, TX, USA), anti-beta-actin (1:1000; HUABIO, Woburn, MA, USA), and anti-CD46 (1:1000, ab108307; Abcam, Cambridge, UK). Grayscale analysis by WB was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). 
Table 1.
 
Primers Used for RT-qPCR
Table 1.
 
Primers Used for RT-qPCR
FCM and ELISA
For FCM, CD4+T cells (1 × 105/500 µL) were stimulated for 6 hours at 37°C with 1 µL of a cell stimulation cocktail containing brefeldin A (BioLegend) and then cultured with surface antigen antibodies for 30 minutes at 4°C in the dark. Following washes with PBS, cells were fixed and permeabilized for 60 minutes with fixation/permeabilization diluent and concentrate (BioLegend) or intracellular fixation buffer (BioLegend). At 4°C in the dark, intracellular marker staining was performed for 30 minutes. FCM was conducted with the following antibodies from BioLegend: anti-human Foxp3-PE (clone 259D) and IL-17-PE (clone BL168). CytExpert software was used to analyze the results. For ELISA, CD4+T cells were seeded in 24-well plates (1 × 105 cells/well), and their supernatants were collected after 72 hours. IL-10 and IL-17 protein expression levels were measured using a DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN, USA). 
Statistical Analysis
All diagram and graph analyses were conducted utilizing Prism 6.0 (GraphPad Software, San Diego, CA, USA). The average value and standard error of the mean (SEM) were utilized to express the data. For comparative analysis of two and more than two groups, paired and unpaired t-tests and paired ANOVA tests were utilized, respectively. P < 0.05 was considered statistically significant. 
Results
BU Plasma-Derived Exosomes Induces an Imbalance in Treg/Th17 Cells
To explore the impacts of plasma-derived exosomes on CD4+T cell, these cells were treated with plasma-derived exosomes. Fluorescence microscopy showed that red-fluorescent-labeled exosomes were internalized in green-fluorescent-labeled CD4+T cells, suggesting uptake of exosomes by CD4+T cells (Fig. 1D). FCM analysis showed no difference in exosome uptake by CD4+T cells between the BU plasma exosome–treated group and healthy control plasma exosome–treated group (Supplemental Fig. S1A). To explore the potential contribution of BU plasma-derived exosomes to Treg/Th17 cell imbalance, the proportion of Treg and Th17 cells in CD4+T cells was detected following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS. A decreased proportion of Treg cells in association with an increased frequency of Th17 cells was discovered in the BU plasma exosome group when compared to the healthy control group (Fig. 1E). At the protein level, a similar finding was discovered (Fig. 1F). 
BU Plasma-Derived Exosomes Have Differential miRNA Expression Profiles
To investigate the mechanisms underlying the contribution of plasma-derived exosomes from patients with active BU to Treg/Th17 cell imbalance, differential miRNA analysis of plasma exosomes from patients with active BU and healthy controls was performed. The result identified 2656 miRNAs (Supplementary Table S4), of which 451 (177 upregulated and 274 downregulated) were markedly different (fold change > 2; P < 0.05) between patients with active BU and healthy controls (Fig. 2A, Supplementary Table S5). Gene Ontology (GO) enrichment analysis showed that the target genes were enriched in 7502 pathways (Fig. 2B, Supplementary Table S6), including T-cell differentiation, activation, and proliferation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these target genes also showed that they were concentrated in 290 functional pathways (Fig. 2C, Supplementary Table S7), including Th17 cell differentiation and the IL-17 signaling pathway. 
Figure 2.
 
The exosomal miRNA profiles of patients with active BU differed from those of healthy controls. (A) Volcano plot comparing expressed exosomal miRNAs in plasma from patients with active BU and from healthy controls. A volcano plot was created using a log2 fold change and –log10 P values of all of the detected miRNAs. (B) GO pathway enrichment analysis of the target genes associated with the differentially expressed miRNAs was performed, and 45 pathways related to biological processes (BPs), cellular components (CCs), and molecular functions (MFs) were identified. (C) KEGG analysis of differential miRNA target genes identified 15 immune-related pathways. (D) Exosomal miR-19b-3p expression in plasma of patients with active BU and healthy controls as detected by RT-qPCR. The patients with active BU did not use immunosuppressive drugs for at least 1 week or used less than 20 mg/d prednisone less than 1 week before blood sampling (n = 8 for each treatment). Experiments were repeated three times. Data were analyzed using an unpaired t-test (***P < 0.001).
Figure 2.
 
The exosomal miRNA profiles of patients with active BU differed from those of healthy controls. (A) Volcano plot comparing expressed exosomal miRNAs in plasma from patients with active BU and from healthy controls. A volcano plot was created using a log2 fold change and –log10 P values of all of the detected miRNAs. (B) GO pathway enrichment analysis of the target genes associated with the differentially expressed miRNAs was performed, and 45 pathways related to biological processes (BPs), cellular components (CCs), and molecular functions (MFs) were identified. (C) KEGG analysis of differential miRNA target genes identified 15 immune-related pathways. (D) Exosomal miR-19b-3p expression in plasma of patients with active BU and healthy controls as detected by RT-qPCR. The patients with active BU did not use immunosuppressive drugs for at least 1 week or used less than 20 mg/d prednisone less than 1 week before blood sampling (n = 8 for each treatment). Experiments were repeated three times. Data were analyzed using an unpaired t-test (***P < 0.001).
CD46 Is a Target Gene of Exosomal miR-19b-3p
The top ten miRNAs that have significant upregulation or downregulation in exosomes are displayed in Table 2 according to fold change and P value. We found a markedly upregulated expression of miR-19b-3p, which is a top-ranked miRNA that plays an important part in immune response regulation.2527 It was therefore selected as a target for further validation using samples from patients with active BU and healthy controls. The results revealed that, consistent with miRNA expression profile results, miR-19b-3p was markedly upregulated in the plasma exosomes of patients with active BU when compared to the healthy control group (Fig. 2D). However, there was no difference in exosome-depleted plasma between patients with active BU and healthy controls (Supplemental Fig. S1B), suggesting that the differential miR-19b-3p expression was mainly derived from plasma exosomal miRNA rather than plasma-free miRNA. 
Table 2.
 
Top 10 Upregulated and 10 Downregulated Exosomal miRNAs in Patients with Active BD as Compared With Healthy Controls
Table 2.
 
Top 10 Upregulated and 10 Downregulated Exosomal miRNAs in Patients with Active BD as Compared With Healthy Controls
To identify the role of exosomal miR-19b-3p in the development of BU, we employed miRTarBase to detect its target genes. A total of 713 target genes were identified (Supplementary Table S8). CD46 was found to be implicated in immune-associated pathways including T-cell activation, lymphocyte activation regulation, and immune effector process regulation, according to the GO pathway enrichment analysis (Fig. 3A, Supplementary Table S9). We further performed experiments to test the possible targeting of CD46 in CD4+T cells by exosomal miR-19b-3p. The results showed that CD46 protein expression was markedly lower in patients with active BU than in healthy controls (Fig. 3B). CD46 3′ UTR was defined as a potential miR-19b-3p target via TargetScan analysis (Fig. 3C). We observed strong binding between miR-19b-3p and the wild-type (WT) CD46 firefly luciferase vector, indicating that CD46 was its direct target (Fig. 3D). To further investigate the inhibitory effect of miR-19b-3p on CD46 expression in vitro, a miR-19b-3p overexpression or inhibition model was constructed in CD4+T cells by transfecting with miR-19b-3p mimic or inhibitor (Fig. 3E). We discovered that miR-19b-3p mimic inhibited CD46 protein expression, whereas miR-19b-3p inhibitor increased the expression of this protein (Fig. 3F). 
Figure 3.
 
CD46 was the target gene of exosomal miR-19b-3p. (A) GO pathway enrichment analysis of CD46 identified 15 pathways related to BPs, CCs, and MFs. (B) Comparison of CD46 protein expression in CD4+T cells from patients with active BU and healthy controls (n = 6 for each treatment). (C) miR-19b-3p binding sites in the CD46 3′ UTR. The binding sequence of miR-19b-3p is located at nucleotides 1393 to 1399 from the 3′ UTR, as predicted by TargetScanHuman 8.0. (D) The relative luciferase activity of the 3′ UTR-CD46-WT or 3′ UTR-CD46-MUT vectors in 293T cells co-transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor. (E) The mRNA levels of miR-19b-3p in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor measured by RT-qPCR (n = 6 for each treatment). (F) WB analysis of CD46 expression in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor (n = 4 for each treatment). All experiments were repeated three times. P values were calculated using the paired t-test to compare two groups. For multiple comparisons, P values were calculated using the paired ANOVA test (*P < 0.05, **P < 0.01); NS, no significance.
Figure 3.
 
CD46 was the target gene of exosomal miR-19b-3p. (A) GO pathway enrichment analysis of CD46 identified 15 pathways related to BPs, CCs, and MFs. (B) Comparison of CD46 protein expression in CD4+T cells from patients with active BU and healthy controls (n = 6 for each treatment). (C) miR-19b-3p binding sites in the CD46 3′ UTR. The binding sequence of miR-19b-3p is located at nucleotides 1393 to 1399 from the 3′ UTR, as predicted by TargetScanHuman 8.0. (D) The relative luciferase activity of the 3′ UTR-CD46-WT or 3′ UTR-CD46-MUT vectors in 293T cells co-transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor. (E) The mRNA levels of miR-19b-3p in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor measured by RT-qPCR (n = 6 for each treatment). (F) WB analysis of CD46 expression in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor (n = 4 for each treatment). All experiments were repeated three times. P values were calculated using the paired t-test to compare two groups. For multiple comparisons, P values were calculated using the paired ANOVA test (*P < 0.05, **P < 0.01); NS, no significance.
Exosomal miR-19b-3p Induces an Imbalance in Treg/Th17 Cells by Targeting CD46
The studies above established the inhibitory influences of exosomal miR-19b-3p on CD46 expression in CD4+T cells. To determine the possible influence of miR-19b-3p in the Treg/Th17 cell imbalance, CD4+T cells of the healthy controls were transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor, and then cultured for 72 hours. The results showed that miR-19b-3p overexpression of CD4+T cells of the healthy controls could decrease the proportion of Treg cells in association with an increased proportion of Th17 cells (Fig. 4A). It could also effectively decrease IL-10 protein levels and increase IL-17 (Fig. 4B). Additionally, the miR-19b-3p inhibitor group had an opposite effect. 
Figure 4.
 
miR-19b-3p transfection in CD4+T cells increased Th17 cell frequency and IL-17 levels while decreasing Treg cell frequency and IL-10 levels. (A) The proportions of Treg and Th17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (B) The protein levels of IL-10 and IL-17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). (C) The proportions of Treg and Th17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (D) The protein levels of IL-10 and IL-17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). All experiments were repeated three times. Data are presented as the mean ± SEM of three independent experiments. For multiple comparisons, the paired ANOVA test was applied (*P < 0.05, **P < 0.01); NS, no significance.
Figure 4.
 
miR-19b-3p transfection in CD4+T cells increased Th17 cell frequency and IL-17 levels while decreasing Treg cell frequency and IL-10 levels. (A) The proportions of Treg and Th17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (B) The protein levels of IL-10 and IL-17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). (C) The proportions of Treg and Th17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (D) The protein levels of IL-10 and IL-17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). All experiments were repeated three times. Data are presented as the mean ± SEM of three independent experiments. For multiple comparisons, the paired ANOVA test was applied (*P < 0.05, **P < 0.01); NS, no significance.
Inhibition of miR-19b-3p in CD4+T Cells From Patients With Active BU Restores the Treg/Th17 Cell Balance
In subsequent experiments, we performed functional rescue experiments to investigate whether miR-19b-3p inhibition in CD4+T cells from patients with active BU could restore the Treg/Th17 cell balance. MiR-19b-3p inhibitor or NC inhibitor were transfected into CD4+T cells from patients with active BU, and cells were cultured for 72 hours. The result revealed that miR-19b-3p inhibition could increase the Treg cell frequency while decreasing the Th17 cell frequency in CD4+T cells from patients with active BU (Fig. 4C). A similar finding was observed concerning the protein level as measured using ELISA (Fig. 4D). 
Discussion
In this study, we found that CD4+T cells can uptake BU plasma-derived exosomes, promote Th17 cell differentiation, and inhibit Treg cell differentiation. We also observed different plasma miRNA profiles between patients with active BU and healthy controls. Additionally, we have provided evidence that higher expression of exosomal miRNA-19b-3p may lead to a Treg/Th17 cell imbalance in BU by targeting CD46. Importantly, miR-19b-3p inhibition in CD4+T cells from patients with active BU could reduce Th17 cell frequency and IL-17 levels in association with the increased proportion of Treg cells and IL-10 levels. 
Exosomes, acting as immune microenvironment factors, play a crucial role in immune responses and the pathogenesis of immune diseases. According to a report, patients with intestinal Behçet’s syndrome had differentially expressed exosomal miRNA as compared to healthy controls.28 Another study showed that plasma exosome samples from patients with intestinal Behçet's syndrome could induce intestinal epithelial cell pyroptosis by activating NLRP3.29 This study explored the possibility of plasma exosomes being involved in the development of BU and found that these exosomes from patients with active BU resulted in an increase in Th17 cells and a reduction in Treg cells. It is widely recognized that an increased Th17 cell frequency and a decreased Treg cell frequency are immune characteristic of BU.6,7 All of these studies suggest that exosomes are engaged in the pathogenesis of BU. 
The role of miRNAs in the pathogenesis of various diseases including cancer, immune-related diseases, and cardiovascular diseases has received increased attention.3032 Previous studies have shown that 11 miRNAs are differentially expressed in peripheral blood or PBMCs of patients with BD in comparison to healthy controls.3338 Except for miR-155, which has been associated with the pathogenesis of BU by targeting TAB2,35 the remaining miRNAs have not been investigated for their functions. miRNAs are the main exosome component that regulates immune cell functions, and they have been found to be involved in the development of multiple immune diseases.1317 This study found an increased miR-19b-3p expression in plasma exosomes from patients with active BU. miR-19b-3p has been demonstrated to be an important regulator of immune homeostasis in various inflammatory and autoimmune diseases.3941 Amjad et al.27 found that high levels of miR-19b-3p expression could produce a large amount of chemokines and proinflammatory cytokines by decreasing TNFAIP3 expression in meningitic Escherichia coli-induced neuroinflammation. Another study has shown that miR-19b-3p can target SOCS-1 to activate nuclear factor kappa B (NF-κB) in macrophages and promote M1 macrophage activation, thus contributing to the development of tubulointerstitial inflammation.12 All of these results suggest that these inflammatory diseases may share a similar pathogenesis in the context of miR-19b-3p expression. 
It is widely acknowledged that miRNAs exert their effect through modulating target genes, but what is the target gene of miR-19b-3p and how does this miRNA exert its function? In this study, we applied WB analysis and luciferase reporter assays to determine that CD46 is a direct target of miR-19b-3p in BU. CD46, a membrane cofactor protein, is a potent human T- lymphocyte costimulatory molecule,4244 and is critical for the induction of Tregs and IL-10 production (anti-inflammatory cytokine).45 Ellinghaus et al.46 found that CD4+T cells activated with anti-CD46 antibodies increased IFN-γ expression while decreasing IL-10 production in systemic lupus erythematosus. Studies on rheumatoid arthritis and multiple sclerosis have shown that a CD46 deficiency in patients can lead to a decreased frequency of T regulatory type 1 cells and a reduction of IL-10.47,48 Other studies have reported that increased IL-17 production is associated with a reduction in CD46 expression.49 In this study, we discovered that elevated miR-19b-3p expression could inhibit CD46 expression but increase the Th17 cell frequency and IL-17 level while reducing the proportion of Treg cells and IL-10 levels. These studies collectively suggest that CD46 could be implicated in the development of Treg/Th17 cell imbalance and the production of related cytokines. 
The present study showed that 451 miRNAs differed markedly between patients with active BU and healthy controls. miR-19b-3p has been reported to be related to various inflammatory and autoimmune diseases.3941 Additionally, we also found that some other miRNAs were associated with immune or inflammatory responses. For example, miR-9-5p could alleviate apoptosis, inflammation, and endoplasmic reticulum stress in spinal cord injury by regulating the histone deacetylase 5/fibroblast growth factor 2 axis.50 miR-145-5p has also been reported to inhibit inflammatory responses of rat mesangial cells by regulating the AKT/GSK pathway.51 In the present study, we focused on the effect of miR-19b-3p on T-cell differentiation and found that it could reduce Treg cell frequency and IL-10 levels in association with an increased proportion of Th17 cells and IL-17 levels. Our study, along with others, suggests that miRNAs may exert their effect in diseases including BU through multiple mechanisms. 
This study has several limitations. First, we identified the differentially expressed exosomal miRNAs from the plasma of patients with active BU but did not test their dynamic changes during this disease. To further understand the relationship between disease activity and exosomal miRNAs, longitudinal studies that include patients with active and inactive BU are required. Second, the strict inclusion criteria limited the number of samples used in this study. Future studies with larger sample sizes should be conducted for further validation. Third, Th1 cells and neutrophils are also implicated in the pathogenesis of BU. The uptake of exosomes by these two cell types and the subsequent functional effects were not explored in this study and should be clarified in the future. Fourth, plasma-derived exosomes are produced by various peripheral immune cells, so further studies are required to investigate the cellular origin of miR-19b-3p and other differentially expressed miRNAs. Fifth, whether targeting genes other than CD46 are involved in the Treg/Th17 cell imbalance should be investigated in future studies. Finally, we found that exosomal miR-19b-3p was associated with the Treg/Th17 cell imbalance in BU; however, its relevance to clinical manifestations requires further study. 
In summary, this study has revealed that plasma-derived exosomal miR-19b-3p exerts inhibitory effects on CD46 expression, resulting in a Treg/Th17 cell imbalance in patients with active BU. These findings suggest that miR-19b-3p might be a potential target for the investigation of BU prevention and treatment. 
Acknowledgments
Supported by grants from the National Natural Science Foundation Key Program (82230032), National Natural Science Foundation Key Program (81930023), Key Project of Chongqing Science and Technology Bureau (CSTC2021jscx-gksb-N0010), National Natural Science Foundation (82101106), Chongqing Outstanding Scientists Project (2019), Natural Science Foundation of Chongqing (cstc2021jcyj-bsh0055), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), and the Chongqing Science & Technology Platform and Base Construction Program (cstc2014pt-sy10002). 
Disclosure: Q. Jiang, None; Q. Wang, None; S. Tan, None; J. Cai, None; X. Ye, None; G. Su, None; P. Yang, None 
References
Deuter CM, Kötter I, Wallace GR, Murray PI, Stübiger N, Zierhut M. Behçet's disease: Ocular effects and treatment. Prog Retin Eye Res. 2008; 27(1): 111–136. [CrossRef] [PubMed]
Yang P, Ohno S, Zierhut M. Editorial: New insights into uveitis: Immunity, genes, and microbes. Front Immunol. 2021; 12: 765377. [CrossRef] [PubMed]
Triolo G, Accardo-Palumbo A, Dieli F, et al. Humoral and cell mediated immune response to cow's milk proteins in Behçet's disease. Ann Rheum Dis. 2002; 61(5): 459–462. [CrossRef] [PubMed]
Mendes D, Correia M, Barbedo M, et al. Behçet's disease – a contemporary review. J Autoimmun. 2009; 32: 178–188. [CrossRef] [PubMed]
Cunningham ET, Jr, Tugal-Tutkun I, Khairallah M, Okada AA, Bodaghi B, Zierhut M. Behçet uveitis. Ocul Immunol Inflamm. 2017; 25(1): 2–6. [CrossRef] [PubMed]
Geri G, Terrier B, Rosenzwajg M, et al. Critical role of IL-21 in modulating TH17 and regulatory T cells in Behçet disease. J Allergy Clin Immunol. 2011; 128(3): 655–664. [CrossRef] [PubMed]
Wang C, Zhou W, Su G, Hu J, Yang P. Progranulin suppressed autoimmune uveitis and autoimmune neuroinflammation by inhibiting Th1/Th17 cells and promoting Treg cells and M2 macrophages. Neurol Neuroimmunol Neuroinflamm. 2022; 9(2): e1133. [CrossRef] [PubMed]
Robbins P, Morelli A. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol. 2014; 14(3): 195–208. [CrossRef] [PubMed]
Zhao Y, Wei W, Liu ML. Extracellular vesicles and lupus nephritis - new insights into pathophysiology and clinical implications. J Autoimmun. 2020; 115: 102540. [CrossRef] [PubMed]
Mori M, Ludwig R, Garcia-Martin R, Brandão B, Kahn C. Extracellular miRNAs: From biomarkers to mediators of physiology and disease. Cell Metab. 2019; 30(4): 656–673. [CrossRef] [PubMed]
Isaac R, Reis F, Ying W, Olefsky J. Exosomes as mediators of intercellular crosstalk in metabolism. Cell Metab. 2021; 33(9): 1744–1762. [CrossRef] [PubMed]
Lv L, Feng Y, Wu M, et al. Exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury. Cell Death Differ. 2020; 27(1): 210–226. [CrossRef] [PubMed]
Tan L, Zhao M, Wu H, et al. Downregulated serum exosomal miR-451a expression correlates with renal damage and its intercellular communication role in systemic lupus erythematosus. Front Immunol. 2021; 12: 630112. [CrossRef] [PubMed]
Wang W, Yue C, Gao S, et al. Promising roles of exosomal microRNAs in systemic lupus erythematosus. Front Immunol. 2021; 12: 757096. [CrossRef] [PubMed]
Kimura K, Hohjoh H, Fukuoka M, et al. Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis. Nat Commun. 2018; 9(1): 17. [CrossRef] [PubMed]
Rozier P, Maumus M, Maria ATJ, et al. Mesenchymal stromal cells-derived extracellular vesicles alleviate systemic sclerosis via miR-29a-3p. J Autoimmun. 2021; 121: 102660. [CrossRef] [PubMed]
Xing Y, Li B, He J, Hua H. Labial gland mesenchymal stem cell derived exosomes-mediated miRNA-125b attenuates experimental Sjögren's syndrome by targeting PRDM1 and suppressing plasma cells. Front Immunol. 2022; 13: 871096. [CrossRef] [PubMed]
International Study Group for Behçet's Disease. Criteria for diagnosis of Behçet's disease. Lancet. 1990; 335(8697): 1078–1080. [PubMed]
Jabs DA, Nussenblatt RB, Rosenbaum JT, Standardization of Uveitis Nomenclature Working G. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. Am J Ophthalmol. 2005; 140(3): 509–516. [PubMed]
Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018; 7(1): 1535750. [CrossRef] [PubMed]
Clayton A, Boilard E, Buzas EI, et al. Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles. J Extracell Vesicles. 2019; 8(1): 1647027. [CrossRef] [PubMed]
Zhou J, Li X, Wu X, et al. Exosomes released from tumor-associated macrophages transfer miRNAs that induce a Treg/Th17 cell imbalance in epithelial ovarian cancer. Cancer Immunol Res. 2018; 6(12): 1578–1592. [CrossRef] [PubMed]
Wang Q, Yi S, Du Z, et al. The Rs12569232 SNP association with Vogt-Koyanagi-Harada disease and Behçet's disease is probably mediated by regulation of Linc00467 expression. Ocul Immunol Inflamm. 2021; 29(7-8): 1464–1470. [CrossRef] [PubMed]
Liu J, Qi X, Wang XH, et al. Downregulation of the LncRNA MEG3 promotes osteogenic differentiation of BMSCs and bone repairing by activating Wnt/β-catenin signaling pathway. J Clin Med. 2022; 11(2): 395. [CrossRef] [PubMed]
Juárez-Vicuña Y, Guzmán-Martín C, Martínez-Martínez L, et al. miR-19b-3p and miR-20a-5p are associated with the levels of antiphospholipid antibodies in patients with antiphospholipid syndrome. Rheumatol Int. 2021; 41(7): 1329–1335. [CrossRef] [PubMed]
Aripova A, Akparova A, Bersimbaev R. The potential role of miRNA-19b-3p and miRNA-320c in patients with moderate bronchial asthma. MicroRNA. 2020; 9(5): 373–377. [CrossRef] [PubMed]
Amjad N, Yang R, Li L, et al. Decrease of miR-19b-3p in brain microvascular endothelial cells attenuates meningitic Escherichia coli-induced neuroinflammation via TNFAIP3-mediated NF-κB inhibition. Pathogens. 2019; 8(4): 268. [CrossRef] [PubMed]
Hou CC, Bao HF, Shen Y, Ye JF, Ma HF, Guan JL. Expression of miRNAs derived from plasma exosomes in patients with intestinal Behçet's syndrome. Clin Exp Rheumatol. 2022; 40(8): 1480–1490. [PubMed]
Hou CC, Ma HF, Ye JF, Luo D, Bao HF, Guan JL. Plasma exosomes derived from patients with intestinal Behçet's syndrome induce intestinal epithelial cell pyroptosis. Clin Rheumatol. 2021; 40(10): 4143–4155. [CrossRef] [PubMed]
Baumjohann D, Ansel KM. MicroRNA-mediated regulation of T helper cell differentiation and plasticity. Nat Rev Immunol. 2013; 13(9): 666–678. [CrossRef] [PubMed]
Lin S, Gregory RI. MicroRNA biogenesis pathways in cancer. Nat Rev Cancer. 2015; 15(6): 321–333. [CrossRef] [PubMed]
Hosen MR, Goody PR, Zietzer A, et al. Circulating microRNA-122-5p is associated with a lack of improvement in left ventricular function after transcatheter aortic valve replacement and regulates viability of cardiomyocytes through extracellular vesicles. Circulation. 2022; 146(24): 1836–1854. [CrossRef] [PubMed]
Jadideslam G, Ansarin K, Sakhinia E, et al. Expression levels of miR-21, miR-146b and miR-326 as potential biomarkers in Behçet's disease. Biomark Med. 2019; 13(16): 1339–1348. [CrossRef] [PubMed]
Ahmadi M, Yousefi M, Abbaspour-Aghdam S, et al. Disturbed Th17/Treg balance, cytokines, and miRNAs in peripheral blood of patients with Behçet's disease. J Cell Physiol. 2019; 234(4): 3985–3994. [CrossRef] [PubMed]
Zhou Q, Xiao X, Wang C, et al. Decreased microRNA-155 expression in ocular Behçet's disease but not in Vogt Koyanagi Harada syndrome. Invest Ophthalmol Vis Sci. 2012; 53(9): 5665–5674. [CrossRef] [PubMed]
El Boghdady NA, Shaker OG. Role of serum miR-181b, proinflammatory cytokine, and adhesion molecules in Behçet's disease. J Interferon Cytokine Res. 2019; 39(6): 347–354. [CrossRef] [PubMed]
Uğurel E, Şehitoğlu E, Tüzün E, Kürtüncü M, Çoban A, Vural B. Increased complexin-1 and decreased miR-185 expression levels in Behçet's disease with and without neurological involvement. Neurol Sci. 2016; 37(3): 411–416. [CrossRef] [PubMed]
Shan J, Zhou P, Liu Z, Zheng K, Jin X, Du L. Association of miRNA-146a gene polymorphism Rs2910164 with Behçet's disease: A meta-analysis. Ocul Immunol Inflamm. 2022; 30(7-8): 1883–1889. [CrossRef] [PubMed]
Wang-Renault S, Boudaoud S, Nocturne G, et al. Deregulation of microRNA expression in purified T and B lymphocytes from patients with primary Sjögren's syndrome. Ann Rheum Dis. 2018; 77(1): 133–140. [CrossRef] [PubMed]
Guggino G, Orlando V, Saieva L, et al. Downregulation of miRNA17-92 cluster marks Vγ9Vδ2 T cells from patients with rheumatoid arthritis. Arthritis Res Ther. 2018; 20(1): 236. [CrossRef] [PubMed]
Oduor C, Kaymaz Y, Chelimo K, et al. Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma. BMC Cancer. 2017; 17(1): 761. [CrossRef] [PubMed]
Dempsey L. Modulation of CD46 in T cells. Nat Immunol. 2017; 18(12): 1287.
Kolev M, Dimeloe S, Le Friec G, et al. Complement regulates nutrient influx and metabolic reprogramming during Th1 cell responses. Immunity. 2015; 42(6): 1033–1047. [CrossRef] [PubMed]
Marie J, Astier A, Rivailler P, Rabourdin-Combe C, Wild T, Horvat B. Linking innate and acquired immunity: Divergent role of CD46 cytoplasmic domains in T cell induced inflammation. Nat Immunol. 2002; 3(7): 659–666. [CrossRef] [PubMed]
Kemper C, Chan AC, Green JM, Brett KA, Murphy KM, Atkinson JP. Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature. 2003; 421(6921): 388–392. [CrossRef] [PubMed]
Ellinghaus U, Cortini A, Pinder CL, Le Friec G, Kemper C, Vyse TJ. Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus. Eur J Immunol. 2017; 47(7): 1200–1210. [CrossRef] [PubMed]
Astier AL, Meiffren G, Freeman S, Hafler DA. Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis. J Clin Invest. 2006; 116(12): 3252–3257. [CrossRef] [PubMed]
Perucha E, Melchiotti R, Bibby JA, et al. The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells. Nat Commun. 2019; 10(1): 498. [CrossRef] [PubMed]
Suzuki H, Lasbury ME, Fan L, et al. Role of complement activation in obliterative bronchiolitis post-lung transplantation. J Immunol. 2013; 191(8): 4431–4439. [CrossRef] [PubMed]
He X, Zhang J, Guo Y, Yang X, Huang Y, Hao D. Exosomal miR-9-5p derived from BMSCs alleviates apoptosis, inflammation and endoplasmic reticulum stress in spinal cord injury by regulating the HDAC5/FGF2 axis. Mol Immunol. 2022; 145: 97–108. [CrossRef] [PubMed]
Wu J, He Y, Luo Y, et al. MiR-145-5p inhibits proliferation and inflammatory responses of RMC through regulating AKT/GSK pathway by targeting CXCL16. J Cell Physiol. 2018; 233(4): 3648–3659. [CrossRef] [PubMed]
Figure 1.
 
Exosomes from patients with active BU induced a Treg/Th17 cell imbalance. (A) Exosomes were isolated from plasma of healthy controls and patients with active BU, and their morphology and size were validated by TEM. Scale bars: 200 nm and 100 nm. (B) NTA was used to evaluate the size and concentration of plasma exosomes. (C) WB analysis was used to detect positive exosome markers (CD9, CD63, and TSG101) and a negative exosome marker (calnexin). (D) Representative fluorescence microscopy images showing the internalization of PKH-26-labeled exosomes (red) by PKH-67-labeled CD4+T cells (green). Scale bars: 10 µm and 1 µm. (E) The proportion of Treg and Th17 in CD4+T cells was detected by FCM following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). (F) The protein levels of IL-10 and IL-17 were measured by ELISA following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). All experiments were repeated three times. The paired ANOVA test was used for statistical analysis (*P < 0.05, **P < 0.01); NS, no significance.
Figure 1.
 
Exosomes from patients with active BU induced a Treg/Th17 cell imbalance. (A) Exosomes were isolated from plasma of healthy controls and patients with active BU, and their morphology and size were validated by TEM. Scale bars: 200 nm and 100 nm. (B) NTA was used to evaluate the size and concentration of plasma exosomes. (C) WB analysis was used to detect positive exosome markers (CD9, CD63, and TSG101) and a negative exosome marker (calnexin). (D) Representative fluorescence microscopy images showing the internalization of PKH-26-labeled exosomes (red) by PKH-67-labeled CD4+T cells (green). Scale bars: 10 µm and 1 µm. (E) The proportion of Treg and Th17 in CD4+T cells was detected by FCM following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). (F) The protein levels of IL-10 and IL-17 were measured by ELISA following 24 hours of treatment with plasma exosomes from patients with active BU, plasma exosomes from healthy controls, or PBS (n = 6 for each treatment). All experiments were repeated three times. The paired ANOVA test was used for statistical analysis (*P < 0.05, **P < 0.01); NS, no significance.
Figure 2.
 
The exosomal miRNA profiles of patients with active BU differed from those of healthy controls. (A) Volcano plot comparing expressed exosomal miRNAs in plasma from patients with active BU and from healthy controls. A volcano plot was created using a log2 fold change and –log10 P values of all of the detected miRNAs. (B) GO pathway enrichment analysis of the target genes associated with the differentially expressed miRNAs was performed, and 45 pathways related to biological processes (BPs), cellular components (CCs), and molecular functions (MFs) were identified. (C) KEGG analysis of differential miRNA target genes identified 15 immune-related pathways. (D) Exosomal miR-19b-3p expression in plasma of patients with active BU and healthy controls as detected by RT-qPCR. The patients with active BU did not use immunosuppressive drugs for at least 1 week or used less than 20 mg/d prednisone less than 1 week before blood sampling (n = 8 for each treatment). Experiments were repeated three times. Data were analyzed using an unpaired t-test (***P < 0.001).
Figure 2.
 
The exosomal miRNA profiles of patients with active BU differed from those of healthy controls. (A) Volcano plot comparing expressed exosomal miRNAs in plasma from patients with active BU and from healthy controls. A volcano plot was created using a log2 fold change and –log10 P values of all of the detected miRNAs. (B) GO pathway enrichment analysis of the target genes associated with the differentially expressed miRNAs was performed, and 45 pathways related to biological processes (BPs), cellular components (CCs), and molecular functions (MFs) were identified. (C) KEGG analysis of differential miRNA target genes identified 15 immune-related pathways. (D) Exosomal miR-19b-3p expression in plasma of patients with active BU and healthy controls as detected by RT-qPCR. The patients with active BU did not use immunosuppressive drugs for at least 1 week or used less than 20 mg/d prednisone less than 1 week before blood sampling (n = 8 for each treatment). Experiments were repeated three times. Data were analyzed using an unpaired t-test (***P < 0.001).
Figure 3.
 
CD46 was the target gene of exosomal miR-19b-3p. (A) GO pathway enrichment analysis of CD46 identified 15 pathways related to BPs, CCs, and MFs. (B) Comparison of CD46 protein expression in CD4+T cells from patients with active BU and healthy controls (n = 6 for each treatment). (C) miR-19b-3p binding sites in the CD46 3′ UTR. The binding sequence of miR-19b-3p is located at nucleotides 1393 to 1399 from the 3′ UTR, as predicted by TargetScanHuman 8.0. (D) The relative luciferase activity of the 3′ UTR-CD46-WT or 3′ UTR-CD46-MUT vectors in 293T cells co-transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor. (E) The mRNA levels of miR-19b-3p in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor measured by RT-qPCR (n = 6 for each treatment). (F) WB analysis of CD46 expression in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor (n = 4 for each treatment). All experiments were repeated three times. P values were calculated using the paired t-test to compare two groups. For multiple comparisons, P values were calculated using the paired ANOVA test (*P < 0.05, **P < 0.01); NS, no significance.
Figure 3.
 
CD46 was the target gene of exosomal miR-19b-3p. (A) GO pathway enrichment analysis of CD46 identified 15 pathways related to BPs, CCs, and MFs. (B) Comparison of CD46 protein expression in CD4+T cells from patients with active BU and healthy controls (n = 6 for each treatment). (C) miR-19b-3p binding sites in the CD46 3′ UTR. The binding sequence of miR-19b-3p is located at nucleotides 1393 to 1399 from the 3′ UTR, as predicted by TargetScanHuman 8.0. (D) The relative luciferase activity of the 3′ UTR-CD46-WT or 3′ UTR-CD46-MUT vectors in 293T cells co-transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor. (E) The mRNA levels of miR-19b-3p in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor measured by RT-qPCR (n = 6 for each treatment). (F) WB analysis of CD46 expression in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor (n = 4 for each treatment). All experiments were repeated three times. P values were calculated using the paired t-test to compare two groups. For multiple comparisons, P values were calculated using the paired ANOVA test (*P < 0.05, **P < 0.01); NS, no significance.
Figure 4.
 
miR-19b-3p transfection in CD4+T cells increased Th17 cell frequency and IL-17 levels while decreasing Treg cell frequency and IL-10 levels. (A) The proportions of Treg and Th17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (B) The protein levels of IL-10 and IL-17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). (C) The proportions of Treg and Th17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (D) The protein levels of IL-10 and IL-17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). All experiments were repeated three times. Data are presented as the mean ± SEM of three independent experiments. For multiple comparisons, the paired ANOVA test was applied (*P < 0.05, **P < 0.01); NS, no significance.
Figure 4.
 
miR-19b-3p transfection in CD4+T cells increased Th17 cell frequency and IL-17 levels while decreasing Treg cell frequency and IL-10 levels. (A) The proportions of Treg and Th17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (B) The protein levels of IL-10 and IL-17 in CD4+T cells transfected with miR-19b-3p mimic or NC mimic or with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). (C) The proportions of Treg and Th17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were detected by FCM (n = 6 for each treatment). (D) The protein levels of IL-10 and IL-17 in CD4+T cells from patients with active BU transfected with miR-19b-3p inhibitor or NC inhibitor were measured by ELISA (n = 6 for each treatment). All experiments were repeated three times. Data are presented as the mean ± SEM of three independent experiments. For multiple comparisons, the paired ANOVA test was applied (*P < 0.05, **P < 0.01); NS, no significance.
Table 1.
 
Primers Used for RT-qPCR
Table 1.
 
Primers Used for RT-qPCR
Table 2.
 
Top 10 Upregulated and 10 Downregulated Exosomal miRNAs in Patients with Active BD as Compared With Healthy Controls
Table 2.
 
Top 10 Upregulated and 10 Downregulated Exosomal miRNAs in Patients with Active BD as Compared With Healthy Controls
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×