UC-BSCs Exosomes Regulate Th17/Treg Balance in Patients with Systemic Lupus Erythematosus via miR-19b/KLF13

Abstract

Umbilical cord blood mesenchymal stem cells (UC-BSCs) are cells with low immunogenicity and differentiation potential, and the transfer of exosomes carried by UC-BSCs can regulate innate and adaptive immunity and affect immune homeostasis. This is an area of focus for autoimmune illnesses such as systemic lupus erythematosus (SLE). The target of this research was to investigate the immunomodulatory effect of exosomes produced from mesenchymal stem cells on SLE and its mechanism. After isolation of peripheral blood mononuclear cells (PBMC) from the SLE group and healthy group and treatment of SLE-derived PBMCs with UC-BSC-derived exosomes, the mRNA levels of corresponding factors in cells under different treatments were determined by RT-PCR, Th17/Treg content was analyzed by FCM (flow cytometry), and the targeted binding of microRNA-19b (miR-19b) to KLF13 was identified by in vitro experiments and bioinformatics analysis. The findings demonstrated that PBMC cells from SLE patients had higher proportions of Th17 subsets than the control group, whereas Treg subgroups with lower percentages were discovered. miR-19b's expression level was markedly reduced, which was inversely associated to the concentration of KLF13. In vitro experiments show that UC-BSC-derived exosome treatment can target KLF13 expression by increasing the miR-19b level, thereby regulating Th17/Treg balance and inhibiting the expression of inflammatory factors. According to the study's findings, SLE patients have dysregulated expression of the genes miR-19b and KLF13, and UC-BSC exosomes could regulate Th17/Treg cell balance and inflammatory factor expression in SLE patients through miR-19b/KLF13.

Keywords: exosomes; inflammatory factors; mathematical and statistical analysis; peripheral blood mononuclear cells; systemic lupus erythematosus; umbilical cord blood mesenchymal stem cells.

Figure 1

Expression degrees of miR-19b in SLE patients and normal controls. (Compared with the control group, ** p < 0.01).

Figure 2

Imbalance of Th17/Treg production in SLE patients. (A) Flow analysis of CD4 + IL-17 + Th17 cells, (B) percentage of CD4 + IL-17 + Th17 cells, (C) flow analysis of CD4 + Foxp3 + Treg cells, (D) CD4 + Foxp3 + Treg percentage of cells, (E) PBMC production of cytokines IL-6, TNF-α, IL-17, IL-10, and TGF-β, compared to control. ** p < 0.01, *** p < 0.001.

Figure 3

miR-19b targets KLF13 expression. (A) Bioinformatics website analysis of the binding site of KLF13 3’-UTR to miR-19b, (B) dual luciferase reporter gene to identify miR-19b and KLF13 binding, (C) Western blot analysis of several transfections protein expression of post-KLF13. Compared with NC mimics group. ** p < 0.01.

Figure 4

Inverse correlation between KLF13 and miR-19b expression in SLE patients. (A) The mRNA expression of KLF13 in SLE patients, (B) connection between KLF13 expression level and miR-19b expression level in SLE group, compared with the control group. *** p < 0.001.

Figure 5

Isolation and characterization of exosomes in UC-MSCs. (A) Transmission electron microscope image of exosome morphology (scale bar: 100 μm), (B) Size distribution of exosomes, (C) CD63 and TSG101 expression, (D) UC-MSC exosomes expression of miR-19b in exosomes, (E) uptake of exosomes by PBMC cells (scale bar = 25 μm) (F) the expression of KLF13 and miR-19 in PBMC cells after UC-MSC exosome treatment for 48 h compared to the control group. ** p < 0.01.

Figure 6

Impact of UC-MSC exosomes on Th17/Treg. (A) Flow analysis of CD4 + IL-17 + Th17 cells, (B) percentage of CD4 + IL-17 + Th17 cells, (C) flow analysis of CD4 + Foxp3 + Treg cells, (D) CD4 + Foxp3 + percentage of Treg cells, (E) PBMC production of cytokines IL-6, TNF-α, IL-17, IL-10, and TGF-β, compared with SLE group. * p < 0.05, ** p < 0.01, *** p < 0.001.