Exosomes secreted by podocytes regulate the differentiation of Th17/Treg cells in idiopathic nephrotic syndrome

Abstract

Background: Previous studies have demonstrated that immune cells release exosomes, which act as antigen-presenting vesicles to activate T cells. In our previous study, we discovered that podocytes, a type of kidney cell, can also exhibit antigen-presenting functions to naïve CD4⁺ T cells in idiopathic nephrotic syndrome (INS). Building upon these findings, the objective of this study was to investigate whether podocytes can regulate the balance between Th17 and Treg cells through the release of exosomes.

Methods: We co-cultured naïve CD4⁺ T cells with LPS-treated bone marrow dendritic cells (LPS-BMDC), LPS-treated mouse podocyte clone 5 (LPS-MPC-5), and exosomes derived from LPS-MPC-5 (LPS-EXO). As a control group, naïve CD4⁺ T cells were cultured with exosomes from untreated MPC-5 (EXO). After 48 h, we analyzed the percentages of Th17 and Treg cells using flow cytometry, measured the concentrations of IL-17A, IL-10, and IL-4 were using ELISA, and examined the expressions of IL-17a, IL-10, RORC, and FOXP3 using RT-qPCR.

Results: We confirmed the presence of exosomes derived from podocytes based on their morphology, size distribution, concentrations, and the levels of exosomes-specific markers. The percentage of Th17 and Treg cells in the LPS-EXO group was significantly higher than that in the control groups, but lower than in the LPS-MPC-5 group. Furthermore, the ratio of Th17/Treg was relatively higher in the LPS-EXO group compared to the LPS-MPC-5 group.

Conclusion: This study indicated further insights into the role of exosomes released from LPS-treated podocytes in regulating the balance between Th17 and Treg cells in INS.

Keywords: Exosomes; Nephrotic syndrome; Podocyte; Th17 cells; Treg cells.

Fig. 1

Characterization of exosomes secreted from MPC-5. (A) Diagram of particle size distribution and concentration in exosomes analyzed by nanoparticles tracking analysis. (B) Representative transmission electron microscopy showing exosomes (arrowheads). Western blots analysis of exosomes markers (CD9, CD63, TSG101) (C) and CD80, MHC II, TLR-4 (D). The control samples were cell culture supernatant.

Fig. 2

Schematic diagram of this study.

Fig. 3

The exosomes secreted from LPS-treated MPC-5 could promote naïve CD4⁺ T cells transformed to Th17 cells. (A)–(E) Percentages of CD4⁺IL17⁺ cells after culturing for 48 h in gated CD4⁺ T cells. F: The percentage of Th17 cells (means ± SD) in each group after simulation. (G) The concentration of IL-17A (means ± SD) in supernatants by ELISA. (H) and (I) The expression of IL-17A and RORC mRNA (means ± SD) by RT-qPCR. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001 (Tukey's test).

Fig. 4

The exosomes secreted from LPS-treated MPC-5 could promote naïve CD4⁺ T cells transformed to Treg cells. (A)–(E) Percentages of CD4⁺CD25⁺FOXP3⁺ cells after culturing for 48 h in gated CD4⁺ T cells. (F) The percentage of Treg cells (means ± SD) in each group after simulation. (G) and (H) The concentration of IL-10 and IL-4 (means ± SD) in supernatants by ELISA. (I) and (J) The expression of IL-10 and FOXP3 mRNA (means ± SD) by RT-qPCR. (K) The Th17/Treg ratio (means ± SD) in each group after culturing for 48 h *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001 (Tukey's test).