Exosomes with FOXP3 from gene-modified dendritic cells ameliorate the development of EAE by regulating the balance of Th/Treg

Abstract

Objective: To investigate the efficiency and potential mechanisms of exosomes from dendritic cells (DCs) transfected with Forkhead box protein P3 (FOXP3) in the development of experimental autoimmune encephalomyelitis (EAE). Method: Mouse bone marrow-derived immature DCs were loaded with adenovirus carrying FOXP3 gene, and exosomes were generated. Then the exosomes with FOXP3 (FOXP3-EXOs) were co-cultured with CD4+T cell in vitro to evaluate their potential on CD4+T cell proliferation and differentiation, and injected into EAE mice to assess their effects on the development of EAE. Result: FOXP3-EXOs were effective to inhibit the CD4⁺T cell proliferation and the production of Interferon gamma (IFN-γ), interleukin (IL)-6, and IL-17, while they promoted the production of IL-10 in vitro. Moreover, FOXP3-EXOs treatment significantly decreased the neurological scores, reduced the infiltration of inflammatory cells into the spinal cord, and decreased demyelination in comparison to saline and Con-EXOs treated EAE mice. Moreover, the FOXP3-EXOs treatment resulted in obvious increases in the levels of regulatory T (Treg) cells and IL-10, whereas levels of T helper 1 (Th1) cells, Th17 cells, IFN-γ, IL-6, and IL-17 decreased significantly in the splenocyte culture of EAE mice. Conclusion: The present study preliminarily investigated the effects and potential mechanisms of FOXP3-EXOs in EAE and revealed that the FOXP3-EXOs could inhibit the production of Th1 and Th17 cells and promote the production of Treg cells as well as ameliorate the development of EAE. The neuroprotective effects of FOXP3-EXOs on EAE are likely due to the regulation of Th/Treg balance.

Keywords: FOXP3; Multiple sclerosis; dendritic cell; exosomes; experimental autoimmune encephalomyelitis.

Figure 1

The morphologic features of imDCs transfected with Ad/Con or Ad/FOXP3. (A) Fluorescence Microscope (Scale bar = 50 µm) and (B) electron microscope (Scale bar = 5 µm) observations of imDCs transfected with Ad/Con. (C) Fluorescence Microscope and (D) electron microscope observations of imDCs transfected with Ad/FOXP3. (E) Western blot analysis of FOXP3 protein expression in the imDCs transfected with Ad/Con or Ad/FOXP3. GADPH was used as the internal control.

Figure 2

Characterization of imDCs derived exosomes. (A) EM analysis of exosomes from imDCs. Scale bar, 100 nm. (B) Size distribution profile analysis of exosomes. (C) Exosomes were analyzed by western blot for the presence of several proteins characteristic of exosomes as well as FOXP3. GADPH was used as the internal control.

Figure 3

The effects of FOXP3-EXOs on CD4⁺T cell proliferation and differentiation in vitro. (A) FOXP3-EXOs significantly inhibited CD4⁺T cell proliferation in a dose-dependent manner assessed by CCK-8 assay. Con-EXO1 and FOXP3-EXO1 indicated 1ug/ml of exosomes of each type were used and Con-EXO2 and FOXP3-EXO2 indicated 2ug/ml of exosomes of each type were used for the assay. N=3, ^*P < 0.05, ^**P < 0.01 vs. the PBS group. (B) FOXP3-EXO obviously suppressed the production of INF-γ, IL-6, IL-17 and promoted the production of IL-10. Unstimulated group indicated cells were not activated by CD3/CD28 antibody beads, and groups of Control, Con-EXO, and FOXP3-EXO indicated cells were activated and treated with PBS, Con-EXOs, and FOXP3-EXOs, respectively. Data shown are expressed as the mean± SEM of each group (n=3). ^*P < 0.05, ^**P < 0.01 vs. the unstimulated group; ^#P < 0.05, ^##P < 0.01 vs. the control group.

Figure 4

FOXP3-EXOs ameliorate the loss of body weight and neuro-behavioral symptoms in EAE mice. (A) Mice in FOXP3-EXOs group showed dramatically higher weights compared to those in the EAE and Con-EXOs groups. (B) FOXP3-EXOs treatment significantly slowed down the increase in clinical scores and reduced severity relative to the EAE mice with saline or Con-EXOs treatment. Control group indicated wildtype C57BL/6 mice treated with saline, and groups of EAE, Con-EXO, and FOXP3-EXO indicated EAE mice treated with saline, Con-EXOs, and FOXP3-EXOs, respectively. Data shown are expressed as the mean± SEM of each group (n=6). ^*P < 0.05 vs. the EAE group; ^#P < 0.05 vs. the Con-EXO group.

Figure 5

FOXP3-EXOs treatment ameliorate inflammatory infiltration and demyelination in the spinal cord of EAE mice. (A) High magnification of HE analysis of the spinal cord sections, Scale bar = 100 µm. (B) High magnification of LFB analysis of the spinal cord sections, Scale bar = 100 µm. (C and D) The mean scores of inflammation and demyelination in the EAE mice, Con-EXOs-treated mice and FOXP3-EXOs-treated mice. Data shown are representative images from each group or expressed as the mean±SEM of each group (n = 6). ^#P < 0.05, ^##P < 0.01 vs. the Control group; ^**P < 0.01 vs. the EAE group.

Figure 6

FOXP3-EXOs treatment modulates immune responses in EAE mice. (A) Flow cytometry analysis of the splenocytes culture of mice in each group (n=6 per group). (B) ELISA analysis of serum cytokines of mice in each group (n=6 per group). Data shown are representative images from each group or expressed as the mean± SEM of each group. ^* P < 0.05 or ^** P< 0.01 vs. the control group; ^# P < 0.05 or ^## P < 0.01 vs. the EAE group.

Figure 6

FOXP3-EXOs treatment modulates immune responses in EAE mice. (A) Flow cytometry analysis of the splenocytes culture of mice in each group (n=6 per group). (B) ELISA analysis of serum cytokines of mice in each group (n=6 per group). Data shown are representative images from each group or expressed as the mean± SEM of each group. ^* P < 0.05 or ^** P< 0.01 vs. the control group; ^# P < 0.05 or ^## P < 0.01 vs. the EAE group.