Dendritic cells mediated by small extracellular vesicles derived from MSCs attenuated the ILC2 activity via PGE2 in patients with allergic rhinitis

Abstract

Background: Mesenchymal stromal cells-derived small extracellular vesicles (MSC-sEVs) have recently attracted considerable attention because of their therapeutic potential in various immune diseases. We previously reported that MSC-sEVs could exert immunomodulatory roles in allergic airway inflammation by regulating group 2 innate lymphoid cell (ILC2) and dendritic cell (DC) functions. Therefore, this study aimed to investigate the indirect effects of MSC-sEVs on ILC2s from patients with allergic rhinitis (AR) via DCs.

Methods: Here, we isolated sEVs from induced pluripotent stem cells-MSCs using anion-exchange chromatography and mature DCs (mDCs) were treated with MSC-sEVs. sEV-mDCs were co-cultured with peripheral blood mononuclear cells from patients with AR or purified ILC2s. The levels of IL-13 and GATA3 in ILC2s were examined by flow cytometry. Bulk RNA sequence for mDCs and sEV-mDCs was employed to further probe the potential mechanisms, which were then validated in the co-culture systems.

Results: sEV-mDCs showed impaired capacity in priming the levels of IL-13 and GATA3 in ILC2s when compared with mDCs. Furthermore, there was higher PGE2 and IL-10 production from sEV-mDCs, and the blockade of them especially the former one reversed the inhibitory effects of sEV-mDCs.

Conclusions: We demonstrated that MSC-sEVs were able to dampen the activating effects of mDCs on ILC2s in patients with AR. Mechanismly, the PGE2-EP2/4 axis played an essential role in the immunomodulatory effects of sEV-mDCs on ILC2s. Herein, we provided new insights into the mechanism underlying the therapeutic effects of MSC-sEVs in allergic airway inflammation.

Keywords: Allergic rhinitis; Dendritic cells; Group 2 innate lymphoid cells; Mesenchymal stromal cells; Prostaglandin E2; Small extracellular vesicles.

Fig. 1

Identification of MSC-sEV. MSC-sEVs were isolated from iPSC-MSCs using anion-exchange chromatography. A. Nanoparticle Tracking Analysis (NTA) showed the particle size distribution and concentration of MSC-sEVs. B. TEM showed morphology and size of MSC-sEVs. C. CD9, CD63 and calnexin expression in MSCs and sEVs were determined by western blotting. The original blots are presented in Additional file 1: Fig. S4. MSC: mesenchymal stromal cells; MW: molecular weight; sEV: small extracellular vesicles; TEM: transmission electron microscope. Scale bar, 200 nm

Fig. 2

MSC-sEVs inhibited the effects of dendritic cells on ILC2s in patients with allergic rhinitis. A. Schematic representation of mDCs and sEV-mDCs generation and co-cultures. B–F. PBMCs from patients with AR were co-cultured with allogeneic mDCs or sEV-mDCs for 3 days. B. The levels of IL-13 in the supernatants were analyzed by ELISA (n = 8). C. Gating strategy of human ILC2s with Lin⁻CRTH2⁺CD127⁺. D-E. Intracellular IL-13 levels in ILC2s were analyzed by flow cytometry (n = 10). F-G. Levels of GATA3 in ILC2s were analyzed by flow cytometry (n = 4). DC: dendritic cell; ILC2: group 2 innate lymphoid cell; MSC: mesenchymal stromal cell; sEV: small extracellular vesicle. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

sEV-mDCs exhibited impaired capacity on purified ILC2 function. A. Schematic of isolation and amplifying purified ILC2s. B–C. Purified ILC2s were co-cultured with allogeneic mDCs or sEV-mDCs for 3 days. Intracellular IL-13 levels in ILC2s were analyzed by flow cytometry (n = 5). *P < 0.05

Fig. 4

Uptake of MSC-sEV by DCs. DCs on day 5 were co-cultured with mCherry-labeled MSC-sEVs for 12 h. A. The cells were photographed by confocal microscopy. Panels showed mCherry (Red) and DAPI (Blue). B–C. mCherry in DCs was analyzed using flow cytometry. MFI: mean fluorescence intensity. Scale bar, 5 μm. Data are shown as mean ± SEM. *P < 0.05

Fig. 5

The expression of PGE2 and IL-10 in sEV-mDCs and mDCs. A–B. GO analysis and KEGG enrichment analysis of differential mRNAs between mDCs and sEV-mDCs. C. Heatmap representation of differential mRNAs involved in ILC2s regulation between mDCs and sEV-mDCs. D. The violin plot showed the differential expression of PTGES, IL10 and TNFSF15 in mDCs and sEV-mDCs. E. The levels of PTGES, IL10 and TNFSF15 were examined by RT-quantitative PCR (n = 6–18). F. The levels of PGE2 in the supernatants of mDCs and sEV-mDCs (n = 16). G-H. The levels of IL-10 in the supernatants of mDCs and sEV-mDCs and co-cultured PBMCs (n = 13–16). PTGES: Prostaglandin E Synthase; IL-10: Interleukin-10; PGE2: Prostaglandin E2. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

PGE2 mediated the impaired function of sEV-mDCs on ILC2s through EP2/4. PBMCs from patients with AR were co-cultured with allogeneic mDCs, sEV-mDCs, MF63-pretreated sEV-mDCs or combined with MF63 (0.1 μM), ONO/PF (1 μM), anti-IL-10 (2 μg/mL), respectively, for 3 days. A–B. Schematic of MF63-sEV-mDCs induction and blocking experiments. C. The levels of PGE2 in the supernatants of mDCs, sEV-mDCs and MF63-sEV-mDCs (n = 7). D. Intracellular IL-13 levels in ILC2s under different condition were analyzed by flow cytometry. E–H. Percentage of IL-13⁺ILC2s in the culture conditions as described (n = 7). MF63: pharmacological inhibitor of prostaglandin E Synthase; ONO/PF: ONO-AE3-208 and PF-04418948, antagonists for EP2/EP4. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01

Fig. 7

Schema for MSC-sEVs indirectly suppress ILC2 function in allergic rhinitis through increasing production of PGE2 from sEV-mDCs