Fig 1: TSG-6 mediates UCMSC suppression of DC maturation, function, and CCR6+ Th17 differentiation. (A) Flow cytometry was used to detect the expression of surface mature markers CD14, CD1a, and CD83 in each group, along with their quantitative statistical graphs (n = 6). (B) Flow cytometry was employed to detect the expression of surface functional markers CD40, CD86, and HLA-DR in each group, together with their quantitative statistical graphs (n = 6). (C) Flow cytometry was utilized to detect the proportion of IL17+CCR6+ Th17 cells in each group of mDC after co-culture with mixed lymphocytes for 3 days, as well as their quantitative statistical graphs (n = 6). (D, E) Immunofluorescence was used to detect representative images and quantitative statistical graphs of the number of peripheral and central DCs in the corneas of each group of mice (n = 6). (F) Flow cytometry was applied to detect the number and quantitative statistical graphs of CD45+CD11c+DC in the corneas, conjunctiva, and draining lymph nodes of each group of mice (n = 6). (G, H) Flow cytometry was used to detect the number and quantitative statistical graphs of CD11c+CD86+ DC and CD11c+MHC-II+ DC in the corneas, conjunctiva, and draining lymph nodes of each group of mice (n = 6). Data are presented as the mean ± SD. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: TSG-6-dependent regulation of CCL20-CCR6 signaling by UCMSCs. (A) The mRNA expression level of CCL20 in HCECs, detected by qRT-PCR with GAPDH as the internal reference (n = 3). (B) CCL20 secretion level in culture supernatants, detected by ELISA (n = 3). (C) The qRT-PCR analysis of CCL20 mRNA in corneal and conjunctival tissues (n = 6). (D) CCL20 secretion level in corneal and conjunctival tissues, detected by an ELISA (n = 6). (E, F) Immunohistochemical staining of the corneal and conjunctival tissues, scale bar 50 µm. (G) Representative images and statistical graphs of CCR6+ Th17 flow cytometry in draining lymph nodes (n = 6). Data are presented as the mean ± SD. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: UCMSCs-derived TSG-6 suppresses CCL20 production via NF-κB inhibition. (A) Western blot analysis and quantification of P-IκBα and IκBα protein levels in HCECs at various time points post high osmotic stimulation (n = 3). (B) Protein levels of P-IκBα and IκBα in HCECs co-cultured with shNC-UCMSCs or shTSG-6-UCMSCs, measured 1 hour after hyperosmotic stimulation, with quantification of relative protein levels (n = 3). (C) Evaluation of phosphorylation levels of MAPK pathway proteins in HCECs at different time points post high osmotic stimulation (n = 3). (D) Phosphorylation levels of MAPK pathway proteins in HCECs co-cultured with shNC-UCMSCs or shTSG-6-UCMSCs, measured 1 hour after hyperosmotic stimulation (n = 3). (E) ELISA detection of CCL20 levels in cell culture supernatants of each group following PDTC pretreatment (n = 3). Data are presented as the mean ± SD. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01.
Fig 4: Schematic representation of the mechanism by which UCMSCs secrete TSG-6 to regulate the Th17-related CCL20-CCR6 chemotaxis axis in the treatment of DED.
Fig 5: TSG-6 mediates the therapeutic effects of UCMSCs in dry eye models. (A) Cell viability of HCECs in different treatment groups, assessed by CCK-8 (n = 8). (B) The qRT-PCR analysis of TNF-α, IL-1β, and IL-6 expression in HCECs, with GAPDH as the internal reference (n = 3). (C) Schematic of mouse model experimental treatment. (D) Schirmer's test for tear secretion volume in mice in at 0, 10, and 20 days post eye drop instillation (n = 6). (E) CFS scoring in mice at 0, 10, and 20 days after eye drop instillation (n = 6). (F) Representative images of fluorescein sodium staining. (G) Representative images and statistical data (n = 6) of goblet cell density in the conjunctival epithelium, scale bar of 50 µm. (H, I) The qRT-PCR of TNF-α, IL-1β, and IL-6 mRNA levels in corneal and conjunctival tissues (n = 6). (J–L) Representative images and statistical graphs of Th17 flow cytometry in cornea, conjunctiva, and draining lymph nodes (n = 6). Data are presented as the mean ± SD. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Human TSG6 ELISA Kit