CD73/adenosine Pathway Contributes To The Amelioration Of Experimental Autoimmune Uveitis(EAU) By Mesenchymal Stem Cells(MSCs)

Objective In the present study, we use an experimental autoimmune uveitis(EAU) model to clarify the therapeutic role mediated by CD73 on MSCs, through clinical observation, histopathological examination and detection of retinal function. Furthermore, we investigate the mechanism of the CD73/adenosine pathway in immune modulation of MSCs by measuring T cell proliferation and differentiation and analyzing the effect of MSCs on CD73 expression of T cells.Methods 1. Cultivation of MSCs: Isolation, cultivation and characterization of adipose tissue derived MSCs from C57BL/6 mice. Isolation and cultivation of adipose tissue derived MSCs from human. 2. Induction of EAU model: For induction of adoptively transferred EAU model, the IRBP1-20 specific T cells were injected intraperitoneally into na?ve C57BL/6 mice. 3. Pretreatment of MSCs: APCP, a CD73 inhibitor, was added into the culture system of MSCs overnight before injection into EAU mice. 4. MSC treatment protocol: In order to determine the therapeutic effects of MSCs on EAU, the MSCs group received a single intravenous injection of MSCs at disease onset(10 or 11 days after transfer). Alternatively, to identify whether CD73 is involved in the immunomodulatory effects of MSCs, the same dose of APCP-pretreated MSCs were used in APCP-pretreated MSCs group. An equal volume of PBS was given to control group. 5. Clinical observation: EAU mice were examined every other day for fundus signs of uveitis by indirect fundoscopy from day 8 to day 60 post transfer. The incidence and severity of inflammation was assessed according to the criteria of Caspi. 6. Histopathological observation: The eyeballs were collected on day 60. The histopathological changes of retina were examined by hematoxylin and eosin staining and assessed using Caspi criteria.7. Retinal function detection: In order to evaluate the retinal function, dark-adapted ERG was recorded on day 25, 35, 45, and 60 after transfer. 8. Assay of T cell proliferation by 5-bromo-2-deoxyuridine(Brdu): In order to assess the suppressive effects of MSCs and APCP-pretreated MSCs on T cells in vivo, T cells and APCs from spleens and lymph nodes were prepared on day 21 post transfer and were treated with various doses of IRBP 1-20(0, 1, 10, and 30 μg/ml). In order to measure the direct suppressive effects of MSCs and APCP-pretreated MSCs on uveitic T cell proliferation in vitro, T cells and APCs from spleens and lymph nodes of untreated EAU mice were isolated. MSCs or APCP-pretreated MSCs were then added at various ratios(MSCs/T cells: 1:5, 1:10, and 1:20). Brd U ELISA kit was used to determine the proliferation index of T cells in several conditions, including CD39 inhibitor(POM-1), CD73 inhibitor(APCP) and the antagonist of the A2 A adenosine receptor(SCH58261). 9. T cell differentiation assays: T cells and APCs from spleens and lymph nodes of untreated EAU mice were prepared on day 21 post transfer and were co-cultured with MSCs and APCP-pretreated MSCs. CD4+IL-17+ and CD4+Foxp3+ cells were analyzed by flow cytometry. 10. Determination of the function of CD73 on MSCs: The malachite green colorimetric assay and high performance liquid chromatography(HPLC) were used to determine the ecto-nucleotidase activity of CD73 and the production of adenosine in the culture supernatants of MSCs. 11. Changes in CD73 expression of T cells: T cells and APCs were prepared from untreated EAU mice, and were cultured with MSCs or MSC-secreted cytokines. CD73 expression on the activated CD4+ T cells was detected by flow cytometry.Results 1. MSCs were successfully cultured and identified. 2. Adoptively transferred EAU model was successfully induced. 3. Clinical observation: A significant decrease in disease scores was observed following MSC treatment(P<0.05), whereas blocking the activity of CD73 on MSCs by APCP significantly decreased the therapeutic effect of MSCs on the progression of EAU(P<0.05).4. Histopathological observation: In line with the clinical observations, histopathological examinations revealed that MSC treatment dramatically reduced the retinal damage(P<0.05). As expected, APCP pretreatment significantly attenuated the protective efficacy of MSCs(P<0.05). 5. Retinal function detection: The retinal function of mice in the MSC-treated group was much better than that of mice in the control group(P<0.05), whereas blocking the activity of CD73 significantly reversed the protective effect of MSCs on retinal function(P<0.05). 6. T cell proliferation assays: MSC treatment resulted in lower proliferation of T cells(P<0.05), whereas the proliferative potential was partially recovered in T cells following APCP-pretreated MSC treatment(P<0.05). When a certain part of CD39/CD73/ adenosine pathway was blocked, a burst of T cell proliferation was observed. 7. T cell differentiation assays: After co-culture with MSCs, the ratio of Foxp3+ T cells/ IL-17+ T cells was significantly increased(P<0.05). 8. Malachite green colorimetric assay and high performance liquid chromatography(HPLC): CD73 on MSCs possessed the function of ecto-nucleotidase and could degrade AMP into adenosine, which could be inhibited by APCP(P<0.05). 9. CD73 expression of T cells: Co-culture with MSCs enhanced CD73 expression on activated T cells(P<0.05), and this could be attributed to TGF-β1 secretion.Conclusion 1. MSC treatment could significantly ameliorate EAU. 2. MSCs could inhibit the autoimmune response partially through the cooperation of CD73 expression on MSCs and CD39/CD73 co-expression on T cells. 3. MSCs could promote the expression of CD73 on activated T cells, which in turn might enhance autoimmune suppression.