CD73 mediates the therapeutic effects of endometrial regenerative cells in concanavalin A-induced hepatitis by regulating CD4+ T cells

Abstract

Background: As a kind of mesenchymal-like stromal cells, endometrial regenerative cells (ERCs) have been demonstrated effective in the treatment of Concanavalin A (Con A)-induced hepatitis. However, the therapeutic mechanism of ERCs is not fully understood. Ecto-5`-nucleotidase (CD73), an enzyme that could convert immune-stimulative adenosine monophosphate (AMP) to immune-suppressive adenosine (ADO), was identified highly expressed on ERCs. The present study was conducted to investigate whether the expression of CD73 on ERCs is critical for its therapeutic effects in Con A-induced hepatitis.

Methods: ERCs knocking out CD73 were generated with lentivirus-mediated CRISPR-Cas9 technology and identified by flow cytometry, western blot and AMPase activity assay. CD73-mediated immunomodulatory effects of ERCs were investigated by CD4⁺ T cell co-culture assay in vitro. Besides, Con A-induced hepatitis mice were randomly assigned to the phosphate-buffered saline treated (untreated), ERC-treated, negative lentiviral control ERC (NC-ERC)-treated, and CD73-knockout-ERC (CD73-KO-ERC)-treated groups, and used to assess the CD73-mediated therapeutic efficiency of ERCs. Hepatic histopathological analysis, serum transaminase concentrations, and the proportion of CD4⁺ T cell subsets in the liver and spleen were performed to assess the progression degree of hepatitis.

Results: Expression of CD73 on ERCs could effectively metabolize AMP to ADO, thereby inhibiting the activation and function of conventional CD4⁺ T cells was identified in vitro. In addition, ERCs could markedly reduce levels of serum and liver transaminase and attenuate liver damage, while the deletion of CD73 on ERCs dampens these effects. Furthermore, ERC-based treatment achieved less infiltration of CD4⁺ T and Th1 cells in the liver and reduced the population of systemic Th1 and Th17 cells and the levels of pro-inflammatory cytokines such as IFN-γ and TNF-α, while promoting the generation of Tregs in the liver and spleen, while deletion of CD73 on ERCs significantly impaired their immunomodulatory effects locally and systemically.

Conclusion: Taken together, it is concluded that CD73 is critical for the therapeutic efficiency of ERCs in the treatment of Con A-induced hepatitis.

Keywords: CD4+ T cell; CD73; Concanavalin A-induced hepatitis; Endometrial regenerative cells; Mice.