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Objective and design: The present study aimed to investigate the relationship between the disturbed balance of CD4+/CD8+, Th17/Treg and the activation of the Notch signaling pathway in experimental autoimmune uveitis (EAU). Methods: An EAU rat model was induced in Lewis rats, and pathology analysis was performed by hematoxylin and eosin (H&E) staining. CD4+, CD8+, Th17, and Treg levels in spleen, lymph nodes and eye tissues were determined by flow cytometry. Meanwhile, the expression of Notch1, DLL4, IL-10, and IL-17 was determined by quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA). In addition, the inhibitory effect of N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT) on Th17 differentiation by Notch signaling in vitro was further investigated using T lymphocytes from EAU rats on day 12 post-immunization by flow cytometry. Results: The pathological results showed that inflammatory cell infiltration occurred in ocular tissues in EAU rats. The CD4+/CD8+ and Th17/Treg ratios in EAU rats were apparently higher than those in normal control individuals. Q-PCR and ELISA analyses indicated the expression of Notch1, DLL4, IL-10, and IL-17 in EAU rats gradually increased on day 6 after immunization, peaked on day 12, and then gradually decreased. The dynamic trends in Notch1 and DLL4 expression in EAU rats were identical to those of CD4+/CD8+ and Th17/Treg levels. DAPT can significantly inhibit the activation of Notch signaling, decrease Th17 cell differentiation, and attenuate the level of the Th17 cell lineage, contributing to the balance of the Th17/Treg ratio. Conclusion: The activation of the Notch signaling pathway can regulate Th17 and Treg cell differentiation, disrupt the CD4+/CD8+ and Th17/Treg balance, and aggravate the severity of EAU; inactivation of the Notch signaling pathway contributes to the CD4+/CD8+ and Th17/Treg balance in EAU rats. Our findings highlighted that the dynamic change in the CD4+/CD8+ and Th17/Treg ratio was consistent with the expression trend of Notch signaling in EAU rats, suggesting that Notch signaling may be a potentially important therapeutic target in clinical practice. [ABSTRACT FROM AUTHOR]

• The pathogenesis of uveitis is associated with the activation of Notch signaling. • Uveitis accompanied by a disturbed balance of CD4+/CD8+ and Th17/Treg ratio. • DAPT can efficiently inhibit Th17 cell response via the Notch signaling pathway. • Inhibiting Notch signaling pathway can restore the CD4+/CD8+, Th17/Treg balance. Uveitis is the most common cause in inflammatory eye diseases that can lead to visual impairment even blindness worldwide. T helper (Th) 17 and regulatory T (Treg) cells are critical mediators for immune response. Notch signaling can regulate the cell differentiation, playing a role in the pathogenesis of the diseases. In this study, we measured the expression levels of Notch1, DLL4, IL-10, IL-17, RORγt and Foxp3 in T cells from lymph node, spleen and eye tissues in experimental autoimmune uveitis (EAU) rats in vitro , determined the ratios of CD4+/CD8+ and Th17/Treg. Moreover, we also investigated the effect of Notch signaling inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanyl))-S-phenylglycine t-Butyl Ester (DAPT) on Notch1, DLL4 expression and on Th17, Treg cell differentiation. The results indicated that the pathogenesis of uveitis accompanied by the elevated expression of Notch1, DLL4, IL-10, IL-17, RORγt, and Foxp3 as well as the imbalanced CD4+/CD8+ and Th17/Treg ratios. By contrast, inhibition of Notch signaling by DAPT can efficiently decrease Th17 cell response, downregulate the expression of Notch1, DLL4, IL-17 and the transcription of RORγt, reduce Th17 levels and restore the CD4+/CD8+, Th17/Treg balance. Moreover, DAPT can also inhibit Th17 cell differentiation in healthy rats, though the inhibitory capacity of Th17, Treg differentiation is less than that in EAU rats. Overall, Notch signaling activation can lead to the disturbed Th17/Treg balance in uveitis, whereas inhibition of Notch signaling can ameliorate the inflammatory response and may be a potential immunoregulatory strategy in patients with uveitis. [ABSTRACT FROM AUTHOR]