Xuewei Yin1, Bin Liu1, Huixia Wei1, Shanshan Wu2, Lijie Guo3, Furu Xu2, TingTing Liu4, Hongsheng Bi5, Dadong Guo6. 1. Postgraduate Student of the Second Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, 250002, China. 2. School of Ophthalmology and Optometry, Shandong University of Traditional Chinese Medicine, Jinan, 250002, China. 3. College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China. 4. Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine, Jinan, 250002, China. 5. Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases, Eye Institute of Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan, 250002, China. 6. Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases, Eye Institute of Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan, 250002, China. dadonggene@163.com.
Abstract
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.
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 EAUrat 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 EAUrats on day 12 post-immunization by flow cytometry. RESULTS: The pathological results showed that inflammatory cell infiltration occurred in ocular tissues in EAUrats. The CD4+/CD8+ and Th17/Treg ratios in EAUrats 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 EAUrats gradually increased on day 6 after immunization, peaked on day 12, and then gradually decreased. The dynamic trends in Notch1 and DLL4 expression in EAUrats 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 EAUrats. 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 EAUrats, suggesting that Notch signaling may be a potentially important therapeutic target in clinical practice.
Entities:
Keywords:
Experimental autoimmune uveitis; Notch signaling pathway; Regulatory T cell; T helper 17 cell