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Activation of the Notch signaling pathway disturbs the CD4+/CD8+, Th17/Treg balance in rats with experimental autoimmune uveitis

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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.

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Abbreviations

BD:

Behcet’s disease

CFA:

Complete Freund’s adjuvant

DAPT:

N-(N-(3,5-Difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester

DMSO:

Dimethyl sulfoxide

EAU:

Experimental autoimmune uveitis

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

H&E:

Hematoxylin and eosin

IFN-γ:

Interferon-γ

IL-17:

Interleukin-17

IRBP:

Interphotoreceptor retinoid-binding protein

MCP-1:

Monocyte chemoattractant protein-1

NC:

Normal control

PBS:

Phosphate buffer saline

Q-PCR:

Quantitative polymerase chain reaction

TB:

Tuberculin

TGF:

Transforming growth factor

Th:

T helper

TNF:

Tumor necrosis factor

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (81873163), the Natural Science Foundation of Shandong Province (ZR2017LH042), Key Development & Research Program of Shandong Province (2016GGB14291), the Development Project of Medicine and Health Science Technology of Shandong Province (2013WS0251), and the Development Project of Science and Technology of Traditional Chinese Medicine of Shandong Province (2015-145).

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Authors and Affiliations

  1. Postgraduate Student of the Second Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, 250002, China

    Xuewei Yin, Bin Liu & Huixia Wei

  2. School of Ophthalmology and Optometry, Shandong University of Traditional Chinese Medicine, Jinan, 250002, China

    Shanshan Wu & Furu Xu

  3. College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China

    Lijie Guo

  4. Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine, Jinan, 250002, China

    TingTing Liu

  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

    Hongsheng Bi & Dadong Guo

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  1. Xuewei Yin
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  2. Bin Liu
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  3. Huixia Wei
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Contributions

DG conceived and designed the study; XY, BL, HW, SW, FX, and LG performed the experiments; XY and TL analyzed the data; HB and DG contributed reagents/materials/analysis tools; XY and DG wrote the paper.

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Correspondence to Dadong Guo.

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Yin, X., Liu, B., Wei, H. et al. Activation of the Notch signaling pathway disturbs the CD4+/CD8+, Th17/Treg balance in rats with experimental autoimmune uveitis. Inflamm. Res. 68, 761–774 (2019). https://doi.org/10.1007/s00011-019-01260-w

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