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RBPJ Knockdown Promotes M2 Macrophage Polarization Through Mitochondrial ROS-mediated Notch1-Jagged1-Hes1 Signaling Pathway in Uveitis

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Abstract

Uveitis is an autoimmune eye disease that can be involved in the entire body and is one of the leading causes of blindness. Therefore, comprehending the mechanisms underlying the development and regulation of ocular immune responses in uveitis is crucial for designing effective therapeutic interventions. In this study, we investigated how RBPJ regulates macrophage polarization in uveitis. We demonstrated that targeted RBPJ knockdown (RBPJKD) promotes M2 macrophage polarization and ameliorates uveitis through the mtROS-mediated Notch1-Jagged1-Hes1 signaling pathway. Real-time quantitative (Q-PCR) analysis revealed that the Notch1-Jagged1-Hes1 signaling pathway was active in the eye tissues of experimental autoimmune uveitis (EAU) rats. Immunofluorescence double staining confirmed enhanced signaling primarily occurring in macrophages, establishing a correlation between the Notch1 signaling pathway and macrophages. Transmission electron microscopy evaluated the morphological and functional changes of mitochondria in each group's eye tissues. It demonstrated significant swelling and disorganization in the EAU group, which were effectively restored upon RBPJ knockdown intervention. Finally, by employing an antioxidant N-acetyl-L-cysteine (NAC) to eliminate mtROS in vivo, we observed a decrease in the M2 macrophage polarization level, which prevented the cytoprotective effect conferred by RBPJKD. These findings underscore the relevance of the Notch signaling pathway to the immune system while highlighting the potential role of mtROS as a therapeutic target for inflammation and other related diseases.

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Data Availability

No datasets were generated or analysed during the current study.

Abbreviations

NC:

Normal control

EAU:

Experimental autoimmune uveitis

IRBP:

Interphotoreceptor retinoid-binding protein

CFA:

Complete Freund's adjuvant

TB:

Tuberculosis

RBPJ:

Recombination signal binding protein-Jκ

NICD:

Notch intracellular domain

Hes1:

Hairy enhancer of split 1

Q-PCR:

Quantitative real-time polymerase chain reaction

Arg-1:

Arginase 1

ELISA:

Enzyme-linked immunosorbent Assay

IL:

Interleukin

mtROS:

Mitochondrial reactive oxygen species

mtDNA:

Mitochondrial DNA

OXPHOS:

Oxidative phosphorylation

GMFG:

Glial cell maturation factor-γ

NOX4:

NADPH oxidase 4

MIF:

Migration inhibitory factor

NAC:

N-acetyl-L-cysteine

References

  1. Agarwal, N., and M. Kothari. 2022. Uveitis, glaucoma, and cataract with mevalonate kinase deficiency. Journal of AAPOS 26 (2): 93–95. https://doi.org/10.1016/j.jaapos.2021.11.009.

    Article  PubMed  Google Scholar 

  2. Apple, D., and M. Boniuk. 1997. Clinical pathological review of Behcet’s. Survey of Ophthalmology 42 (2): 157–162.

    Google Scholar 

  3. Rong, H., H. Shen, Y. Xu, and H. Yang. 2016. Notch signaling suppresses regulatory T-cell function in murine experimental autoimmune uveitis. Immunology 149 (4): 447–459. https://doi.org/10.1111/imm.12663.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bhuyan, Z.A., et al. 2014. Abrogation of Rbpj attenuates experimental autoimmune uveoretinitis by inhibiting IL-22-producing CD4+ T cells. PLoS ONE 9 (2): 2–8. https://doi.org/10.1371/journal.pone.0089266.

    Article  CAS  Google Scholar 

  5. Uttarkar, S., N.C. Brembilla, and W.H. Boehncke. 2019. Regulatory cells in the skin: Pathophysiologic role and potential targets for anti-inflammatory therapies. The Journal of Allergy and Clinical Immunology 143 (4): 1302–1310. https://doi.org/10.1016/j.jaci.2018.12.1011.

    Article  CAS  PubMed  Google Scholar 

  6. Deng, L., et al. 2023. Macrophage Polarization: An Important Candidate Regulator for Lung Diseases. Molecules 28 (5): 1–19. https://doi.org/10.3390/molecules28052379.

    Article  CAS  Google Scholar 

  7. Dong, T., et al. 2022. Mitochondrial metabolism mediated macrophage polarization in chronic lung diseases. Pharmacology and Therapeutics 239: 108208. https://doi.org/10.1016/j.pharmthera.2022.108208.

    Article  CAS  Google Scholar 

  8. Wang, Y., N. Li, X. Zhang, and T. Horng. 2021. Mitochondrial metabolism regulates macrophage biology. Journal of Biological Chemistry 297 (1): 1–11. https://doi.org/10.1016/j.jbc.2021.100904.

    Article  CAS  Google Scholar 

  9. Caliceti, C.,  P. Nigro, P. Rizzo, and R. Ferrari. 2014. ROS, Notch, and Wnt signaling pathways: Crosstalk between three major regulators of cardiovascular biology. Biomed Research International. https://doi.org/10.1155/2014/318714.

  10. Aerbajinai, W., et al. 2019. Glia maturation factor-g regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS. Blood Advances 3 (8): 1211–1225. https://doi.org/10.1182/bloodadvances.2018026070.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Han, C., et al. 2022. NOX4 promotes mucosal barrier injury in inflammatory bowel disease by mediating macrophages M1 polarization through ROS. International Immunopharmacology 104: 108361. https://doi.org/10.1016/j.intimp.2021.108361.

  12. Cotzomi-Ortega, I., et al. 2021. Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization. Cellular Signalling 86. https://doi.org/10.1016/j.cellsig.2021.110075.

  13. Ko, J.H., S.O. Yoon, H.J. Lee, and J.Y. Oh. 2017. Rapamycin regulates macrophage activation by inhibiting NLRP3 inflammasome-p38 MAPK-NFκB pathways in autophagy- and p62-dependent manners. Oncotarget 8 (25): 40817–40831. https://doi.org/10.18632/oncotarget.17256.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chen, W., et al. 2021. The Notch signaling pathway regulates macrophage polarization in liver diseases. International Immunopharmacology 99: 107938. https://doi.org/10.1016/j.intimp.2021.107938.

  15. Lai, E.C. 2004. Notch signaling: Control of cell communication and cell fate. Development 131 (5): 965–973. https://doi.org/10.1242/dev.01074.

    Article  CAS  PubMed  Google Scholar 

  16. Tanigaki, K., K. Kuroda, H. Han, and T. Honjo. 2003. Regulation of B cell development by Notch/RBP-J signaling. Seminars in Immunology 15 (2): 113–119. https://doi.org/10.1016/S1044-5323(03)00008-3.

    Article  CAS  PubMed  Google Scholar 

  17. Di Vincenzo, S., D.K. Ninaber, C. Cipollina, M. Ferraro, P.S. Hiemstra, and E. Pace. 2022. Cigarette smoke impairs airway epithelial wound repair: role of modulation of epithelial-mesenchymal transition processes and Notch-1 signaling. Antioxidants 11(10). https://doi.org/10.3390/antiox11102018.

  18. Xu, J., et al. 2015. NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation. The Journal of Clinical Investigation 125 (4): 1579–1590. https://doi.org/10.1172/JCI76468.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Chan, C.C., R.B. Nussenblatt, B. Wiggert, T.M. Redmond, L.S. Fujikawa, G.J. Chader, and I. Gery. 1987. Immunohistochemical analysis of experimental autoimmune uveoretinitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) in the rat. Immunol Investigations 16 (1): 63–74. https://doi.org/10.3109/08820138709055713.

    Article  CAS  Google Scholar 

  20. Chen, Z., et al. 2021. Induction of antigen-specific Treg cells in treating autoimmune uveitis via bystander suppressive pathways without compromising anti-tumor immunity. eBioMedicine 70: 103496. https://doi.org/10.1016/j.ebiom.2021.103496.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Agarwal, RK, P.B. Silver, R.R. Caspi. 2012. Rodent models of experimental autoimmune uveitis. Methods in Molecular Biology 900: 443–69. https://doi.org/10.1007/978-1-60761-720-4_22.

  22. Li, X., et al. 2022. Nrf2 Activation mediates antiallodynic effect of electroacupuncture on a rat model of complex regional pain syndrome type-i through reducing local oxidative stress and inflammation. Oxidative Medicine and Cellular Longevity 2022. https://doi.org/10.1155/2022/8035109.

  23. Ng, T.F.,  K. Dawit, and A. W. Taylor. 2022. Melanocortin receptor agonists suppress experimental autoimmune uveitis. Experimental Eye Research 218: 108986. https://doi.org/10.1016/j.exer.2022.108986.

  24. Li, H., et al. 2022. Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice. Protein and Cell 13 (6): 422–445. https://doi.org/10.1007/s13238-021-00882-3.

    Article  CAS  Google Scholar 

  25. Wu, X., et al. 2022. Macrophage polarization toward M1 phenotype through NF-κB signaling in patients with Behçet’s disease. Arthritis Research and Therapy 24 (1): 1–12. https://doi.org/10.1186/s13075-022-02938-z.

    Article  CAS  Google Scholar 

  26. Jordan, J., et al. 2014. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please c. The Laryngoscope 44: 2–31. https://doi.org/10.1002/eji.201445427.This.

    Article  Google Scholar 

  27. Lin, Y., et al. 2020. Gallic acid alleviates gouty arthritis by inhibiting NLRP3 inflammasome activation and pyroptosis through enhancing Nrf2 signaling. Frontiers in Immunology 11 (December): 1–13. https://doi.org/10.3389/fimmu.2020.580593.

    Article  CAS  Google Scholar 

  28. Wu, X., et al. 2021. NLRP3 deficiency protects against intermittent hypoxia-induced neuroinflammation and mitochondrial ros by promoting the pink1-parkin pathway of mitophagy in a murine model of sleep apnea. Frontiers in Immunology 12 (February): 1–16. https://doi.org/10.3389/fimmu.2021.628168.

    Article  CAS  Google Scholar 

  29. Bai, Q., et al. 2022. Sesamin alleviates asthma airway inflammation by regulating mitophagy and mitochondrial apoptosis. Journal of Agriculture and Food Chemistry 70 (16): 4921–4933. https://doi.org/10.1021/acs.jafc.1c07877.

    Article  CAS  Google Scholar 

  30. Naik, E., and V.M. Dixit. 2011. Mitochondrial reactive oxygen species drive proinflammatory cytokine production. Journal of Experimental Medicine 208 (3): 417–420. https://doi.org/10.1084/jem.20110367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Formentini, L., et al. 2017. Mitochondrial ROS production protects the intestine from inflammation through functional M2 macrophage polarization. Cell Reports 19 (6): 1202–1213. https://doi.org/10.1016/j.celrep.2017.04.036.

    Article  CAS  PubMed  Google Scholar 

  32. Shiau, D.J., et al. 2020. Hepatocellular carcinoma-derived high mobility group box 1 triggers M2 macrophage polarization via a TLR2/NOX2/autophagy axis. Science and Reports 10 (1): 1–14. https://doi.org/10.1038/s41598-020-70137-4.

    Article  CAS  Google Scholar 

  33. Hsu, S.M. et al. 2020. Suppression of the reactive oxygen response alleviates experimental autoimmune uveitis in mice. International Journal of Molecular Science 21 (9). https://doi.org/10.3390/ijms21093261.

  34. Wu, L., Y. Li, M. Yu, F. Yang, M. Tu, and H. Xu. 2018. Notch signaling regulates microglial activation and inflammatory reactions in a rat model of temporal lobe epilepsy. Neurochemical Research 43 (6): 1269–1282. https://doi.org/10.1007/s11064-018-2544-5.

    Article  CAS  PubMed  Google Scholar 

  35. Cao, Q., et al. 2008. Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells. Glia 56 (11): 1224–1237. https://doi.org/10.1002/glia.20692.

    Article  PubMed  Google Scholar 

  36. Dailey, D.D., et al. 2013. HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors. BMC Veterinary Research 9: 1–14. https://doi.org/10.1186/1746-6148-9-130.

    Article  CAS  Google Scholar 

  37. Qu, R., et al. 2023. MiR-223-3p attenuates M1 macrophage polarization via suppressing the Notch signaling pathway and NLRP3-mediated pyroptosis in experimental autoimmune uveitis. European Journal of Pharmacology 960 (48). https://doi.org/10.1016/j.ejphar.2023.176139.

  38. Li, H., et al. 2018. M1-polarized macrophages promote self-renewing phenotype of hepatic progenitor cells with Jagged1-Notch signaling involved: Relevance in primary sclerosing cholangitis. Journal of Immunology Research 2018. https://doi.org/10.1155/2018/4807145.

  39. Liu, C., et al. 2022. Dental pulp stem cell-derived exosomes suppress M1 macrophage polarization through the ROS-MAPK-NFκB P65 signaling pathway after spinal cord injury. J. Nanobiotechnology 20 (1): 1–19. https://doi.org/10.1186/s12951-022-01273-4.

    Article  CAS  Google Scholar 

  40. Fan, J., et al. 2020. Bruceine D induces lung cancer cell apoptosis and autophagy via the ROS/MAPK signaling pathway in vitro and in vivo. Cell Death and Disease 11 (2).https://doi.org/10.1038/s41419-020-2317-3.

  41. Yazaki, K., et al. 2021. ROS-Nrf2 pathway mediates the development of TGF-β1-induced epithelial-mesenchymal transition through the activation of Notch signaling. European Journal of Cell Biology 100 (7–8): 151181. https://doi.org/10.1016/j.ejcb.2021.151181.

    Article  CAS  PubMed  Google Scholar 

  42. Xu, J., F. Chi, and H. Tsukamoto. 2015. Notch signaling and M1 macrophage activation in obesity-alcohol synergism. Clinics and Research in Hepatology and Gastroenterology 39: S24–S28. https://doi.org/10.1016/j.clinre.2015.05.016.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Keewan, E.,  and S.A. Naser. 2020. The role of Notch signaling in macrophages during inflammation and infection: implication in rheumatoid arthritis? Cells 9 (1). https://doi.org/10.3390/cells9010111.

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Funding

This work was supported by the Key Project of Natural Science Foundation of Shandong Province (ZR2020KC024), the National Natural Science Foundation of China (No. 81873163), and “Taishan Scholar” Project Special Fund (tsqnz20231252).

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Authors and Affiliations

  1. Shandong University of Traditional Chinese Medicine, Jinan, 250002, China

    Ruyi Qu, Yuan Peng, Shuqin Xu, Mengxian Zhou & Bin Liu

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

    Xuewei Yin & Hongsheng Bi

  3. Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases, Shandong Academy of Eye Disease Prevention and Therapy, Medical College of Optometry and Ophthalmology, Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan, 250002, China

    Dadong Guo

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Contributions

Q.R and G.D wrote the main manuscript. Others were responsible for date analysis. All authors reviewed the manuscript.

Corresponding author

Correspondence to Dadong Guo.

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Ethical Approval

The research program and experimental design were approved by the Experimental Animal Management Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Number: AWE-2022-017). All processes involving experimental animals in the experiment followed the ARVO statement on the use of animals in ophthalmic and visual studies.

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Qu, R., Peng, Y., Xu, S. et al. RBPJ Knockdown Promotes M2 Macrophage Polarization Through Mitochondrial ROS-mediated Notch1-Jagged1-Hes1 Signaling Pathway in Uveitis. Inflammation (2024). https://doi.org/10.1007/s10753-024-02053-y

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