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. 2018 Sep 9;8(17):4795-4804.
doi: 10.7150/thno.26093. eCollection 2018.

Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis

Bo Youn Choi  1 , Yuri Choi  1 , Jong-Sung Park  1 , Li-Jung Kang  2 , Seung Hyun Baek  1 , Jin Su Park  1   3 , Gahee Bahn  1 , Yoonsuk Cho  1 , Hark Kyun Kim  1 , Jihoon Han  1 , Jae Hoon Sul  1 , Sang-Ha Baik  1   4 , Dong Hoon Hyun  5 , Thiruma V Arumugam  1   4 , Siyoung Yang  2 , Jeung-Whan Han  1 , Young Mo Kang  6 , Yong-Woo Cho  7 , Jae Hyung Park  3   8   9 , Dong-Gyu Jo  1   3   9
Affiliations

Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis

Bo Youn Choi et al. Theranostics. .

Abstract

Inhibition of Notch signalling has shown anti-inflammatory properties in vivo and in vitro models of rheumatoid arthritis (RA). The objective of this study was to determine whether Notch1 might play a role in regulating T-regulatory cells (Tregs) in animal models of RA. Methods: Collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) were induced in C57BL/6, Notch1 antisense transgenic (NAS) or DBA1/J mice. We examined whether pharmacological inhibitors of γ-secretase (an enzyme required for Notch1 activation) and antisense-mediated knockdown of Notch1 could attenuate the severity of inflammatory arthritis in CIA and CAIA mice. Proportions of CD4+CD25+Foxp3+ Treg cells were measured by flow cytometry. To assess the suppressive capacity of Treg toward responder cells, CFSE-based suppression assay of Treg was performed. Results: γ-secretase inhibitors and antisense-mediated knockdown of Notch1 reduced the severity of inflammatory arthritis in both CIA and CAIA mice. Pharmacological and genetic inhibition of Notch1 signalling induced significant elevation of Treg cell population in CIA and CAIA mice. We also demonstrated that inhibition of Notch signalling suppressed the progression of inflammatory arthritis through modulating the expansion and suppressive function of regulatory T (Treg) cells. Conclusion: Pharmacological and genetic inhibition of Notch1 signalling suppresses the progression of inflammatory arthritis through modulating the population and suppressive function of Treg cells in animal models of RA.

Keywords: CAIA; CIA; Notch1; Treg; rheumatoid arthritis; γ-secretase.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
γ-secretase inhibitors ameliorate symptoms of CIA-induced RA. (A-B) Severity of arthritis was assessed using a visual arthritis scoring system in CIA mice treated with vehicle, L-685,458 or RO4929097. Values are mean ± SEM. *P < 0.05. (C) Bead-based cytokine analysis of serum using a Luminex 100 system at 47 days after primary immunization. Values are mean ± SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 versus controls; *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle-treated controls. (D) Representative photomicrographs of H&E-stained tissue sections of knee joints of control mice and CIA mice treated with vehicle, L-685,458, or RO4929097. Original magnification, 200x. Scale bar: 100 μm. (E) Quantification of synovial inflammation, cartilage erosion, and neutrophil infiltration. Values are mean ± SEM. **P < 0.01, ***P < 0.001.
Figure 2
Figure 2
Administration of γ-secretase inhibitors induces Treg population in CIA-induced DBA1/J mice and cytokines are altered by γ-secretase inhibitors in human RA patients' synoviocytes. (A) Proportions of CD4+CD25+Foxp3+ cells among splenocytes of vehicle-, L-685,458-, or RO4929097-treated mice at 46 days after primary immunization evaluated by flow cytometry. Numbers indicate percentages of CD25+Foxp3+ cells among CD4+ cells. (B) Statistical analysis of CD4+CD25+Foxp3+ cells in splenocytes. Values are mean ± SEM. **P < 0.01. (C) Quantitative real-time PCR analyses of relative mRNA expression levels in RA patients' synoviocytes treated with 5 μM DAPT or 5 μM L-685,458 for 24 h. Value are mean ± SEM (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 versus controls.
Figure 3
Figure 3
Treg population is increased in CIA-induced NAS mice. (A) Proportions of CD4+CD25+Foxp3+ cells among splenocytes of NAS CIA or WT CIA mice at 28 days after primary immunization evaluated by flow cytometry. Numbers indicate percentages of CD25+Foxp3+ cells among CD4+ cells. (B) Statistical analysis of CD4+CD25+Foxp3+ cells. Values are mean ± SEM. ***P < 0.001. (C) At 28 days after primary immunization, both CD4 and CD8 T cells from splenic lymphocytes of NAS or WT CIA mice were analysed by flow cytometry. (D) Statistical data shown as bar graphs. Values are mean ± SEM. ***P < 0.001.
Figure 4
Figure 4
Therapeutic effect of Notch1 inhibition in CAIA. (A-B) Severity of arthritis was assessed using a visual arthritis scoring system in WT CAIA and NAS CAIA mice treated with vehicle, adalimumab, or L-685,458 (arrow). Values are mean ± SEM. *P < 0.05, **P < 0.01. (C) H&E-stained representative joint sections from the experiment shown in (A). Original magnification, 200x. Scale bar: 100 μm. (D) Quantification of synovial inflammation, cartilage erosion, and neutrophil infiltration. Values are mean ± SEM. **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
(A, C) Notch inhibition increases Treg population in the CAIA model. Proportions of CD4+Foxp3+ Treg cells among splenocytes of indicated mice at 9 days after αCII-IA induction were evaluated by flow cytometry. Numbers indicate percentages of CD4+Foxp3+ cells. Values are mean ± SEM. **P < 0.01. (B, D) Proportions of T effector cells among splenocytes. Numbers indicate percentages of CD62LlowCD44high cells. *P < 0.05 versus control. (E) Ratios between effector T cells and regulatory T cells (Teff/Treg). Values are mean ± SEM. *P < 0.05 versus control (WT/Vehicle). (F) Suppressive effects of Treg cells were anlayzed by CFSE-based suppression assay. Values are mean ± SEM. ##P < 0.01 versus WT mice; *P < 0.05 versus NAS mice. (G, H) Synovial tissues of CAIA were used for immunohistochemical staining analysis. Consecutive sections of synovial tissues from WT CAIA and NAS CAIA mice were DAB-stained for CD3 and Foxp3. Quantitative analysis is shown in the bar graph. Values are mean ± SEM. *P < 0.05. Scale bar: 25 μm.
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