Blockade of Notch Signaling Ameliorates Murine Collagen-Induced Arthritis via Suppressing Th1 and Th17 Cell Responses

The American Journal of Pathology, Vol. 184, No. 4, April 2014

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IMMUNOPATHOLOGY AND INFECTIOUS DISEASES

Blockade of Notch Signaling Ameliorates Murine Collagen-Induced Arthritis via Suppressing Th1 and Th17 Cell Responses Zhijun Jiao,* Wenhong Wang,y Shenghao Hua,* Meihong Liu,* Hui Wang,* Xuhui Wang,* Yan Chen,* Huaxi Xu,z and Liwei Lux From the Zhenjiang Key Laboratory of Medical Immunology,* Department of Laboratory Medicine, Affiliated Hospital of Jiangsu University, Zhenjiang; the Departments of Pathogenic Biology,y and Immunology,z School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang; and the Department of Pathology and Center of Infection and Immunology,x The University of Hong Kong, Hong Kong, China Accepted for publication December 30, 2013. Address correspondence to Zhijun Jiao, Ph.D., Zhenjiang Key Laboratory of Medical Immunology, Department of Laboratory Medicine, Affiliated Hospital of Jiangsu University, 438 Jiefang Rd., Zhenjiang 212001, China; or Liwei Lu, Ph.D., Department of Pathology and Center of Infection and Immunology, The University of Hong Kong, Hong Kong, China. E-mail: [email protected] or [email protected].

Recent studies have demonstrated that Notch signaling is critically involved in the regulation of immune response and contributes to autoimmune pathogenesis. Here, Notch signaling was found to be activated in CD4þ T cells and synovial tissue from collagen-induced arthritis mice. In vivo administration of the g-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) substantially reduced the severity of arthritic symptoms and joint damage in collageninduced arthritis mice. Notably, DAPT treatment significantly suppressed Th1- and Th17-cell responses in spleen and lymph nodes and reduced IFN-g and IL-17 levels in plasma. In polarization culture, DAPT treatment markedly reduced Th17 cell expansion from naïve T cells, whereas fusion protein of the Notch receptor ligand delta-like 3 significantly increased the frequency and absolute number of Th17 cells. These results suggest a novel therapeutic strategy for treatment of human rheumatoid arthritis by targeting Notch signaling using g-secretase inhibitors. (Am J Pathol 2014, 184: 1085e1093; http://dx.doi.org/10.1016/j.ajpath.2013.12.010)

Numerous studies have demonstrated that Notch signaling is critically involved in the regulation of immune response. An increasing body of evidence indicates that dysregulated Notch activation contributes to autoimmune pathogenesis. Notch signaling is initiated by ligand engagement of Notch receptors. To date, four Notch receptors (Notch 1 to 4) and five of their ligands (delta-like 1, 3, and 4 and Jagged-1 and -2) have been identified in mammals. Upon ligand binding, the intracellular domain (ICD) of the receptor is proteolytically cleaved by g-secretase and translocated into the nucleus, where it associates with the RBP-Jk transcription factor and regulates the expression of various target genes, including members of the hairy enhancer of split (Hes) or hairy related (Hey and Hrt) gene families. Although Notch signaling is known to be critical primarily in tissue and organ formation during embryonic development, accumulating evidence also suggests that it plays an important Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2013.12.010

role in the pathogenesis of various disorders, including tumors and autoimmune diseases.1e4 Rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease of the synovial joints, is characterized by leukocyte infiltration and synoviocyte activation, leading to destruction of cartilage and bone.5 Recent findings suggest the potential role of Notch signaling in the pathogenesis of RA. In one study, the expression pattern of Notch homologs among synovium from RA patients differed from that of healthy subjects.6 Our research group has previously characterized the different expression profiles of Notch-related Supported by grants from the Natural Science Foundation of China (30872335, 81172871, 81373217) and the National Basic Research Program of China (2010 CB529100) and by a special grant for clinical medicine from Jiangsu Province (BL2013034). L.L. is a recipient of a Croucher Senior Research Fellowship award from the Croucher Foundation, Hong Kong. Disclosures: None declared.

Jiao et al molecules in peripheral T helper cells of RA patients and healthy control subjects.7 Others have shown that g-secretase inhibitors (GSIs) can effectively block the signaling from Notch receptors by preventing the release of ICD and transcriptional activation, which suggests the potential application of a GSI as a novel therapeutic approach for diseases with constitutive Notch activation.8,9 In addition, Notch signaling has been shown to be essential for the differentiation and function of various immune cells, including T cells, B cells, dendritic cells, and macrophages, that are involved in driving the chronicity and destructiveness of autoimmune arthritis.10e13 Injection of a soluble Jagged1-encoding plasmid before or even after initial collagen type II immunization has been shown to suppress the disease severity in collagen-induced arthritis (CIA) mice by providing a negative signal in CD8þ T cells without affecting CD4þ T cells.14 It has become clear that distinct Notch ligands have differential abilities in modulating T cell differentiation. Interestingly, our previous in vitro experiments showed that activation of Notch signaling with its ligand delta-like 1 promotes Th1/Th17 responses.15 Many studies, including our recent work, have highlighted a critical role of Th17 cells during the pathogenesis of RA and CIA in both human and mouse.16 Upon interaction with synovial fibroblasts, Th17 cells (but not Th1 cells) from patients with early RA are potent inducers of matrix metalloproteinases and proinflammatory cytokines, including autocrine IL-17A.17 In the present study, we addressed the question of how the blockade of Notch signaling in vivo affects Th1/Th17 cell responses and the development of autoimmune arthritis. We tested the effect of N-[N-(3,5-difluorophenacetyl-Lalanyl)]-S-phenylglycine t-butyl ester (DAPT), a GSI, on the development of CIA in mice. We demonstrated the activation of Notch signaling in CD4þ T cells of CIA mice and found that in vivo treatment with the GSI DAPT ameliorated arthritis development via suppressing Th1 and Th17 responses. These results indicate that the targeting of Notch signaling by GSIs may prove to be beneficial for the treatment of autoimmune arthritis.

Materials and Methods Mice Male DBA/1J mice (8 to 10 weeks of age) were supplied by the Model Animal Research Center of Nanjing University (Nanjing, China) and were maintained in a specific pathogene free animal facility at Jiangsu University. All animal experiments were conducted with approval of the Scientific Investigation Board of Jiangsu University.

Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO), followed 21 days later by a boost with 100 mg of the same bovine collagen type II (Chondrex) emulsified in Freund’s incomplete adjuvant (Sigma-Aldrich) via the same route, as described previously.11,12 With the same protocol, but using PBS in place of collagen type II, adjuvant-treated DBA/1J mice served as controls. For assessing the severity of arthritis, clinical symptoms were evaluated on a five-point scale: grade 0 Z no swelling; grade 1 Z paw with detectable swelling in a single digit; grade 2 Z paw with swelling in more than one digit; grade 3 Z paw with swelling of all digits and instep; and grade 4 Z severe swelling of the paw and ankle.

Treatment of CIA Mice with DAPT DAPT, an inhibitor of Notch signaling, was suspended in dimethyl sulfoxide, and 1.0 mg/kg and 5.0 mg/kg DAPT (or dimethyl sulfoxide alone as vehicle; 0 mg/kg DAPT) was administered intraperitoneally on the day before the first immunization, followed by administration every other day throughout the course of disease (n Z 5 mice per group). On day 36, mice were sacrificed and ankle joints were harvested and examined radiographically and histologically. Cell suspensions of spleen and draining inguinal lymph nodes (LNs) were prepared for frequency analysis of T cell subsets by flow cytometry and for CD4þ T-cell purification by magnetic bead separation. CD4þ T cells were purified by depleting non-CD4þ T cells using a CD4þ T-cell isolation kit (Miltenyi Biotec, Auburn, CA). Plasma samples were also collected for measuring cytokine concentration.

Western Blot Analysis of Notch Intracellular Domain For protein extraction, CD4þ T cells were lysed and denatured by boiling in Laemmli sample buffer (62.5 mmol/L Tris-HCl, glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) for 10 minutes. Cells were then centrifuged in a cold room (4 C to 8 C) at 12,000  g for 10 minutes. The supernatant was used for Western blot (WB) analysis. Denatured proteins (5 mg) were resolved by 10% SDSePAGE and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA), followed by overnight incubation at 4 C with antibody against cleaved Notch intracellular domain (NICD) (Val 1744; Cell Signaling Technology, Danvers, MA). After incubation with horseradish peroxidaseelabeled secondary antibody, the labeled products were visualized using a Pierce enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA) and quantitated using a chemiluminescence and fluorescence imaging system (Beijing Sage Creation Science, Beijing, China). Antieb-actin (Sigma-Aldrich) was used as a loading control.

Induction of CIA For CIA induction, DBA/1J mice were first injected subcutaneously in the base of the tail with 100 mg of bovine collagen type II (Chondrex, Redmond, WA) emulsified in

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Histopathologic Analysis of Joint Tissue Hind-paw ankle joints were fixed in 10% phosphatebuffered formalin for 72 hours, then decalcified in 10%

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Notch Signaling in CIA nitric acid for 48 hours before paraffin embedding. Tissue sections (4 mm thick) were prepared for H&E staining.

of each respective cytokine was calculated using FlowCytomix Pro 2.1 software.

IHC Staining of NICD

Th17 Cell Expansion in Vitro

Paraffin sections of mouse ankle joint tissue were rehydrated and treated with 15% hydrogen peroxide. Samples were blocked with 10% rat serum and then were incubated with primary antibody against cleaved NICD (Val 1744; Cell Signaling Technology) and secondary antibody (horseradish peroxidaseelabeled anti-rabbit IgG). Diaminobenzidine (DAB) substrate was used for color reaction.

CD4þ T cells were purified from spleen of naïve mice using magnetic activated cell sorting (Miltenyi Biotec); purity was routinely >95%. Cells were cultured with plate-bound antiCD3 and anti-CD28 antibodies, TGF-b, IL-6, and TNF-a to induce Th17 cell expansion in vitro. At the beginning of culture, 10 mmol/L of DAPT or of the fusion proteins of Notch ligands delta-like 1 (D1) and delta-like 3 (D3) was added at the beginning of culture. Cells were harvested on day 7 and were assayed for intracellular cytokines. Culture supernatants were analyzed for cytokine concentrations by cytometric bead array.

Radiological Examination Radiography of mouse hindlimbs was performed using a mammographic imager (GIOTTO; I.M.S., Bologna, Italy). All images were obtained using exposure settings of 22 kVp (peak voltage) and 6 mAs (exposure) and were evaluated by two radiologists who were masked to the experimental conditions.

Statistical Analysis Two-tailed Student’s t-test and analysis of variance test were used for determining significant differences between groups. P < 0.05 was considered statistically significant.

Flow Cytometric Analysis For analysis of Th1/Th2 and Th17 cells, cell suspensions were stimulated for 5 hours using 50 ng/mL of phorbol myristate acetate (Sigma-Aldrich) and 1 mg/mL ionomycin (Sigma-Aldrich) in the presence of 5 mg/mL brefeldin A (Sigma-Aldrich) at 37 C and in a 5% CO2 atmosphere. Cells were first stained extracellularly with anti-CD4efluorescein isothiocyanate (eBioscience, San Diego, CA), then were fixed and permeabilized with IntraPrep permeabilization reagent (Beckman Coulter, Brea, CA), and finally were stained intracellularly with antieIFN-gephycoerythrin, antieIL-4e phycoerythrin, or antieIL-17Aephycoerythrin. For analysis of regulatory T (Treg) cells, the cell suspensions were surface-labeled with anti-CD4efluorescein isothiocyanate without phorbol myristate acetate and ionomycin stimulation, followed by fixation and permeabilization and intracellular staining with anti-FoxP3ephycoerythrin. Labeled cells were washed and analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software version 3.2 (BD Biosciences). As control, each staining was compared with the appropriately labeled isotype control antibody.

Determination of Plasma Cytokines by Cytometric Bead Array The levels of plasma IFN-g, IL-4, IL-10, IL-17, and IL-22 were determined by cytometric bead array, using a mouse Th1/Th2/Th17/Th22 13-plex FlowCytomix multiplex kit (eBioscience, San Diego, CA), and were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Standard curves were determined for each cytokine from a range of 27 to 20,000 pg/mL. The quantity (pg/mL)

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Results Notch Signaling Is Increased in CD4þ T Cells and Synovial Tissue from CIA Mice To determine whether Notch signaling is activated in CD4þ T cells from CIA mice, we examined and compared the expression of NICD in CD4þ T cells from spleen and LNs of CIA and control mice using WB to detect endogenous levels of activated NICD only when released by cleavage between Gly1743 and Val1744. NICD expression was substantially up-regulated in CD4þ T cells of spleen and LNs from CIA mice, compared with control mice (Figure 1, A and B). In addition, up-regulation of NICD was detected in the synovial tissue from CIA mice, relative to control mice (Figure 1C). These results indicate increased activation of Notch signaling in CD4þ T cells during development of autoimmune arthritis.

Inhibition of Notch Signaling Ameliorates Arthritis Development in CIA Mice To determine whether inhibition of Notch signaling in vivo can affect the development of autoimmune arthritis, we administered DAPT, an inhibitor of Notch signaling, during the induction of CIA. DAPT treatment significantly reduced the severity of arthritic symptoms in a dose-dependent manner, as indicated by markedly decreased incidence of disease development and clinical scores of arthritis in CIA mice, compared with vehicle-treated CIA controls (Figure 2, A and B). Notably, differences in disease severity scores were statistically significance at 7 days after the second immunization. Moreover, radiological examination of the

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Jiao et al did not affect the clinical score in this model (Supplemental Figure S1).

Inhibition of Notch Signaling Suppresses Th1/Th17 Responses in CIA Mice Because helper T cells are known to play a potent proinflammatory role in autoimmune arthritis, we next evaluated the potential effect of Notch inhibition on the generation of T-cell subsets in CIA mice. We first compared the NICD levels in purified CD4þ T cells from CIA mice with or without DAPT treatment and found that DAPT treatment decreased NICD levels in CD4þ T cells from spleen and LNs of CIA mice (Figure 3, A and B). Next, we determined the populations of Th1/Th2, Treg, and Th17 cells in both spleen and LNs from CIA mice, with and without DAPT treatment. As expected, Th1 and Th17 cell populations were greatly increased in both spleen and LNs from CIA mice, compared with normal controls, whereas DAPT treatment significantly decreased the frequency of Th1 and Th17 cells in both spleen and LNs in CIA mice (Figure 3, C and D and Figure 4, A and B). No obvious difference in the frequency of Th2 cells was observed among the three groups. Collagen type II immunization increased the Treg population in both spleen and LNs in CIA mice; however, this was not downregulated by DAPT treatment. Consistent with the reduced frequency of Th1 and Th17 cells, the absolute number of both T-cell subsets was also markedly decreased in both spleen and LNs from CIA mice with DAPT inhibition of Notch (Figure 5, A and B). Taken together, these findings indicate that blocking of Notch signaling in vivo suppresses the generation of Th1 and Th17 cell in CIA mice.

Inhibition of Notch Signaling Reduces Levels of IFN-g and IL-17 in the Peripheral Blood of CIA Mice

Figure 1 Notch signaling is activated in CD4þ T cells and synovial tissue of CIA mice. DBA/1J mice were first immunized with bovine collagen type II in Freund’s complete adjuvant and given booster injections in Freund’s incomplete adjuvant on day 21 to induce CIA. Mice were sacrificed on day 36. CD4þ T cells were purified from spleen and LNs of CIA mice and normal control mice. NICD expressed by CD4þ T cells and synovial tissue was determined by WB and IHC, respectively. A: Representative WB results of three independent experiments. B: Quantitative analysis of NICD expression in each group, normalized to b-actin. C: Representative IHC results of NICD expression in synovium from CIA and control mice of three independent experiments. Data are expressed as means  SD. n Z 5 mice per group. *P < 0.05. Original magnification, 400.

ankle joints 2 weeks after the second immunization revealed that bone erosion was substantially reduced in the joints of DAPT-treated CIA mice (Figure 2C). Histopathological analysis of the joints treated with DAPT confirmed greatly reduced synovial hyperplasia, pannus formation, and cartilage destruction, compared with those in control groups (Figure 2D). However, DAPT treatment after onset of CIA

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To further examine effect of Notch inhibition on cytokine production, we examined cytokine expression in the peripheral blood of CIA mice by cytometric bead array. Levels of plasma IFN-g and IL-17 were increased in CIA mice treated with vehicle alone, compared with normal mice (P < 0.05) (Figure 5C). However, levels of plasma IFN-g and IL-17 were significantly decreased in DAPT-treated CIA mice (P < 0.05). No significant change in IL-4, IL-10, or IL22 levels was observed among the three groups (P > 0.05).

Notch Signaling Regulates Th17 Cell Differentiation in Vitro To confirm the involvement of Notch signaling in Th17 cell differentiation, we established in vitro culture for murine Th17 cell expansion, using a combination of cytokines (TGF-b, IL-1b, and IL-6) in the presence or absence of the GSI DAPT. Treatment of naïve T cells with DAPT markedly reduced the frequency of Th17 cells in polarized Th17 differentiation condition (Figure 6A). Both the total number

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Figure 2 Blockade of Notch signaling by the GSI DAPT ameliorates disease in CIA mice. DBA/1J mice were immunized by collagen type II to induce arthritis. The mice were administered DAPT [1.0 (open squares) or 5.0 mg/kg (white circles) i.p.] or vehicle (black squares) on the day before primary immunization, followed by administration every other day. A: The incidence of arthritis development in mice with or without DAPT. B: Evaluation of the severity of arthritis in terms of clinical arthritis score. C and D: On day 36, ankle joints were prepared for radiological examination (C) and for histopathological examination by H&E staining (D). Data are expressed as means  SD. n Z 10 (A) or 5 (B) mice per group. *P < 0.05 versus vehicle-treated CIA mice. Original magnification, 100.

of Th17 cells and the level of IL-17 in supernatant were also significantly decreased in DAPT-treated T-cell cultures (Figure 6, B and C). These data indicate the involvement of Notch signaling in Th17 cell differentiation in vitro.

We then investigated whether the fusion proteins of Notch ligands can enhance the differentiation of Th17 cells. Splenic CD4þ T cells from normal mice were purified and cultured for 7 days under a polarized Th17 differentiation

Figure 3

DAPT treatment decreases the frequency of Th1 and Th17 cells in spleen of CIA mice. The mice were administered DAPT (5.0 mg/kg i.p.) or vehicle on the day before primary immunization, followed by administration every other day. CD4þ T cells were purified from spleen and inguinal LNs from CIA mice treated with DAPT (CIAþD) or with vehicle (CIA). A: NICD expressed by CD4þ T cells was determined by WB. B: Quantitative analysis of NICD expression in each group, normalized to b-actin. C: The frequency of Th1/Th2, Treg, and Th17 cells in spleen was determined by flow cytometry. D: Representative flow cytometry data for T-cell subsets gated on CD4þ T cells in spleen. Data are expressed as means  SD from three independent experiments. n Z 5 mice per group. *P < 0.05. FITC, fluorescein isothiocyanate.

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Figure 4

DAPT treatment decreases the frequency of Th1 and Th17 cells in LNs of CIA mice. The mice were administered DAPT (5.0 mg/kg i.p.) or vehicle on the day before primary immunization, followed by administration every other day. A: The frequency of Th1/Th2, Treg, and Th17 cells in LNs was determined by flow cytometry. B: Representative flow cytometry data for T-cell subsets gated on CD4þ T cells in LNs. Data are expressed as means  SD from three independent experiments. n Z 5 mice per group. *P < 0.05.

condition in the presence of the fusion proteins of Notch ligands D1 and D3. Both the frequency of Th17 cells and their absolute number were significantly increased on addition of D3, consistent with elevated level of IL-17 in supernatant; however, D1 treatment did not show any obvious effect on Th17 cell expansion (Figure 6, DeF). These results further suggest that Notch signaling mediated by different ligands may play differential roles in the differentiation of Th17 cells.

A

Discussion In the present study, we detected enhanced activation of Notch signaling in CD4þ T cells from both spleen and LNs, as well as in synovial tissue of CIA mice, consistent with our previous findings that peripheral T helper cells from RA patients exhibit significantly altered expression profiles of Notch receptors and enhanced activation of Notch signaling, compared with healthy controls.7 Given that dysfunction of

B

C

Figure 5 DAPT treatment decreases the number of Th1 and Th17 cells in spleen and LNs, as well as plasma cytokines, of CIA mice. The mice were administered DAPT (5.0 mg/kg i.p.) or vehicle on the day before primary immunization, followed by administration every other day. A and B: The absolute number of Th1 cells (A) and Th17 cells (B) in spleen and LNs was recorded. C: Plasma levels of cytokines were determined by cytometric bead array. Data are expressed as means  SD from three independent experiments. n Z 5 mice per group. *P < 0.05.

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Notch signaling regulates Th17 cell expansion in vitro. Th17 cells were expanded from mouse CD4þ T cells in vitro in the presence of several cytokines (TGF-b, IL-1b, and IL-6) and simultaneously treated with GSI, DAPT (AeC) or the fusion proteins of Notch ligands D1 and D3 (DeF). The frequency of Th17 cells was determined by flow cytometry (A and D), the absolute number of Th17 cells was enumerated (B and E), and IL-17 levels in culture supernatant were determined by cytometric bead array (C and F). Data are expressed as means  SD from three independent experiments. n Z 5 mice per group. *P < 0.05. Cks, cytokines.

Figure 6

CD4þ T cells has been implicated in the development of RA, these results suggest that inhibition of Notch signaling might suppress the disease course of RA. The effect of Notch signaling inhibition by GSI on the course of several autoimmune inflammatory disorders has been addressed in several studies. Kavian et al18 reported the activation of Notch signaling in skin, lung, and splenocytes from a mouse model of systemic sclerosis and in the skin of patients with systemic sclerosis. In mice with experimental systemic sclerosis, inhibition of Notch signaling by the GSI DAPT limited both fibrosis and autoimmune activation. Blockade of Notch signaling using a GSI effectively abrogated Th1 polarization and altered disease progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.10,19 In models of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus, inhibition of Notch signaling with the GSI DAPT can profoundly and safely decrease all disease parameters.20 In the present study, we used the GSI DAPT to block Notch signaling in a CIA mouse model and demonstrated that in vivo administration of DAPT effectively reduces disease severity and joint damage. Taken together, these findings demonstrate the effectiveness and safety of DAPT in animal models and strongly suggest that inhibition of Notch signaling could be an effective strategy for the treatment of autoimmune diseases. GSIs have been actively tested in clinical trials for Alzheimer disease for their potential in blocking the generation of Ab peptide,21,22 and further experiments are needed to identify other mechanisms

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of DAPT action in vivo. Given that DAPT treatment after onset did not affect the clinical score in our CIA model, the best time window for DAPT therapy of CIA (eg, day 7, day 14, or day 21) needs to be further examined. To elucidate the possible mechanism underlying the therapeutic effect of DAPT in targeting autoimmune arthritis in this CIA model, we analyzed the changes of Th1/Th2, Treg, and Th17 cells in both spleen and LNs of CIA mice on DAPT treatment. Th17 cells are a well-recognized proinflammatory helper T-cell subset in the development of CIA, and Th17 cells play a similar role in human RA pathogenesis. Th17 cells that secrete the cytokines IL-17A and IL17F and express the lineage-specific transcription factor RORC (RORgt in mice) represent a distinct lineage of CD4þ T cells. In addition to their role in RA, Th17 cells and their effector molecules are also associated with the pathogenesis of several autoimmune and inflammatory diseases, including systemic lupus erythematosus, multiple sclerosis, psoriasis, inflammatory bowel disease (IBD), allergy, and asthma.23 In the present study, DAPT treatment decreased the level of NICD in CD4þ T cells from spleen and LNs of CIA mice, which suggests effective suppression of Notch signaling in CD4þ T cells in vivo. Furthermore, Notch inhibition by DAPT reduced the frequency and total number of Th1 and Th17 cells in spleen and LNs, together with plasma levels of IFN-g and IL-17, in CIA mice. In a recent study, GSI blocking of Notch signaling ameliorated the severity of experimental autoimmune encephalomyelitise induced inflammation and Th17 differentiation in vivo.10

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Jiao et al Notch signaling is also critical in the differentiation not only of Th1 and Th17 cells, but also of Treg cells.24,25 Interestingly, we did not observe any significant changes in Treg cells in spleen and LNs in CIA mice with DAPT treatment. Thus, these findings indicate that suppression of experimental arthritis by blocking of Notch signaling is due in part to the inhibition of Th1 and Th17 cell differentiation. Although the role of Notch signaling in the pathogenesis of RA is yet to be fully elucidated, we have recently reported that inhibition of Notch signaling by DAPT or Notch 3 antibody decreases collagen-specific T-cell proliferation and attenuates Th1- and Th17-type responses, whereas treatment with Notch ligand D1 promotes Th1/Th17 responses.15 In vitro, GSIs extinguish expression of Notch, IFN-g, and Tbx21 in Th1-polarized CD4þ cells, whereas ectopic expression of activated Notch1 restores Tbx21 transcription. In vivo, administration of GSIs substantially impedes Th1mediated disease progression in a mouse experimental autoimmune encephalomyelitis model of multiple sclerosis.26 In the present study, we assessed the effect of Notch signaling on Th17 polarization in vitro, using several cytokines other than antigen-specific stimulation. Treatment with DAPT markedly reduced the frequency and total number of Th17 cells, as well as IL-17 production in polarized Th17 differentiation condition. These results are in accord with findings of a previous study, in which IL-CHO and compound E (two chemically distinct GSIs) were used to inhibit g-secretase.10 Our present findings also provide further evidence that Notch signaling plays an important role in the Th17 differentiation, because addition of the Notch ligand D3 significantly increased the frequency and total number of Th17 cells, as well as the supernatant level of IL-17 in Th17 polarization condition. Taken together, the present results identify the Notch signaling pathway as a potential therapeutic target in the treatment of autoimmune arthritis.

Conclusion With the present study, we have demonstrated that Notch signaling is activated in CD4þ T cells during the development of autoimmune arthritis and that inhibition of Notch signaling by DAPT, a GSI, suppresses the disease severity of CIA in mice. Our results also reveal that Notch signaling plays an important role in Th1 and Th17 cell differentiation. These findings support a role of Notch signaling in the development of experimental arthritis, which may facilitate the validation of targeting Notch signaling using GSIs as a novel therapeutic strategy for the treatment of human RA.

Acknowledgments We thank all members of the Central Laboratory of the Affiliated Hospital of Jiangsu University for technical assistance (including evaluation of radiographic images) and Prof. Kuang-Yen Chou for helpful comments and discussion.

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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2013.12.010.

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