Amelioration of DSS-induced colitis by BJ-2223 through the inhibition of Th17 cell differentiation

DOI 10.17480/psk.2018.62.5.297

Amelioration of DSS-induced colitis by BJ-2223 through the inhibition of Th17 cell differentiation

Maheshwor Timilshina and Jae-Hoon Chang

College of Pharmacy, Yeungnam University, Gyeongsan 38541

(Received September 5, 2018; Revised September 13, 2018; Accepted September 13, 2018)

Abstract — CD4

T cells differentiate into T helper (Th) 1 and Th17 cells, which are key mediators of several inflam- matory and autoimmune diseases. Inhibition of Th1 and Th17 cell differentiation helps attenuate several inflammatory con- ditions. Th17 cells are highly pro-inflammatory cells that orchestrate tissue inflammation and ulcerative colitis (UC). Thus, novel compounds that inhibit Th17 cell differentiation are required for the effective treatment of inflammatory and auto- immune diseases. By extensive screening of several synthetic compounds, we found that BJ-2223 inhibits Th17 cell dif- ferentiation without affecting Th1 and regulatory T (Treg) cell differentiation in vitro. Furthermore, BJ-2223 does not induce apoptosis and does not affect T cell proliferation and viability. Mechanistically, BJ-2223 inhibits Th17 cell differentiation through inhibition of the Janus-activated kinase (JAK)/signal transducer and activator of transcription protein (STAT) sig- naling pathway. Moreover, BJ-2223 ameliorates dextran sulfate sodium (DSS)-induced colitis, protects intestinal tissue, and decreases the percentage of Th17 cells in vivo. Thus, BJ-2223 is a novel compound that inhibits in vitro Th17 differentiation and attenuates DSS-induced colitis by reducing the percentage of Th17 cells.

Keywords BJ-2223, Th17 cell, Differentiation, JAK/STAT, DSS-induced colitis

Introduction

Upon encountering an antigen/MHC complex, CD4

T cells are activated and polarized to Th1, Th17, and Treg cells, depending on the cytokine milieu of the microenvironment.

Th1 and Th17 cells are involved in eliciting immune responses against extracellular pathogens and bacterial and fungal infec- tion.

T cell receptor (TCR) activation in the presence of specific cytokine guidance, induces the proliferation and differ- entiation of naïve CD4

T cells into several lineages of Th cells that can be distinguished by their unique cytokine profile and functions.

The binding of interferon-(IFN)-γ and interleu- kin (IL)-12 activates signal transducer and activator of tran- scription protein (STAT)1 and STAT4, thereby activating Th1- specific T-box transcription factor (T-bet) and drives the differ- entiation of naïve CD4

T cells into Th1 cells.

Differentia- tion of Th17 cells is driven by STAT3-activating cytokines,

such as IL-6, IL-21, and IL-23, which upregulate the expres- sion of the transcription factor retinoid-related orphan recep- tor (ROR) γt.

IL-2-mediated STAT5 activation leads to the differentiation of naïve CD4

T cells to Foxp3

Treg cells, which are necessary for the prevention of autoimmune dis- eases and maintenance of immune homeostasis.

However, the abnormal activation and differentiation of Th1 and Th17 cells lead to different autoimmune inflammatory diseases.

Inflammatory bowel diseases (IBDs), characterized by ulcer- ative colitis (UC) and Crohn’s disease (CD), are progressive and chronic inflammatory disorders that are caused due to an imbalanced mucosal immune system.

IBD pathogenesis is associated with the increase in the concentration of inflamma- tory mediators and cytokines, such as IFN- γ and IL-6.

IL-6 is the key cytokine that promotes the generation and differen- tiation of Th17 cells.

Moreover, IL-6 intensifies IBD by enhancing T cell survival and apoptosis resistance at the site of inflammation.

Th17 cells are widely present throughout the intestinal lamina propria and provide protection against the pathogens in the mucosal surrounding, but sustained activa- tion of Th17 cells causes colitis.

Dextran sulfate sodium (DSS) is the most widely studied mouse model of IBD because

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Corresponding Author Jae-Hoon Chang

College of Pharmacy, Yeungnam University, Gyeongsan 38541 Tel.: 053-810-4654 Fax.: 053-810-2833

E-mail: [email protected]

Short Report

its etiology and pathogenesis are similar to those of human UC.

Genome-wide association studies have shown that Th17 cells are important for colitis as they recruit and stimulate IL- 17A and IL-17F in colonic mucosa.

Previous studies have shown that mice administered with DSS for 7 days exhibit increased percentage of IL-17.

Therefore, modulation of IL-17-producing Th17 cells helps to mitigate the exacerbation of disease severity.

Janus-activated kinase (JAK)/STAT signaling pathways are implicated in the pathogenesis of IBD, and JAK inhibitor decreases inflammation in UC.

Studies have indicated that STAT3 activation in mucosal T cells plays a crucial role in the pathogenesis of colitis.

Activation of the IL-6/STAT3 pathway results in the induction of Th17 cell differentiation.

Moreover, activation of the IL-6/STAT3 pathway in the lamina propria induces prolonged survival of pathogenic T cells, including Th17 cells, thereby activating and exacerbating colitis.

Suppression of the IL-6/STAT3 pathway could be the potential target for the treatment of colitis.

In this study, we investigated the effect of BJ-2223 in the dif- ferentiation of Th cells. We observed that BJ-2223 inhibited Th17 cell differentiation without extensive effects on Th1 and Treg cells. Furthermore, in vivo study showed that BJ-2223 ameliorated DSS-induced colitis by decreasing the percentage of Th17 cells through the inhibition of JAK1/JAK2 and STAT3 phosphorylation. Thus, our finding suggests that BJ-2223 could be a promising drug for the treatment of Th17-mediated inflam- matory diseases.

Experimental Methods

Mice

C57BL/6 mice and ovalbumin (OVA)-specific T cell receptor transgenic (OT-II) mice were purchased from The Jackson Labo- ratory and housed under specific pathogen-free conditions at the Yeungnam University animal care center. All animal experi- ments were performed in accordance with guidelines for care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee at Yeungnam University.

Flow Cytometry

For intracellular cytokine staining, isolated cells were stimu- lated with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL;

Sigma) and ionomycin (750 ng/mL; Calbiochem) with the pro-

tein transport inhibitor GolgiStop (BD Biosciences) and incu- bated in 5% CO

at 37

C for 4 h. Cell surface staining was performed with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (GK1.5; Biolegend), phycoerythrin (PE)-Cyanine7- conjugated anti-B220 (RA3-6B2; Biolegend), allophycocyanin (APC)-conjugated anti-CD3 ε (145-2C11 Biolegend), and PE- Cyanine7-conjugated anti-CD8a (53-6.7; Biolegend). Cells were fixed and permeabilized using fixation and permeabilization buffer (BD Biosciences). For Foxp3 staining, cells were fixed and permeabilized using fixation and permeabilization buffer (eBioscience) according to the manufacturer’s instructions.

Antibodies used for cell staining included anti-IFN- γ (PE, XMG;

Biolegend), anti-IL-17-A (APC, TC11-18H10.1; Biolegend), anti- CD25 (PE, PC61; Biolegend), and anti-Foxp3 (APC, FJK-16s;

eBioscience). Antibodies used for immunoblotting were phos- pho-JAK1 (Y1022/1023; Cell signaling), JAK1 (3332S, Cell sig- naling), phospho-JAK2 (Y1007/1008; Cell signaling), JAK2 (D2E12, Cell signaling), phopho-STAT3 (Tyr705), and STAT3 (124H6, Cell signaling). Samples were analyzed in BD FACS Verse flow cytometer (BD Biosciences), and data were ana- lyzed using the FlowJo software.

In vitro differentiation of CD4

T cells

Naïve CD4

T cells isolated from the spleen and lymph node were purified by positive selection using CD4-conju- gated magnetic beads (MiltenyiBiotec). CD4

T cells (2 × 10

/ well) were cultured in vitro for 3 or 4 days (37

C, 5% CO

) in flat bottom 96-well plates coated with anti-CD3 (5 μg/mL; 145- 2C11; Biolegend) and anti-CD28 (1 μg/mL; 37.51; eBiosci- ence) in the presence of tofacitinib or BJ-2223. OVA-specific naïve CD4

T cells isolated from the spleen and lymph node of OT-II mice were co-cultured with irradiated antigen present- ing cells (APCs; 1 × 10

/well) in the presence of IL-12 (10 ng/

mL; Biolegend) and anti-IL4 (5 μg/mL; Biolegend) as the blocking antibody. For Th17 cell differentiation, TGF- β1 (1 ng/

mL; R&D system), IL-6 (10 ng/mL; R&D system), anti-IL-4 (5 μg/mL; Biolegend), and anti-IFN-γ (5 μg/mL; Biolegend) were used. For Treg differentiation, IL-2 (10 ng/mL; Bioleg- end), TGF- β1 (10 ng/mL; R&D system), anti-IL-4 (5 μg/mL;

Biolegend), and anti-IFN- γ (5 μg/mL; Biolegend) were used and cultured in 5% CO

at 37

C for 72 h.

T cell proliferation assay

MACS-sorted naïve CD4

T cells isolated from the spleen

and lymph node were labeled with 5(6)-carboxyfluorescein diace- tate N-succinimidyl ester (CFSE; eBioscience). Cells were then stimulated with plate-bound anti-CD3, anti-CD-28 antibody in the presence of BJ- 2223 in Th17 polarizing condition. Prolifera- tion of cell was assessed after 72 h using flow cytometry. For Ki- 67 detection, isolated naïve CD4

T cells were cultured under Th17 differentiation condition and stained with PE-conjugated Ki-67 (Biolegend) after 72 h of culture.

Apoptosis assay

Purified naïve CD4

T cells were cultured for 72 h in 96- well plates coated with anti-CD3, anti-CD28 with or without BJ-2223 under Th1 differentiation condition. Cells were then stained with annexin V–FITC and propidium iodide (PI)–PE and analyzed by flow cytometry.

MTS assay

Naïve CD4

T cells isolated from the spleen and lymph node were cultured in 96-well plate with RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/

streptomycin in the presence of varying concentrations of BJ-2223 and incubated at 37

C in 5% CO

for 72 h. MTS [3-(4,5-dimeth- ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium] reagent (20 μL/well; Promega) was added and incubated for 2 h. After incubation, absorbance was measured at 490 nm.

DSS-induced colitis

Acute colitis was induced in C57BL/6 mice by oral feeding 2.5% (w/v) DSS (36–50 kDa; MP Biomedicals, USA) dissolved in drinking water for six days. BJ-2223 (5 mg/kg) was adminis- tered intraperitoneally every day. Only water was provided to control group. The body weight, stool condition, and histopa- thology were determined as described previously.

On day 9, the mice were sacrificed and analyzed.

Immunoblot analysis

Purified naïve CD4

T cells were cultured under Th17 con- dition for 72 h with or without BJ-2223. Cells were further stimulated with or without anti-CD3, anti-CD28 for 30 min.

Cell samples were lysed in RIPA buffer containing protease and phosphatase inhibitors and quantified using the BCA pro- tein assay reagent. Protein samples were resolved in sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membrane for 1 h, followed by blocking for 1 h with 5% bovine serum albumin in Tris-buffered saline (TBS), 0.1% Tween-20 (TBST). The membranes were then incubated overnight with primary monoclonal antibody at 4

C. On the next day, membranes were washed with TBST and incubated with secondary antibody for 1 h at 25

C. Protein bands were detected using the ECL reagent kit (Pierce) under a che- miluminescent image analyzer.

Statistical analysis

All data were presented as mean ± SEM and were the rep- resentative of three independent experiments. One way analy- sis of variance (ANOVA) followed by Newman–Keul’s post hoc test was performed to compare multiple groups. Paired Stu- dent’s t-test was used to compare between two groups. P <

0.05 was considered as statistically significant. FACS data were analyzed using the FlowJo software.

Results

BJ-2223 inhibits in vitro Th17 cell differentiation Th1 and Th17 cells play the main role in pathogenesis of inflammatory conditions. Inhibition of Th17 cell proliferation helps to ameliorate the severity of colon inflammation. To find novel compounds for inhibiting inflammatory conditions, we screened several synthetic compounds using an in vitro Th dif- ferentiation assay and found an inhibitory compound named BJ-2223. To investigate the inhibitory effect of BJ-2223 on in vitro T cell differentiation, naive CD4

T cells were differenti- ated under Th1, Th17, and Treg skewing cytokine conditions.

We found that treatment with BJ-2223 significantly reduced in vitro Th17 differentiation, similar to that by tofacitinib (Fig. 1a, b). However, unlike tofacitinib, BJ-2223 did not inhibit Th1 dif- ferentiation. Tofacitinib strongly reduced Treg cell differentia- tion, whereas BJ-2223 had no effect on Treg cells (Fig. 1a, b).

To further investigate the effect of BJ-2223 on CD4

T cells,

different doses of BJ-2223 were treated under Th17 skewing

cytokine conditions. It was found that BJ-2223 decreased Th17

cell differentiation percentage in a dose-dependent manner

(Fig. 1c, d). Thus, BJ-2223 decreased Th17 differentiation

without having a broad effect on Th1 and Treg cells.

BJ-2223 inhibits antigen-specific Th17 cell differentia- tion

To further investigate whether BJ-2223 inhibited Th cell dif- ferentiation with antigen specificity, OT-II mice were used, which recognized OVA

by class II MHC molecules.

Naïve CD4

T cells were cultured in Th1, Th17, and Treg dif- ferentiation conditions in the presence of OVA peptide and APCs. Our in vitro results showed that BJ-2223 exhibited no effect on Th1 and Treg differentiation, but tofacitinib strongly inhibited Th1 and Treg cell differentiation (Fig. 2a, b). BJ-2223 treatment significantly decreased Th17 differentiation, similar to that by commercial tofacitinib (Fig. 2a, b). Moreover, BJ- 2223 reduced Th17 cell differentiation percentage in a dose- dependent manner (Fig. 2c, d). Thus, BJ-2223 inhibited Th17 cell differentiation with antigen specificity.

BJ-2223 exhibited no negative effect on T cell prolifer- ation and viability

The regulatory effect of BJ-2223 on Th17 cell differentiation can be mediated by cytotoxicity or reduced proliferation of T cells. To investigate whether low proliferation of CD4

T cells decreased Th17 differentiation by BJ-2223, CFSE-labeled CD4

T cells were cultured with varying doses of BJ-2223 under Th17

polarizing conditions for 72 h. Based on CFSE dilution, treat- ment with different doses of BJ-2223 exhibited no effect on Th17 cell proliferation (Fig. 3a). Furthermore, the level of the nuclear protein Ki-67 was not altered by BJ-2223 treatment, indicating that BJ-2223 exhibited no effect on proliferation (Fig.

3b). To investigate the cytotoxicity of the compound, CD4

T cells were cultured in Th17 differentiation condition with or without BJ-2223 and stained with annexin V and PI. The per- centage of viable cells was similar to that in the control group, suggesting that BJ-2223 exhibited no cytotoxic effect even at higher concentration of up to 10 μM (Fig. 3c, d). Moreover, the MTS assay showed that treatment with BJ-2223 exhibited no effect on cell viability, indicating the safety of this compound (Fig. 3e). Thus, our findings suggest that BJ-2223 does not affect T cell proliferation and does not induce T cell apoptosis.

BJ-2223 reduced Th17 differentiation via inhibition of JAK/STAT signaling

The JAK–STAT signaling pathway is the major pathway to modulate T cell differentiation via cytokine receptor signal- ing.

IL-6 cytokine binds to IL-6 receptor and activates JAK1/

JAK2 and STAT3 to induce Th17 differentiation.

To investi- gate whether BJ-2223 regulates JAK–STAT activation, naïve Fig. 1 − BJ-2223 inhibits in vitro Th17 cells differentiation.

CD4

T cells were isolated form spleen and lymph nodes from normal C57BL/6 mice. Purified naïve CD4

T cells were stimulated

with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) under Th1, Th17 and T

differentiation conditions with or without BJ-2223 (1 μM)

or tofacitinib (1 μM). (a) The percentage of IFN-γ

Th1, IL-17A

Th17 and Foxp3

T

cells were analyzed by flow cytometry. (b)

Bar diagram shows the percentage of Th1, Th17 and Treg differentiation. (c) Different concentration of BJ-2223 was used to check

its effect in Th17 differentiation and analyzed by flow cytometry. (d) Bar diagram shows the dose dependent effect of Th17 cell

differentiation. Data represent three independent experiments. Mean±SEM of the triplicates are shown. **P<0.01, ***P<0.001.

Fig. 2 − BJ-2223 inhibits antigen specific Th17 cells differentiation.

Naive CD4

T cells were isolated from 8-10 weeks OT-II mice and co-cultured with irradiated antigen presenting cells under Th1, Th17 and T

polarizing conditions in the presence of OVA

(0.1 μM). (a) The percentage of IFN-γ

Th1, IL-17A

Th17 and Foxp3

T

cells was analyzed by flow cytometry. (b) Bar diagram shows the percentage of Th1, Th17 and Treg differentiation. (c) Different concentration of BJ-2223 were cultured with OT-II CD4

T cells under Th17 differentiation condition and analyzed by flow cytometry. (d) Bar diagram shows the dose dependent effect of Th17 cell differentiation. Data represent three independent experiments. Mean±SEM of the triplicates are shown. **P<0.01, ***P<0.001.

Fig. 3 − Effect of BJ-2223 on CD4

T cell proliferation, viability and apoptosis.

(a) Naïve CD4

T cells were labelled with CFSE and cultured in the presence or absence of BJ-2223 (1 μM) under Th17 differentiation

condition for 72 hour and Cell proliferation was assessed by CFSE dilution. (b) CD4

T cell proliferation assessed by the detection

by of Ki-67 protein expression. (c), (d) Naïve CD4

T cells were cultured under Th17 differentiation condition in the presence or

absence of BJ-2223 and the percentage of live cells were detected by Annexin-V and PI staining. (e) Splenic lymphocytes were isolated

and cultured with the different concentrations of BJ-2223 under Th17 polarizing condition and Cell viability was measured by MTS

assay. Data are the representative of three independent experiments. Mean±SEM of the triplicates are shown.

CD4

T cells were stimulated with anti-CD3, anti-CD28 under Th17 differentiation conditions, and isolated cells were ana- lyzed by western blot. Our result showed that BJ-2223 reduced the phosphorylation of JAK1 and JAK2 without affecting total JAK1 and JAK2 levels (Fig. 4). Next, we analyzed the activa- tion of STAT3 protein expression, which is the downstream target of JAK. Immunoblotting results showed that BJ-2223 decreased phosphorylation of STAT3 without affecting total STAT3 protein expression (Fig. 4). Thus, BJ-2223 reduced Th17 cell differentiation through inhibition of the JAK1/JAK2–STAT3 signaling pathway.

BJ-2223 ameliorated DSS-induced colitis

Th1 and Th17 cells have been implicated in the pathogene- sis of experimental colitis.

DSS-induced colitis is the most common mouse model for human IBD. We induced colitis in B6 mice using DSS to evaluate the therapeutic potential of BJ- 2223. Mice treated with BJ-2223 exhibited fewer colitis symp- Fig. 4 − BJ-2223 inhibits Th17 cells differentiation through the

modulation of JAK/STAT pathway.

Naïve CD4

T cells isolated from spleen and lymph nodes were cultured with or without BJ-2223 under Th17 condition and re stimulated with anti-CD3 (1 μg/ml)/CD28 (1 μg/ml) with or without IL-6 for 30 min. Phosphorylated and total form of JAK1/2 and STAT3 were analyzed by immunoblotting. Data are the representative of three independent experiments.

Fig. 5 − BJ-2223 attenuates DSS induced colitis.

Acute colitis was induced in C57BL/6 mice by oral administration with 2.5% (w/v) DSS in drinking water for 7 days. BJ-2223 was orally

administered every day (n=5) mice in each group. (a) Body weight of water, DSS and BJ-2223 treated mice, (b) Bleeding score, (c)

total no. of cells in MLN and spleen of mice given with water, DSS and BJ-2223, (d) Colon length of the mice in each group. (e) The

percentage of Th1 and Th17 cells was analyzed by the stimulation of cells from MLN with PMA, ionomycin and golgistop for 4 h and

analyzed by flow cytometry. (f) The percentage of Foxp3

Treg cells was analyzed from spleen and MLN. (g) H&E staining of colonic

section from water, DSS and BJ-2223 treated mice were shown in 40X and 100X magnification. Data represent three independent

experiments. Mean±SEM of the triplicates are shown. *p< 0.05, **P< 0.01.

toms, including loss of body weight, rectal bleeding, decreased cell count in the lymphoid organ, and shortening of colon, com- pared to the control mice (Fig. 5a–d). Next, we analyzed the effect of BJ-2223 on generation of Th1, Th17, and Treg cells in vivo. CD4

T cells from the mesenteric lymph node (MLN) were isolated and stimulated with PMA, ionomycin, and golgis- top for 4 h to analyze IFN-γ- and IL-17A-producing CD4

T cells. In comparison to the DSS-treated group, a slight decrease in Th1 percentage and a significant decrease in the percentage of Th17 cell generation were observed in the BJ- 2223 treated group (Fig. 5e). Moreover, BJ-2223 treatment exhibited no effect on Foxp3

Treg cell percentage in the spleen and MLN compared to that in the control group (Fig. 5f).

Histological observations of the colon by hematoxylin and eosin (H&E) staining showed that water-fed mice without DSS exhib- ited normal colon histology with intact epithelium and no infiltra- tion of inflammatory cells (Fig. 5g). DSS-fed mice showed massive mucosal ulceration, crypt damage, and severe inflamma- tion, and BJ-2223 treatment significantly ameliorated DSS- induced histological damages of the colon tissues (Fig. 5g).

These findings suggest that BJ-2223 ameliorated DSS-induced colitis through decreasing the percentage of Th17 cells.

Discussion

There is compelling evidence that Th17 is an appealing tar- get for the treatment of several inflammatory diseases.

Here, we demonstrated that BJ-2223 efficiently decreased dif- ferentiation of IL-17A-producing Th17 cells in vitro without demonstrating broad effect on other Th cells. Moreover, BJ- 2223 exhibited therapeutic potential for amelioration of DSS- induced colitis. Thus, we report BJ-2223 as a novel synthetic compound capable for suppressing in vitro Th17 cell differenti- ation and in vivo DSS-induced colitis.

The compound BJ-2223, along with many other compounds, was initially screened and selected by an in vitro Th cell differ- entiation assay. Polarization of Th17 cells in vitro was pro- pelled by the presence of cytokines IL-6 and TGF- β in the culture medium, which induced the expression of ROR γt for Th17 differentiation. Based on the screening results, we found that BJ-2223 treatment inhibited IL-17A-producing Th17 cell differentiation without any impact on Th1 and Treg cell polar- ization. Furthermore, BJ-2223 exhibited no effect on CD4

T cell proliferation and apoptosis.

A complicated network of cytokine and transcription factor is of great importance to determine the differentiation and func- tion of CD4

T cells. The major signaling pathway induced by numerous cytokines in T cell differentiation is the JAK/STAT pathway, which plays essential roles in the expansion and polar- ization of Th cells. JAK1 and JAK2 activation regulates STAT1 and STAT3 activation, which are important for Th1 and Th17 cell differentiation. IFN- γ binds to the IFN-γ receptor expressed on naïve CD4

T cells and triggers the activation of STAT1, and IL-6 binds to the IL-6 receptor and triggers the activation of STAT3. During differentiation of a series of Th cells from naïve CD4

T cells, STAT molecules are upregulated, which subse- quently induce target gene expression and cytokine production.

STAT3 plays an important role in Th17-mediated inflamma- tion.

Thus, inhibition of JAK1/2–STAT3 activation by BJ-2223 significantly decreased Th17 cell differentiation.

DSS-induced colitis model resembles IBD in humans, which is characterized by diarrhea, bloody feces, weight loss, colon shortening, mucosal ulceration, and accumulation of T cells in the colon.

IBD is a result of impaired function of Th cells, but recent advances have suggested an important role of Th17 and Treg cells. In particular, Th17 cells are the major factor for pathogenesis of colitis.

Our in vivo result showed ameliora- tion of DSS-induced colon damage, improvement in body weight, and colon length because of Th17 cell inhibition by BJ- 2223 treatment.

Several series of novel synthetic compounds have been shown to be effective for autoimmune diseases and DSS- induced colitis.

Azathioprine, cyclosporine, 6-mercapto- purine, tacrolimus, and methotrexate have been used in DSS- induced colitis as immunosuppressive agents.

However, all these drugs present several side-effects and are ineffective for patients. Therefore, novel drugs for DSS-induced colitis that do not exhibit side effects are needed. Tofacitinib, an oral JAK inhibitor, has been used for DSS-induced colitis therapy.

Inhi- bition of STAT3 activation has shown better treatment efficacy for DSS-induced colitis. In fact, gene associated with retinoid interferon-induced mortality 19 (Grim 19), an endogenous spe- cific inhibitor of STAT3, and metformin have demonstrated better therapeutic effects in DSS-induced colitis by decreasing STAT3 activation.

BJ-2223 inhibited the activation of JAK1/

JAK2 and its downstream target molecule STAT3 and may,

therefore, be a good candidate for the treatment of DSS-

induced colitis.

Conclusion

Our findings suggest that BJ-2223, a novel anti-inflamma- tory agent, inhibits Th17 differentiation in vitro and is effective for the treatment of DSS-induced colitis via the suppression of Th17 cells percentage in vivo. Mechanistically, BJ-2223 inhib- its the activation of JAK1/JAK2 and STAT3. However, it is pre- mature to conclude whether inhibition of JAK–STAT activation by BJ-2223 is a direct or indirect effect of the drug. Therefore, further study is needed to determine the precise role of this compound and its specific target. Our current study revealed the novel anti-inflammatory property of BJ-2223 and its effi- cacy in the treatment of colitis, which could be beneficial for the development of immunomodulatory therapies.

Acknowledgment

This study was supported by a Yeungnam University Research Grant, 217A380014, (http://www.yu.ac.kr/index.php). The funders had no role in the study design, data collection and analysis, deci- sion to publish, or preparation of the manuscript.

Conflict of Interest

The authors declare that they have no conflicts of interest.

References

1) Dittel, B. N. CD4 T cells: balancing the coming and going of autoimmune-mediated inflammation in the CNS. Brain, behavior, and immunity 22, 421-430 (2008).

2) Devarajan, P. & Chen, Z. Autoimmune effector memory T cells:

the bad and the good. Immunologic research 57, 12-22 (2013).

3) Kebir, H. et al. Human T H 17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation.

Nature medicine 13, 1173 (2007).

4) Zhou, L., Chong, M. M. & Littman, D. R. Plasticity of CD4+

T cell lineage differentiation. Immunity 30, 646-655 (2009).

5) Afkarian, M. et al. T-bet is a STAT1-induced regulator of IL- 12R expression in naive CD4+ T cells. Nature immunology 3, 549 (2002).

6) Ivanov, I. I. et al. The orphan nuclear receptor ROR γt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121-1133 (2006).

7) Sadlack, B. et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75, 253-261 (1993).

8) Kwon, H. S., Oh, S. M. & Kim, J. K. Glabridin, a functional compound of liquorice, attenuates colonic inflammation in mice with dextran sulphate sodium-induced colitis. Clinical &

Experimental Immunology 151, 165-173 (2008).

9) Atreya, R. & Neurath, M. F. Involvement of IL-6 in the pathogenesis of inflammatory bowel disease and colon cancer.

Clinical reviews in allergy & immunology 28, 187-195 (2005).

10) Rojas-Cartagena, C., Flores, I. & Appleyard, C. B. Role of tumor necrosis factor receptors in an animal model of acute colitis. Cytokine 32, 85-93 (2005).

11) Yang, X. O. et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. Journal of Biological Chemistry 282, 9358-9363 (2007).

12) Mudter, J. & Neurath, M. F. Il-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance.

Inflammatory bowel diseases 13, 1016-1023 (2007).

13) Kaser, A., Martínez-Naves, E. & Blumberg, R. S. Endoplasmic reticulum stress: implications for inflammatory bowel disease pathogenesis. Current opinion in gastroenterology 26, 318 (2010).

14) Solomon, L. et al. The dextran sulphate sodium (DSS) model of colitis: an overview. Comparative Clinical Pathology 19, 235- 239 (2010).

15) Maynard, C. L. & Weaver, C. T. Intestinal effector T cells in health and disease. Immunity 31, 389-400 (2009).

16) Acharya, S. et al. Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation.

Scientific reports 8, 7799 (2018).

17) Alex, P. et al. Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflammatory bowel diseases 15, 341-352 (2008).

18) Coskun, M., Salem, M., Pedersen, J. & Nielsen, O. H.

Involvement of JAK/STAT signaling in the pathogenesis of inflammatory bowel disease. Pharmacological research 76, 1-8 (2013).

19) Lovato, P. et al. Constitutive STAT3 activation in intestinal T cells from patients with Crohn's disease. Journal of Biological Chemistry 278, 16777-16781 (2003).

20) Nishihara, M. et al. IL-6–gp130–STAT3 in T cells directs the development of IL-17+ Th with a minimum effect on that of Treg in the steady state. International immunology 19, 695-702 (2007).

21) Robertson, J. M., Jensen, P. E. & Evavold, B. D. DO11. 10 and OT-II T cells recognize a C-terminal ovalbumin 323–339 epitope.

The Journal of Immunology 164, 4706-4712 (2000).

22) Murray, P. J. The JAK-STAT signaling pathway: input and

output integration. The Journal of Immunology 178, 2623-2629

(2007).

23) Feng, T. et al. Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production. The Journal of Immunology, 1001454 (2011).

24) Owaga, E. et al. Th17 cells as potential probiotic therapeutic targets in inflammatory bowel diseases. International journal of molecular sciences 16, 20841-20858 (2015).

25) Kim, J.-k. et al. Grim19 attenuates DSS induced colitis in an animal model. PloS one 11, e0155853 (2016).

26) Xavier, R. & Podolsky, D. Unravelling the pathogenesis of inflammatory bowel disease. Nature 448, 427 (2007).

27) Raza, A. et al. Th17 cells: interactions with predisposing factors in the immunopathogenesis of inflammatory bowel disease. Expert review of clinical immunology 8, 161-168 (2012).

28) Timilshina, M. et al. BJ-3105, a 6-Alkoxypyridin-3-ol Analog, impairs T Cell differentiation and prevents experimental autoimmune encephalomyelitis disease progression. PloS one 12, e0168942 (2017).

29) Kang, Y., Timilshina, M., Nam, T.-g., Jeong, B.-S. & Chang, J.-H.

BJ-1108, a 6-Amino-2, 4, 5-trimethylpyridin-3-ol analogue,

regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis. Biological research 50, 8 (2017).

30) You, Z., Timilshina, M., Jeong, B. S. & Chang, J. H. BJ?2266 ameliorates experimental autoimmune encephalomyelitis through down?regulation of the JAK/STAT signaling pathway.

European journal of immunology 47, 1488-1500 (2017).

31) Triantafillidis, J. K., Merikas, E. & Georgopoulos, F. Current and emerging drugs for the treatment of inflammatory bowel disease. Drug design, development and therapy 5, 185 (2011).

32) Vuitton, L., Koch, S. & Peyrin-Biroulet, L. Janus kinase inhibition with tofacitinib: changing the face of inflammatory bowel disease treatment. Current drug targets 14, 1385-1391 (2013).

33) Lee, S.-Y. et al. Metformin ameliorates inflammatory bowel

disease by suppression of the STAT3 signaling pathway and

regulation of the between Th17/Treg balance. PloS one 10,

e0135858 (2015).