Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

Interferon-γ (IFN-γ), a critical inflammatory cytokine, is primarily produced by T helper 1 (Th1) cells and accelerates the pathogenesis of inflammatory colitis. Pharmacological suppression of IFN-γ production attenuates dysregulated inflammatory responses and may be beneficial for treating inflammatory disease. In this study, we aimed to discover potent anti-inflammatory compounds that suppress IFN-γ production and found that the novel benzoxazole derivatives, 2-((3,4-dichlorophenyl) amino) benzo[d]xazol-5-ol (DCPAB) and 2-((3,4-hydroxyphenyl) amino) benzo[d]xazol-5-ol (HPAB), suppressed IFN-γ production by T cells. Treatment of CD4+ T cells with DCPAB and HPAB selectively inhibited Th1 cell development, and DCPAB more potently suppressed IFN-γ than HPAB did. Interestingly, DCPAB and HPAB significantly suppressed the expression of T-box containing protein expressed in T cells (T-bet) that activates IFN-γ gene transcription. DCPAB additionally suppressed transcriptional activity of T-bet on IFN-γ gene promoter, whereas HPAB had no effect on T-bet activity. IFN-γ suppressive activity of DCPAB and HPAB was impaired in the absence of T-bet but was retrieved by the restoration of T-bet in T-bet-deficient T cells. Furthermore, DCPAB and HPAB attenuated inflammatory colitis development that was induced by CD4+ T cells in vivo. We suggest that the novel benzoxazole derivatives, DCPAB and HPAB, may have therapeutic effects on inflammatory colitis.

derivatives have shown several biological activities, including antimicrobial, antibacterial, antiviral 9,10 , anti-cancer [11][12][13][14] , anti-tuberculosis 15 , and anti-inflammatory activity 16 . Recently, benzoxazole derivatives were reported to have the potential to control depression and neurological diseases 17,18 . Anti-inflammatory benzoxazole derivatives inhibited cyclooxygenase and 5-lipoxygenase activities, leading to the suppression of inflammatory responses 16,19 . Furthermore, benzoxazole derivatives with a nitrogen-containing heterocyclic substituent suppressed irritable bowel syndrome through the suppression of 5-HT3 receptor activity 20 . However, whether benzoxazole derivatives modulate inflammatory cytokine production and have therapeutic effects in inflammatory bowel diseases require clarification.

Novel benzoxazole derivatives DCPAB and HPAB suppress IFN-γ production by splenic T cells.
In an attempt to isolate potent anti-inflammatory compounds that inhibits IFN-γ production, we synthesized novel benzoxazole derivatives and purified using preparative column chromatography, followed by cell-based assay using splenic T cells. Two novel benzoxazole derivatives, DCPAB and HPAB, were isolated to suppress IFN-γ production. The structures of DCPAB and HPAB were characterized and shown in Fig. 1a. Spleen cells were treated with increasing amounts of DCPAB and HPAB after T cell receptor stimulation. Interestingly, DCPAB and HPAB suppressed IFN-γ produced by splenic T cells in a dose-dependent manner (Fig. 1b). DCPAB and HPAB had no effect on the viability of splenic T cells (Fig. 1c).

DCPAB and HPAB inhibit IFN-γ production by CD4+ T cells. We then examined whether DCPAB
and HPAB affected IFN-γ production by CD4+ T cells. CD4+ T cells were stimulated through a T cell receptor activation and subsequently treated with DCPAB or HPAB. IFN-γ production by activated CD4+ T cells was significantly suppressed by treatment with DCPAB or HPAB, whereas IL-2 production was not affected by both compounds (Fig. 2a). To examine the selective functions of DCPAB and HPAB, we treated CD4+ T cells with DCPAB or HPAB for 48 h under different skewing conditions. Under Th1-skewing conditions, DCPAB and HPAB significantly suppressed IFN-γ production (Fig. 2b). IFN-γ expression was suppressed by DCPAB more strongly than by HPAB (Fig. 2b). Under either Th2-skewing or Th17-skewing conditions, both DCPAB and HPAB had no effect on IL-4 and IL-17 expression (Fig. 2c,d), suggesting a specific inhibitory activity of DCPAB and HPAB on IFN-γ , not IL-4 nor IL-17, production.
Th1 cell differentiation is inhibited by treatment with DCPAB and HPAB. In order to confirm the specific activity of DCPAB and HPAB on IFN-γ expression, effects of DCPAB and HPAB on Th1 cell differentiation were investigated. We induced the naive CD4+ T cells to differentiate into Th1 cells and incubated the cells with DCPAB and HPAB during Th1 cell differentiation (Fig. 3a). Fully differentiated Th1 cells were determined by intracellular IFN-γ staining, followed by flow cytometry analysis. DCPAB and HPAB dose-dependently decreased IFN-γ -producing Th1 cell populations (Fig. 3b). Further analysis of the secreted IFN-γ cytokine in cell supernatants confirmed that DCPAB and HPAB significantly suppressed IFN-γ production (Fig. 3c). A quantitative and comparative analysis of DCPAB and HPAB demonstrated that DCPAB more potently suppressed IFN-γ than HPAB did (Fig. 3d).

DCPAB suppresses both T-bet expression and activity while HPAB decreases T-bet expression.
To investigate the molecular mechanisms underlying IFN-γ suppression and inhibition of Th1 cell differentiation by DCPAB and HPAB, we examined the effects DCPAB and HPAB on T-box containing protein expressed in T cells (T-bet) that is an essential Th1-specific transcription factor for inducing IFN-γ gene transcription 21,22 . We first verified that T-bet expression during Th1 cell differentiation was significantly suppressed by DCPAB and HPAB in a concentration-dependent manner (Fig. 4a). Quantitative analysis demonstrated that DCPAB potently suppressed T-bet expression. HPAB also significantly suppressed T-bet expression, but to a lesser extent (Fig. 4a). However, T-bet mRNA level was not affected by both DCPAB and HPAB (Fig. 4b), indicating the post-transcriptional regulation of T-bet by DCPAB and HPAB. We next examined whether DCPAB or HPAB affected the transcriptional activity of T-bet on IFN-γ gene promoter. Enforced T-bet expression in 394 T cells was detected by immunoblotting but its expression was not affected by treatment with DCPAB and HPAB (Fig. 4c). The IFN-γ gene promoter activity was enhanced by ectopic T-bet expression, and T-bet-induced IFN-γ promoter activity was subsequently decreased by treatment with DCPAB, but not affected by HPAB (Fig. 4d), indicating that DCPAB, not HPAB, was capable of inhibiting the transcriptional activity of T-bet. Abs and subsequently incubated with DCPAB and HPAB for 48 h. (a) Cell supernatants were collected and used for measuring IFN-γ and IL-2 by ELISA. (b-d) CD4+ T cells were stimulated and additionally cultured under skewing conditions: IL-12 (2 ng/mL) and anti-IL-4 Ab (5 μ g/mL) for Th1-skewing (b), IL-4 and anti-IFN-γ Ab (5 μ g/mL) for Th2-skewing (c), and TGF-β (10 ng/mL) and IL-6 (10 ng/mL) for Th17-skewing (d) for 48 h. Total RNA was prepared and subjected to reverse transcription and real time-PCR to determine the relative expression level of IFN-γ (b), IL-4 (c), and IL-17 (d), respectively. Relative transcript level was calculated after normalization with actin level and is expressed as a value of fold-induction. *P < 0.05, **P < 0.005, ***P < 0.0005.
Scientific RepoRts | 7:42144 | DOI: 10.1038/srep42144 DCPAB and HPAB require T-bet for IFN-γ suppression. As our results showed that DCPAB and HPAB suppressed IFN-γ production through regulation of T-bet, we attempted to verify the T-bet-dependency of DCPAB and HPAB functions in IFN-γ suppression. CD4+ T cells were isolated from wild-type (WT) and T-bet knockout (KO) mice and induced to differentiate into Th1 cells in the presence of DCPAB or HPAB. In order to restore T-bet in T-bet-null T cells, we also isolated CD4+ T cells from double transgenic in KO (DTg/KO) mice that restore T-bet expression in a doxycycline-dependent and a T cell-dependent manner 23 and treated the cells with doxycycline ( Fig. 5a). T-bet expression in WT, not KO, Th1 cells was decreased by treatment with DCPAB and HPAB (Fig. 5b). In addition, restored T-bet expression in DTg/KO cells that were treated with doxycycline was decreased by DCPAB or HPAB treatment (Fig. 5b). IFN-γ production was accordingly inhibited in the presence of DCPAB and HPAB in WT Th1 cells, as evidenced by ELISA and intracellular staining (Fig. 5c,d). Although IFN-γ production was dramatically declined in T-bet deficient cells, a fair amount of IFN-γ was still produced in a T-bet-independent way but was not suppressed by treatment with DCPAB and HPAB (Fig. 5c,d), indicating a T-bet-dependent inhibitory functions of DCPAB and HPAB. Furthermore, IFN-γ production was induced by the restoration of T-bet expression in T-bet KO background and the robust production of IFN-γ was subsequently decreased by DCPAB and HPAB (Fig. 5c,d).

DCPAB and HPAB attenuate T cell-induced inflammatory colitis in vivo.
As DCPAB and HPAB suppressed inflammatory IFN-γ production and Th1 cell development in vitro, we evaluated the anti-inflammatory activity of DCPAB and HPAB in vivo. Inflammatory colitis was established in immune-deficient recombinase activating gene (RAG) KO mice by adoptively transferring naive CD4+ T cells 24 . Although the disease activity index of colitis was increased in CD4+ T/RAG KO mice, the administration of DCPAB and HPAB attenuated colitis development (Fig. 6a). Daily administration of DCPAB and HPAB significantly suppressed histopathological phenotypes in the inflamed colon (Fig. 6b). IFN-γ production was increased in the spleen of CD4+ T/RAG KO mice but was significantly decreased after the administration of DCPAB and HPAB (Fig. 6c,d). We found that DCPAB and HPAB ameliorated T cell-induced inflammatory colitis in vivo through suppression of IFN-γ production.

Discussion
Our results demonstrated that the novel benzoxazole derivatives, DCPAB and HPAB suppressed the development of Th1 cells by inhibiting T-bet-induced IFN-γ expression, thereby ameliorating inflammatory colitis induced by inflammatory Th1 cells in vivo.
IFN-γ , a typical pro-inflammatory cytokine, is predominantly produced by activated T cells. A robust production of IFN-γ was observed in chronic inflammatory disease conditions and its deficiency ameliorated the development of inflammatory colitis 4-7 , indicating its indispensable role in the initiation and aggravation of inflammatory colitis. IFN-γ expression is mainly regulated at transcriptional level, and T-bet is known to be a master transcription factor for IFN-γ gene transcription. T-bet deficiency impairs IFN-γ production in developing Th cells and prevents their differentiation into mature Th1 cells and attenuates autoimmune responses 22 . The novel benzoxazole derivatives, DCPAB and HPAB, were found to suppress IFN-γ production through inhibition of T-bet expression. DCPAB and HPAB decreased the protein level of T-bet but did not affect T-bet mRNA level, indicating the post-transcriptional or translational regulation of T-bet by DCPAB and HPAB. Indeed, multiple post-translational modification of T-bet including phosphorylation and ubiquitination resulted in differential regulation of Th cell development. T-bet suppression induced by DCPAB was inhibited by MG132 treatment, suggesting the ubiquitination-proteasomal degradation of T-bet protein by DCPAB. It is yet to be clarified that whether DCPAB and HPAB controlled the phosphorylation of T-bet protein or the mRNA stability of T-bet [25][26][27] . Interestingly, DCPAB, but not HPAB, suppressed the transcriptional activity of T-bet on the IFN-γ gene promoter. It is possible that DCPAB may inhibit the DNA-binding activity of T-bet or transcription complex formation. It also remains to be studied whether and how DCPAB controlled the DNA-binding activity of T-bet and how the structural difference between DCPAB and HPAB affected the transcriptional activity of T-bet. Due to such differences in T-bet regulation, DCPAB more potently suppressed IFN-γ production than HPAB did.
Structurally, DCPAB and HPAB contain the same phenyl amino benzoxazolol, but have different groups in the phenyl ring (two chloride groups and one hydroxyl group, respectively). Because the structures of DCPAB and HPAB are related to their function, the dichlorophenyl ring of DCPAB may be critical for the control of T-bet stability. Although T-bet has been extensively studied and determined to play multiple roles in T cell development, inflammation, and tumor immunity, the structure of T-bet protein has not yet been fully characterized. Structural identification of T-bet protein modifications and DNA-T-bet binding complex will help to identify the functional Protein blots were incubated with anti-T-bet Ab, followed by detection using ECL and densitometry analysis (a). *P < 0.05, **P < 0.005, ***P < 0.0005. Total RNA was independently prepared using TRIzol reagent and subjected to reverse transcription, followed by the quantitative analysis of T-bet mRNA (b). (c,d) HEK 293 T cells were transfected with T-bet expression vector together with INF-γ promoter-linked reporter gene (pIFN-γ -luc). The β -galactosidase expression vector, pCMVβ was also transfected for normalizing transfection efficiency. Transfected cells were incubated with DCPAB or HPAB for an additional 24 h. T-bet expression was determined by immunoblot analysis (c). Relative luciferase activity was calculated after normalization with β -galactosidase activity and is expressed as a fold induction from four independent experiments (d). **P < 0.005, ***P < 0.0005.
Scientific RepoRts | 7:42144 | DOI: 10.1038/srep42144 mechanisms of DCPAB and HPAB underlying IFN-γ suppression in a T-bet-dependent manner. In the future, the structural correlation between the dichlorophenyl group of DCPAB and T-bet binding to the IFN-γ gene promoter should be verified. CD4+ T cells differentiate into not only Th1 cells, but also into Th2 and Th17 cells upon antigenic stimulation 28 . Th2 and Th17 cells produce IL-4 and IL-17 and control allergic and autoimmune responses, respectively 28,29 . Dysregulated and imbalanced generation of Th2 and Th17 cells and overproduction of their signature cytokines cause development of chronic inflammatory and autoimmune diseases in vivo 30 . We observed that DCPAB and HPAB specifically suppressed Th1, but not Th2 or Th17, cells. Selective suppression of Th1 cells by DCPAB and HPAB may have beneficial effects on the control of the Th1-mediated inflammatory response, including on inflammatory colitis. However, in addition to IFN-γ , many other cytokines, such as TNF-α , IL-17, IL-6, and IL-23, affect inflammatory colitis development 6,31-34 . Daily injection of DCPAB and HPAB into a T cell-induced colitis model significantly ameliorated disease severity. These in vivo results suggested that DCPAB and HPAB may function as potent immune suppressors by suppressing inflammatory responses coordinated by innate and adaptive immune cells. We therefore concluded that DCPAB and HPAB may have therapeutic and preventative benefits in the control of inflammatory diseases. To further evaluate the use of benzoxazole derivatives as anti-inflammatory agents, the broad range effects of DCPAB and HPAB on the differentiation of all subsets of T cells, including regulatory T cells, and on chronic inflammatory and autoimmune diseases should be examined.

Materials and Methods
Reagents. All cytokines were purchased from BD Pharmingen (San Diego, CA). Abs against cytokines, T-bet,  were purified with 95% purity (endotoxin-free) and dissolved in 100% DMSO. DCPAB and HPAB was freshly diluted by a factor of 1,000 for treating cells and 0.1% DMSO was used as the control (vehicle).

In vitro culture of spleen cells and differentiation of CD4+ T cells into Th1 cells.
Single-cell suspensions were obtained from spleen of WT (male, 6 to 8 weeks of age) mice and cultured in RPMI 1640 media (HyClone Laboratories, Logan, UT). Cells (5 × 10 6 cells/well for 6-well plate) were stimulated with anti-CD3 Ab (1 μ g/mL, BD Pharmingen) in the presence of vehicle (0.1% DMSO), DCPAB (10 μ M), or HPAB (10 μ M) for 24 h. CD4+ T cells were isolated from single-cell suspensions of lymph node and spleen using miniMACS CD4 microbeads (Miltenyi Biotec Inc., San Diego, CA). Cells were stimulated with plate bound anti-CD3 (2 μ g/ml) and anti-CD28 (1 μ g/ml) for 24 h and additionally treated with recombinant human IL-2 (10 U/mL), IL-12 (2 ng/mL), and anti-IL-4 Ab (5 μ g/ml) for Th1 differentiation. Instead of IL-12 and anti-IL-4 Ab, cells were treated with IL-4 and anti-IFN-γ Ab for Th2 cell differentiation, or skewed into Th17 cells by treatment with TGF-β and IL-6. Cells were additionally incubated with DCPAB or HPAB for an additional 4-5 days and were stimulated with anti-CD3 (1 μ g/ml) for 24 h. Cell supernatants were analyzed by ELISA and cells were used for intracellular cytokine staining analysis or total RNA preparation. Cytotoxicity assay. Spleen cells were stimulated with anti-CD3 and incubated with either DCPAB or HPAB for 24 h. Cells were additionally treated with EZ-Cytox reagent (EZ-CYTOX cell viability assay kit, Daeil lab, Seoul, Korea) for 2 h. Cell supernatants were harvested and measured using microplate reader at 450 nm according to the manufacturer's instructions. Cell viability was calculated and expressed as percentage after normalization with vehicle-treated control.
Cytokine measurement by ELISA. An ELISA plate was pre-coated with purified anti-IFN-γ Ab (1 μ g/ml) and incubated with cell supernatants for 1 h. The plate was washed and loaded with biotinylated anti-IFN-γ (1 μ g/ml) Ab, and subsequently incubated with streptavidin-conjugated alkaline phosphatase (1 μ g/ml). The substrate for phosphatase was added and color development was detected at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The known amount of standard cytokine was used to construct a standard curve. Intracellular cytokine staining. Differentiated Th1 cells were pre-treated with monensin (4 μ M) for 3 h and collected, followed by fix in 4% paraformaldehyde. Cells were treated with permeablization buffer (0.1% saponin, 0.1% sodium azide, and 1% fetal bovine serum in phosphate buffered saline) and incubated with phycoerythrin (PE)-conjugated anti-IFN-γ Ab (2 μ g/ml) for 30 min on ice in dark. After washing, the cells were analyzed by flow cytometry and CellQuest software (BD Pharmingen).

Reporter gene assay. Highly transfectable human embryonic kidney (HEK) 293 T cells were transfected
with an IFN-γ promoter-linked reporter gene (pIFN-γ -luc) and T-bet expression vector. The pCMVβ reporter gene that expresses β -galactosidase activity was also transfected for normalization of transfection efficiency. Luciferase and β -galactosidase activity were measured using a luciferase assay kit (Promega) and a Galacto-Light (Tropix), respectively. Relative luciferase activity was calculated after normalization with β -galactosidase. Mock transfection was set to 1 by fold induction.

Conclusion
The novel benzoxazole derivatives, DCPAB and HPAB, share a common amino phenyl benzoxazole structure and specifically suppressed IFN-γ production and Th1 cell differentiation through the inhibition of T-bet expression. DCPAB additionally inhibited T-bet activity and thus more potently suppressed IFN-γ production than HPAB did. DCPAB and HPAB attenuated inflammatory colitis induced by T cells in vivo, indicating the therapeutic effects of DCPAB and HPAB on chronic inflammatory diseases.