BJ-3105, a 6-Alkoxypyridin-3-ol Analog, Impairs T Cell Differentiation and Prevents Experimental Autoimmune Encephalomyelitis Disease Progression

CD4+ T cells are essential in inflammation and autoimmune diseases. Interferon-γ (IFN-γ) secreting T helper (Th1) and IL-17 secreting T helper (Th17) cells are critical for several autoimmune diseases. To assess the inhibitory effect of a given compound on autoimmune disease, we screened many compounds with an in vitro Th differentiation assay. BJ-3105, a 6-alkoxypyridin-3-ol analog, inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells which were activated by T cell receptor (TCR) engagement. BJ-3105 ameliorated the experimental autoimmune encephalomyelitis (EAE) model by reducing Th1 and Th17 generation. Notably, Th cell differentiation was significantly suppressed by BJ-3105 treatment without inhibiting in vitro proliferation of T cells or inducing programmed cell death. Mechanistically, BJ-3105 inhibited the phosphorylation of JAK and its downstream signal transducer and activator of transcription (STAT) that is critical for Th differentiation. These results demonstrated that BJ-3105 inhibits the phosphorylation of STAT in response to cytokine signals and subsequently suppressed the differentiation of Th cell responses.


Introduction
CD4 + T cells are pivotal in mediating adaptive immunity. The major function of adaptive immunity is to mount a specific response to a pathogen while minimizing self-reactivity [1]. Naïve T cells differentiate into effector cells with functional potential for orchestrating pathogen clearance under the guidance of cytokines produced by innate immune cells [2]. Differentiation of the naïve CD4 + T cells require antigenic stimulation through T cell receptor (TCR) and CD4 as a co-receptor with major histocompatibility complex class-II (MHC-II) molecule presented by antigen presenting cells [3]. During the TCR activation and antigenic stimulation a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 draining lymph nodes and CNS of EAE inflicted mice. We further investigated the molecular mechanism underlying the BJ-3105 mediated effect and found that BJ-3105 attenuated differentiation of naïve CD4 + T cells by inhibiting phosphorylation of JAK1 and JAK2 and its downstream STAT1 and STAT4 in Th1 cell and phosphorylation of STAT3 in Th17 cell. This study therefore revealed a novel immune regulatory function of BJ-3105 by inhibiting Th1 and Th17 by modulating the JAK-STAT pathway.

Materials and Methods Mice
C57BL/6 mice and OT-II mice were purchased from Jackson Laboratory and housed under specific pathogen-free conditions and animal health were monitored every day in the animal care facility at the Yeungnam University animal care center. Experiments were carried out according to the institutional Guide for Care and Use of Laboratory Animals. CO 2 inhalation using gradual fill method was used as method of euthanasia to minimize potential pain. No animals were died during the study. The experiment protocol was reviewed and approved by Institutional Animal Care and Use Committee at Yeungnam University.

Apoptosis assays
Naive CD4 + T cells from spleen and lymph nodes were purified by immunomagnetic positive selection and isolated cells were stimulated in 96 well culture plates with plate bound anti-CD3 (5 μg/mL) and anti-CD-28 (1 μg/mL) in the presence of DMSO or BJ-3105 in Th1 (IL-12, 10 ng/ml plus anti-IL-4, 5 μg/mL) differentiation conditions in a cell incubator with 5% CO 2 at 37˚C for 72 h. Cells were stained with Annexin V-APC and Propidium Iodide as per the manufacturer's protocols (BD Biosciences) and analyzed by flow cytometry. EAE induction, prevention, and treatment C57BL/6 mice (8-12 weeks old) were immunized subcutaneously with 6 mg/mL synthetic peptide of myelin oligodendrocyte glycoprotein (MOG  ) peptide (MEVGWYRSPFSRVVH-LYRNGK, AnaSpec). The immunization was prepared by mixing MOG  peptide in CFA containing 10 mg/mL heat-killed H37Ra, strain of Mycobacterium tuberculosis (Difco Laboratories). In addition, the animals received 250 ng pertussis toxin (List Biological Laboratories) intraperitoneally (I.P.) on days of immunization and 48 h later. For treatment of EAE, BJ-3105 or DMSO (Sigma-Aldrich) as vehicle control were diluted in 1× PBS and administered at 3 mg/kg on day 0, and continued for every other day. The severity of EAE was monitored and graded on a clinical score of 0 to 5 with 0.5 for intermediate scores: 0 = no clinical signs; 1 = flaccid tail; 2 = paraparesis (weakness, incomplete paralysis of one or two hind limbs); 3 = paraplegia (complete paralysis of two hind limbs); 4 = paraplegia with forelimb weakness or paralysis; 5 = moribund or dead.

Immunoblotting
Naive CD4 + T cells from spleens and lymph nodes were isolated by immunomagnetic positive selection from 6-10 weeks C57BL/6 mice. The cells were cultured in Th1 and Th17 differentiation conditions for 72 h with indicated concentration of compound. Splenocytes and CNS mononuclear cells were obtained from drug treated and untreated EAE mice. Cell pellets were harvested and protein samples were prepared using RIPA buffer containing protease and phosphatase inhibitors. BCA protein assay reagents were used to quantify protein content. Equal amounts of protein were separated by SDS-PAGE and transferred to Polyvinylidene Difluoride (PVDF) membrane for 1 h. Membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS)-Tween 20 (TBS-T) for phosphorylated and total protein for 1 h followed by incubation with primary antibodies for overnight at 4˚C. The membranes were washed and incubated with secondary antibody in BSA for 1 h at room temperature. The protein bands were detected using ECL kit (Pierce) under a luminescent image analyzer. The membranes were reprobed with β-actin antibody for loading control.

Cytokine binding assay
Naïve CD4 + T cells from spleens and lymph nodes were activated and cultured with plate bound anti-CD3 (5 μg/mL) and anti-CD-28 (1 μg/mL) in Th1, Th2, Th9 and Th17 differentiation conditions for 72 h with indicated dose of BJ-3105. Cell pellets were washed with PBS and rested for 24 h with media containing 10% FBS at 37˚C, 5% CO 2 . Cells were stimulated with PMA (50 ng/mL) and ionomycin (750 ng/mL) for 24 h, and harvested proteins were quantified with cytokine binding assay kit (740005; Biolegend) by flow cytometer.

Histopathology
To assess the degree of CNS inflammation and demyelination, immunized C57BL/6 mice treated with vehicle or BJ-3105 were anesthetized and perfused by intracardiac injection of PBS containing 4% paraformaldehyde. Paraffin embedded 3 μm section of brain and spinal cord were stained with H&E and Luxol fast blue and then examined by light microscopy.

BrdU incorporation in EAE mice
C57BL/6 mice were treated with 0.8 mg/ml 5-Bromo-2'-deoxyuridine (BrdU) in their drinking water. BrdU solution was prepared in sterile water, protected from light and changed daily. During the continuous labeling phase, EAE induced mice received BrdU up to 15 days. The mice were sacrificed, splenocytes and CNS infiltrated mononuclear cells were analyzed BrdU incorporation using the BrdU Flow Kit (BD Pharmingen) according to the manufacturer's instructions.

Generation of bone marrow-derived dendritic cells
Bone marrow cells were flushed from the femurs and tibias of female C57BL/6 mice (6-8 wk) and depleted of red blood cells. Cells were cultured at 2×10 5 cells per well in 60×15 mm culture discs in medium supplemented with 20 ng/ml recombinant mouse granulocyte macrophage colony stimulation factor (GM-CSF). Nonadherent cells were removed and fresh medium was added every 3 days. On day 8, nonadherent cells released spontaneously from proliferating cell clusters were collected and cultured with BJ-3105 or vehicle under the condition of 200 ng/ml lipopolysaccharide (LPS) stimulation for 4 h.

Quantitative real time PCR
Total RNA was obtained using Trizol reagent (Invitrogen) and cDNA was synthesized with Goscript Reverse Transcription system (# A5001, Promega Corporation, WI, USA). Level of mRNA expression of each gene was measured with Real-Time PCR system using QuantiTect SYBR Green PCR kit (Qiagen). The following primer pairs were used: Il12a,

Statistical analysis
Data are presented as mean±S.E.M. of three independent experiments. Statistical significance was calculated by Student's t test or one-way ANOVA (Graph Pad Prism 5.0 software, San Diego, CA, USA). FACS data were analyzed with Flowjo software. Null hypotheses of no difference were rejected if p-values were less than 0.05.

BJ-3105 decreases the percentage of Th1 and Th17 cells differentiation
CD4 + T cells are essential in the immune response, and Th1 and Th17 cells are most extensively studied for understanding inflammation and autoimmune diseases [32,33]. Inhibition of IFN-γ producing Th1 and IL-17 producing Th17 cells helps to mitigate autoimmune disease [6]. Exploring the inhibitory effects of candidate compounds on autoimmune disease, we screened with an in vitro Th differentiation assay. Our screening revealed potential in a small molecule named BJ-3105 ( Fig 1A). To determine whether BJ-3105 influences T cell differentiation, CD4 + T cells were purified and cultured under Th1, Th17 and Treg skewing cytokine conditions with or without BJ-3105. We found that BJ-3105 (1 μM) treatment significantly reduced IFN-γ and IL-17 production like commercial tofacitinib at day 3 after in vitro stimulation with TCR and cytokine ( Fig 1B). In contrast, tofacitinib critically reduced Treg differentiation but BJ-3105 has very less impact on Treg differentiation. The amount of IFN-γ and IL-17 cytokine produced by CD4 + T cells were decreased drastically by BJ-3105 treatment ( Fig 1C). The amount of IL-4, IL-5 and IL-13 cytokines produced by CD4 + T cells were significantly reduced by BJ-3105 treatment in Th2 polarizing condition, but IL-9 cytokine was increased by BJ-3105 in Th9 polarizing condition (S1 Fig). So, the effect of BJ-3105 is different with that of tofacitinib on T cell biology because tofacitinib inhibited all Th generation including Th1, Th2, Th9, Th17 and Treg (Fig 1B and S1 Fig). To further investigate the immune modulatory effects of BJ-3105 on the differentiation of CD4 + T cells, varying doses of BJ-3105 were added into TCR and cytokines stimulated CD4 + T cells. The levels of IFN-γ and IL-17A were all decreased by this compound in a dose-dependent manner (Fig 1D and 1E). To rule out the possible effect of BJ-3105 on innate myeloid cells, we generated the bone marrow-derived dendritic cells (BMDC) in vitro. BMDC was cultured in the presence of BJ-3105 or vehicle under condition of LPS stimulation for 4 hr and found that there was no significant effect of BJ-3105 on mRNA expression of several inflammatory cytokines produced by DCs ( S2 Fig). Altogether, we concluded that BJ-3105 significantly and selectively regulate the expression of pro-inflammatory cytokines in CD4 + T cells stimulated with TCR and cytokines in vitro.

BJ-3105 decreases the percentage of antigen-specific Th1 and Th17 cell differentiation
To clarify whether BJ-3105 directly inhibits Th differentiation regardless of antigen type, we used ovalbumin (OVA)-specific OT-II T cell receptor (TCR) transgenic mice, which recognizes OVA 323-339 in the context of class-II MHC [34]. An in vitro culture of naïve OT-II CD4 + T cells with tofacitinib and BJ-3105 in the presence of OVA peptide and APCs significantly suppressed the generation of both IFN-γ and IL-17A-secreting cells except Treg generation (Fig 2A). To further characterize the immune modulatory effects of BJ-3105 on the OVA-specific The Inhibitory Effect of BJ-3105 on T Cell Differentiation differentiation of CD4 + T cells, varying doses of BJ-3105 were added into OVA peptide and cytokines stimulated OT-II CD4 + T cells. The levels of IFN-γ and IL-17A were all decreased by this compound in a dose-dependent manner (Fig 2B). It is notable that the BJ-3105-mediated suppression of IL-17A seemed more sensitive than that of IFN-γ because high dose (20 μM) BJ-3105 decreased Th1 by 50%, and decreased Th17 differentiation by more than 80% (Fig 2C). Thus, BJ-3015 compound directly inhibits T cell differentiation without Ag specificity.

BJ-3105 slightly regulates T cell proliferation without inducing cell death
To investigate whether the reduced generation of Th cells by BJ-3105 was caused by a reduced proliferation or increased apoptosis, CFSE-labeled CD4 + T cells were cultured with serial doses of BJ-3105 in Th differentiation condition for three days. Based on CFSE dilution, proliferation of IL-12 cytokine treated group was enhanced compared with that of no cytokinetreated group while proliferation of BJ-3105-treated group was slightly decreased dose-dependently (Fig 3A). Similarly, IL-6 and TGF-β increased the proliferation of CD4 + T cells with TCR stimulation in vitro, but BJ-3105 weakly affected the proliferation of primed T cells by TCR and cytokines (Fig 3B). For analyzing DNA level for proliferation, we used a bromodeoxyuridine (BrdU) cell proliferation assay. Consistently, in vitro proliferation by thymidine analog BrdU labeling assay demonstrated that BJ-3105 treatment induced a slight decrease of proliferation under Th1 polarizing condition (Fig 3C). Ki-67, a nuclear protein that is related with cell proliferation, was analyzed after T cell activation with TCR and cytokines. BJ-3105 compound reduced Ki-67 expression rate less than 10% compared with no compound-treated group (Fig 3D). Thus, BJ-3105 may slightly regulate the proliferation of T cells but this effect is less than the inhibitory effect on T cell differentiation by BJ-3105. Next we investigated whether the apoptosis of primed CD4 + T cells causes a reduction in T cell differentiation by BJ-3105. CD4 + T cells were cultured with TCR and cytokines in the presence of the BJ-3105 or PBS. On day 3 after activation, apoptosis was assayed by PI and Annexin-V staining. The percentage of viable cells was comparable between PBS and BJ-3105 treated group (Fig 3E). To further explore the effect of BJ-3105 on apoptosis, we cultured CD4 + T cells with TCR and cytokines in the presence of serial doses of BJ-3105. Still, there was no significant deleterious effect of high dose compound on in vitro T cell activation (Fig 3F). We conclude that although BJ-3105 slightly affects the proliferation of CD4 + T cells, the inhibition of T cell differentiation by BJ-3105 is not caused by less proliferation or more apoptosis.

Treatment of BJ-3105 ameliorates EAE by decrease of inflammatory T cells
The inhibition of Th1 and Th17 cell differentiation by BJ-3105 in in vitro prompted us to examine if this compound also affects antigen-specific Th cell responses in vivo. To test this, we used the EAE model, because Th1 and Th17 cells are critical for progression and pathology of EAE [35]. Mice were induced for EAE and disease scores were analyzed for the inhibitory effect of BJ-3105. Of note, most BJ-3105-treated mice were resistant to the development of EAE compared with PBS-treated group (Fig 4A). The total cell number from spleen and CNS were also decreased in drug treated EAE mice (Fig 4B). Since autoreactive CD4 + T cells, especially Th1 and Th17, are critical to induce EAE, we analyzed Th cells in EAE mice. To this end, mononuclear cells in brain and spinal cord were enriched by density gradient and analyzed by flow cytometry. Significantly fewer infiltrated CD4 + and CD8 + T cells were present in the brain and spinal cord of the BJ-3105-treated EAE mice than in the same tissue of PBS-treated EAE mice (Fig 4C). Th cells were further analyzed in spleen, draining lymph node (dLN) and central nervous system (CNS) of EAE mice with and without BJ-3105 treatment. As expected, Th1 and Th17 cells were predominant in the spleen and dLN of EAE-induced mice when compared with the untreated wild-type mice (data not shown). Of note, treatment with BJ-3105 significantly reduced the numbers of Th cells in the spleen, dLN and CNS of EAE-induced mice (Fig 4D). The proportion of Th1 and Th17 cells from dLN were strongly decreased in BJ-3105 treated-EAE mice. Histological characterization of the brain and spinal cord revealed less inflammatory infiltrates and less demyelination in the BJ-3105 treated EAE mice (S3A Fig). To find out whether the decrease in Th1 and Th17 cells is due to decrease in proliferation, we use in vivo labeling of BrdU through drinking water in BJ-3105 treated and untreated EAE mice. In contrast with previous in vitro proliferation data, drug treatment reduced BrdU + CD4 + T cells

in spleen and CNS mononuclear cells (S3B Fig). But we analyzed BrdU + CD4 + T cells at 14 day after EAE induction, so less proliferation of T cells in BJ-3105 treated EAE mice may be related with less inflammation not by the direct effect of BJ-3105.
Various studies reported that the CXCR3-CXCL11 and CCR6-CCL20 axis plays an essential role in controlling the entry of Th1 and Th17 cells into the CNS and hence mediates the initiation of EAE [19,36]. In our study we also found significantly reduced percentage of CD4 + T cells in to CNS following BJ-3105 treatment (Fig 4C). To investigate the effect of BJ-3105 on the migration of Th1 and Th17 cells to CNS, we analyzed the CXCR3 expression in Th1 and CCR6 expression in Th17 polarizing conditions. The level of CXCR3 expression on Th1-polarizing condition and CCR6 expression on Th17-polarizing condition was slightly down in drug-treated group (Fig 4E) but this inhibitory effect of CXCR3 and CCR6 expression by BJ-3105 is very less than inhibitory effect of Th differentiation. So, reduced number of T cells in CNS is mediated by less differentiation of T cells in dLN not by less expression of chemokine receptors by BJ-3105. Taken together, we concluded that BJ-3105 compound negatively controls the differentiation of CD4 + T cells in the EAE inductive phase and prevents EAE disease progression.

BJ-3105 regulates CD4 + T cell differentiation through modulating JAK-STAT pathway
Consistent with the observed efficacy of BJ-3105 in EAE as a marked decrease in the percentage of Th1 and Th17 cells in EAE mice, we investigated whether this compound regulates signaling pathway in T cell differentiation. JAK-STAT signaling pathway is indispensable for controlling cytokine production and differentiation of T cells [37,38]. We first hypothesized that BJ-3105 exerted regulatory effect on Th1 and Th17 development through direct inhibition of JAK/STAT pathway in T cells. To this end, CD4 + T cells were cultured with TCR stimulation and IL-12 plus BJ-3105 for three days. Autocrine IFN-γ and added IL-12 can induce the The Inhibitory Effect of BJ-3105 on T Cell Differentiation phosphorylation of STAT1 and STAT4 respectively [10]. In this condition, BJ-3105 inhibited the phosphorylation of both of STAT1 and STAT4 on day 3 after stimulation ( Fig 5A). Inhibitory intensity of phosphorylation of STATs by BJ-3105 is comparable with that of JAK inhibitor, tofacitinib. Phosphorylation of STAT3 on Th17 polarizing condition was also inhibited by The Inhibitory Effect of BJ-3105 on T Cell Differentiation BJ-3105 and tofacitinib. To further explore molecular mechanisms, we analyzed the JAK which is the upstream molecule of STAT protein. As shown in the Fig 5B and 5C, consistent with the reduction in Th1 and Th17 cells, expressions of p-JAK1/2, p-STAT1 and p-STAT4 for Th1 cells and p-STAT3 for Th17 cells was decreased by BJ-3105. To further delineate the in vivo effect of BJ-3105, we analyzed the splenocytes and CNS mononuclear cells from BJ-3105 treated and untreated EAE mice. The result demonstrated that BJ-3105 treatment inhibit the phosphorylation of JAK1/2, p-STAT1, p-STAT3 and p-STAT4 without altering total JAK and STAT in EAE mice (Fig 5D). From the above findings, we concluded that BJ-3105 affects the JAK/STAT signaling pathway for negatively regulating the development of Th1 and Th17 cells.

Discussion
In this study, we demonstrated that BJ-3105 had unique anti-inflammatory properties and therapeutic potential for autoimmune inflammatory conditions. We recently reported that 6-amino-2,4,5-trimethylpyridin-3-ols, first designed as antioxidants, showed good to excellent inhibitory effects against angiogenesis [39,40]. We also found that certain 6-amino-2,4,5-trimethylpyridin-3-ols potently alleviated a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis [unpublished results]. However their roles in immune system and, more specifically, in the differentiation and function of Th cell subsets in autoimmune disease settings are still elusive. BJ-3105 has a close structural similarity to 6-amino-2,4,5-trimethylpyridin-3-ols but contains an alkoxy moiety at 6-position of pyridine ring instead. The compound was selected in a primary screening using in vitro Th differentiation assay along with many kinds of pyridin-3-ols. Our initial series of experiments on differentiation of the naïve CD4 + T cell into effector lineages utilizes specifically formulated in vitro system where TCR signaling and cytokine co-stimulation can drive polarization in Th1 and Th17 cell lineages. Using this approach, we found that BJ-3105 treatment significantly impaired differentiation of naïve CD4 + T cell into Th1, Th2 and Th17 subsets whereas the percentage of Treg cells were not affected and IL-9 production is increased by this compound. Thus, BJ-3105 has an advantage for regulating inflammatory T cells but not all T cells. Innate immune system is important for protecting host but also can be involved in inflammatory diseases. So, we evaluate the effect of BJ-3105 on innate cell activation. But BJ-3105 compound did not affect mRNA level of several cytokines of BMDC by LPS ( S2 Fig). Based on our results, we believe BJ-3105 may be restricted to inflammatory T cells, Th1, Th2 and Th17.
The effector mechanism of inflammation in EAE process mostly depends on the expression of proinflammatory cytokines and chemokines. Autoimmune diseases like MS, inflammatory bowel disease and rheumatoid arthritis are the prototypic Th1 and Th17 mediated autoimmune disorders [41][42][43]. The EAE model recapitulates several aspects of human MS, with increased proinflammatory cytokines, particularly IFN-γ and IL-17A [35]. Although the exact role of Th1 and Th17 cells in EAE development is not well established, these effector T cells cause CNS inflammation and demyelination [6]. IFN-γ producing Th1 cells and IL-17A producing Th17 cells are very critical to autoimmune disease by inducing inflammation [44][45][46]. Therefore investigating specific drugs targeting Th1 and Th17 cell for management of autoimmune disease has clinical significance. We provide the in vitro and in vivo evidence that BJ-3105 represses the development of Th1 and Th17 cells and ameliorates EAE. BJ-3105 treatment significantly reduced the CD4 + cell population in spleen and CNS and proportion of both IFN-γ secreting Th1 and IL-17A secreting Th17 cells at the peak of disease in the spleen, draining lymph nodes, and CNS of EAE mice. The reduction of inflammatory lesion and demyelination in brain and spinal cord of BJ-3105 treated EAE mice is caused by the inhibition of Th1 and Th17 generation. These results substantially boost our understanding of the beneficial effect of BJ-3105 on management of EAE, which in turn helps to manage the effective preventive therapeutic approach to combat T cell mediated autoimmune diseases.
Encephalitogenic antigen myelin oligodendrocyte glycoprotein (MOG) has clinical relevance especially in MS [47]. Tolerance mediated treatment strategies through injection of MOG into anterior chamber of eye could be used as therapeutic tool in the treatment of MS [48,49]. Several studies showed that autoimmune disease like multiple sclerosis can be attenuated by administration of granulocyte-macrophage-colony-stimulation factor (GM-CSF) [50]. GM-CSF acts as pro-inflammatory or regulatory cytokine that can differentiate bone-marrow precursor into tolerogenic dendritic cells which in turn can increase Treg cell number and function [51,52]. However our compound is unique by its properties that BJ-3105 specifically inhibits Th1 and Th17 without altering Treg generation and mRNA expression of major cytokines produced by bone marrow derived dendritic cells. Therefore, compounds like BJ-3105 should receive a great deal of attention due to its excellent anti-inflammatory activities.
In cellular mechanisms, we examined the proliferation and apoptosis of T cells after BJ-3105 treatment. But BJ-3105 compound didn't affect the activation of T cells as well as proliferation and apoptosis. In in vitro system, anti-CD3 Ab and anti-CD28 Ab can stimulate TCR on T cells and these T cells can be activated via CD25 expression. Tofacitinib, JAK inhibitor, suppressed the expression of CD25 after TCR stimulation but BJ-3105 didn't (Figs 1B and 2A). Thus, tofacitinib may regulate TCR signaling but BJ-3105 do not. So, early activation and proliferation or apoptosis were not affected by BJ-3105 treatment. But in vivo T cell proliferation was less in BJ-3105 treated EAE mice and total cell number is also reduced in BJ-3105 treated EAE mice. In vivo condition is more complicated and we believe that less T cell population in BJ-3105 treated EAE mice is related with less inflammatory cytokines not by less TCR signaling or IL-2R signaling by this compound. So, based on these results, we focused on cytokine signaling.
Several studies showed that most cytokines engaged in EAE, comprising IFN-γ, IL-17A, IL-12, IL-23, use JAK/STAT signaling pathway to incite the biological response [31,53]. Naive CD4 + T cells constitutively express IFN-γ receptor (IFN-γR) where IFN-γ bind and results in STAT1 phosphorylation (p-STAT1). Likewise, IL-12 binds to IL-12 receptor and results in phosphorylation of STAT4 (p-STAT4). Finally p-STAT1 and p-STAT4 bind IFN-γ promoter and drive Th1 differentiation [6]. IFN-γ-STAT1/STAT4 signaling is important in T cell mediated immune response [54]. This study demonstrated that BJ-3105 treatment inhibited IFN-γ-STAT1/STAT4 signaling pathway, consequently reducing Th1 cell differentiation. However, time point inhibition of phosphorylation in STAT1 and STAT4 was different. In time-dependent approach, BJ-3105 inhibited the phosphorylation of STAT1 at early time (12 h after stimulation) but STAT4 phosphorylation was highly suppressed at 24 h after stimulation (S4 Fig)  and the inhibition of STAT3 by BJ-3105 was less at 12 h but high since then 24 h after stimulation (S4 Fig). In addition, phosphorylation of JAK was also affected by BJ-3105. So this compound regulate JAK/STAT signaling pathway. However, still it is unclear whether inhibition of phosphorylation is a direct or indirect effect of the drug on JAK and STAT molecules. Compared with tofacitinib, JAK inhibitor, the effect of BJ-3105 is different with tofacitinib on T cell activation. Therefore, BJ-3105 may have an unknown target for regulating JAK/STAT signaling.
BJ-3105 not only regulates the priming of pathogenic Th1 and Th17 in early stage of EAE but also suppresses these subsets at the peak of disease through inhibition of JAK/STAT pathways. BJ-3105, a novel anti-inflammatory agent, inhibits Th1 and Th17 differentiation in vitro, prevents onset, and ameliorates disease progression in EAE inflicted mice through sustained inhibition of JAK1, JAK2, STAT1, STAT4 and STAT3 phosphorylation. Our findings suggest that BJ-3105 is a promising therapeutic compound for the management of Th1 and Th17 mediated inflammation and autoimmune disease.