BJ-1108, a 6-Amino-2,4,5-trimethylpyridin-3-ol analogue, regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis

Abstract Background CD4+ T cells play an important role in the initiation of an immune response by providing help to other cells. Among the helper T subsets, interferon-γ (IFN-γ)-secreting T helper 1 (Th1) and IL-17-secreting T helper 17 (Th17) cells are indispensable for clearance of intracellular as well as extracellular pathogens. However, Th1 and Th17 cells are also associated with pathogenesis and contribute to the progression of multiple inflammatory conditions and autoimmune diseases. Results In the current study, we found that BJ-1108, a 6-aminopyridin-3-ol analogue, significantly inhibited Th1 and Th17 differentiation in vitro in a concentration-dependent manner, with no effect on proliferation or apoptosis of activated T cells. Moreover, BJ-1108 inhibited differentiation of Th1 and Th17 cells in ovalbumin (OVA)-specific OT II mice. A complete Freund’s adjuvant (CFA)/OVA-induced inflammatory model revealed that BJ-1108 can reduce generation of proinflammatory Th1 and Th17 cells. Furthermore, in vivo studies showed that BJ-1108 delayed onset of disease and suppressed experimental autoimmune encephalomyelitis (EAE) disease progression by inhibiting differentiation of Th1 and Th17 cells. Conclusions BJ-1108 treatment ameliorates inflammation and EAE by inhibiting Th1 and Th17 cells differentiation. Our findings suggest that BJ-1108 is a promising novel therapeutic agent for the treatment of inflammation and autoimmune disease

Role of Intestinal Microbiota in Baicalin-Induced Drug Interaction and Its Pharmacokinetics

Since many glycoside compounds in natural products are hydrolyzed by intestinal microbiota when administered orally, it is of interest to know whether their pharmacological effects are derived from the glycoside itself or from the aglycone form in vivo. An interesting example is baicalin versus baicalein, the aglycone of baicalin, which is contained in some herbs from Labiatae including Scutellaria baicalensis Georgi and Scutellaria lateriflora Linne. The herbs have been extensively used for treatment of inflammatory diseases in Asia. Although there have been numerous reports regarding the pharmacological effects of baicalin and baicalein in vivo and in vitro, some reports indicated that the glycoside form would hardly be absorbed in the intestine and that it should be hydrolyzed to baicalein in advance for absorption. Therefore, the role of metabolism by intestinal microbiota should also be considered in the metabolism of baicalin. In addition, baicalin contains a glucuronide moiety in its structure, by which baicalin and baicalein show complex pharmacokinetic behaviors, due to the interconversion between them by phase II enzymes in the body. Recently, concerns about drug interaction with baicalin and/or baicalein have been raised, because of the co-administration of Scutellaria species with certain drugs. Herein, we reviewed the role of intestinal microbiota in pharmacokinetic characteristics of baicalin and baicalein, with regards to their pharmacological and toxicological effects

JNK signaling activity regulates cell-cell adhesions via TM4SF5-mediated p27Kip1 phosphorylation

Transmembrane 4 L six family member 5 (TM4SF5) can regulate cell–cell adhesion and cellular morphology via cytoplasmic p27Kip1-mediated changes in RhoA activity. However, how TM4SF5 causes cytosolic p27Kip1 stabilization remains unknown. In this study we found that TM4SF5-mediated Ser10 phosphorylation of p27Kip1 required for cytosolic localization was not always correlated with Akt activity. Inhibition or suppression of c-Jun Nterminal kinase (JNK) in TM4SF5-expressing cells decreased Ser10 phosphorylation of p27Kip1 and rescued expression levels and localization of adherence junction molecules to cell–cell contacts. These observations suggest involvement of JNKs in TM4SF5-mediated p27Kip1 Ser10 phosphorylation and localization during epithelial–mesenchymal transition.OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000003910/2SEQ:2PERF_CD:SNU2012-01EVAL_ITEM_CD:102USER_ID:0000003910ADJUST_YN:NEMP_ID:A078142DEPT_CD:375CITE_RATE:4.238FILENAME:63HJK-CL.pdfDEPT_NM:약학과EMAIL:[email protected]:

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

BJ-3105 treatment suppresses development of EAE by decreasing autoreactive Th1 and Th17 generation.

<p>Active EAE was elicited in 8–12 weeks female C57BL/6 mice by immunization with MOG_35-55. The mice were subcutaneously treated with vehicle or BJ-3105 (3 mg/kg/every other day) from day 1 post-immunization as detailed under materials and methods. Mice were monitored for signs and symptoms of EAE. (A) Clinical score were recorded every day. (B) Total cell count in spleen and CNS of drug treated and untreated EAE mice. (C) At 24 days after challenge, total mononuclear cell obtained from the brains and spinal cord of MOG_35–55 immunized wild-type mice and BJ-3105-treated mice. Total percent of infiltrated CD4⁺ T cells and CD8⁺ T cells in CNS. (D) After 24 days of EAE, lymphocytes were isolated from the spleen, lymph nodes and spinal cord of mice. The fraction of IFN-γ and IL-17A producing CD4⁺ T cells from spleen, draining lymph node and CNS were determined by intracellular staining and analyzed by flow cytometry. Percentage of Th1 and Th17 cells were shown. (E) Flow cytometry analysis of CXCR3 expression on activated T cells under Th1 and CCR6 expression under Th17-polarizing conditions with or without BJ-3105 (1 μM). Bar diagram showing the mean fluorescence intensity (MFI) of CXCR3 and CCR6. Mean±SEM of multiple mice are shown. <i>*p</i> < 0.05, compared with drug untreated group.</p

BJ-3105 decreases the percentage of Th1 and Th17 cells differentiation.

<p>(A) Chemical Structure of BJ-3105 (2,4,5-trimethyl-6-(3-phenylpropoxy)pyridin-3-ol). (B) Purified naïve mouse CD4⁺ T cells from spleens and draining lymph nodes were stimulated in Th1 and Th17 polarizing conditions for 72 h and Treg for 96 h in the presence of BJ-3105 (1 μM) or tofacitinib (1 μM). CD4⁺ T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. Percentage of IFN-γ⁺ Th1 cells, IL-17A⁺ Th17 cells and Foxp3⁺ Treg cells were shown in bar diagram. (C) IFN-γ and IL-17A cytokine produced by CD4⁺ T cells were quantified by cytokine binding assay. (D) Th1, Th17 differentiation with different dose of BJ-3105. (E) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. <i>*p<</i>0.05, <i>**p</i><0.001 compared with drug untreated group. Data shown are mean±SEM.</p

BJ-3105 controls T cell differentiation through inhibiting phosphorylation of JAK-STAT signaling pathway.

<p>Naïve CD4⁺ T cells from spleens and draining lymph nodes were isolated and cells were cultured in Th1 and Th17 cells differentiation conditions with BJ-3105. Drug and cytokine untreated groups were used as control. (A) Naïve CD4⁺ T cells were polarized to Th1 cells with anti-CD3 (2 μg/mL), anti CD28 (1 μg/mL) and IL-12 (10 ng/mL) plus anti-IL4 Ab (5 μg/mL) for 72 h of culture. Th17 cells were differentiated with anti-CD3 (2 μg/mL), anti CD28 (1 μg/mL), TGF-β (1 ng/mL), IL-6 (10 ng/mL) plus anti-IL4 Ab (5 μg/mL) and anti-IFN-γ Ab (5 μg/mL) for 72 h of culture. Phosphorylated STAT1 and STAT4 from Th1 cells and STAT3 from Th17 cells were analyzed by flow cytometer. (B) Phosphorylated and total JAK1/2, STAT1 and STAT4 were detected by immuno-blotting under Th1 polarizing condition. (C) Phosphorylated and total JAK1/2 and STAT3 were detected by immuno-blotting under Th17 polarizing condition. (D) Western blot analysis of phosphorylated and total JAK1, JAK2, STAT1, STAT3 and STAT4 proteins in splenocytes and CNS infiltrated mononuclear cells from EAE induced mice. β-actin as loading control were detected by immune-blotting. Representative results of three experiments are shown.</p

BJ-3105 decreases the percentage of antigen-specific Th1 and Th17 cell differentiation.

<p>(A) Naïve CD4⁺ T cells and antigen presenting cells from spleens and lymph nodes were isolated by immunomagnetic positive selection from 6–10 weeks OT-II mice. CD4⁺ T cells and irradiated antigen presenting cells were cultured in Th1, Th17 and Treg cells differentiation conditions in the presence of OVA_323–339 (0.1 μM) with BJ-3105 (1 μM) or tofacitinib (1 μM). CD4⁺ T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. (B) The cells were cultured in Th1 and Th17 cells differentiation conditions for 72 h with different concentration of BJ-3105 and analyzed by flow cytometer. (C) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. <i>*p</i><0.05, compared with drug untreated group. Data shown are mean±SEM.</p