Scholarly article on topic 'Endothelin regulates function of IL-17-producing T cell subset'

Endothelin regulates function of IL-17-producing T cell subset Academic research paper on "Biological sciences"

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{ET-1 / "Multiple sclerosis" / IL-17 / Th17}

Abstract of research paper on Biological sciences, author of scientific article — Kensuke Tanaka, Kento Yoshioka, Koichiro Tatsumi, Sadao Kimura, Yoshitoshi Kasuya

Abstract Aims Although endothelin (ET) is known to play pleiotropic roles in various pathological conditions, its relation to autoimmune disease has not been elucidated. Here, we focused on interleukin (IL)-17, which is closely related to the pathogenesis of multiple sclerosis, and investigated the effect of ET receptor blockers on the production of IL-17 by T lymphocytes. Main methods Lymph node cells from mice at 8days post-immunization with MOG35–55 were stimulated in vitro with MOG35–55 in the presence or absence of an ET receptor blocker (BQ123 for ETA or BQ788 for ETB). Naïve T cells from mice were subjected to an in vitro model of Th17 differentiation, and ET-mediated IL-17 production was investigated under the states of Th17 differentiation and activation. Key findings ELISA revealed that MOG35–55-induced IL-17 production was significantly inhibited by BQ123 but not BQ788. Consistent with the ELISA results for IL-17, the frequency of CD4+ T cells producing IL-17 but not IFN-γ was reduced by BQ123. Under the differentiating state from naïve T cells to Th17 cells, the spontaneous release of IL-17 from CD4+ T cells was increased, which was insensitive to BQ123, indicating that ET/ETA signaling did not affect Th17 differentiation. After the time period of Th17 differentiation, however, the increase in IL-17 production by restimulation of the cells with anti-CD3 plus anti-CD28 antibodies was significantly inhibited by BQ123. Significance We demonstrated that ET/ETA signaling plays a crucial role in IL-17 production by Th17. BQ123 might be expected to be a future therapeutic drug for multiple sclerosis.

Academic research paper on topic "Endothelin regulates function of IL-17-producing T cell subset"

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Life Sciences

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Endothelin regulates function of IL-17-producing T cell subset

Kensuke Tanaka a,b, Kento Yoshioka a, Koichiro Tatsumib, Sadao Kimura a, Yoshitoshi Kasuya a'*

a Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, Chiba 2608670, Japan b Department of Respirology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan

ARTICLE INFO

ABSTRACT

Article history: Received 25 October 2013 Accepted 31 January 2014 Available online xxxx

Keywords: ET-1

Multiple sclerosis

Aims: Although endothelin (ET) is known to play pleiotropic roles in various pathological conditions, its relation to autoimmune disease has not been elucidated. Here, we focused on interleukin (IL)-17, which is closely related to the pathogenesis of multiple sclerosis, and investigated the effect of ET receptor blockers on the production of IL-17 by T lymphocytes.

Main methods: Lymph node cells from mice at 8 days post-immunization with MOG35-55 were stimulated in vitro with MOG35-55 in the presence or absence of an ET receptor blocker (BQ123 for ETA or BQ788 for ETB). Naïve T cells from mice were subjected to an in vitro model of Th17 differentiation, and ET-mediated IL-17 production was investigated under the states of Th17 differentiation and activation.

Key findings: ELISA revealed that MOG35-55-induced IL-17 production was significantly inhibited by BQ123 but not BQ788. Consistent with the ELISA results for IL-17, the frequency of CD4+ T cells producing IL-17 but not IFN-y was reduced by BQ123. Under the differentiating state from naïve T cells to Th17 cells, the spontaneous release of IL-17 from CD4+ T cells was increased, which was insensitive to BQ123, indicating that ET/ETA signaling did not affect Th17 differentiation. After the time period of Th17 differentiation, however, the increase in IL-17 production by restimulation of the cells with anti-CD3 plus anti-CD28 antibodies was significantly inhibited by BQ123. Significance: We demonstrated that ET/ETA signaling plays a crucial role in IL-17 production by Th17. BQ123 might be expected to be a future therapeutic drug for multiple sclerosis.

© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction

Endothelin-1 (ET-1), an extremely potent vasoconstrictor released mainly by endothelial cells, exerts many pleiotropic roles including effects on the autoimmune system (Barton and Yanagisawa, 2008). Being the only cells that promote differentiation of naïve T cells to effector T cells, dendritic cells (DC) contribute importantly to the development of autoimmune diseases (Banchereau et al., 2000). It has been shown that the maturation of DC is associated with the production of large amounts of ET-1, with concomitant upregulation of both ETA and ETB receptors (ETAR and ETBR; Guruli et al., 2004). The incubation of immature DC with an ETA receptor antagonist, BQ123, inhibited DC maturation and interleukin (IL)-12 production, which promotes a type 1 T helper cell (Th1) response.

Multiple sclerosis (MS) is a severe autoimmune demyelinating disease of the central nervous system (CNS). Though its clinical features were first depicted by Robert Carswell in 1838 (Compston, 1988), the exact pathogenesis of and effective curative therapies for MS still remain to be established. An autoimmune rodent model of experimental

allergic encephalomyelitis (EAE) has proved useful for research on MS (Lisak and Behan, 1975). EAE is initiated by immunization with myelin peptide and complete Freund's adjuvant (CFA). Myelin-specific T cells are activated by antigen presenting cells (APC) including DC in lymph nodes, and cross the blood brain barrier (BBB) into the brain and spinal cord where they are restimulated by APC to promote myelin destruction and trigger inflammation, astrogliosis and the production of chemotac-tic cytokines (Rodgers and Miller, 2012). Recently, the view thatTh1 cytokines were the main cause of EAE has been modified in light of evidence showing a crucial participation of Th17 cytokines in its pathogenic mechanisms (O'Garra et al., 1997; Kuwabara et al., 2009). IL-17, which is mainly secreted by Th17, induces permeability of the BBB and promotes recruitment of additional CD4+ T lymphocytes into the CNS, leading to the development of EAE (Kebir et al., 2007).

Here, we have used an in vitro T cell differentiation/activation system to investigate if ET-1, signaling through ET receptors (ETR), might contribute to the pathogenesis of EAE.

Materials and methods

* Corresponding author at: Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel.: +81 43 226 2193; fax: +81 43 226 2196. E-mail address: kasuya@faculty.chiba-u.jp (Y. Kasuya).

Animals

C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan). All animal procedures conformed to the Japanese regulations for animal

http://dx.doi.org/10.1016/j.lfs.2014.01.084

0024-3205/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Please cite this article as: Tanaka K, et al, Endothelin regulates function of IL-17-producingT cell subset, Life Sci (2014), http://dx.doi.org/10.1016/ j.lfs.2014.01.084

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Fig. 1. Upregulation ofIL-17 in MOG35-55-specific LNC mediated by ET-1/ETR signaling. LNC isolated from MOG35-55-immunized mouse at 8 DPI were restimulated in vitro with MOG35-55 as an antigen-specific response (gray columns) or anti-CD3/CD28 antibodies as an APC-free response (black columns) in the presence or absence of BQ123 or BQ788 for 72 h. As a negative control, LNC were cultured for 72 h without restimulation (white columns). A) MOG35-55-induced IL-17 in LNC. Supernatants were subjected to ELISA for IL-17. Data are shown as mean ± S.E.M. (n = 3). *, P< 0.05 (ANOVA followed by Tukey test). B) Detection of Th17 cells byFACScan.Th17 cells were defined as CD4+IL17+IFNy- cells. The quantification of Th17 cells was performed with Flowjo software. Data are shown as mean ± S.E.M. (n = 3). *, P < 0.05 (ANOVA followed by Tukey test).

care and use, following the Guidelines for Animal Experimentation of the Japanese Association for Laboratory of Animal Science, and were approved by the Animal Care and Use Committee of Chiba University.

MOG peptide

A synthetic peptide derived from myelin-oligodendrocyte-glycoprotein sequence 35-55 (MEVGWYRSPFSRVVHLYRNGK, MOG35_55) was synthesized by TORAY Research Center Inc. (Tokyo, Japan).

Characterization ofMOG35-55-specific LNC

WT mice were immunized subcutaneously in four flanks with 100 |ag MOG35_55 emulsified 1:1 in complete Freund's adjuvant (Difco, Franklin Lakes, NJ, USA) supplemented with 500 |ag Mycobacterium tuberculosis H37Ra on day 0. A single cell suspension of lymph node cells (LNC) was prepared from the axillary, inguinal, and cervical lymph nodes of mice at 8 days post-immunization (DPI). Then, the cells were stimulated with 20 |ag/ml MOG35_55 or 2 ^g/ml anti-CD3 plus anti-CD28 in LNC medium (RPMI1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS, 500 |jM 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 |ag/ml)) at 37 °C in a humidified atmosphere (5% CO2) in the presence or absence of BQ123 or BQ788 (1 x 10-5 M) for 72 h. As negative controls, LNC were incubated in LNC medium without any stimulator in the presence or absence of BQ123 or BQ788 for 72 h. The resulting supernatants and LNC were subjected to enzyme-linked immunosorbent assay (ELISA) and intracellular cytokine staining followed by flow cytometric analysis, respectively. The ELISA was performed according to the manufacturer's instructions for mouse 1L-17A ELISA MAX (Biolegend, San Diego, CA, USA). Cells were stained with APC-conjugated CD4 antibody (Ab) (Biolegend). After fixing and permeabilizing (Fix/Perm buffer, Biolegend), the cells were further stained with phycoerythrin-conjugated anti-lL-17 Ab and FITC-conjugated anti-lFN-Y Ab (BD Biosciences). Analysis was performed with a FACS Canto ll flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

In vitro model of Thl7 differentiation

A single suspension of LNC was prepared from the axillary, inguinal, and cervical lymph nodes of mice. Then, the purification of CD4+ T cells was performed with anti-CD4 magnetic microbeads (Miltenyi Biotech, Gladbach, Germany) in combination with MACS columns. CD4+ T cells were incubated in LNC medium containing anti-CD3 plus anti-CD28 Abs (1 |ag/ml each) and Th17-polarizing agents (30 ng/ml lL-6,1 ng/ml TGF-

ß,10 |Jg/ml anti-mouse 1L-4 and 10 |Jg/ml anti-mouse IFN-y) for 72 h as a Th17-polarizing period. As a negative control, 72-h incubation was conducted without the Th17-polarizing agents. Then, the cells were restimulated with anti-CD3 plus anti-CD28 Abs for another 6 h as an activation period of Th17 cells. To evaluate the effect of BQ123 or BQ788 (1 x 10-5 M), each agent was added to the medium for the Th17-polarizing period or activation period of Th17 cells. The supernatants and cells under each condition were subjected to ELISA and RNA preparation, respectively.

RT-PCR for RORyt

In order to assess Th17 cell differentiation, we carried out assays to quantify mRNA expression of the orphan nuclear receptor ROR^t, a transcription factor which directs this process. Single strand cDNA was synthesized from prepared total RNA (1 |ag), with MMLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using an oligo (dT) primer (Invitrogen) in a total volume of 20 pl. The resultant cDNA sample (1 |al) was subjected to PCR for amplification of mouse ROR^t and mouse peptidylprolyl isomerase A (PPIA as an internal control) using specific primers (for ROR^t, sense primer, 5'-CCGCTGAGAGGGCTTC AC-3'; antisense primer, 5'-TGCAGGAGTAGGCCACATTACA-3', for PPIA, sense primer, 5'-AGGTCCATCTACGGAGAG-3'; antisense primer, 5'-TCAGTCTTGGCAGTGCAG-3'). The settings of the thermal cycler were 22 cycles of 15 s at 98 °C, 30 s at 60 °C, 45 s at 68 °C, and 7 min at 68 °C for RORYt and 27 cycles of 15 s at 98 °C, 30 s at 55 °C, 45 s at 68 °C for PPIA. The amplified products were separated in 1.2% agarose gel and visualized with ethidium bromide staining under UV radiation. The specific amplification of the expected size (mouse ROR^t, 230 bp; PPIA, 134 bp) was observed.

Statistics

Data are expressed as mean ± S.E.M. Statistical analysis was conducted using Graphpad Prism Version 6 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey test, and P values < 0.05 were considered significant.

Results and discussion

It has been demonstrated that plasma ET-1 level is elevated in patients with MS, suggesting the possible involvement of ET-1 in the pathogenesis of MS (Haufschild et al., 2001). Very recently, a correlation between ET-1 and MS development associated with a decrease in cerebral blood flow (CBF) has been reported (D'haeseleer et al., 2013). In

Please cite this article as: Tanaka K, et al, Endothelin regulates function of IL-17-producing T cell subset, Life Sci (2014), http://dx.doi.org/10.1016/ j.lfs.2014.01.084

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Fig. 2. Effect of BQ123 on in vitro Th17 cell differentiation. CD4+ naïve T cells were induced to differentiate to Th17 in vitro by incubation with anti-CD3/CD28 Abs concomitantly with (PC) or without Th17-polarizing agents (NC) for 72 h (Th17-polarizing period), followed by another 6-h restimulation with anti-CD3/CD28Abs (activation period ofTh17); positive control (PC). As a negative control for activation period ofTh17, CD4 + naïve T cells were incubated with anti-CD3/CD28 Abs during Th17-polarizing period, followed by incubated with vehicle during activation period. To evaluate the effects ofBQ123 on the Th17-polarizing period and activation period of Th17, BQ123 was added to the culture medium of positive control during each period. A) BQ123 affected Th17-derivedIL17 production during the activation period ofTh17 but not the Th17-polarizing period. Data are shown as mean ± S.E.M. (n = 3). *, P< 0.05 (ANOVA followed by Tukey test). B) The induction of ROR^t mRNA during Th17-polarizing period was insensitive to BQ123. As a negative control (NC), mRNA from the CD4+ naïve T cells was used. As an internal control, RT-PCR was performed for peptidylprolyl isomerase A (PPIA).

that report, vasoconstriction of intracerebral arterioles by ET-1 released from reactive astrocytes in MS plaques was shown to lead to a reduction of CBF in MS patients. Likewise, administration of bosentan (a nonselec-tive ET-1 antagonist) to patients with MS can restore CBF to a normal level. These findings suggest that ET-1 may be one of the therapeutic targets for MS.

Considering the immune system, however, there is no report about the contribution of ET-1 to the pathogenesis of MS. Th17 cell-derived IL-17 is known to be a major mediator in EAE and MS (Komiyama et al., 2006; Wang et al., 2011). In the present study, we therefore investigated whether ET-1 regulates the function of the IL-17-producing T cell subset.

To evaluate whether ET-1/ETR signaling participates in IL-17 production by encephalitogenic antigen-specific effector T cells, LNC from MOG35-55-immunized mice were subjected to in vitro restimulation in the presence or absence of an ETR antagonist. As shown in Fig. 1A, restimulation of LNC with MOG35-55 induced IL-17 production, which was significantly inhibited by BQ123 but not BQ788. This finding suggests that ET-1/ETAR signaling can regulate T cell-derived IL-17 production closely related to the pathogenesis of EAE. This notion was supported by flow cytometric analysis of intracellular IL-17 in CD4+ T cells. Consistent with the ELISA results for IL-17, the frequency of CD4+ T cells producing IL-17 in LNC treated with BQ123 was significantly lower than that in control LNC in the case of stimulation with MOG35-55 (Fig. 1B). On the other hand, the stimulation of LNC with anti-CD3/CD28 Abs, mimicking antigen contact via APC also upregulat-ed both IL-17 production and the frequency of CD4+ T cells producing IL-17, which were sensitive to BQ123 treatment (Fig. 1A & B). This fact indicates that ET-1/ETAR signaling can regulate CD4+ T cell-derived IL-17 production even in an APC-free system. Although the existence of the ET-1/ETR system in macrophages has been shown, ET-1/ETAR signaling seems not to affect the antigen-presenting capacity of macrophages in the activation of effector T cells (Ruetten and Thiemermann, 1997). Taken together, these results suggest the possibility that ET-1/ ETAR signaling affects Th17 differentiation or the IL-17-producing activity ofTh17 cells.

To assess these possibilities, isolated naive T cell was subjected to a Th17-polarizing condition. Prior to examination, the mRNA expression of preproET-1 and ETAR in naive T cells was confirmed (data not shown). As shown in Fig. 2A, IL-17 production was induced during the Th17-polarizing period even in the presence of BQ123, suggesting that ET-1/ETAR signaling is not involved in Th17 differentiation. This notion was strongly supported by the finding that the Th17-polarizing agents-induced mRNA level of the orphan nuclear receptor ROR^t was not suppressed by BQ123 (Fig. 2B). ROR^t is a transcription factor directing the

differentiation of IL-17-producing inflammatory T cells, and is used as a marker ofTh17 differentiation (Ivanov et al., 2006). On the other hand, restimulation of Th17-polarized CD4+ T cells with anti-CD3/CD28 Abs markedly induced IL-17 production, which was significantly inhibited by BQ123. These results suggest that ET-1/ETAR signaling regulates IL-17 production by Th17 cells in an autocrine/paracrine manner.

Currently, the first-line treatments for control of MS are glatiramer acetate, interferon (IFN) (3-1a, IFN(3-1b and the new orally active sphingosine-1-phosphate receptor modulator fingolimod, and some monoclonal antibodies are in late stages of development (Jeffery, 2013). Likewise, G-protein-coupled receptors (GPCRs) were recently recognized as therapeutic targets for MS. In addition to chemokine receptor modulators under clinical investigation for MS, GPCRs for bioac-tive peptides are also expected to become new therapeutic targets for this disease in the near future (Du and Xie, 2012). Both the previous report by D'haeseleer et al. (2013) and our present study place ETRs (particularly the ETAR) in the list of GPCRs that are potentially relevant therapeutic targets for MS.

Conclusion

The present study indicated the presence of an ET-1/ETAR signaling loop in IL-17-producing T lymphocytes. Although further study is needed to evaluate the therapeutic effect of ETR antagonists using an in vivo MS model, our study might provide a new insight into MS treatment.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Acknowledgments

This work was supported in part by the Grants-in-Aid for Scientific Research ((B), 24390137 to Y.K.) and for the Challenging Exploratory Research (25670256 to Y.K.), and by the Takeda Science Foundation for Visionary Research (to Y.K.). We thank Dr. Wendy Gray for editing our manuscript.

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