Lipid mediators foster the differentiation of T follicular helper cells

Accepted Manuscript Title: Lipid Mediators Foster the Differentiation of T Follicular Helper Cells Author: Tomonori Nagaya Koji Kawata Ryuta Kamekura Sumito Jitsukawa Terufumi Kubo Motonari Kamei Noriko Ogasawara Ken-ichi Takano Tetsuo Himi Shingo Ichimiya PII: DOI: Reference:

S0165-2478(16)30268-1 http://dx.doi.org/doi:10.1016/j.imlet.2016.11.006 IMLET 5950

To appear in:

Immunology Letters

Received date: Revised date: Accepted date:

26-1-2016 23-10-2016 8-11-2016

Please cite this article as: Nagaya Tomonori, Kawata Koji, Kamekura Ryuta, Jitsukawa Sumito, Kubo Terufumi, Kamei Motonari, Ogasawara Noriko, Takano Ken-ichi, Himi Tetsuo, Ichimiya Shingo.Lipid Mediators Foster the Differentiation of T Follicular Helper Cells.Immunology Letters http://dx.doi.org/10.1016/j.imlet.2016.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Lipid Mediators Foster the Differentiation of T Follicular Helper Cells

Tomonori Nagaya1,2,*, Koji Kawata1,*, Ryuta Kamekura1,2, Sumito Jitsukawa1,2, Terufumi Kubo3, Motonari Kamei1, Noriko Ogasawara2, Ken-ichi Takano2, Tetsuo Himi2, Shingo Ichimiya1

1

Department of Human Immunology, Research Institute for Frontier Medicine,

Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan 2

Department of Otolaryngology, Sapporo Medical University School of Medicine,

Sapporo 060-8556, Japan 3

Department of Pathology, Sapporo Medical University School of Medicine,

Sapporo 060-8556, Japan

Address for correspondence and reprint requests: Shingo Ichimiya, Department of Human Immunology, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan; Phone: +81-11-611-2111 (ext. 2420), Fax: +81-11-688-5354, E-mail address: [email protected]

*These authors equally contributed to this work.

1

Highlights 

Naïve T cells have certain expression of the lipoxin 4 (LXA4) receptor FPR2 at mRNA and protein level.



LXA4 as well as leukotriene B4 (LTB4) facilitated the differentiation of naïve CD4+ T cells into T follicular helper cells.



Direct activation of FPR2 by LXA4 enhances Tfh cell differentiation.

Abstract Lipid mediators such as leukotrienes and lipoxines broadly regulate innate and acquired immunity, and their dysfunction causes various immune-mediated disorders. We previously reported a salient feature of arachidonate 5-lipoxyganase (Alox5), which is responsible for the production of such lipid mediators, in the regulation of high affinity antibodies in vivo. The aim of this study was to determine the functional significance of Alox5-related lipid mediators during the processes of acquired humoral responses. The results of in vitro experiments using lymphocytes in tonsils and blood specimens showed that lipoxin A4 (LXA4) and leukotriene B4 (LTB4) have the capacity to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells, which activate naïve B cells to form germinal centers. Such a function of LXA4 was further supported by results of in vitro studies using BML-111 and BOC-2, which are an agonist and an antagonist, respectively, of FPR2 of an LXA4-specific cell-surface receptor. The results suggest that such lipid mediators have a potential role in the development of lymphoid follicles through the regulation of Tfh cell differentiation. Abbreviations used in this article: Tfh cells, T follicular helper cells; LXA4, lipoxin A4; LTB4, leukotriene B4; formyl peptide receptor 2, FPR2 Key words: lipid mediators, T follicular helper cells,

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1. Introduction Lymphoid organs such as lymph nodes, tonsils, and the spleen control immune responses to efficiently inactivate and eliminate pathogens [1]. In addition

to

a

unique

capacity

to

concentrate

foreign

antigens

and

antigen-presenting cells (APCs), such lymphoid organs characteristically contain a representative site of lymphoid follicles surrounded by interfollicular regions and T cell zones harboring variable numbers of B cells and CD4+ T cells. T follicular helper (Tfh) cells actively move around lymphoid follicles and encourage cognate interaction with B cells, which are eventually activated to form germinal centers to produce high-affinity antibodies and memory B cells [2, 3, 4]. This elaborated process is linked to the initiation of antigen-specific immune responses and generation of long-lived protective immunity. The structures of lymphoid organs are often functionally altered by infection, aging and various immune-related disorders [5]. Thus, an understanding of lymphoid follicles providing cardinal foci of immune cells is important to recognize physiological and pathological immune conditions. Our previous study focusing on lymphoid tissues showed that arachidonate 5-lipoxygenase (Alox5) expressed in mantle zone B cells around germinal centers has a unique role in the establishment of antigen-specific antibody responses [6]. Alox5 is an enzyme responsible for the production of lipid mediators, including leukotrienes and lipoxins, and is present in large amounts in resting B cells and APCs such as dendritic cells and macrophages [6, 7]. Experimental evidence obtained by using Alox5-deficient mice suggests that Alox5-related lipid mediators support T follicular helper (Tfh) cells in the spleen [6]. Furthermore, the number of follicular B cells in Alox5-deficient mice is significantly decreased compared to that in wild-type mice, indicating that Alox5-related lipid mediators assist T-cell-mediated B-cell responses [6]. Leukotrienes and lipoxins of Alox5-related lipid mediators widely operate innate and acquired immune responses [7, 8]. However, little is known about the functional significance of Alox5-related lipid mediators in immune cells of lymphoid tissues. In this study, we examined the role of leukotrienes and lipoxins associated with Alox5 in the regulation of Tfh cell function. When receptors

3

specific to lipid mediators in CD4+ T cells were investigated, it was found that naïve

CD4+

T

cells

(CD3+CD4+CD45RA+CD45RO-CCR7+)

preferentially

expressed FPR2 (also named ALXR) of a lipoxin A4 (LXA4) receptor at the mRNA level and, to a lesser extent, BLT1 and BLT2 of a leukotriene B4 (LTB4) receptor [9, 10]. Experiments using in vitro differentiation models of Tfh cells further revealed that LXA4 as well as LTB4 intensified the differentiation of naïve CD4+ T cells to Tfh cells (CD3+CD4+PD-1+CXCR5+) but not to Th1 and Th2 cells. The results indicate that Alox5-related lipid mediators affect the development of Tfh cells. Further investigation focusing on lipid mediators would lead to a better understanding of the regulation of Tfh cell differentiation and the pathogenesis of autoimmunity and allergies associated with specific antibody production. 2. Materials and methods 2.1. Tissues and blood samples Surgically resected specimens of tonsils were obtained from patients admitted to Sapporo Medical University Hospital. Some of the tissues were stored in OCT compound (Sakura, Tokyo, Japan) at -80°C for frozen tissue sections. Heparinized blood was obtained from healthy volunteers. All tissues were obtained after receiving informed consent and with the approval of the institutional review boards of Sapporo Medical University. 2. 2. Reagents Anti-human mAbs including APC-anti-CD3 (UCHT1), PE-anti-CD3 (UCHT1), APC-Cy7-anti-CD4 (RPA-T4), FITC-anti-CD4 (RPA-T4), PE-anti-PD-1 (EH12.1),

PE-anti-Bcl6

PE-Cy7-anti-CCR7

(K112-91),

(3D12),

PE-Cy7-anti-PD-1

(EH12.1),

PerCP-Cy5.5-anti-CXCR5

(RF8B2),

PerCP-Cy5.5-anti-CD24 (ML5), FITC-anti-CD27 (M-T271), FITC-anti-CD45RA (HI100),

APC-anti-CD45RO

BV421-anti-IFNγ

(4S.B3),

(UCHL1),

APC-Cy7-anti-CD19

BV421-anti-IL-4

(8D4-8),

(SJ25C1),

BV421-anti-IL-17A

(N49-653) (BD Biosciences), BV421-anti-CXCR5 (J252D4; Biolegend) and APC-anti-FPR2 (304455; R&D SYSTEMS) were used for flow cytometry. Lipid mediators (LTB4, LTC4, LTD4, LTE4 and LXA4), FPR2/ALX, an agonist of BML-111

(5[S]-6[R]-7-trihydroxyheptanoic-

4

acid-methyl-ester)

(Cayman

Chemicals), and FPR2/ALX, an antagonist of BOC-2 (Bachem), were used for in vitro culture experiments. 2.3. Flow cytometry and cell sorting Human tonsil tissues were mechanically disrupted, and lymphocytes in single cell suspensions were prepared by density gradient centrifugation with Lympholyte (CEDARLANE). Heparinized PBMCs from fresh blood specimens were also isolated with Lympholyte. After standard staining with specific surface markers, cells were analyzed using FACSCanto II or FACSAria II for careful cell sorting (BD Biosciences). In each experiment, specimens were analyzed for singlet events with doublet discrimination, and the purity of FACS-sorted cells reached 95% after validation by reanalysis. To analyze the expression of a Bcl6 transcription factor, IFNγ, IL-4, IL-17 and FPR2, intracellular staining was performed according to the protocol of the Transcription Factor Buffer Set (BD Biosciences), and then cells were counted by FACSCanto II. Data were examined by using FACSDiVA software (BD Biosciences). 2.4. Cell culture For naïve CD4+ T cell culture, FACS-sorted human naive CD4+ T cells (1X105 cells) from human peripheral blood were stimulated under a Tfh polarization condition supplemented with anti-CD3 mAb (OKT3; 10 μg/ml), anti-CD28 mAb (15E8; 10 μg/ml), IL-6 (25 ng/ml), IL-12 (1 ng/ml), IL-21 (25 ng/ml), TGF-β (5 ng /ml), anti-IL-4 mAb (10 μg/ml), and anti-IFNγ mAb (10 μg/ml) as described previously [12] in the presence of lipid mediators as described above. After 5 days, polarized Tfh cells were detected by FACS analyses for PD-1 and CXCR5. All of the cytokines used in this study were obtained from Peprotech, and anti-IL-4 and anti-IFNγ antibodies were obtained from Milteny Biotech.

Serum-free

AIM-V

medium

(Invitrogen)

containing

50

μg/ml

streptomycin and 100 U/ml penicillin was used in all experiments, and all experiments were performed at 37°C in a humidified atmosphere with 5% carbon dioxide. 2.5. Gene analysis For quantitative PCR analysis, total RNA extracted by TRIZOL reagent

5

was reverse-transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed with a Step One Real-Time PCR System of Assay-on-Demand probes according to the instructions of the manufacturer (Applied Biosystems). The levels of expression of target genes were calculated using CT and comparative methods after normalization

to

glyceraldehyde-3-phosphate

dehydrogenase

(GAPDH)

expression. 2.6. Immunohistochemistry Immunohistochemistry was conducted as described previously [6]. In brief, tissue sections of tonsils were stained with primary Abs in a moisture box at 4°C overnight and reacted with secondary goat pAbs conjugated to Alexa Fluor dyes (Invitrogen). After staining with DAPI, tissues slides were analyzed using an immunofluorescence microscope (IX71; Olympus) or a confocal laser scanning microscope with image examiner software (LMS510META; Carl Zeiss). 2.7. Cell viability assay Tfh cell viability was assessed by staining with annexin V-FITC (ImmunoChemistry Technologies) and 7-AAD (BD Biosciences) according to the manufacturer’s protocol. 2.8. Statistical analysis Results are expressed as means and SD. The unpaired Student’s t-test was used to compare experimental groups with P < 0.05 considered significant.

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3. Results 3.1. Alox5-releted lipid mediators regulate the differentiation of Tfh cells. We initially focused on analysis of various types of Alox5-releted lipid mediators responsible for Tfh cell differentiation from naïve CD4+ T cells. When the mRNA expression profiles of receptors specific to lipid mediators on CD4+ T cells of tonsils were investigated, it was found that naïve CD4+ T cells (CD3+CD4+CD45RA+CD45RO-CCR7+) highly expressed FPR2, which is a receptor for LXA4, in comparison to the levels of expression on Tfh cells (CD3+CD4+PD-1+CXCR5+) and non-Tfh cells (CD3+CD4+PD-1-CXCR5-) (Fig. 1A). To a lesser extent, naïve CD4+ T cells expressed BLT1 and BLT2, receptors for LTB4, which were also expressed by Tfh cells as well as non-Tfh cells at levels similar to those in naïve CD4+ T cells. CysLTR1 and CysLTR2, which are receptors for cysteinyl leukotrienes including LTC4, LTD4, and LTE4, were detected in naïve CD4+ T cells, but their expression levels were lower than those of LXA4 and LTB4. We further compared FPR2 protein expression levels in naïve CD4+ T cells, Tfh cells, Th1 cells, Th2 cells and Th17 cells by a FACS analyses. Although all T cell subsets examined in this study expressed a certain level of FPR2 protein, the levels in Tfh cells were significantly higher than those in naïve CD4+ T cells. In addition, no significant difference was found between the expression levels of FRP in naïve CD4+ T cells and the other three T cell subsets. 3.2. LXA4 and LTB4 facilitate differentiation of Tfh cells. We next investigated the percentages of Tfh cells in naïve CD4+ T cells in the presence of a series of Alox5-related lipid mediators with stimuli for Tfh cell differentiation (Tfh-polarizing condition) [12]. As shown in Fig. 1, the Tfh-polarizing condition supplemented with LXA4 or LTB4 clearly facilitated the differentiation of naïve CD4+ T cells (CD3+CD4+CD45RA+CD45RO-CCR7+) into Tfh cells (PD-1+CXCR5+ cells within naïve CD4+ T cells), which express Bcl6, a key regulator for Tfh cell development (Figs. 2A, 2B, and 2C) [13]. These results were also confirmed by an increased concentration of Tfh-characteristic cytokine, IL-21, in the supernatant from culture supplemented with LXA4 or LTB4 (Fig. 2D). We further verified a potential role of LXA4 during Tfh cell differentiation by using BML-111 and BOC-2, which are an agonist and an antagonist specific to FPR2,

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respectively [11]. As expected, BML-111 augmented the differentiation of naïve CD4+ T cells to Tfh cells, and its action was effectively blocked by BOC-2 (Fig. 2E). Collectively, the results of these in vitro studies suggest that LXA4 and LTB4 can induce Tfh cell differentiation. We did not observe any significant effects of LXA4 and LTB4 on the differentiation into Th1 and Th2 cells, suggesting differentiation stimulatory effects of these lipid mediators on naïve CD4+ T cells are specific for differentiation to Tfh cells (Fig. 2F). 3.3. Viability of Tfh cells in the presence of LXA4 and LTB4 Given the positive influence of LXA4 and LTB4 on Tfh cell differentiation, we further investigated their activities for the proliferation and maintenance of Tfh cells. As shown in Fig. 3A, the condition supplemented with each lipid mediator did not expand total CD4+ T cells up to 4 days with stimuli for Tfh cell differentiation, and significant differences were not observed in their apoptosis levels (Fig. 3A and 3B). In addition, when tissue-resident Tfh cells isolated from tonsils were cultured in a serum-free AIM-V medium with or without LXA4 and LTB4 for up to 7 days, we did not observe any significant effects of LXA4 and LTB4 on the viability of Tfh cells (Fig. 3C, lower panel). Even under the condition of stimulation by CD3, the viability of Tfh cells was not affected by LXA4 and LTB4 (Fig. 3C, upper panel). These results suggest that Tfh cells cannot simply prolong the proliferation and survival by LXA4 and LTB4, even during the activation of T cell receptors. 4. Discussion In this study, we first showed potential roles of LXA4 and, to a lesser extent, LTB4 for attenuation of Tfh cell differentiation. Tfh cells of specialized effector CD4+ T cells establish initial and recall phases of specific humoral immunity. Functional defects of Tfh cells underlie the pathogenesis of immune-related disorders including bronchial asthma, rheumatoid arthritis, systemic lupus erythematodes, and cancer [2, 15]. Thus, elucidation of the mechanism of differentiation of human Tfh cells would lead to a further understanding of the pathology associated with Tfh cells. The results of our real-time PCR and FACS experiments revealed that

8

naïve T cells have high mRNA levels of the FPR2 and certain levels of FPR2 protein. Subsequent in vitro culture experiments showed that polarization of naïve T cells to Tfh cells was facilitated in the presence of LXA4. These results were supported by the results of experiment using the FPR2-specific agonist BML-111, and its effect on Tfh differentiation was blocked by the FPR2 antagonist BOC-2, suggesting that direct activation of FPR2 by LXA4 enhances Tfh cell differentiation. In this context, the results of our study showing that LXA4, which has an anti-inflammatory potential, regulates Tfh cell differentiation indicate the existence of a new class of molecules that act on Tfh cell differentiation in addition to cytokine species such as IL-12, IL-21, and TGFβ. It has been reported that direct activation of FRPL1, which is a member of the receptor family to which FRP2 belongs, increased T cell proliferation and skewing of Th1 cells [16]. Thus, there are FPR-mediated pathways for regulating the polarization of T cell subsets. How LXA4 works on Tfh cell differentiation in collaboration with the main signals for STAT3 and STAT4 transcription factors has not been elucidated. LXA4 can modulate several intracellular signaling pathways in various cells such as NF-kB activation [17, 18]. In T cells, LXA4 analogs inhibit TNF-α production through blocking extracellular signaling-regulated kinase (ERK) [19]. Tfh cell differentiation is a multistep process, being initiated at the time of priming of dendritic cells in the T cell zone followed by interactions with B cells at the T-B border [2, 20]. LXA4 is secreted from most myeloid cells such as neutrophils, macrophages and dendritic cells [21]. In addition, Alox5, an enzyme responsible for the production of lipoxin, is highly expressed in mantle zone B cells [6, 14]. It is thus thought that LXA4 produced by dendritic cells and B cells acts as a facilitating factor for Tfh cell differentiation during the activation of naïve CD4+ T cells and the interaction with B cells. LTB4, which is another lipid mediator that was shown to facilitate the differentiation of Tfh cells in this study, has been reported to regulate the recruitment of T cells and T cell activation and to dose-dependently enhance the differentiation of Th17 cells [10, 22, 23]. Although little is known about the mechanism of its action on Tfh cell differentiation, blocking agents against LTB4 as therapeutic drugs may inhibit the production of Tfh cells in situ, as well as the recruitment of lymphocytes, in immune-related disorders such as allergies and autoimmune disorders [24]. Similarly, an antagonist of LXA4 may also be used to

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reduce the number of Tfh cells by controlling their activation in immune diseases. A previous study using human lymphocytes from peripheral blood revealed that the LXA4 receptor FPR2 is expressed at higher levels in activated and memory CD4 T+ cells than in resting or naïve CD4 T+ cells [25]. The results of our study using human tonsillar CD4+ T cells showed that FPR2 and BLT1/BLT2 were present at certain mRNA levels in Tfh cells and that protein levels of FPR2 in Tfh cells were higher than those in naïve CD4+ T cells, Th1 cells, Th2 cells and Th17 cells.

Our results suggest that the previously reported higher FPR2

expression in activated CD4+ T cells is specific to Tfh cells. Other studies have shown that activation of cysteinyl leukotriene receptor 1 by LTD4, LTC4, or LTE4 led to the induction of calcium signaling in Th2 cells and that LTD4 can also act as a chemoattractant for Th2 cells [26]. Similarly, LXA4 might regulate the activities of Tfh cells directly, but further investigation is required to address this issue [2, 27]. In summary, we demonstrated a new role of lipid mediators in the differentiation of Tfh cells as assessed by culture of naïve CD4+ T cells under a Tfh polarization condition. It has been reported that patients with systemic lupus erythematosus, rheumatoid arthritis or allergic diseases have an increased proportion of ICOS+CD4+ T cells, suggesting a pathological expansion of Tfh cells [15, 28]. Taken together with the results of a previous study showing that synovial fluid from rheumatoid arthritis patients contains high levels of LXA4 [29], our results indicate that lipid mediators may be closely involved in Tfh-related disorders such as autoimmunity and allergies. The results of this study may contribute to an understanding of immune homeostasis and pathogenesis of autoimmunity effected by Tfh cell function. Conflict of interest The authors declare no conflict of interest.

Acknowledgements This work was supported by grants from JSPS to TN (No. 26861400), to KK (No. 15K20214), RK (No. 15K10787) and SI (No. 26670178).

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Figure legends Figure 1. Leukotriene receptor expression in T cells. (A) Quantitative RT-PCR analysis of leukotriene receptor expression in T cells. Freshly isolated naïve T, Tfh and non-Tfh cells were examined. The data represent relative levels of expression compared with that of total lymphocytes. Each expression level presented was normalized by GAPDH. Bar graphs show means ± SD of results from three independent experiments. *P < 0.05, **P < 0.01,***P < 0.005, NS, not significant (P > 0.05); unpaired Student's t-test. (B) FPR2 expression examined by FACS analysis in five Th subsets. Human tonsillar Tfh cells and naïve T cells were detected as shown in Supplementary Fig. S1, and the other three subsets were detected by intracellular FACS analyses for IFN-γ, IL-4 and IL-17 in cells activated with PMA/ionomycin (upper panels). Histograms show FPR2 expression as a comparison between naïve T cells and other T cell subsets (lower panels). The data are from a single experiment representative of three independent experiments with one tonsil sample per experiment.

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Figure 2. Lipid mediators help differentiation of Tfh cells. (A) Effect of lipid mediators on Tfh polarization. Naïve T cells (1X105) purified from human tonsils were stimulated under a Tfh-polarizing condition in the presence of a series of lipid mediators in 200 μl of medium. After 5 days, the Tfh-cell phenotype was analyzed by FACS. The data shown are representative of three independent experiments. (B) Percentage of in vitro polarized Tfh cells in total CD4+ T cells as examined in (A). (C) Bcl6 expression in in vitro polarized Tfh cells. The cells identified as shown in (A) were also analyzed by intracellular staining for the expression of Bcl6. (D) IL-21 production by polarized Tfh cells. Naïve T cells (1X105) were stimulated and polarized as shown in (A). After 10 days, cells were washed and treated with PMA (50 ng/ml) ionomycin (1 μg/ml) in 200 μl of medium. After 24 h, IL-21 concentrations in culture supernatants were measured by ELISA. (E) Effect of an ALX agonist on Tfh polarization. Tfh cells from human tonsils were stimulated in the presence of the FPR2/ALX agonist BML-111 (1 μg/ml) and antagonist BOC-2 and analyzed as described in (A). (F) Effects of lipid mediators on Th1 and Th2 polarization. Naïve T cells (1X105) purified from human tonsils were stimulated under a Th1-polarizing condition (anti-CD3/CD28 mAbs (4 μg/ml), anti-IL-4 mAb (10 μg/ml) and IL-12 (25 ng/ml)) or a Th2-polarizing condition (anti-CD3/CD28 mAbs (4 μg/ml), anti-IFNγ mAbs (10 μg/ml) and IL-4 (25 ng/ml)) in the presence of LXA4 or LTB4 in 200 μl of medium. After 7 days, cells were subjected to intracellular FACS analysis for detecting IFNγ (Th1 cells) and IL-4 (Th2 cells). Bar graphs (B, D and E) show means ± SD of results from three independent experiments. FACS data (A, C and F) are from a single experiment representative of three independent experiments *P < 0.05, **P < 0.01,***P < 0.005, NS, not significant (P > 0.05); unpaired Student's t-test.

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Figure 3. Cell viability and apoptosis level of Tfh cells in the presence of LXA4 and LTB4. (A) Effect of lipid mediators on the viability of Tfh polarized cells. Naïve T cells (1X105) were stimulated and polarized as shown in Figure 2A. After 5 days, viable cells were counted by the trypan blue dye exclusion assay. (B) Naïve T cells were cultured and stimulated as in (A) and stained with annexin V and 7-amino-actinomycin D (7-AAD) to detect early and late apoptotic cells. The data shown are representative of three independent experiments. (C) Tfh cells isolated from human tonsils were cultured with LXA4 or LTB4 in the presence or absence of a stimulating anti-CD3 mAb. Viable cells were counted by annexin V and 7-AAD assay. Bar graphs (A and C) show means ± SD of results from three independent experiments.

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