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Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice
Journal of Pharmacological Sciences xxx (xxxx) xxx
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Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice Masaya Matsuda a, Kana Doi a, Tatsuya Tsutsumi a, Miki Inaba a, Junpei Hamaguchi a, Tetsuya Terada b, Ryo Kawata b, Kazuyuki Kitatani a, Takeshi Nabe a, * a b
Laboratory of Immunopharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan Department of Otolaryngology, Head and Neck Surgery, Osaka Medical College, Osaka, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2019 Received in revised form 19 October 2019 Accepted 28 October 2019 Available online xxx
Type 1 regulatory T (Tr1) cells are CD4þ T cells that produce a large amount of IL-10, an antiinflammatory cytokine. However, it has not been fully elucidated whether Tr1 cells suppress allergic asthma. In this study, the effects of adoptive transfer of in vitro-induced Tr1 cells on allergic asthma were evaluated. Splenocytes from ovalbumin (OVA)-sensitized BALB/c mice were cultured with OVA, IL-21, IL27, and TGF-b. After culture, IL-10-producing CD4þ T cells were isolated by Dynabeads mouse CD4 and IL10 secretion assay, and analyzed by flow cytometry. Purified Tr1 cells (IL-10þ CD4þ T cells) were intravenously injected into OVA-sensitized BALB/c mice. The recipient mice were intratracheally challenged with OVA. Airway hyperresponsiveness to methacholine was assessed by the forced oscillation technique, followed by bronchoalveolar lavage (BAL). Almost all of the induced IL-10-producing CD4þ T cells were negative for interferon-g, IL-4, IL-17A, and forkhead box P3, suggesting that the cells were Tr1 cells. The adoptive transfer of Tr1 cells significantly suppressed the development of airway hyperresponsiveness, and increases in IL-5, eosinophils, and neutrophils in BAL fluid. In conclusion, we demonstrated that Tr1 cells suppressed allergic asthma in mice. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Asthma Airway hyperresponsiveness IL-10 Immunotherapy T cell
1. Introduction Regulatory T (Treg) cells play a key role in promoting immune tolerance to allergens and preventing allergic disorders.1 Treg cells are divided into two groups, thymus-derived Treg cells and peripherally induced Treg cells.1 Moreover, they can be classified into two subsets based on the expression of forkhead box P3 (Foxp3).2 Foxp3þ Treg cells express the high-affinity T cell receptor against endogenous self-antigens and are involved in autoimmune tolerance.3 On the other hand, type 1 regulatory T (Tr1) cells were identified as CD4þ T cells that do not express Foxp3, but highly produce an anti-inflammatory cytokine, interleukin (IL)-10.2 Tr1 cells, upon activation, secrete high levels of IL-10, variable amounts * Corresponding author. Laboratory of Immunopharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotouge-cho, Hirakata, Osaka 573-0101, Japan. Fax: þ81 72 807 6074. E-mail address: [email protected] (T. Nabe). Peer review under responsibility of Japanese Pharmacological Society.
of IFN-g, and minimal amounts of IL-4 and IL-17A.2 Tr1 cells suppress excessive immune responses by reacting with specific antigens and producing IL-10.4 Based on these characteristics, Tr1 cells are important for peripheral tolerance to exogenous antigens. Allergen immunotherapy, such as subcutaneous immunotherapy (SCIT), using allergen extracts is a causative treatment for allergic diseases.5 Recently, we developed a murine airway allergic model to assess the effects of SCIT.6 Ovalbumin (OVA) þ Al(OH)3sensitized BALB/c mice received subcutaneous OVA at 1 mg/animal for SCIT, and were then intratracheally challenged with OVA at 5 mg/ animal. The number of Tr1 cells was significantly increased in the lungs by SCIT, whereas that of Foxp3þ Treg cells was not increased in this model.6 More recently, we demonstrated that Tr1 cells could be increased by treatment with SCIT in clinical situation: The number of Tr1 cells in peripheral blood mononuclear cells of SCITtreated pollinosis patients was significantly higher than that of patients non-treated with SCIT.7 However, it has not been fully elucidated whether Tr1 cells suppress allergic reactions.
https://doi.org/10.1016/j.jphs.2019.10.004 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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We previously induced Tr1 cells in vitro by stimulating splenic cells isolated from OVA-sensitized mice in the presence of OVA and a mixture of cytokines, IL-21, IL-27, and TGF-b for seven days.8 The cultured CD4þ T cells isolated from cultured splenic cells were activated by OVA stimulation to produce a large amount of IL-10 in vitro and in vivo.8 Adoptive transfer of the cultured CD4þ T cells suppressed allergic airway eosinophilia and airway remodeling.8 However, the cultured CD4þ T cells contained not only Tr1 cells, but also Foxp3þ Treg cells and IL-4-producing cells.8 Therefore, the effects of purified Tr1 cells from cultured CD4þ T cells on allergic airway inflammation should be analyzed. In this study, in order to elucidate whether the induced Tr1 cells suppress allergic asthma, we first analyzed phenotypes of the induced Tr1 cells to develop a method to purify Tr1 cells. Second, we assessed whether adoptive transfer of the purified Tr1 cells can suppress the development of airway hyperresponsiveness, in addition to airway eosinophilia and neutrophilia in vivo. 2. Materials and methods 2.1. Sensitization and challenge of mice For the induction of Tr1 cells, 5-week-old female BALB/c mice (Japan SLC, Hamamatsu, Japan) were sensitized by i.p. injections of OVA (Grade V; SigmaeAldrich, St. Louis, MO, USA) þAl (OH)3 suspension (50 mg OVA absorbed on 2 mg Al(OH)3/animal) on days 0, 14, and 28.6,8e15 The preparation of Al(OH)3 was conducted as previously reported.16 To prepare the recipient mice, the i.p. sensitization of OVA þ Al (OH)3 was conducted on day 0, and then the mice were intratracheally challenged with 500 mg OVA/mouse on days 7, 8, 9, and 12. T cells from BALB/c mouse preferentially produce type 2 cytokines such as IL-4, IL-5 and IL-13.17 Type 2 cytokines play important roles in IgE production, airway eosinophilia and airway remodeling.18 Thus, BALB/c mouse has been regarded as a Th2-dominant mouse strain, and commonly used for allergic airway inflammation model.19e21 This study was approved by the Experimental Animal Research Committees of Setsunan University. 2.2. Culture of spleen cells The method of induction of CD4þ T cells, including Tr1 cells, was previously reported.8 In brief, after the 3rd sensitization, the splenic cells were collected, and then disrupted on a cell strainer in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 50 mM 2mercaptoethanol, 100 mM MEM non-essential amino acids, 100 U/ ml of penicillin, 100 mg/ml of streptomycin, and 10% FBS (all from Thermo Fisher Scientific, Waltham, MA, USA). Collected splenic cells were re-suspended in medium at 3 106 cells/ml, followed by incubation in 5% CO2 at 37 C for 7 days in the presence of OVA and a mixture of cytokines, mouse IL-21 and IL-27 (both 2.5 109 g/ ml, R&D Systems, Minneapolis, MN, USA), and TGF-b (3 109 g/ ml, Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2.3. Isolation of Tr1 cells from cultured splenic cells CD4þ cells in the cultured splenic cells were isolated using Dynabeads mouse CD4 and DETACHaBEAD mouse CD4 (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. The isolated CD4þ cells were stimulated with a mixture of phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin calcium salt (500 ng/ml) (SigmaeAldrich) or PBS at 37 C for 4 h. After incubation, IL-10-producing CD4þ cells were labeled by PE using the mouse IL-10 secretion assay (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the protocol provided by the manufacturer. PE-labeled IL-10-producing CD4þ cells were sorted by FACSAria™ Fusion and FACSDiva™ version 8.0.1 (BD Biosciences, San Diego, CA, USA). 2.4. Analyses of phenotypes of the induced Tr1cells In order to analyze the phenotypes of the induced Tr1 cells, the isolated CD4þ cells (106 cells/ml) from cultured splenic cells were incubated with a mixture of PMA (50 ng/ml) and ionomycin calcium salt (500 ng/ml) at 37 C for 6 h in the presence of a protein transport inhibitor, monensin sodium salt (2 mM, Calbiochem, San Diego, CA, USA). After incubation with anti-Fcg receptor II/III (2.4G2) for Fc blocking, the cells were incubated with FITC anti-CD3 molecular complex (17A2) (BD Biosciences), PerCP/Cy5.5 anti-lymphocyte activation gene 3 (LAG3) (C9B7W), and/or APC anti-CD49b (HMa2) (all from BioLegend, San Diego, CA, USA) for 20 min. After washing, the cells were incubated with Fixation/Permeabilization Concentrate (Thermo Fisher Scientific) for 15 h, and then washed with Permeabilization Buffer (Thermo Fisher Scientific), followed by incubation with PE anti-IL-10 (JES5-16E3), eFluor660® anti-Foxp3 (FJK-16s), PE anti-IFN-g (XMG1.2), PE anti-IL-4 (11B11), PE anti-IL-17A (eBio17B7) (all from Thermo Fisher Scientific), and/ or APC anti-IL-10 (JES5-16E3) (BioLegend) for 40 min. Each sample was analyzed using FACSAria™ Fusion and FACSDiva™ version 8.0.1. 2.5. Adoptive transfer of the induced Tr1 cells In our previous study, OVA-induced eosinophilic infiltration into the lung was suppressed by the CD4þ T cells originating from cultured splenic cells at 3 107 cells/animal.8 As shown in Fig. 1A, approximately 1% of the cultured splenic CD4þ cells were Tr1 cells. Thus, in this study, Tr1 cells (IL-10-producing CD4þ cells) or nonTr1 cells (non-IL-10-producing CD4þ cells) at 2.5 105 cells/animal were injected intravenously into mice 6 days after sensitization with OVA þ Al(OH)3. The recipient mice were challenged intratracheally with OVA on days 7, 8, 9, and 12. At 24 h after the 4th challenge, the recipient mice were anesthetized by i.p injection of pentobarbital (70 mg/kg) and xylazine (12 mg/kg). After measurement of pulmonary function as described below, bronchoalveolar lavage fluid (BALF) was collected from the right lung lobes to analyze airway eosinophilia and neutrophilia, as previously reported.11,12,14,15 The numbers of total cells, eosinophils, neutrophils, and mononuclear cells in the BALF were measured after treatment with ACK lysis buffer. For differential cell counts, cells were transferred onto a glass slide by centrifugation in a cell settling chamber (Neuro Probe, Gaithersburg, MD, USA). Subsequently, cells were stained by Diff-Quik solution (Sysmex International Reagent, Kobe, Japan). 2.6. Quantitative analyses of cytokines in the BALF IL-5 and IL-17A were measured using Ready Set Go! ELISA kits (Thermo Fisher Scientific). Macrophage inflammatory protein-2 (MIP-2) was measured using Quantikine ELISA Kit (R&D Systems). 2.7. Measurement of airway hyperresponsiveness Airway hyperresponsiveness (AHR) to methacholine was measured by the forced oscillation technique using FlexiVent (SCIREQ, Montreal, Canada). At 24 h after the 4th challenge, the recipient mice were anesthetized by i.p injection of pentobarbital (70 mg/kg) and xylazine (12 mg/kg). A 19-gauge blunt needle was
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 1. Analyses of the phenotypes of the induced-IL-10-producing CD4þ cells. (A) IL-10-producing CD4þ cells in purified CD4þ cells. (B) IL-10-producing CD4þ T cells in IL-10producing CD4þ cells. (C, D, E, and F) IFN-g-, IL-4-, IL-17A, and Foxp3- cells in IL-10-producing CD4þ T cells, respectively. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 6 h, followed by FACS analyses. Each value is the mean ± SEM of 3 experiments. FMO: fluorescence minus one.
inserted into the trachea and connected to a mechanical small animal ventilator, FlexiVent. Mice were ventilated with a tidal volume of 10 ml/kg at a rate of 150 breaths/min and a positive endexpiratory pressure of 3 cmH2O by a computerized FlexiVent System. First, “deep inflation” was carried out to open any closed airways twice, followed by methacholine (acetyl-b-methylcholine chloride, SigmaeAldrich) challenges. Methacholine was successively administered using the aeroneb lab nebulizer (SCIREQ) in concentrations ranging from 0 to 50 (0, 6.25, 12.5, 25 and 50) mg/ml for 12 s. After each methacholine challenge, eight repeats of alternating “SnapShot-150” (single frequency forced oscillation waveform) and “Quick Prime-3” (multi-frequency forced oscillation waveform) scans were conducted to measure changes in respiratory function. Resistance of the respiratory system (Rrs) and elastance of the respiratory system (Ers) were acquired by “SnapShot150”. Tissue damping (G) and tissue elastance (H) were assessed by “Quick Prime-3”. Rrs represents the respiratory resistance and reflects total airway resistance. G is also known as tissue resistance, and reflects viscous dissipation of energy in the respiratory tissues and airflow heterogeneity. Ers is the respiratory elastance and reflects total airway elastance. H is also known as elastance and reflects the elastic energy stored in the tissues. Rrs, G, Ers, and H are shown as maximal values after each methacholine challenge.
performed using JMP® Pro (Version 11.2.0, SAS Institute Japan, Tokyo, Japan). 3. Results 3.1. Phenotypes of the in vitro-induced IL-10-producing CD4þ T cells In our previous study, cytokine profiles of the induced IL-10producing CD4þ T cells were analyzed by stimulating cultured splenic cells with PMA þ ionomycin.8 In this study, the phenotypes of the induced Tr1 cells were further analyzed by stimulating cultured splenic CD4þ T cells with PMA þ ionomycin. As shown in Fig. 1A, approximately 1% of the cultured splenic CD4þ cells were positive for IL-10. Almost all of the IL-10-producing CD4þ cells were positive for CD3 (Fig. 1B). Moreover, more than 93% of the IL-10-producing CD4þ T cells were negative for IFN-g, IL-4, IL17A, and Foxp3, demonstrating that almost all of the IL-10producing CD4þ T cells were Tr1 cells (Fig. 1CeF). This suggested that Tr1 cells can be purified by sorting IL-10-producing CD4þ cells from cultured splenic CD4þ cells. Thus, IL-10-producing CD4þ cells (Tr1 cells) were labeled by the IL-10 secretion assay and separated from non-IL-10-producing CD4þ cells (non-Tr1 cells), as shown in Fig. 2. After sorting, the purity of Tr1 cells was 94.3 ± 2.7% (n ¼ 3).
2.8. Statistical analyses Two groups were compared using the unpaired t-test. To compare the four groups, one-way ANOVA was performed, followed by Dunnett's multiple comparison test. A P-value less than 0.05 was considered significant. Statistical analyses were
3.2. Effects of adoptive transfer of Tr1 cells on antigen-induced airway eosinophilia and neutrophilia In comparison with the non-challenged mice, the total number of leukocytes was significantly higher in the BALF of sensitized-
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 2. Sorting of Tr1 cells and non-Tr1 cells. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, the IL-10-producing CD4þ cells and non-IL-10-producing CD4þ cells were sorted by FACS. The IL-10-producing CD4þ cells and non-IL-10producing CD4þ cells were identified as Tr1 cells and non-Tr1 cells, respectively. The purity of IL-10-producing CD4þ cells is described as the mean ± SEM of 3 experiments.
challenged mice (Fig. 3A). Approximately 50% and 6% of the infiltrated leukocytes consisted of eosinophils and neutrophils, respectively (Fig. 3AeC). The infiltration of eosinophils and neutrophils was significantly suppressed by the adoptive transfer of Tr1 cells (Fig. 3B, C). On the other hand, the infiltration of neutrophils was slightly suppressed by the adoptive transfer of non-Tr1 cells, but the effects were not significant (Fig. 3C). As for mononuclear cells, there was no difference between respective groups (Fig. 3D).
3.3. Effects of adoptive transfer of Tr1cells on antigen-induced cytokine production In comparison with the non-challenged group, the level of IL-5, an eosinophil chemotactic factor, was significantly increased in BALF after multiple antigen challenges (Fig. 4A). Antigen-induced IL-5 production was not significantly affected by the adoptive transfer of non-Tr1 cells (Fig. 4A), whereas the
Fig. 3. Effects of the adoptive transfer of Tr1 cells on multiple antigen challenge-induced increases in (A) total cells, (B) eosinophils, (C) neutrophils, and (D) mononuclear cells in the BALF in recipient sensitized mice. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, Tr1 cells and non-Tr1 cells were sorted by FACS. Tr1 cells or non-Tr1 cells were adoptively transferred before the 1st challenge, followed by four OVA challenges. BALF was obtained at 24 h after the 4th challenge. Each column represents the mean ± SEM of 5e15 animals. ** and ***: P < 0.01 and 0.001, respectively. y: P < 0.05. z and zz: P < 0.05 and 0.01, respectively.
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 4. Effects of the adoptive transfer of Tr1 cells on antigen-induced production of (A) IL-5, (B) IL-17A, and (C) MIP-2 in BALF in recipient sensitized mice. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, Tr1 cells and non-Tr1 cells were sorted by FACS. Tr1 cells or non-Tr1 cells were adoptively transferred before the 1st challenge, followed by four OVA challenges. BALF was obtained at 24 h after the 4th challenge. Each column represents the mean ± SEM of 5e15 animals. ***: P < 0.001. yy: P < 0.01. z: P < 0.05.
adoptive transfer of Tr1cells significantly suppressed IL-5 production (Fig. 4A). IL-17A is a neutrophil chemotactic factor.22 Only a minimal level of IL-17A was detected in BALF after multiple OVA challenges (Fig. 4B). A significant difference in IL-17A production was not observed (Fig. 4B). Neutrophilic chemotaxis is induced by not only IL-17A but also macrophage inflammatory protein (MIP)-2, a human IL-8 homolog.23 As shown in Fig. 4C, the amount of MIP-2 in BALF was significantly increased by the multiple OVA challenges. However, MIP-2 production was not suppressed by the adoptive transfer of Tr1 cells (Fig. 4C). 3.4. Effects of adoptive transfer of Tr1 cells on AHR to methacholine In order to clarify the effects of the adoptive transfer of Tr1 cells on respiratory function, AHR to methacholine was measured by the forced oscillation technique using FlexiVent. AHR to methacholine was developed in OVA-challenged mice, when resistance of the respiratory system (Rrs), tissue damping (G), elastance of the respiratory system (Ers), and tissue elastance (H) were assessed as parameters of airway function (Fig. 5AeD). The adoptive transfer of Tr1 cells significantly suppressed the development of AHR (Fig. 5AeD). On the other hand, no significant effect was observed after the adoptive transfer of non-Tr1 cells (Fig. 5AeD). 4. Discussion We previously reported that Tr1 cells can be induced in vitro by stimulating splenic cells isolated from OVA-sensitized mice in the presence of OVA with a mixture of cytokines, IL-21, IL-27, and TGF-b for 7 days.8 The adoptive transfer of cultured splenic CD4þ T cells, including Tr1 cells, significantly suppressed eosinophilic infiltration into BALF.8 However, the cultured splenic CD4þ T cells contained not only Tr1 cells but also Foxp3þ Treg cells and IL-4-producing
cells. Thus, whether the induced Tr1 cells can suppresses allergic asthma has not been fully elucidated. In this study, we isolated Tr1 cells from cultured CD4þ cells and analyzed their suppressive functions. Specific biomarkers for Tr1 cells have not been identified, limiting experimental and clinical studies. In recent years, Gagliani et al24 reported that CD49b and lymphocyte activation gene 3 (LAG3) are expressed on mouse and human Tr1 cells. On the other hand, Huang et al25 found that the co-expression of CD49b and LAG3 is not restricted to Tr1 cells, and is also observed in Foxp3þ Treg cells and CD8þ T cells that produce IL-10 using an IL-10GFP/ Foxp3RFP dual reporter transgenic murine model. In this study, only 20% of the induced IL-10-producing CD4þ T cells were doublepositive for CD49b and LAG3 (data not shown). Therefore, CD49b and LAG3 were not suitable to mark the in vitro-induced Tr1 cells in this study. As shown in Fig. 1, most of the IL-10-producing CD4þ cells were T cells, and negative for IFN-g, IL-4, IL-17A, and Foxp3, suggesting that the cells were Tr1 cells. Therefore, IL-10-producing CD4þ cells were considered Tr1 cells in this study. In this study, the adoptive transfer of Tr1 cells significantly suppressed the development of AHR, and increases in IL-5 and eosinophils in BAL fluid. IL-5 is produced by Th2 cells and type 2 innate lymphoid cells (ILC2), and plays a role in the infiltration of eosinophils into the lung.26 In our previous study, when cultured CD4þ T cells containing Tr1 cells were transferred into asthmatic mice, and then the recipient mice were challenged with OVA, IL-10 production was markedly augmented in vivo.8 IL-5 production from mouse Th2 cells and ILC2 was reported to be significantly inhibited by the addition of IL-10 in vitro.27,28 Therefore, the adoptive transfer of Tr1 cells may have suppressed AHR by regulating Th2 cells and ILC2 via IL-10 production. It has been recently suggested that Tr1 cells suppress immunity through CD39 on the cell surface in mice and humans.29 Mascanfroni et al30 reported that the deficiency of CD39 reduced the
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 5. Effects of the adoptive transfer of Tr1 cells on antigen-induced airway hyperresponsiveness (AHR) in recipient sensitized mice. Rrs (resistance of the respiratory system, A), G (tissue damping, B), Ers (elastance of the respiratory system, C), and H (tissue elastance, D) are shown as the maximum values after each methacholine challenge. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, Tr1 cells and non-Tr1 cells were sorted by FACS. Tr1 cells or non-Tr1 cells were adoptively transferred before the 1st challenge, followed by four OVA challenges. Airway hyperresponsiveness to methacholine was analyzed at 24 h after the 4th challenge. Each dot represents the mean ± SEM of 5e15 animals. *, ** and ***: P < 0.05, 0.01 and 0.001, respectively. y: P < 0.05 versus challenged mice. z: P < 0.05 versus challenged mice administered non-Tr1 cells.
suppressive activity of mouse Tr1 cells in vitro. Inflammatory immune reactions are accompanied by the release of large amounts of adenosine triphosphate (ATP) in the extracellular space where it is converted into adenosine monophosphate (AMP) by CD39 expressed on Tr1 cells, and then dephosphorylated to adenosine by CD73 expressed on effector T cells.31 Recently, it was reported that adenosine exhibits strong anti-inflammatory function on ILC2, preferentially by activating adenosine A2B receptors on the cell membrane, leading to a decrease in the release of IL-5 and IL-13 from activated ILC2.32 Taken together, the suppressive effects of the adoptive transfer of Tr1 cells on the increase in IL-5, airway eosinophilia, and development of AHR may be associated with CD39-mediated anti-inflammatory function. In our previous study, neutrophilic infiltration into the lungs was not suppressed by the adoptive transfer of the cultured CD4þ T cells.8 The CD4þ T cells contained not only Tr1 cells but also inflammatory cells such as IFN-g-producing cells and IL-4-producing cells.8 These inflammatory cells in the adoptively transferred CD4þ T cells may have limited the regulatory function of Tr1cells. In this study, the infiltration of neutrophils into the lungs was significantly suppressed by the adoptive transfer of purified Tr1 cells. However, the production of MIP-2, a neutrophil chemotactic factor, was not suppressed by the adoptive transfer of Tr1 cells, suggesting that regulatory action of Tr1 cells on the neutrophilic infiltration was MIP-2 independent. On the other hand, neutrophil extravasation from blood vessels to the inflamed tissue requires several paired molecular interactions between neutrophils and vascular endothelial cells such as integrins of neutrophils and endothelial intercellular adhesion molecule-1 (ICAM-1).33 IL-10 was reported to significantly inhibit the increase in TNF-a-induced ICAM-1 expression on human umbilical vein endothelial cells. Moreover,
blocking antibodies against ICAM-1 inhibited the migration of neutrophils in PMA-induced rabbit lung inflammation.34 Therefore, Tr1 cells may have downregulated the expression of ICAM-1 in pulmonary vascular endothelial cells via IL-10 production, followed by the suppression of neutrophilic migration into the lungs. Tr1 cells used in this study contained a small population of Foxp3þ Treg cells, suggesting that contained Foxp3þ Treg cells may also be partially related to the suppression of airway inflammation. However, because 93.5% of IL-10-producing cells transferred were Foxp3-, IL-10þ Foxp3- CD4þ T cells could be more likely to contribute to the suppression of allergic airway inflammation than IL-10þ Foxp3þ CD4þ T cells. In conclusion, we established a method to purify Tr1 cells by sorting IL-10-producing cells from CD4þ T cells originating from splenic cells cultured in the presence of OVA, and a mixture of IL-21, IL-27, and TGF-b. The adoptive transfer of Tr1 cells significantly suppressed airway eosinophilia, neutrophilia, and allergic AHR. The Tr1 cell transfer strategy is expected to be a new therapy for allergic diseases.
Declaration of Competing Interest The authors have no conflicts of interest.
Acknowledgement This study was supported by a research support project of Setsunan University and JSPS KAKENHI Grant Number 19K16385 (to M.M.).
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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19. Watanabe H, Numata K, Ito T, Takagi K, Matsukawa A. Innate immune response in Th1- and Th2-dominant mouse strains. Shock. 2004;22(5):460e466. €hm L, Maxeiner J, Meyer-Martin H, et al. IL-10 and regulatory T cells coop20. Bo erate in allergen-specific immunotherapy to ameliorate allergic asthma. J Immunol. 2015;194(3):887e897. 21. Hesse L, Brouwer U, Petersen AH, et al. Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma. Clin Exp Allergy. 2018;48(8):1035e1049. 22. Ray A, Kolls JK. Neutrophilic inflammation in asthma and association with disease severity. Trends Immunol. 2017;38(12):942e954. 23. Qin C-C, Liu Y-N, Hu Y, Yang Y, Chen Z. Macrophage inflammatory protein-2 as mediator of inflammation in acute liver injury. World J Gastroenterol. 2017;23(17):3043e3052. 24. Gagliani N, Magnani CF, Huber S, et al. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med. 2013;19(6): 739e746. 25. Huang W, Solouki S, Carter C, Zheng SG, August A. Beyond type 1 regulatory t cells: Co-expression of LAG3 and CD49b in IL-10-producing t cell lineages. Front Immunol. 2018;9:1e11. 26. Larose M, Archambault A, Provost V, Laviolette M, Flamand N. Regulation of eosinophil and group 2 innate lymphoid cell trafficking in asthma. Front Med. 2017;4:1e12. 27. Coomes SM, Kannan Y, Pelly VS, et al. CD4þ Th2 cells are directly regulated by IL-10 during allergic airway inflammation. Mucosal Immunol. 2017;10(1): 150e161. 28. Morita H, Arae K, Unno H, et al. An Interleukin-33-mast cell-interleukin-2 axis suppresses papain-induced allergic inflammation by promoting regulatory T cell numbers. Immunity. 2015;43(1):175e186. 29. Roncarolo MG, Gregori S, Bacchetta R, Battaglia M, Gagliani N. The biology of T regulatory type 1 cells and their therapeutic application in immune-mediated diseases. Immunity. 2018;49(6):1004e1019. 30. Mascanfroni ID, Takenaka MC, Yeste A, et al. Metabolic control of type 1 regulatory T cell differentiation by AHR and HIF1-a. Nat Med. 2015;21(6): 638e646. G. CD39 and CD73 in immunity and 31. Antonioli L, Pacher P, Vizi ES, Hasko inflammation. Trends Mol Med. 2013;19(6):355e367. 32. Csoka B, Nemeth ZH, Duerr CU, Fritz JH, Pacher P, Hasko G. Adenosine receptors differentially regulate type 2 cytokine production by IL-33-activated bone marrow cells, ILC2s, and macrophages. FASEB J. 2018;32(2):829e837. 33. Liu J, Hou M, Yan M, et al. ICAM-1-dependent and ICAM-1-independent neutrophil lung infiltration by porcine reproductive and respiratory syndrome virus infection. Am J Physiol Lung Cell Mol Physiol. 2015;309(3): L226eL236. 34. Barton RW, Rothlein R, Ksiazek J, Kennedy C. The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation. J Immunol. 1989;143(4):1278e1282.
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice Masaya Matsuda a, Kana Doi a, Tatsuya Tsutsumi a, Miki Inaba a, Junpei Hamaguchi a, Tetsuya Terada b, Ryo Kawata b, Kazuyuki Kitatani a, Takeshi Nabe a, * a b
Laboratory of Immunopharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan Department of Otolaryngology, Head and Neck Surgery, Osaka Medical College, Osaka, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2019 Received in revised form 19 October 2019 Accepted 28 October 2019 Available online xxx
Type 1 regulatory T (Tr1) cells are CD4þ T cells that produce a large amount of IL-10, an antiinflammatory cytokine. However, it has not been fully elucidated whether Tr1 cells suppress allergic asthma. In this study, the effects of adoptive transfer of in vitro-induced Tr1 cells on allergic asthma were evaluated. Splenocytes from ovalbumin (OVA)-sensitized BALB/c mice were cultured with OVA, IL-21, IL27, and TGF-b. After culture, IL-10-producing CD4þ T cells were isolated by Dynabeads mouse CD4 and IL10 secretion assay, and analyzed by flow cytometry. Purified Tr1 cells (IL-10þ CD4þ T cells) were intravenously injected into OVA-sensitized BALB/c mice. The recipient mice were intratracheally challenged with OVA. Airway hyperresponsiveness to methacholine was assessed by the forced oscillation technique, followed by bronchoalveolar lavage (BAL). Almost all of the induced IL-10-producing CD4þ T cells were negative for interferon-g, IL-4, IL-17A, and forkhead box P3, suggesting that the cells were Tr1 cells. The adoptive transfer of Tr1 cells significantly suppressed the development of airway hyperresponsiveness, and increases in IL-5, eosinophils, and neutrophils in BAL fluid. In conclusion, we demonstrated that Tr1 cells suppressed allergic asthma in mice. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Asthma Airway hyperresponsiveness IL-10 Immunotherapy T cell
1. Introduction Regulatory T (Treg) cells play a key role in promoting immune tolerance to allergens and preventing allergic disorders.1 Treg cells are divided into two groups, thymus-derived Treg cells and peripherally induced Treg cells.1 Moreover, they can be classified into two subsets based on the expression of forkhead box P3 (Foxp3).2 Foxp3þ Treg cells express the high-affinity T cell receptor against endogenous self-antigens and are involved in autoimmune tolerance.3 On the other hand, type 1 regulatory T (Tr1) cells were identified as CD4þ T cells that do not express Foxp3, but highly produce an anti-inflammatory cytokine, interleukin (IL)-10.2 Tr1 cells, upon activation, secrete high levels of IL-10, variable amounts * Corresponding author. Laboratory of Immunopharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotouge-cho, Hirakata, Osaka 573-0101, Japan. Fax: þ81 72 807 6074. E-mail address: [email protected] (T. Nabe). Peer review under responsibility of Japanese Pharmacological Society.
of IFN-g, and minimal amounts of IL-4 and IL-17A.2 Tr1 cells suppress excessive immune responses by reacting with specific antigens and producing IL-10.4 Based on these characteristics, Tr1 cells are important for peripheral tolerance to exogenous antigens. Allergen immunotherapy, such as subcutaneous immunotherapy (SCIT), using allergen extracts is a causative treatment for allergic diseases.5 Recently, we developed a murine airway allergic model to assess the effects of SCIT.6 Ovalbumin (OVA) þ Al(OH)3sensitized BALB/c mice received subcutaneous OVA at 1 mg/animal for SCIT, and were then intratracheally challenged with OVA at 5 mg/ animal. The number of Tr1 cells was significantly increased in the lungs by SCIT, whereas that of Foxp3þ Treg cells was not increased in this model.6 More recently, we demonstrated that Tr1 cells could be increased by treatment with SCIT in clinical situation: The number of Tr1 cells in peripheral blood mononuclear cells of SCITtreated pollinosis patients was significantly higher than that of patients non-treated with SCIT.7 However, it has not been fully elucidated whether Tr1 cells suppress allergic reactions.
https://doi.org/10.1016/j.jphs.2019.10.004 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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We previously induced Tr1 cells in vitro by stimulating splenic cells isolated from OVA-sensitized mice in the presence of OVA and a mixture of cytokines, IL-21, IL-27, and TGF-b for seven days.8 The cultured CD4þ T cells isolated from cultured splenic cells were activated by OVA stimulation to produce a large amount of IL-10 in vitro and in vivo.8 Adoptive transfer of the cultured CD4þ T cells suppressed allergic airway eosinophilia and airway remodeling.8 However, the cultured CD4þ T cells contained not only Tr1 cells, but also Foxp3þ Treg cells and IL-4-producing cells.8 Therefore, the effects of purified Tr1 cells from cultured CD4þ T cells on allergic airway inflammation should be analyzed. In this study, in order to elucidate whether the induced Tr1 cells suppress allergic asthma, we first analyzed phenotypes of the induced Tr1 cells to develop a method to purify Tr1 cells. Second, we assessed whether adoptive transfer of the purified Tr1 cells can suppress the development of airway hyperresponsiveness, in addition to airway eosinophilia and neutrophilia in vivo. 2. Materials and methods 2.1. Sensitization and challenge of mice For the induction of Tr1 cells, 5-week-old female BALB/c mice (Japan SLC, Hamamatsu, Japan) were sensitized by i.p. injections of OVA (Grade V; SigmaeAldrich, St. Louis, MO, USA) þAl (OH)3 suspension (50 mg OVA absorbed on 2 mg Al(OH)3/animal) on days 0, 14, and 28.6,8e15 The preparation of Al(OH)3 was conducted as previously reported.16 To prepare the recipient mice, the i.p. sensitization of OVA þ Al (OH)3 was conducted on day 0, and then the mice were intratracheally challenged with 500 mg OVA/mouse on days 7, 8, 9, and 12. T cells from BALB/c mouse preferentially produce type 2 cytokines such as IL-4, IL-5 and IL-13.17 Type 2 cytokines play important roles in IgE production, airway eosinophilia and airway remodeling.18 Thus, BALB/c mouse has been regarded as a Th2-dominant mouse strain, and commonly used for allergic airway inflammation model.19e21 This study was approved by the Experimental Animal Research Committees of Setsunan University. 2.2. Culture of spleen cells The method of induction of CD4þ T cells, including Tr1 cells, was previously reported.8 In brief, after the 3rd sensitization, the splenic cells were collected, and then disrupted on a cell strainer in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 50 mM 2mercaptoethanol, 100 mM MEM non-essential amino acids, 100 U/ ml of penicillin, 100 mg/ml of streptomycin, and 10% FBS (all from Thermo Fisher Scientific, Waltham, MA, USA). Collected splenic cells were re-suspended in medium at 3 106 cells/ml, followed by incubation in 5% CO2 at 37 C for 7 days in the presence of OVA and a mixture of cytokines, mouse IL-21 and IL-27 (both 2.5 109 g/ ml, R&D Systems, Minneapolis, MN, USA), and TGF-b (3 109 g/ ml, Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2.3. Isolation of Tr1 cells from cultured splenic cells CD4þ cells in the cultured splenic cells were isolated using Dynabeads mouse CD4 and DETACHaBEAD mouse CD4 (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. The isolated CD4þ cells were stimulated with a mixture of phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin calcium salt (500 ng/ml) (SigmaeAldrich) or PBS at 37 C for 4 h. After incubation, IL-10-producing CD4þ cells were labeled by PE using the mouse IL-10 secretion assay (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the protocol provided by the manufacturer. PE-labeled IL-10-producing CD4þ cells were sorted by FACSAria™ Fusion and FACSDiva™ version 8.0.1 (BD Biosciences, San Diego, CA, USA). 2.4. Analyses of phenotypes of the induced Tr1cells In order to analyze the phenotypes of the induced Tr1 cells, the isolated CD4þ cells (106 cells/ml) from cultured splenic cells were incubated with a mixture of PMA (50 ng/ml) and ionomycin calcium salt (500 ng/ml) at 37 C for 6 h in the presence of a protein transport inhibitor, monensin sodium salt (2 mM, Calbiochem, San Diego, CA, USA). After incubation with anti-Fcg receptor II/III (2.4G2) for Fc blocking, the cells were incubated with FITC anti-CD3 molecular complex (17A2) (BD Biosciences), PerCP/Cy5.5 anti-lymphocyte activation gene 3 (LAG3) (C9B7W), and/or APC anti-CD49b (HMa2) (all from BioLegend, San Diego, CA, USA) for 20 min. After washing, the cells were incubated with Fixation/Permeabilization Concentrate (Thermo Fisher Scientific) for 15 h, and then washed with Permeabilization Buffer (Thermo Fisher Scientific), followed by incubation with PE anti-IL-10 (JES5-16E3), eFluor660® anti-Foxp3 (FJK-16s), PE anti-IFN-g (XMG1.2), PE anti-IL-4 (11B11), PE anti-IL-17A (eBio17B7) (all from Thermo Fisher Scientific), and/ or APC anti-IL-10 (JES5-16E3) (BioLegend) for 40 min. Each sample was analyzed using FACSAria™ Fusion and FACSDiva™ version 8.0.1. 2.5. Adoptive transfer of the induced Tr1 cells In our previous study, OVA-induced eosinophilic infiltration into the lung was suppressed by the CD4þ T cells originating from cultured splenic cells at 3 107 cells/animal.8 As shown in Fig. 1A, approximately 1% of the cultured splenic CD4þ cells were Tr1 cells. Thus, in this study, Tr1 cells (IL-10-producing CD4þ cells) or nonTr1 cells (non-IL-10-producing CD4þ cells) at 2.5 105 cells/animal were injected intravenously into mice 6 days after sensitization with OVA þ Al(OH)3. The recipient mice were challenged intratracheally with OVA on days 7, 8, 9, and 12. At 24 h after the 4th challenge, the recipient mice were anesthetized by i.p injection of pentobarbital (70 mg/kg) and xylazine (12 mg/kg). After measurement of pulmonary function as described below, bronchoalveolar lavage fluid (BALF) was collected from the right lung lobes to analyze airway eosinophilia and neutrophilia, as previously reported.11,12,14,15 The numbers of total cells, eosinophils, neutrophils, and mononuclear cells in the BALF were measured after treatment with ACK lysis buffer. For differential cell counts, cells were transferred onto a glass slide by centrifugation in a cell settling chamber (Neuro Probe, Gaithersburg, MD, USA). Subsequently, cells were stained by Diff-Quik solution (Sysmex International Reagent, Kobe, Japan). 2.6. Quantitative analyses of cytokines in the BALF IL-5 and IL-17A were measured using Ready Set Go! ELISA kits (Thermo Fisher Scientific). Macrophage inflammatory protein-2 (MIP-2) was measured using Quantikine ELISA Kit (R&D Systems). 2.7. Measurement of airway hyperresponsiveness Airway hyperresponsiveness (AHR) to methacholine was measured by the forced oscillation technique using FlexiVent (SCIREQ, Montreal, Canada). At 24 h after the 4th challenge, the recipient mice were anesthetized by i.p injection of pentobarbital (70 mg/kg) and xylazine (12 mg/kg). A 19-gauge blunt needle was
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 1. Analyses of the phenotypes of the induced-IL-10-producing CD4þ cells. (A) IL-10-producing CD4þ cells in purified CD4þ cells. (B) IL-10-producing CD4þ T cells in IL-10producing CD4þ cells. (C, D, E, and F) IFN-g-, IL-4-, IL-17A, and Foxp3- cells in IL-10-producing CD4þ T cells, respectively. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 6 h, followed by FACS analyses. Each value is the mean ± SEM of 3 experiments. FMO: fluorescence minus one.
inserted into the trachea and connected to a mechanical small animal ventilator, FlexiVent. Mice were ventilated with a tidal volume of 10 ml/kg at a rate of 150 breaths/min and a positive endexpiratory pressure of 3 cmH2O by a computerized FlexiVent System. First, “deep inflation” was carried out to open any closed airways twice, followed by methacholine (acetyl-b-methylcholine chloride, SigmaeAldrich) challenges. Methacholine was successively administered using the aeroneb lab nebulizer (SCIREQ) in concentrations ranging from 0 to 50 (0, 6.25, 12.5, 25 and 50) mg/ml for 12 s. After each methacholine challenge, eight repeats of alternating “SnapShot-150” (single frequency forced oscillation waveform) and “Quick Prime-3” (multi-frequency forced oscillation waveform) scans were conducted to measure changes in respiratory function. Resistance of the respiratory system (Rrs) and elastance of the respiratory system (Ers) were acquired by “SnapShot150”. Tissue damping (G) and tissue elastance (H) were assessed by “Quick Prime-3”. Rrs represents the respiratory resistance and reflects total airway resistance. G is also known as tissue resistance, and reflects viscous dissipation of energy in the respiratory tissues and airflow heterogeneity. Ers is the respiratory elastance and reflects total airway elastance. H is also known as elastance and reflects the elastic energy stored in the tissues. Rrs, G, Ers, and H are shown as maximal values after each methacholine challenge.
performed using JMP® Pro (Version 11.2.0, SAS Institute Japan, Tokyo, Japan). 3. Results 3.1. Phenotypes of the in vitro-induced IL-10-producing CD4þ T cells In our previous study, cytokine profiles of the induced IL-10producing CD4þ T cells were analyzed by stimulating cultured splenic cells with PMA þ ionomycin.8 In this study, the phenotypes of the induced Tr1 cells were further analyzed by stimulating cultured splenic CD4þ T cells with PMA þ ionomycin. As shown in Fig. 1A, approximately 1% of the cultured splenic CD4þ cells were positive for IL-10. Almost all of the IL-10-producing CD4þ cells were positive for CD3 (Fig. 1B). Moreover, more than 93% of the IL-10-producing CD4þ T cells were negative for IFN-g, IL-4, IL17A, and Foxp3, demonstrating that almost all of the IL-10producing CD4þ T cells were Tr1 cells (Fig. 1CeF). This suggested that Tr1 cells can be purified by sorting IL-10-producing CD4þ cells from cultured splenic CD4þ cells. Thus, IL-10-producing CD4þ cells (Tr1 cells) were labeled by the IL-10 secretion assay and separated from non-IL-10-producing CD4þ cells (non-Tr1 cells), as shown in Fig. 2. After sorting, the purity of Tr1 cells was 94.3 ± 2.7% (n ¼ 3).
2.8. Statistical analyses Two groups were compared using the unpaired t-test. To compare the four groups, one-way ANOVA was performed, followed by Dunnett's multiple comparison test. A P-value less than 0.05 was considered significant. Statistical analyses were
3.2. Effects of adoptive transfer of Tr1 cells on antigen-induced airway eosinophilia and neutrophilia In comparison with the non-challenged mice, the total number of leukocytes was significantly higher in the BALF of sensitized-
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 2. Sorting of Tr1 cells and non-Tr1 cells. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, the IL-10-producing CD4þ cells and non-IL-10-producing CD4þ cells were sorted by FACS. The IL-10-producing CD4þ cells and non-IL-10producing CD4þ cells were identified as Tr1 cells and non-Tr1 cells, respectively. The purity of IL-10-producing CD4þ cells is described as the mean ± SEM of 3 experiments.
challenged mice (Fig. 3A). Approximately 50% and 6% of the infiltrated leukocytes consisted of eosinophils and neutrophils, respectively (Fig. 3AeC). The infiltration of eosinophils and neutrophils was significantly suppressed by the adoptive transfer of Tr1 cells (Fig. 3B, C). On the other hand, the infiltration of neutrophils was slightly suppressed by the adoptive transfer of non-Tr1 cells, but the effects were not significant (Fig. 3C). As for mononuclear cells, there was no difference between respective groups (Fig. 3D).
3.3. Effects of adoptive transfer of Tr1cells on antigen-induced cytokine production In comparison with the non-challenged group, the level of IL-5, an eosinophil chemotactic factor, was significantly increased in BALF after multiple antigen challenges (Fig. 4A). Antigen-induced IL-5 production was not significantly affected by the adoptive transfer of non-Tr1 cells (Fig. 4A), whereas the
Fig. 3. Effects of the adoptive transfer of Tr1 cells on multiple antigen challenge-induced increases in (A) total cells, (B) eosinophils, (C) neutrophils, and (D) mononuclear cells in the BALF in recipient sensitized mice. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, Tr1 cells and non-Tr1 cells were sorted by FACS. Tr1 cells or non-Tr1 cells were adoptively transferred before the 1st challenge, followed by four OVA challenges. BALF was obtained at 24 h after the 4th challenge. Each column represents the mean ± SEM of 5e15 animals. ** and ***: P < 0.01 and 0.001, respectively. y: P < 0.05. z and zz: P < 0.05 and 0.01, respectively.
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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Fig. 4. Effects of the adoptive transfer of Tr1 cells on antigen-induced production of (A) IL-5, (B) IL-17A, and (C) MIP-2 in BALF in recipient sensitized mice. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, Tr1 cells and non-Tr1 cells were sorted by FACS. Tr1 cells or non-Tr1 cells were adoptively transferred before the 1st challenge, followed by four OVA challenges. BALF was obtained at 24 h after the 4th challenge. Each column represents the mean ± SEM of 5e15 animals. ***: P < 0.001. yy: P < 0.01. z: P < 0.05.
adoptive transfer of Tr1cells significantly suppressed IL-5 production (Fig. 4A). IL-17A is a neutrophil chemotactic factor.22 Only a minimal level of IL-17A was detected in BALF after multiple OVA challenges (Fig. 4B). A significant difference in IL-17A production was not observed (Fig. 4B). Neutrophilic chemotaxis is induced by not only IL-17A but also macrophage inflammatory protein (MIP)-2, a human IL-8 homolog.23 As shown in Fig. 4C, the amount of MIP-2 in BALF was significantly increased by the multiple OVA challenges. However, MIP-2 production was not suppressed by the adoptive transfer of Tr1 cells (Fig. 4C). 3.4. Effects of adoptive transfer of Tr1 cells on AHR to methacholine In order to clarify the effects of the adoptive transfer of Tr1 cells on respiratory function, AHR to methacholine was measured by the forced oscillation technique using FlexiVent. AHR to methacholine was developed in OVA-challenged mice, when resistance of the respiratory system (Rrs), tissue damping (G), elastance of the respiratory system (Ers), and tissue elastance (H) were assessed as parameters of airway function (Fig. 5AeD). The adoptive transfer of Tr1 cells significantly suppressed the development of AHR (Fig. 5AeD). On the other hand, no significant effect was observed after the adoptive transfer of non-Tr1 cells (Fig. 5AeD). 4. Discussion We previously reported that Tr1 cells can be induced in vitro by stimulating splenic cells isolated from OVA-sensitized mice in the presence of OVA with a mixture of cytokines, IL-21, IL-27, and TGF-b for 7 days.8 The adoptive transfer of cultured splenic CD4þ T cells, including Tr1 cells, significantly suppressed eosinophilic infiltration into BALF.8 However, the cultured splenic CD4þ T cells contained not only Tr1 cells but also Foxp3þ Treg cells and IL-4-producing
cells. Thus, whether the induced Tr1 cells can suppresses allergic asthma has not been fully elucidated. In this study, we isolated Tr1 cells from cultured CD4þ cells and analyzed their suppressive functions. Specific biomarkers for Tr1 cells have not been identified, limiting experimental and clinical studies. In recent years, Gagliani et al24 reported that CD49b and lymphocyte activation gene 3 (LAG3) are expressed on mouse and human Tr1 cells. On the other hand, Huang et al25 found that the co-expression of CD49b and LAG3 is not restricted to Tr1 cells, and is also observed in Foxp3þ Treg cells and CD8þ T cells that produce IL-10 using an IL-10GFP/ Foxp3RFP dual reporter transgenic murine model. In this study, only 20% of the induced IL-10-producing CD4þ T cells were doublepositive for CD49b and LAG3 (data not shown). Therefore, CD49b and LAG3 were not suitable to mark the in vitro-induced Tr1 cells in this study. As shown in Fig. 1, most of the IL-10-producing CD4þ cells were T cells, and negative for IFN-g, IL-4, IL-17A, and Foxp3, suggesting that the cells were Tr1 cells. Therefore, IL-10-producing CD4þ cells were considered Tr1 cells in this study. In this study, the adoptive transfer of Tr1 cells significantly suppressed the development of AHR, and increases in IL-5 and eosinophils in BAL fluid. IL-5 is produced by Th2 cells and type 2 innate lymphoid cells (ILC2), and plays a role in the infiltration of eosinophils into the lung.26 In our previous study, when cultured CD4þ T cells containing Tr1 cells were transferred into asthmatic mice, and then the recipient mice were challenged with OVA, IL-10 production was markedly augmented in vivo.8 IL-5 production from mouse Th2 cells and ILC2 was reported to be significantly inhibited by the addition of IL-10 in vitro.27,28 Therefore, the adoptive transfer of Tr1 cells may have suppressed AHR by regulating Th2 cells and ILC2 via IL-10 production. It has been recently suggested that Tr1 cells suppress immunity through CD39 on the cell surface in mice and humans.29 Mascanfroni et al30 reported that the deficiency of CD39 reduced the
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
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M. Matsuda et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
Fig. 5. Effects of the adoptive transfer of Tr1 cells on antigen-induced airway hyperresponsiveness (AHR) in recipient sensitized mice. Rrs (resistance of the respiratory system, A), G (tissue damping, B), Ers (elastance of the respiratory system, C), and H (tissue elastance, D) are shown as the maximum values after each methacholine challenge. Splenocytes isolated from sensitized mice were incubated with OVA in the presence of IL-21, IL-27, and TGF-b for 7 days, followed by purification of CD4þ cells using Dynabeads mouse CD4. After purification, CD4þ cells were stimulated with PMA þ ionomycin for 4 h and used in the IL-10 secretion assay. After conjugation of IL-10-producing CD4þ cells, Tr1 cells and non-Tr1 cells were sorted by FACS. Tr1 cells or non-Tr1 cells were adoptively transferred before the 1st challenge, followed by four OVA challenges. Airway hyperresponsiveness to methacholine was analyzed at 24 h after the 4th challenge. Each dot represents the mean ± SEM of 5e15 animals. *, ** and ***: P < 0.05, 0.01 and 0.001, respectively. y: P < 0.05 versus challenged mice. z: P < 0.05 versus challenged mice administered non-Tr1 cells.
suppressive activity of mouse Tr1 cells in vitro. Inflammatory immune reactions are accompanied by the release of large amounts of adenosine triphosphate (ATP) in the extracellular space where it is converted into adenosine monophosphate (AMP) by CD39 expressed on Tr1 cells, and then dephosphorylated to adenosine by CD73 expressed on effector T cells.31 Recently, it was reported that adenosine exhibits strong anti-inflammatory function on ILC2, preferentially by activating adenosine A2B receptors on the cell membrane, leading to a decrease in the release of IL-5 and IL-13 from activated ILC2.32 Taken together, the suppressive effects of the adoptive transfer of Tr1 cells on the increase in IL-5, airway eosinophilia, and development of AHR may be associated with CD39-mediated anti-inflammatory function. In our previous study, neutrophilic infiltration into the lungs was not suppressed by the adoptive transfer of the cultured CD4þ T cells.8 The CD4þ T cells contained not only Tr1 cells but also inflammatory cells such as IFN-g-producing cells and IL-4-producing cells.8 These inflammatory cells in the adoptively transferred CD4þ T cells may have limited the regulatory function of Tr1cells. In this study, the infiltration of neutrophils into the lungs was significantly suppressed by the adoptive transfer of purified Tr1 cells. However, the production of MIP-2, a neutrophil chemotactic factor, was not suppressed by the adoptive transfer of Tr1 cells, suggesting that regulatory action of Tr1 cells on the neutrophilic infiltration was MIP-2 independent. On the other hand, neutrophil extravasation from blood vessels to the inflamed tissue requires several paired molecular interactions between neutrophils and vascular endothelial cells such as integrins of neutrophils and endothelial intercellular adhesion molecule-1 (ICAM-1).33 IL-10 was reported to significantly inhibit the increase in TNF-a-induced ICAM-1 expression on human umbilical vein endothelial cells. Moreover,
blocking antibodies against ICAM-1 inhibited the migration of neutrophils in PMA-induced rabbit lung inflammation.34 Therefore, Tr1 cells may have downregulated the expression of ICAM-1 in pulmonary vascular endothelial cells via IL-10 production, followed by the suppression of neutrophilic migration into the lungs. Tr1 cells used in this study contained a small population of Foxp3þ Treg cells, suggesting that contained Foxp3þ Treg cells may also be partially related to the suppression of airway inflammation. However, because 93.5% of IL-10-producing cells transferred were Foxp3-, IL-10þ Foxp3- CD4þ T cells could be more likely to contribute to the suppression of allergic airway inflammation than IL-10þ Foxp3þ CD4þ T cells. In conclusion, we established a method to purify Tr1 cells by sorting IL-10-producing cells from CD4þ T cells originating from splenic cells cultured in the presence of OVA, and a mixture of IL-21, IL-27, and TGF-b. The adoptive transfer of Tr1 cells significantly suppressed airway eosinophilia, neutrophilia, and allergic AHR. The Tr1 cell transfer strategy is expected to be a new therapy for allergic diseases.
Declaration of Competing Interest The authors have no conflicts of interest.
Acknowledgement This study was supported by a research support project of Setsunan University and JSPS KAKENHI Grant Number 19K16385 (to M.M.).
Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004
M. Matsuda et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
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Please cite this article as: Matsuda M et al., Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/ j.jphs.2019.10.004