Full Length
Arthritis & Rheumatology DOI 10.1002/art.39037
Th1 and Th17 cytokines drive Takayasu Arteritis inflammation
D Saadoun1*, M Garrido1*, C Comarmond1, AC Desbois1, F Domont1, L Savey1, B Terrier1, G Geri1, M Rosenzwajg1, D Klatzmann1, P Fourret2, P Cluzel3, L Chiche4, J Gaudric4, F Koskas4, P Cacoub1.
1
From the Laboratory I3 « Immunology, Immunopathology, Immunotherapy », UMR CNRS 7211, INSERM U959 and Department of Internal Medicine and Clinical Immunology Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France, DHU I2B, inflammation, immunopathology, biotherapy, UPMC, Paris VI, France; Centre national de reference des maladies autoimmunes et systémiques rares.
2
Department of Pathology, Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France. 3
Department of Radiology, Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France. 4
Department of Vascular Surgery, Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France.
The authors had nothing to disclose.
Running title: Th1 and Th17 in TA * co-first authors Key words: Takayasu arteritis, inflammation, Th17, Th1, vasculitis. Words 2949, 4 figures, 1 Table, 18 references and 23 pages.
Correspondence to David Saadoun MD, PhD, Department of Internal Medicine and clinical immunology and Laboratory I3 « Immunology, immunopathology, immunotherapy » UMR 7211 (CNRS/UPMC) INSERM U959, Hôpital Pitié-Salpétrière, 47-83 boulevard de l’Hôpital, 75013 Paris. Phone: + (33) 1 42 17 80 09. Fax: + (33) 1 42 17 80 33. E-mail: [email protected].
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.39037 © 2015 American College of Rheumatology Received: Jun 16, 2014; Revised: Nov 12, 2014; Accepted: Jan 13, 2015 This article is protected by copyright. All rights reserved.
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Abstract Objective: Takayasu arteritis (TA) is a large-vessel vasculitis inducing damage of the aorta and its branches. Glucocorticoids remain the gold standard of therapy in TA. However, the nature of T cell driving vascular inflammation and the effects of glucocorticoids on the systemic components of TA are not understood. Patients & Methods: T cell homeostasis and cytokines production were analyzed in peripheral blood and aorta inflammatory lesions using Luminex, flow cytometry, and immunohistochemistry analysis. The study included 41 TA patients fulfilling the ACR criteria [17 active (aTA) and 24 in remission (rTA)], 30 giant cell arteritis (GCA) patients, 39 patients with Behcet’s disease (BD) (diseases controls) and 20 age and sex-matched controls. Results: We found marked increase in Th1 and Th17 cells that correlates with TA activity. The addition of serum from active TA patients in sorted CD4+ T cells culture of healthy donors induced a significant production of IFN-γ and IL-17A. We demonstrated the presence of IFN-γ, IL-6 and IL17A producing T cells within vascular inflammatory infiltrates of TA. Glucocorticoid therapy was associated to decreased circulating Th1 cytokines with lower IL-2 (mean±SD; 5079±5300 vs. 7359±3197 pg/ml), IFN-γ (2592±3072 vs. 8393±3392 pg/ml) and TNF-α (847±724 vs. 1491±392 pg/ml) in steroid treated TA compared to steroid free TA patients, respectively. However, glucocorticoids essentially left unaffected Th17 cytokines (i.e. IL-1R, IL-17 and IL-23). Conclusion: Th17 and Th1 pathways contribute to the systemic and vascular manifestations of TA. Glucocorticoids suppressed Th1 cytokines and spared Th17 cytokines in TA. Words: 244
This article is protected by copyright. All rights reserved.
2
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Introduction Takayasu arteritis (TA), is a chronic inflammatory vasculitis of unknown etiology with segmental involvement of the aorta and its main branches (1). Initially described in Japan, TA is ubiquitous but is seen with greater frequency in Asia, South America and the Mediterranean countries. In Western countries, epidemiological data show an annual incidence of about 2 to 3 cases per million inhabitants. Takayasu arteritis affects young woman in 80-90% of cases. Vessel inflammation may lead to segmental stenosis,
occlusion, dilatation, and/or aneurysm formation. Histologically, the vascular lesions may affect the 3 tunics but predominate in the media and adventicia. It is usual to distinguish an acute period, called pre-occlusive, from an occlusive phase characterized by ischemic events. These two phases can be separated by an asymptomatic period or overlap (2). There are currently no reliable criteria for measuring the progression of Takayasu arteritis. It is therefore crucial to find appropriate markers to monitor disease
activity. The nature of T cells driving TA is unresolved. Histological findings of inflammatory cell infiltration of the arterial vascular cells strongly suggest that cell-mediated immunity plays an important role in the pathogenetic sequence leading to the lesions (3, 4). In TA, inflammatory cell infiltration tends to be localized in the adventitia and outer part of the media, involving the vasa vasorum. Infiltrating cells consist mainly of γδ T lymphocytes, natural killer (NK) cells, macrophages, cytotoxic T lymphocytes, and T helper cells (3-5). In animal models, deficient mice in IRF-4-binding protein, a protein that inhibits interleukin (IL)-17A production by controlling the activity of IRF-4 transcription factor, rapidly developed a large-vessel vasculitis due to an inappropriate synthesis of IL-17A (6). Few studies have documented quantitative and qualitative alterations in cytokine production in TA (5, 7-9). Herein, T cell homeostasis and cytokines production were analyzed in peripheral blood and aorta inflammatory lesions of TA patients using Luminex, flow cytometry, and immunohistochemistry analysis.
This article is protected by copyright. All rights reserved.
3
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Methods Study population The study population consisted of 41 consecutive adult TA patients (24.2% men and 75.8% women, median age at diagnosis was 36 (18-57) years, referred to the Department of Internal medicine (Groupe hospitalier Pitie-Salpétrière, Paris, France) from 2009 to 2013 (Table 1). All the TA patients fulfilled the American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis (10). Potential confounding conditions (Giant cell arteritis, or Behçet’s disease) were excluded. Disease activity was assessed according to the international criteria of the National Institute of Health (NIH) (11). 17 patients were in an active phase when they were enrolled in the study, and 24 were in remission. Blood samples from 20 age and sex-matched (5 men and 15 women; median age 37 (26–56) years) healthy donors (HD) were obtained from Etablissement Français du Sang (Hôpital Pitié-Salpêtrière) and from 30 patients with giant cell arteritis (GCA) [23% men and 77% women; median age: 72 (60–88) years] and from 39 patients with Behcet’s disease (BD) [77% men and 23% women; median age: 36 (22-75) years] served as diseases controls. The study was approved by our institutional ethics review board and was performed according to the Helsinki declaration. All donors gave informed consent. Analysis of cytokine production Peripheral blood mononuclear cells (PBMCs) from the 41 TA, 30 GCA and 39 BD patients and the 20 HD were stimulated for 4 hours with 0,05µg/ml Phorbol 12-myristate 13-acetate (PMA) and 1 mM (1µg/ml) ionomycin (Sigma-Aldrich) in the presence or not of brefeldin A (BD Pharmingen). Culture supernatants in the absence of brefeldin A were harvested and immediately frozen at -80°C. Cells stimulated in the presence of brefeldin A were then permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen), stained with IFN-γ-FITC (BD Pharmingen), and IL17A-Alexa Fluor 647 (eBiosciences), and acquired. Quantitative determination of 20 cytokines or chemokines (CCL-11 (Eotaxin), GM-CSF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-6, IL-7, IL-8, IL-12, IL-15, IL-
This article is protected by copyright. All rights reserved.
5
Saadoun et al
Th1 and Th17 in Takayasu arteritis
17, CXCL-10 (IP-10), CCL-2 (MCP-1), CXCL-9 (MIG), CCL-3 (MIP-1α), CCL-4 (MIP-1β), and TNF-α) was performed in culture supernatant using Human Cytokine 25-Plex (Invitrogen, Cergy Pontoise, France) in accordance with the manufacturer protocol. Level IL-23 (R&D systems) was performed using enzyme-linked immunosorbent assay (ELISA). Analysis of cell surface markers and FoxP3 expression PBMCs were stained with the following conjugated monoclonal antibodies, at predetermined optimal dilutions, for 30 minutes at 4°C: CD3-ECD, CD4-PCy7, CD4-ECD, CD8-PCy7, CD8-APC, CD28-FITC, CD45RO-FITC, CD45RA-APC, HLA-DR-PCy7 (Beckman Coulter), CD25-PE, CD62L-FITC (BD Pharmingen), CCR7-PE (R&D systems), CD127-FITC (eBiosciences). Intracellular detection of FoxP3 was performed on fixed and permeabilized cells using appropriate buffer (eBioscience). Data were acquired using a Navios flow cytometer and analyzed with the CXP analysis software (Beckman Coulter). Purification of CD4+ T lymphocytes from TA patients Peripheral total CD4+ T cells were isolated from PBMCs using immunomagnetic depletion (Miltenyi Biotec) with a purity of each population >95%. Purified CD4+ T cell populations were cultured in X-vivo 20 supplemented with 2% penicillin-streptomycin (1 x 106 cells/ml) and stimulated in 48-well plates coated with anti-CD3/CD28 monoclonal antibodies. FoxP3 expression was analyzed using flow cytometry as previously described, and intracellular cytokine production was analyzed after re-stimulation with PMA/ionomycin and flow cytometry. The frequency of IL17A, and IFN-γ producing T cells infiltrating the vessel wall in an active TA patient was examined after tissue digestion with pulmozyme (2.5ml, Sanofi-Aventis) and collagenase (2.5ml, Roche). The tissue was incubated at 37° for 45 minutes and filtrated (cell strainer, 70µm Nylon, BD Biosciences). Cells were harvested by centrifugation for 5 min at 4°and stimulated 4 hours with PMA/ionomycin in the presence of brefeldin A. Intracellular cytokine production was analyzed by flow cytometry.
This article is protected by copyright. All rights reserved.
6
Saadoun et al
Th1 and Th17 in Takayasu arteritis
T cell differentiation with sera from TA patients Peripheral total CD4+ T cells from HD were isolated from PBMCs using immunomagnetic depletion (Miltenyi Biotec) with a purity of each population >95%. Purified CD4+ T cell populations were cultured in X-vivo 20 supplemented with 2% penicillin-streptomycin (1 x 106 cells/ml) and stimulated in 48-well plates coated with anti-CD3/CD28 monoclonal antibodies with or without 10% serum from aTA (n=4) or rTA (n=4). After 5 days of culture, cells were collected and re-stimulated for 4 hours with PMA/ionomycin, and analysis of intracellular IFN-γ and IL-17A production was performed. Immunohistochemical analysis Detection of CD3, IL-17A+, IFN-γ+, and IL-6+ cells was performed on fixed, paraffin-embedded samples (aorta) from 3 TA patients with active inflammatory arterial lesions, 3 TA patients without inflammatory arterial lesions and 3 atherosclerosis patients. Dewaxed slides were submitted to antigen retrieval by heating in citrate buffer pH 6.0. Before incubation with primary antibodies, Fc receptor was blocked with normal goat or donkey serum 3,3%. Slides were incubated over night with monoclonal mouse anti-human CD3 (dilution 1:50, Abcam) and polyclonal goat anti-human IL-17A (dilution 1:10, R&D systems) or polyclonal rabbit anti-human IFN-γ (dilution 1:50, Abcam) or polyclonal rabbit anti-human IL-6 (dilution 1:500, Abcam) antibodies, or with isotype control : polyclonal Rabbit IgG (undiluted, Abcam) or polyclonal Goat IgG (dilution 1:100, R&D). Slides were then incubated for 2 hours at room temperature with Cy3-conjugated goat anti-mouse (working dilution 1:1000, Jackson Immunoresearch) and Alexa 488 donkey anti-goat or anti- rabbit (working dilution 1:1000, Life Technologies), mounted in Mowiol, and evaluated under fluorescence microscopy. Statistical analysis Data are presented as mean (SD) for continuous variables and percentage for qualitative variables. Fisher’s exact test was used to compare qualitative variables, and non-parametric Mann–Whitney
This article is protected by copyright. All rights reserved.
7
Saadoun et al
Th1 and Th17 in Takayasu arteritis
and Wilcoxon tests to compare continuous variables as appropriate. A p-value < 0.05 was considered significant. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA).
This article is protected by copyright. All rights reserved.
8
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Results Th1 and Th17-related cytokines are increased in TA patients and correlate with disease activity Comparisons of cytokines between TA patients and GCA and BD (disease controls) patients and HD are presented in Figure 1. TA patients had significantly increased levels of IL-2 (mean±SD ; 3267±3654 in TA vs. 928.1±883.4 in GCA vs. 1476±1738 in BD vs. 1069±817.5 in HD pg/ml; P=0.0022, P=0.023 and P=0.039, respectively), IL-2R (235.5±274.6 vs. 67.1±50.7 vs. 60.6±40.5 vs. 69.8±30 pg/ml; P=0.0002, P
Arthritis & Rheumatology DOI 10.1002/art.39037
Th1 and Th17 cytokines drive Takayasu Arteritis inflammation
D Saadoun1*, M Garrido1*, C Comarmond1, AC Desbois1, F Domont1, L Savey1, B Terrier1, G Geri1, M Rosenzwajg1, D Klatzmann1, P Fourret2, P Cluzel3, L Chiche4, J Gaudric4, F Koskas4, P Cacoub1.
1
From the Laboratory I3 « Immunology, Immunopathology, Immunotherapy », UMR CNRS 7211, INSERM U959 and Department of Internal Medicine and Clinical Immunology Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France, DHU I2B, inflammation, immunopathology, biotherapy, UPMC, Paris VI, France; Centre national de reference des maladies autoimmunes et systémiques rares.
2
Department of Pathology, Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France. 3
Department of Radiology, Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France. 4
Department of Vascular Surgery, Groupe Hospitalier Pitié-Salpetrière, Université Pierre et Marie Curie, Paris 6, Paris, France.
The authors had nothing to disclose.
Running title: Th1 and Th17 in TA * co-first authors Key words: Takayasu arteritis, inflammation, Th17, Th1, vasculitis. Words 2949, 4 figures, 1 Table, 18 references and 23 pages.
Correspondence to David Saadoun MD, PhD, Department of Internal Medicine and clinical immunology and Laboratory I3 « Immunology, immunopathology, immunotherapy » UMR 7211 (CNRS/UPMC) INSERM U959, Hôpital Pitié-Salpétrière, 47-83 boulevard de l’Hôpital, 75013 Paris. Phone: + (33) 1 42 17 80 09. Fax: + (33) 1 42 17 80 33. E-mail: [email protected].
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.39037 © 2015 American College of Rheumatology Received: Jun 16, 2014; Revised: Nov 12, 2014; Accepted: Jan 13, 2015 This article is protected by copyright. All rights reserved.
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Abstract Objective: Takayasu arteritis (TA) is a large-vessel vasculitis inducing damage of the aorta and its branches. Glucocorticoids remain the gold standard of therapy in TA. However, the nature of T cell driving vascular inflammation and the effects of glucocorticoids on the systemic components of TA are not understood. Patients & Methods: T cell homeostasis and cytokines production were analyzed in peripheral blood and aorta inflammatory lesions using Luminex, flow cytometry, and immunohistochemistry analysis. The study included 41 TA patients fulfilling the ACR criteria [17 active (aTA) and 24 in remission (rTA)], 30 giant cell arteritis (GCA) patients, 39 patients with Behcet’s disease (BD) (diseases controls) and 20 age and sex-matched controls. Results: We found marked increase in Th1 and Th17 cells that correlates with TA activity. The addition of serum from active TA patients in sorted CD4+ T cells culture of healthy donors induced a significant production of IFN-γ and IL-17A. We demonstrated the presence of IFN-γ, IL-6 and IL17A producing T cells within vascular inflammatory infiltrates of TA. Glucocorticoid therapy was associated to decreased circulating Th1 cytokines with lower IL-2 (mean±SD; 5079±5300 vs. 7359±3197 pg/ml), IFN-γ (2592±3072 vs. 8393±3392 pg/ml) and TNF-α (847±724 vs. 1491±392 pg/ml) in steroid treated TA compared to steroid free TA patients, respectively. However, glucocorticoids essentially left unaffected Th17 cytokines (i.e. IL-1R, IL-17 and IL-23). Conclusion: Th17 and Th1 pathways contribute to the systemic and vascular manifestations of TA. Glucocorticoids suppressed Th1 cytokines and spared Th17 cytokines in TA. Words: 244
This article is protected by copyright. All rights reserved.
2
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Introduction Takayasu arteritis (TA), is a chronic inflammatory vasculitis of unknown etiology with segmental involvement of the aorta and its main branches (1). Initially described in Japan, TA is ubiquitous but is seen with greater frequency in Asia, South America and the Mediterranean countries. In Western countries, epidemiological data show an annual incidence of about 2 to 3 cases per million inhabitants. Takayasu arteritis affects young woman in 80-90% of cases. Vessel inflammation may lead to segmental stenosis,
occlusion, dilatation, and/or aneurysm formation. Histologically, the vascular lesions may affect the 3 tunics but predominate in the media and adventicia. It is usual to distinguish an acute period, called pre-occlusive, from an occlusive phase characterized by ischemic events. These two phases can be separated by an asymptomatic period or overlap (2). There are currently no reliable criteria for measuring the progression of Takayasu arteritis. It is therefore crucial to find appropriate markers to monitor disease
activity. The nature of T cells driving TA is unresolved. Histological findings of inflammatory cell infiltration of the arterial vascular cells strongly suggest that cell-mediated immunity plays an important role in the pathogenetic sequence leading to the lesions (3, 4). In TA, inflammatory cell infiltration tends to be localized in the adventitia and outer part of the media, involving the vasa vasorum. Infiltrating cells consist mainly of γδ T lymphocytes, natural killer (NK) cells, macrophages, cytotoxic T lymphocytes, and T helper cells (3-5). In animal models, deficient mice in IRF-4-binding protein, a protein that inhibits interleukin (IL)-17A production by controlling the activity of IRF-4 transcription factor, rapidly developed a large-vessel vasculitis due to an inappropriate synthesis of IL-17A (6). Few studies have documented quantitative and qualitative alterations in cytokine production in TA (5, 7-9). Herein, T cell homeostasis and cytokines production were analyzed in peripheral blood and aorta inflammatory lesions of TA patients using Luminex, flow cytometry, and immunohistochemistry analysis.
This article is protected by copyright. All rights reserved.
3
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Methods Study population The study population consisted of 41 consecutive adult TA patients (24.2% men and 75.8% women, median age at diagnosis was 36 (18-57) years, referred to the Department of Internal medicine (Groupe hospitalier Pitie-Salpétrière, Paris, France) from 2009 to 2013 (Table 1). All the TA patients fulfilled the American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis (10). Potential confounding conditions (Giant cell arteritis, or Behçet’s disease) were excluded. Disease activity was assessed according to the international criteria of the National Institute of Health (NIH) (11). 17 patients were in an active phase when they were enrolled in the study, and 24 were in remission. Blood samples from 20 age and sex-matched (5 men and 15 women; median age 37 (26–56) years) healthy donors (HD) were obtained from Etablissement Français du Sang (Hôpital Pitié-Salpêtrière) and from 30 patients with giant cell arteritis (GCA) [23% men and 77% women; median age: 72 (60–88) years] and from 39 patients with Behcet’s disease (BD) [77% men and 23% women; median age: 36 (22-75) years] served as diseases controls. The study was approved by our institutional ethics review board and was performed according to the Helsinki declaration. All donors gave informed consent. Analysis of cytokine production Peripheral blood mononuclear cells (PBMCs) from the 41 TA, 30 GCA and 39 BD patients and the 20 HD were stimulated for 4 hours with 0,05µg/ml Phorbol 12-myristate 13-acetate (PMA) and 1 mM (1µg/ml) ionomycin (Sigma-Aldrich) in the presence or not of brefeldin A (BD Pharmingen). Culture supernatants in the absence of brefeldin A were harvested and immediately frozen at -80°C. Cells stimulated in the presence of brefeldin A were then permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen), stained with IFN-γ-FITC (BD Pharmingen), and IL17A-Alexa Fluor 647 (eBiosciences), and acquired. Quantitative determination of 20 cytokines or chemokines (CCL-11 (Eotaxin), GM-CSF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-6, IL-7, IL-8, IL-12, IL-15, IL-
This article is protected by copyright. All rights reserved.
5
Saadoun et al
Th1 and Th17 in Takayasu arteritis
17, CXCL-10 (IP-10), CCL-2 (MCP-1), CXCL-9 (MIG), CCL-3 (MIP-1α), CCL-4 (MIP-1β), and TNF-α) was performed in culture supernatant using Human Cytokine 25-Plex (Invitrogen, Cergy Pontoise, France) in accordance with the manufacturer protocol. Level IL-23 (R&D systems) was performed using enzyme-linked immunosorbent assay (ELISA). Analysis of cell surface markers and FoxP3 expression PBMCs were stained with the following conjugated monoclonal antibodies, at predetermined optimal dilutions, for 30 minutes at 4°C: CD3-ECD, CD4-PCy7, CD4-ECD, CD8-PCy7, CD8-APC, CD28-FITC, CD45RO-FITC, CD45RA-APC, HLA-DR-PCy7 (Beckman Coulter), CD25-PE, CD62L-FITC (BD Pharmingen), CCR7-PE (R&D systems), CD127-FITC (eBiosciences). Intracellular detection of FoxP3 was performed on fixed and permeabilized cells using appropriate buffer (eBioscience). Data were acquired using a Navios flow cytometer and analyzed with the CXP analysis software (Beckman Coulter). Purification of CD4+ T lymphocytes from TA patients Peripheral total CD4+ T cells were isolated from PBMCs using immunomagnetic depletion (Miltenyi Biotec) with a purity of each population >95%. Purified CD4+ T cell populations were cultured in X-vivo 20 supplemented with 2% penicillin-streptomycin (1 x 106 cells/ml) and stimulated in 48-well plates coated with anti-CD3/CD28 monoclonal antibodies. FoxP3 expression was analyzed using flow cytometry as previously described, and intracellular cytokine production was analyzed after re-stimulation with PMA/ionomycin and flow cytometry. The frequency of IL17A, and IFN-γ producing T cells infiltrating the vessel wall in an active TA patient was examined after tissue digestion with pulmozyme (2.5ml, Sanofi-Aventis) and collagenase (2.5ml, Roche). The tissue was incubated at 37° for 45 minutes and filtrated (cell strainer, 70µm Nylon, BD Biosciences). Cells were harvested by centrifugation for 5 min at 4°and stimulated 4 hours with PMA/ionomycin in the presence of brefeldin A. Intracellular cytokine production was analyzed by flow cytometry.
This article is protected by copyright. All rights reserved.
6
Saadoun et al
Th1 and Th17 in Takayasu arteritis
T cell differentiation with sera from TA patients Peripheral total CD4+ T cells from HD were isolated from PBMCs using immunomagnetic depletion (Miltenyi Biotec) with a purity of each population >95%. Purified CD4+ T cell populations were cultured in X-vivo 20 supplemented with 2% penicillin-streptomycin (1 x 106 cells/ml) and stimulated in 48-well plates coated with anti-CD3/CD28 monoclonal antibodies with or without 10% serum from aTA (n=4) or rTA (n=4). After 5 days of culture, cells were collected and re-stimulated for 4 hours with PMA/ionomycin, and analysis of intracellular IFN-γ and IL-17A production was performed. Immunohistochemical analysis Detection of CD3, IL-17A+, IFN-γ+, and IL-6+ cells was performed on fixed, paraffin-embedded samples (aorta) from 3 TA patients with active inflammatory arterial lesions, 3 TA patients without inflammatory arterial lesions and 3 atherosclerosis patients. Dewaxed slides were submitted to antigen retrieval by heating in citrate buffer pH 6.0. Before incubation with primary antibodies, Fc receptor was blocked with normal goat or donkey serum 3,3%. Slides were incubated over night with monoclonal mouse anti-human CD3 (dilution 1:50, Abcam) and polyclonal goat anti-human IL-17A (dilution 1:10, R&D systems) or polyclonal rabbit anti-human IFN-γ (dilution 1:50, Abcam) or polyclonal rabbit anti-human IL-6 (dilution 1:500, Abcam) antibodies, or with isotype control : polyclonal Rabbit IgG (undiluted, Abcam) or polyclonal Goat IgG (dilution 1:100, R&D). Slides were then incubated for 2 hours at room temperature with Cy3-conjugated goat anti-mouse (working dilution 1:1000, Jackson Immunoresearch) and Alexa 488 donkey anti-goat or anti- rabbit (working dilution 1:1000, Life Technologies), mounted in Mowiol, and evaluated under fluorescence microscopy. Statistical analysis Data are presented as mean (SD) for continuous variables and percentage for qualitative variables. Fisher’s exact test was used to compare qualitative variables, and non-parametric Mann–Whitney
This article is protected by copyright. All rights reserved.
7
Saadoun et al
Th1 and Th17 in Takayasu arteritis
and Wilcoxon tests to compare continuous variables as appropriate. A p-value < 0.05 was considered significant. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA).
This article is protected by copyright. All rights reserved.
8
Saadoun et al
Th1 and Th17 in Takayasu arteritis
Results Th1 and Th17-related cytokines are increased in TA patients and correlate with disease activity Comparisons of cytokines between TA patients and GCA and BD (disease controls) patients and HD are presented in Figure 1. TA patients had significantly increased levels of IL-2 (mean±SD ; 3267±3654 in TA vs. 928.1±883.4 in GCA vs. 1476±1738 in BD vs. 1069±817.5 in HD pg/ml; P=0.0022, P=0.023 and P=0.039, respectively), IL-2R (235.5±274.6 vs. 67.1±50.7 vs. 60.6±40.5 vs. 69.8±30 pg/ml; P=0.0002, P
