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Regulatory T Cell Dysfunction in Idiopathic, Heritable and Connective Tissue-Associated Pulmonary Arterial Hypertension
Accepted Manuscript Regulatory T cell dysfunction in idiopathic, heritable and connective tissue-associated pulmonary arterial hypertension Alice Huertas, MD, PhD, Carole Phan, MSc, Jennifer Bordenave, MSc, Ly Tu, PhD, Raphaël Thuillet, Morane Le Hiress, PhD, Jérôme Avouac, MD, PhD, Yuichi Tamura, MD, PhD, Yannick Allanore, MD, PhD, Roland Jovan, MD, Olivier Sitbon, MD, PhD, Christophe Guignabert, PhD, Marc Humbert, MD, PhD PII:
S0012-3692(16)00461-X
DOI:
10.1016/j.chest.2016.01.004
Reference:
CHEST 242
To appear in:
CHEST
Received Date: 6 October 2015 Revised Date:
27 November 2015
Accepted Date: 4 January 2016
Please cite this article as: Huertas A, Phan C, Bordenave J, Tu L, Thuillet R, Le Hiress M, Avouac J, Tamura Y, Allanore Y, Jovan R, Sitbon O, Guignabert C, Humbert M, Regulatory T cell dysfunction in idiopathic, heritable and connective tissue-associated pulmonary arterial hypertension, CHEST (2016), doi: 10.1016/j.chest.2016.01.004. 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.
Text word count: 2762
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Abstract word count: 242
Regulatory T cell dysfunction in idiopathic, heritable and connective tissue-associated pulmonary arterial hypertension Running title: Regulatory T lymphocytes in PAH
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Alice Huertas, MD, PhD1, 2, 3, Carole Phan, MSc1, 2, Jennifer Bordenave MSc1, 2, Ly Tu, PhD1, 2, Raphaël Thuillet1, 2, Morane Le Hiress, PhD1, 2, Jérôme Avouac, MD, PhD4, Yuichi Tamura, MD, PhD1, 2, Yannick Allanore, MD, PhD4, Roland Jovan, MD1, 2, 3, Olivier Sitbon, MD, PhD1, 2, 3, Christophe Guignabert, PhD1, 2 and Marc Humbert, MD, PhD1, 2, 3
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Affiliations: Inserm UMR_S 999, Hôpital Marie Lannelongue, Le Plessis Robinson, France; 2. Univ. Paris-Sud, Faculté de Médecine, Université Paris-Saclay, Le Kremlin Bicêtre, France; 3. AP-HP, Service de Pneumologie, Hôpital Bicêtre, Le Kremlin Bicêtre, France; 4. Cochin Hospital, Paris Descartes University, INSERM U1016 and CNRS UMR8104, AP-HP, Paris, France.
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Correspondence to: Dr Alice Huertas, MD, PhD INSERM UMR_S 999, Hôpital Marie Lannelongue, 133 Avenue de la Résistance, 92350 Le PlessisRobinson, FRANCE. E-mail: [email protected]
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Disclosures: AH, CP, JB, LT, RT, MLH, JA, YT, RJ and CG have no conflict of interest to declare. YA has/had consultancy relationship and/or has received research funding in relationship with the treatment of systemic sclerosis from Actelion, Bayer, Biogen Idec, Bristol-Myers Squibb, Genentech/ Roche, Inventiva, Medac, Pfizer, Sanofi/Genzyme, Servier and UCB. OS has relationships with pharmaceutical companies including Actelion Pharmaceuticals, Bayer HealthCare, GlaxoSmithKline, Pfizer and United Therapeutics. In addition to being an investigator in trials involving these companies, relationships include consultancy service and membership of scientific advisory boards. MH has relationships with drug companies including Actelion, Bayer Schering, GSK, Lilly, Novartis, Pfizer and United Therapeutics. In addition to being an investigator in trials involving these companies, relationships include consultancy service and membership of scientific advisory boards. Funding footnotes: This research was supported by grants from the French National Institute for Health and Medical Research (INSERM, the French National Agency for Research: grant no. ANR_12_JSV1_0004_01), the Association des Sclérodermiques de France (ASF) and in part by the Département Hospitalo-Universitaire (DHU) Thorax Innovation (TORINO) MLH was supported by the LabEx LERMIT (grant no ANR-10-LABX-0033-LERMIT). YT received a personal ERS/EU Marie Curie grant post-doctoral fellowship (RESPIRE2). CP is supported by the Fonds de Dotation "Recherche en Santé Respiratoire" - Fondation du Souffle.
ABSTRACT
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Pulmonary arterial hypertension (PAH) encompasses a group of conditions with distinct causes. Dysimmunity is a common feature of all forms of PAH and contributes to both disease susceptibility and/or progression. Regulatory T lymphocytes (Treg) are dysfunctional in idiopathic PAH (iPAH) patients in a
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leptin-dependent manner. However, it is not known whether these abnormalities are specific to iPAH. Hence, we hypothesized that (1) Treg dysfunction is also present in heritable (hPAH) and connective tissue disease-associated PAH (CTD-PAH); (2) defective leptin-dependent signaling is present in hPAH and CTD-
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PAH and could contribute to Treg dysfunction; (3) modulating leptin axis in vivo could protect against Treg dysfunction; (4) restoration of Treg activity could limit and/or reverse experimental chronic hypoxia (Hx)-
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induced pulmonary hypertension (PH) in vivo. We analysed 62 patients with PAH (30 iPAH, 18 hPAH and 14 CTD-PAH), 10 CTD patients without PAH and 20 healthy controls. Our results indicate that Treg are dysfunctional in all PAH forms tested, as well as in CTD patients without PAH. Importantly, leptin axis is crucial in Treg dysfunction in iPAH and CTD patients (with or without PAH), whereas in hPAH, Treg are
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altered in a leptin independent manner. Using leptin receptor (ObR)-deficient rats which develop less severe Hx-PH, we found that ObR-deficient rats are protected against decreased Treg function after Hx exposure. Taken altogether, our results suggest that Treg dysfunction is common to all forms of PAH and may
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contribute to the development and/or the progression of the disease.
Keywords: Pulmonary arterial hypertension; Leptin; Lymphocytes; Scleroderma
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INTRODUCTION Pulmonary arterial hypertension (PAH, Group 1, is a severe cardiopulmonary disorder characterized by
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chronic pre-capillary pulmonary hypertension (PH), defined by a mean pulmonary artery pressure at rest ≥ 25 mm Hg, in the presence of a normal pulmonary capillary wedge pressure (≤ 15 mm Hg)1,2. Despite rapid development of specific therapies, PAH carries a high mortality rate3,4. PAH may occur in a number of
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different clinical contexts: without an identifiable underlying cause (idiopathic or iPAH), or induced by drugs and toxins, or associated with other diseases, including connective tissue disease (CTD-PAH), portal
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hypertension, human immunodeficiency virus (HIV) infection2. When PAH occurs in a familial context, germline mutations in the Bone Morphogenetic Protein Receptor type 2 (BMPR2) gene are detected in at least 70% of cases, and around 20% of so-called idiopathic cases carry a BMPR2 mutation (heritable PAH or hPAH)5. Although different forms of PAH could reflect distinct physiopathological mechanisms, current
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evidence strongly suggests that inflammation and autoimmune disorders are common to all forms6,7. In particular, it is now widely accepted that immunological disorders contribute to both disease susceptibility and progression of vascular remodeling in PAH8-12. Nevertheless, the initiating triggers of dysimmunity and
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the exact mechanisms by which altered immune responses contribute to the disease development and/or progression of PAH remain unknown. Furthermore, to which extent immune disorders are implicated in the
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different forms of PAH is still an open question. In the immune system, CD4+ CD25+ Foxp3+ regulatory T lymphocytes (Treg) represent a key cell type: they are potent immunomodulators of the adaptive immune system, the proinflammatory cytokine responses and the activation of monocytes/macrophages13,14. Association between autoimmune disorders and PAH has been recognized for many years15-21. The absence of Treg cells in athymic nude rats leads to activated macrophage recruitment in lungs and exaggerated pulmonary vascular remodeling, following vascular injury with the vascular endothelial growth factor (VEGF) receptor inhibitor SU-541622,23. It has been
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demonstrated that Treg cells are dysfunctional in patients with iPAH24 and that this adaptive immune
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dysregulation may be partly explained by the excessive pulmonary endothelial cell (EC)-derived leptin present in these patients24, suggesting that leptin could represent a missing link between pulmonary endothelial dysfunction and immunological disorders in iPAH. The significance of this metabolic factor in other forms of PAH is still unexplored, in particular in patients who exhibit dysimmunity such as hPAH with
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BMPR2 mutations or systemic sclerosis (SSc), the most frequent autoimmune disease associated with PAH25. Indeed, recent publications underline a strong link between the presence of a BMPR2 mutation and an inappropriate inflammation within the context of PAH26-28. It has been shown that mice heterozygous for
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a null allele in Bmpr2 are more susceptible to develop PH under inflammatory stress when compared to control mice26 and that loss of endothelial Bmpr2 is sufficient to predispose to PH in association with
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perivascular inflammatory infiltration28. Finally, it has recently been demonstrated that this link is partly due to a p38-MK2 mechanism27.
In this study, we determined whether: 1) Treg dysfunction is identified in hPAH due to BMPR2 mutations and in CTD-PAH patients with SSc; 2) defective leptin-dependent signal transduction is present in hPAH
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and CTD-PAH and could contribute to Treg dysfunction as in iPAH; 3) modulating leptin axis in vivo could protect against Treg dysfunction; 4) the effective restoration of normal Treg activity could limit or even
Subjects
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METHODS
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reverse experimental chronic hypoxia-induced PH in vivo.
Adult PAH patients had a diagnosis confirmed by right-heart catheterization and a stable clinical and hemodynamic status for the last 3 months. Adult SSc patients had clinical stability for the last 3 months (with no need of chronic immunosuppressive and/or corticosteroid therapy) and exclusion of PAH by means of right-heart catheterization. Exclusion criteria for all patient groups were diabetes, metabolic syndrome and immunosuppressive and/or corticosteroid therapy in the last 3 months (Tables 1 and 2). Characteristics at diagnosis and follow-up were stored in the Registry of the French Network of PH in agreement with 4
French bioethics laws (Commission Nationale de l'Informatique et des Libertés). This study was approved
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by the local ethics committee (CPP Ile-de-France VII, Paris, France) and all patients signed written informed consent.
Animal models and hemodynamic measurements
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Zucker diabetic fatty (ZDF, Charles River Laboratories) male (4 weeks old) Sprague-Dawley rats (ZDF/Lepr fa/fa) and age matched lean control rats (ZDF/Lean Ctrl) were exposed to normoxia or normobaric hypoxia (FiO2 = 10 %) for 3 consecutive weeks. As previously described29, animals were
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anesthetized with isoflurane for catheterization. A polyvinyl catheter was introduced into the right jugular vein and pushed through the right ventricle into the pulmonary artery. Another polyethylene catheter was
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inserted into the right carotid artery. After measurement of hemodynamic parameters, blood was removed for peripheral blood mononuclear cell (PBMC) analyses. Animal studies were approved by the administrative panel on animal care from Université Paris-Sud (France).
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Flow cytometry analyses
After blood samples withdrawal, PBMCs from humans (patients and controls) or rats were obtained by standard Ficoll gradient centrifugation. The cells were carefully washed with PBS and resuspended in a
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staining buffer containing 10% of human serum. The cells were then fluorescently labeled with the following antibodies under non-permeabilized or permeabilized conditions (IntraPrep, following
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manufacturer’s instructions, Beckman Coulter), to enable surface or intracellular staining, respectively: in humans, fluorophore conjugated monoclonal anti-CD4, monoclonal anti-CD25 and monoclonal anti-CD127 (Treg cocktail), monoclonal anti-phosphorylated STAT3 (Y705 site) (Becton Dickinson) and fluorophore conjugated monoclonal anti-ObR (R&D Systems). In rats, fluorophore conjugated monoclonal anti-CD4, anti-CD25 and anti-FoxP3 (eBioscience) and monoclonal anti-phosphorylated STAT3 (Becton Dickinson). Flow cytometry gating conditions and the mean fluorescence intensity (MFI) were set and normalized, respectively, against isotype- and fluorophore-matched non-immune IgGs. Human Treg phenotype was defined as CD4+ CD25+ CD127low cells and rat Treg phenotype was defined as CD4+ CD25+ Foxp3+ cells 5
and was expressed as the percentage of total CD4+ cells. Flow cytometry data were acquired with a flow
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cytometer (MACSQuant) and analyzed by FlowJo software program (Tree Star, Inc). Statistical analyses Results are expressed as means ± SEM. A p<0.05 level of statistical significance was used for all analyses.
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All between-groups comparisons were assessed using One Way ANOVA in humans; post-hoc analysis of significant variables was performed using Bonferroni correction with all pairwise multiple comparisons. Differences between two selected groups (SSc-PAH and SSc patients) were compared using unpaired t-test.
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All between-groups comparisons in rats were assessed using non parametric Kruskal-Wallis analysis with Dunn’s correction. Pearson correlations were used to establish associations between the dependent variables
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and relevant independent variables. All statistical procedures were carried out using GraphPad Prism version 5.0 (GraphPad Software Inc.)
Patient populations
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RESULTS
Blood samples were collected from 62 patients with PAH, corresponding to iPAH (n=30), hPAH due to a
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BMPR2 mutation (n=18) and SSc-PAH (n=14) (Table 1). Seven SSc patients without PAH and 20 control subjects were also studied. Tables 1 and 2 summarize patient anthropometric, functional and hemodynamic
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characteristics.
Dysfunction of Treg cells in idiopathic, heritable and connective tissue disease-associated pulmonary arterial hypertension
To determine the functional status of Treg function, we measured Treg STAT3 phosphorylation (pSTAT3) as STAT3 represents an important signaling pathway for Treg activity30. We measured pSTAT3 levels in iPAH and hPAH patients carrying a BMPR2 mutation, SSc-PAH and SSc patients without PAH, as well as in control subjects. After Treg permeabilization, pSTAT3 quantification by flow cytometry revealed that the number of Treg-pSTAT3+ in the whole Treg population as well as the Treg-STAT3+ in the Treg 6
subpopulation expressing ObR were markedly decreased in all patients compared to controls. Interestingly,
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the number of Treg-pSTAT3 was similar among all groups of PAH as well as in SSc patients without PAH (Figure 1 and Table 3). Importantly, flow cytometry analyses revealed a normal Treg cell count in all groups, controls, PAH (iPAH, hPAH and SSc-PAH) and SSc patients, expressed as a percentage of CD4+ cells (Figure 2 and Table 3). These results are consistent with our previous work24, while others have
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suggested an increase in Treg cell number in iPAH patients31. Although influences of genetic and environmental factors cannot be excluded in these studies32, it is important to underlie that Ulrich et al. described an increase in FoxP3 in the total CD4+ cell population, whereas it is known that FoxP3 expression
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is no longer restricted to Treg and hence is not sufficient to identify Treg33,34. Interestingly, and consistently with our findings, Ulrich and coworkers did not find differences in FoxP3 expression in the Treg population
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between iPAH and controls31. Our present results indicate that Treg cell counts are normal but that Treg are substantially dysfunctional in all forms of PAH studied, to the same degree as autoimmune diseases, such as SSc.
Leptinemia in iPAH, hPAH and SSc-PAH compared with control and SSc patients
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Our recent work in iPAH patients has showed that dysfunctional pulmonary endothelium synthetizes abnormally high levels of leptin that contribute to altered functional status of circulating Treg24. However, the levels of leptinemia in hPAH and in SSc patients without PAH have not been investigated so far. First,
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we measured circulating levels of leptin in patients with iPAH, hPAH and SSc-PAH compared with control subjects and/or SSc patients. Of note, all patients and controls displayed similar body mass index (BMI). As
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previously published24, the leptinemia was higher in iPAH and SSc-PAH (with no difference between these groups) compared to controls. By contrast, hPAH patients and control subjects had normal leptinemia (Figure 3 and Table 1). We then compared SSc patients with or without PAH and there was no difference within these two groups (Figure 3 and Table 2). Since no correlation was found between serum leptin levels and BMI, disease severity and pro-inflammatory cytokine levels in our previous study, we did not perform these analyses. Our results indicate here that hPAH patients had a normal level of circulating leptin whereas in iPAH and SSc patients, with or without PAH, circulating leptin levels are highly increased compared to controls. 7
ObR expression on Treg cell membrane isolated from iPAH, hPAH and SSc-PAH compared with control
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subjects and SSc patients We next determined the expression level of leptin receptor ObR on Treg cell surface. We selected Treg cells among the fluorescently tagged PBMCs by flow cytometry analyses after peripheral venous blood withdrawal in controls and PAH patients. ObR expression on Treg membrane was markedly and similarly
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increased in iPAH and SSc-PAH patients, and even more in SSc patients without PAH. By contrast, hPAH patients displayed a normal ObR expression level on Treg with no difference compared to controls (Figure 4A-B and Table 3). Interestingly, the expression of ObR on Treg cell membrane did not correlate with
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serum leptin levels or BMI (Figure 4C-F and Table 3). Taken together, these findings suggest that leptin and its receptor expression on circulating Treg are not altered in hPAH as opposed to iPAH and SSc-PAH.
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Importantly, our findings indicate that the level of ObR expression is independent of the patients’ BMI or serum leptin levels.
Leptin axis modulates decrease Treg function in chronic hypoxia-induced PH in rats We then questioned to which extent leptin could play a role on Treg function in vivo and whether the
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presence of functional Treg could limit or even reverse experimental chronic hypoxia-induced PH. To this aim, we used rats lacking ObR, called Zucker Diabetic Fatty (ZDF) rats (ZDF/Lepr fa/fa) and exposed them to chronic hypoxia (FiO2 10%) or normoxia for three weeks and compared them to their wildtype littermates
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(ZDF/Lepr Crl), also exposed to chronic hypoxia or normoxia. Wildtype rats exposed to chronic hypoxia
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displayed decreased Treg function with decreased pSTAT3 level compared to wildtype normoxic animals. Interestingly, ZDF rats displayed a normal pSTAT3 level in normoxia and in hypoxia (Figure 5A and Table 4). Of note, Treg count was normal in all groups (Figure 5B). Overall, these results, together with our recent data indicating that leptin pathway contributes to chronic hypoxia-induced PH susceptibility35, suggest that decrease Treg function may contribute to chronic hypoxia-induced PH onset and/or progression.
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DISCUSSION
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To address if similar or distinct pathomechanisms contribute to Treg dysfunction in PAH, we analyzed Treg cell numbers and functional activity in iPAH, hPAH and SSc-PAH patients and compared our observations with those obtained in control and SSc patients with no evidence of PAH. We show, for the first time, that Treg cells are altered in all these PAH subgroups. In addition, our data highlight the
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differences in pathophysiological mechanisms leading to Treg dysfunction among iPAH, hPAH and SScPAH. Indeed, our results indicate that in iPAH and SSc-PAH patients, the leptin axis is crucial in Treg dysfunction whereas in hPAH, Treg cells are altered independently of the leptin signaling system. Consistent
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with these human findings, we are demonstrating here that ObR-deficient rats, which develop less severe PH35, are protected against decrease Treg function when exposed to chronic hypoxia. Taken together, our
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results support the concept that in the absence of appropriate Treg activity, pulmonary vascular injury may lead to progressive elevations of pulmonary artery pressure and PH.
Although it is now clear that PAH patients display inflammatory and immunologic disorders and that some inflammatory biomarkers predict mortality36-38, the causes of dysimmunity in the different forms of
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PAH development and/or progression are still not fully understood8,11,17,18,39-43. Our study presented here is the first to provide direct experimental evidence that Treg are equally dysfunctional regardless of the cause
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of PAH and that different pathomechanisms may explain this abnormality. Although STAT3 represents an important transcription factor for Treg activity30 and the main leptin-dependent signaling pathway44,45,
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further studies are needed to better characterize the phenotypic and functional status of human Treg cells in healthy individuals and PAH patients by in vitro suppression assay. Whereas it is well known that Treg control immune homeostasis by maintaining self-tolerance and suppressing a variety of physiological and pathological immune responses to non-self antigens13, the exact mechanisms by which they exert their function are not completely understood46,47. It has also been described that Treg are critical in limiting the extent of inflammation and in protecting effects against vascular remodeling13,22,23,32. We are indicating here new aspects of immune homeostasis as we are showing that leptin down-regulates Treg ability to control other immune cells in iPAH and SSc-PAH but not in hPAH due to BMPR2 mutations. While we suggest a 9
role for leptin in iPAH and SSc-PAH, Treg dysfunction in hPAH is not well understood. It needs to be noted
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that different biologic outcomes depend upon the influence from the local cytokine milieu32,48,49 thus we cannot exclude that our findings may be influenced by the local cytokine milieu and the differentiation status of the Treg. However, BMPRII-dependent signaling pathways might be implicated in Treg regulation acting on the level of STAT3 phosphorylation through classical Smad 1/5/8 signaling or p38 mitogen-activated
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protein kinase (MAPK) pathway, as it has been shown that hPAH patients display defective Smad signaling compensated for by an activation of p38MAPK signaling50. Further studies are needed to test these hypotheses and address this specific question. Interestingly, recent data indicate that BMPR-II deficiency
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promotes an exaggerated inflammatory response which can instigate PH development51.
Our recent study performed in iPAH patients and in rodent PH models indicate that increased
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leptinemia and leptin local production by dysfunctional pulmonary ECs contribute to vascular remodeling by acting on pulmonary ECs, pulmonary artery smooth muscle cell and perivascular monocyte/macrophage accumulation24,35. Despite these findings, the effect of pulmonary EC-derived leptin on systemic dysimmunity remains unclear. The present data highlight another role for pulmonary EC-derived leptin,
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suggesting a crosstalk between pulmonary dysfunctional ECs and Treg dysfunction21. Using ObR deficient rats, which are protected against PH development in chronic hypoxia35, we have shown a possible connection between pulmonary EC-derived leptin, dysimmunity and susceptibility of PH development.
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While pulmonary EC dysfunction may lead to altered Treg via excessive leptin secretion, the effects of
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dysfunctional Treg on pulmonary vascular remodeling is still unclear. Interestingly, it has been demonstrated that T cell-deficient athymic rats develop exaggerated PH and display excessive inflammation and leukotriene B4-secreting macrophage recruitment23,52,53. Furthermore, it has been reported that the pulmonary vasculature is sensitive to autoimmune stimuli, such as autoantibodies directed against pulmonary vasculature components, and that the vascular wall can respond by developing abnormal proliferative and apoptotic resistant phenotype16. Consistent with these notions, Treg dysfunction in PAH could contribute to disease onset and/or progression.
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However, several questions remain unanswered and in particular whether dysimmunity represents a
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cause or an effect of PAH onset is still unclear. In summary, our study indicates that hPAH patients display dysfunctional Treg regardless of the leptin level whereas in iPAH and SSc-PAH patients, Treg leptin-dependent dysfunction could represent a pivotal and central element in disease onset and/or progression, as a possible consequence of dysfunctional
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pulmonary endothelium.
Acknowledgments:
Author Contribution: Conception and design: AH, LT, CG and MH. Analysis and interpretation: all. Drafting manuscript: AH, CG and MH. Guarantor of the paper: AH.
The authors thank Prof. Florent Soubrier, Dr. Mélanie Eyries, Dr. Barbara Girerd and Dr David Montani for
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their help in the study.
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26 Song Y, Jones JE, Beppu H, et al. Increased susceptibility to pulmonary hypertension in heterozygous BMPR2mutant mice. Circulation 2005; 112:553-562 ACCEPTED MANUSCRIPT 27 Sawada H, Saito T, Nickel NP, et al. Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension. J Exp Med 2014; 211:263-280 28 Hong KH, Lee YJ, Lee E, et al. Genetic ablation of the BMPR2 gene in pulmonary endothelium is sufficient to predispose to pulmonary arterial hypertension. Circulation 2008; 118:722-730 29 Tu L, De Man FS, Girerd B, et al. A critical role for p130Cas in the progression of pulmonary hypertension in humans and rodents. Am J Respir Crit Care Med 2012; 186:666-676 30 Pallandre JR, Brillard E, Crehange G, et al. Role of STAT3 in CD4+CD25+FOXP3+ regulatory lymphocyte generation: implications in graft-versus-host disease and antitumor immunity. J Immunol 2007; 179:7593-7604 31 Ulrich S, Nicolls MR, Taraseviciene L, et al. Increased regulatory and decreased CD8+ cytotoxic T cells in the blood of patients with idiopathic pulmonary arterial hypertension. Respiration 2008; 75:272-280 32 Chaudhry A, Samstein RM, Treuting P, et al. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity 2011; 34:566-578 33 Gavin MA, Torgerson TR, Houston E, et al. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc Natl Acad Sci U S A 2006; 103:6659-6664 34 Wang J, Ioan-Facsinay A, van der Voort EI, et al. Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur J Immunol 2007; 37:129-138 35 Huertas A, Tu L, Thuillet R, et al. Leptin signalling system as a target for pulmonary arterial hypertension therapy. European Respiratory Journal 2015; 45:1066-1080 36 Cracowski JL, Chabot F, Labarere J, et al. Proinflammatory cytokine levels are linked to death in pulmonary arterial hypertension. European Respiratory Journal 2014; 43:915-917 37 Heresi GA, Aytekin M, Hammel JP, et al. Plasma interleukin-6 adds prognostic information in pulmonary arterial hypertension. European Respiratory Journal 2014; 43:912-914 38 Soon E, Holmes AM, Treacy CM, et al. Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension. Circulation 2010; 122:920-927 39 Humbert M, Monti G, Brenot F, et al. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med 1995; 151:1628-1631 40 Perros F, Dorfmuller P, Souza R, et al. Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension. European Respiratory Journal 2007; 29:462-468 41 Tuder RM, Groves B, Badesch DB, et al. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am J Pathol 1994; 144:275-285 42 Isern RA, Yaneva M, Weiner E, et al. Autoantibodies in patients with primary pulmonary hypertension: association with anti-Ku. Am J Med 1992; 93:307-312 43 Hautefort A, Girerd B, Montani D, et al. T-helper 17 cell polarization in pulmonary arterial hypertension. Chest 2015; 147:1610-1620 44 Fruhbeck G. Intracellular signalling pathways activated by leptin. Biochem J 2006; 393:7-20 45 La Cava A, Matarese G. The weight of leptin in immunity. Nat Rev Immunol 2004; 4:371-379 46 Shalev I, Schmelzle M, Robson SC, et al. Making sense of regulatory T cell suppressive function. Semin Immunol 2011; 23:282-292 47 Li MO, Flavell RA. TGF-beta: a master of all T cell trades. Cell 2008; 134:392-404 48 Ruby CE, Yates MA, Hirschhorn-Cymerman D, et al. Cutting Edge: OX40 agonists can drive regulatory T cell expansion if the cytokine milieu is right. J Immunol 2009; 183:4853-4857 49 Lam E, Choi SH, Pareek TK, et al. Cyclin-dependent kinase 5 represses Foxp3 gene expression and Treg development through specific phosphorylation of Stat3 at Serine 727. Mol Immunol 2015; 67:317-324 50 Dewachter L, Adnot S, Guignabert C, et al. Bone morphogenetic protein signalling in heritable versus idiopathic pulmonary hypertension. European Respiratory Journal 2009; 34:1100-1110 51 Soon E, Crosby A, Southwood M, et al. Bone Morphogenetic Protein Receptor Type II Deficiency and Increased Inflammatory Cytokine Production. A Gateway to Pulmonary Arterial Hypertension. Am J Respir Crit Care Med 2015; 192:859-872 52 Miyata M, Sakuma F, Ito M, et al. Athymic nude rats develop severe pulmonary hypertension following monocrotaline administration. Int Arch Allergy Immunol 2000; 121:246-252 53 Taraseviciene-Stewart L, Nicolls MR, Kraskauskas D, et al. Absence of T cells confers increased pulmonary arterial hypertension and vascular remodeling. Am J Respir Crit Care Med 2007; 175:1280-1289 13
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Figure legends
Figure 1. Regulatory T lymphocytes (Treg) are dysfunctional in patients with idiopathic, heritable and connective tissue disease-associated pulmonary arterial hypertension
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A: Group data of flow cytometry analysis of intracellular staining with anti-phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3) antibody in Treg called pSTAT3+ cells, expressed as percentage of the total Treg population, in control subjects (n=20), idiopathic PAH (iPAH, n=30), heritable
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PAH (hPAH, n=18) and SSc associated PAH (SSc-PAH, n=14) patients. B: Group data of flow cytometry analysis of pSTAT3+ cells, expressed as percentage of the total Treg population, in SSc-PAH (n=14) and
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SSc (n=7) patients. C: Group data of flow cytometry analysis of intracellular staining with antiphosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3) antibody in Treg expressing leptin receptor (ObR) called pSTAT3+ cells, expressed as percentage of the total Treg ObR+ population, in control subjects (n=20), idiopathic PAH (iPAH, n=30), heritable PAH (hPAH, n=18) and SSc associated
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PAH (SSc-PAH, n=14) patients. D: Group data of flow cytometry analysis of pSTAT3+ cells, expressed as percentage of the total Treg ObR+ population, in SSc-PAH (n=14) and SSc (n=7) patients. Values are expressed as mean ± sem. *: p<0.05. **: p<0.01. NS: non significant.
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Figure 2. Normal regulatory T lymphocytes (Treg) count in patients with idiopathic, heritable and
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connective tissue disease-associated pulmonary arterial hypertension A: Group data of circulating Treg cell count, expressed as percentage of the total CD4+ lymphocyte population, in control subjects (n=20), idiopathic PAH (iPAH, n=30), heritable PAH (hPAH, n=18) and SSc associated PAH (SSc-PAH, n=14) patients. B: Group data of circulating Treg cell count, expressed as percentage of the total CD4+ lymphocyte population, in SSc-PAH (n=14) and SSc (n=7) patients. Values are expressed as mean ± sem. NS: non significant.
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Figure 3. Circulating leptin is increased in idiopathic (i) and scleroderma (SSc) associated pulmonary
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arterial hypertension (PAH) patients but not in heritable PAH (hPAH) patients A: Group data of leptin level in serum from controls (n=20), iPAH (n=30), hPAH (n=18) and SSc-PAH (n=14) patients. B: Group data of leptin level in serum from SSc-PAH (n=14) and SSc (n=7) patients.
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Values are expressed as mean ± sem. *: p<0.05. **: p<0.01. NS: non significant. Figure 4. Leptin receptor (ObR) is differentially expressed in patients with pulmonary arterial hypertension (PAH) and scleroderma (SSc)
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A: Group data of ObR expression on circulating regulatory T lymphocytes (Treg), called ObR+ cells expressed as percentage of the total Treg population, in control subjects (n=20), idiopathic PAH (iPAH,
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n=30), heritable PAH (hPAH, n=18) and SSc associated PAH (SSc-PAH, n=14) patients. B: Group data of ObR expression on Treg, called ObR+ cells expressed as percentage of the total Treg population, in SScPAH (n=14) and SSc (n=7) patients. C-D: Correlation between ObR+ cells, expressed as percentage of the total Treg population and leptin in iPAH (n=30), hPAH (n=18), SSc-PAH (n=14) and SSc (n=7) patients (C)
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and in controls (n=20) (D). E-F: Correlation between ObR+ cells, expressed as percentage of the total Treg population and body mass index (BMI) in iPAH (n=30), hPAH (n=18), SSc-PAH (n=14) and SSc (n=7) patients (E) and in controls (n=20) (F). Values are expressed as mean ± sem. ***: p<0.001. NS: non
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significant.
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Figure 5. Leptin inhibition attenuates decrease regulatory T lymphocytes (Treg) function in chronic hypoxia-induced PH in rats
A: Group data of flow cytometry analysis of intracellular staining with anti-phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3) antibody in Treg called pSTAT3+ cells, expressed as percentage of the total Treg population, in wildtype and transgenic rats lacking leptin receptor (ZDF), exposed to either exposed to normoxia or 3 weeks of chronic hypoxia (hypoxia). B: Group data of Treg cell count, expressed as percentage of the total CD4+ lymphocyte population, in wildtype and ZDF rats, exposed to either normoxia or hypoxia. Values are expressed as mean ± sem. * p<0.05. NS: non significant. 15
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Table 1. Characteristics of idiopathic and heritable pulmonary arterial hypertension patients and controls
hPAH n=18 51 ± 3.3* 6 / 12 (0.43)
25 ± 1
26 ± 1
25 ± 1
NA NA NA NA
10 2 11 2
0 2 6 3
2
2
NA NA
3 0
5 0
NA NA NA NA
3 18 9 0
6 8 4 0
NA NA NA NA NA NA
95 ± 24 461 ± 18 47.3 ± 2.5 5.8 ± 0.3 7.3 ± 0.6 8.4 ± 0.5
75 ± 29 484 ± 20 46.3 ± 2.6 5.1 ± 0.4 8.5 ± 1.2 7.9 ± 0.7
2
BMI, Kg/m
Specific PAH Therapy - ERA - PDE5i - ERA + PDE5i - ERA + PGI2 - PDE5i + PGI2 - ERA + PDE5i + PGI2 - No Treatment
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BNP, ng/L 6-MWD, m mPAP, mmHg CO, L/min PVR, Wood units PCWP, mmHg
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NYHA functional class, n class I class II class III class IV
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iPAH n=30 55 ± 2.5* 6 / 24 (0.25)
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Age, years Sex, M/F (ratio)
Controls n=20 47 ± 2.4 9 / 11 (0.82)
*=statistical significance compared to controls; 6-MWD=6-minute walk distance; BMI=body mass index; BNP=brain natriuretic peptide; CO=cardiac output; ERA=endothelin receptor antagonists; hPAH=heritable pulmonary arterial hypertension; iPAH=idiopathic pulmonary arterial hypertension; M/F=male/female; mPAP=mean pulmonary artery pressure; NA = not applicable; NYHA=New York Heart Association; PDE5i=Phophodiesterase 5 inhibitors; PCWP=pulmonary capillary wedge pressure; PGI2 =prostaglandin I2; PVR= pulmonary vascular resistance.
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Table 2. Characteristics of scleroderma associated pulmonary arterial hypertension patients, scleroderma patients ACCEPTED MANUSCRIPT and controls
Specific PAH Therapy - ERA - PDE5i - ERA + PDE5i - ERA + PGI2
25 ± 1
26 ± 1
24 ± 1
4 2 4 0
NA NA NA NA
1
NA
3 0 0
NA NA 0
NA NA NA NA
1 5 6 2
NA NA NA NA
NA NA NA NA NA NA
239 ± 76 320± 36 42.2 ± 3.0 5.7 ± 0.5 6.2 ± 0.9 10.1 ± 1.3
NA NA NA NA NA NA
NA NA NA NA
- PDE5i + PGI2
NA
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NYHA functional class, n class I class II class III class IV
NA NA NA
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- ERA + PDE5i + PGI2 - No Treatment Corticosteroids and/or immunosuppresants
BNP, ng/L 6-MWD, m mPAP, mmHg CO, L/min PVR, Wood units PCWP, mmHg
SSc n=7 64 ± 3.6* 0 / 7 (0.0)
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BMI, Kg/m
SSc-PAH n=14 71 ± 2.9* 2 / 12 (0.17)
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Age, years Sex, M/F (ratio)
Controls n=20 47 ± 2.4 9 / 11 (0.82)
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*=statistical significance compared to controls; 6-MWD=6-minute walk distance; BMI=body mass index; BNP= brain natriuretic peptide; CO=cardiac output; ERA=endothelin receptor antagonists; iPAH=idiopathic pulmonary arterial hypertension; M/F=male/female; mPAP=mean pulmonary artery pressure; NA = not applicable; NYHA= New York Heart Association; PDE5i=Phophodiesterase 5 inhibitors; PCWP=pulmonary capillary wedge pressure; PGI2=prostaglandin I2; PVR= pulmonary vascular resistance; SSc=scleroderma; SSc-PAH=scleroderma associated pulmonary arterial hypertension
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+
Treg number (% CD4 cells) +
Treg ObR cells (% Treg) +
pSTAT3 cells (% Treg) +
+
pSTAT3 cells (% Treg ObR )
iPAH n=30
hPAH n=18
SSc-PAH n=14
SSc n=7
5.0 ± 0.4
4.2 ± 0.4
5.2 ± 0.6
3.5 ± 0.3
4.3 ± 0.6
9.2 ± 2.2
37.6 ± 3.2*
8.5 ± 1.5
37.7 ± 6.9*
60.4 ± 1.7*
7.3 ± 1.1
3.5 ± 0.5*
4.2 ± 0.6*
2.9 ± 0.3*
4.0 ± 0.5*
11.7 ± 1.5
4.7 ± 0.6*
6.3 ± 0.6*
4.5 ± 0.9*
6.6 ± 0.7*
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Controls n=20
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*=statistical significance compared to controls; ObR= leptin receptor; pSTAT3=phosphorylated signal tranducer and activator of transcription 3; Treg= regulatory T lymphocytes
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Table 4. Characteristics of wildtype and ZDF rats in normoxia and hypoxia
Wildtype
ZDF
Normoxia n=4
Hypoxia n=4
Normoxia n=5
Hypoxia n=8
217 ± 4
213 ± 4
254 ± 4
252 ± 5
18.2 ± 0.6
32.8 ± 0.5 *
17.3 ± 0.2
27.7 ± 0.9
#,$
0.2 ± 0.02
0.6 ± 0.12 *
0.1 ± 0.01
0.4 ± 0.04
#,$
100 ± 6
49 ± 7 *
128 ± 2
79 ± 7
1.3 ± 0.2
2.2 ± 0.2 *
1.1 ± 0.1
1.7 ± 0.1
Muscularized pulmonary arteries, %
13 ± 4
71 ± 4 *
15 ± 5
48 ± 3
Wall thickness, %
27 ± 2
34 ± 2 *
19 ± 2
23 ± 2
1.9 ± 0.3
7.8 ± 0.5 *
2.4 ± 0.4
3.8 ± 0.3
5.7 ± 1.9
4.5 ± 1.5
4.8 ± 0.7
5.8 ± 0.9
8.3 ± 0.6
3.5 ± 0.2 *
5.3 ± 1.2
4.5 ± 0.3
mPAP, mmHg PVR, mmHg/mL/min CO, ml/min
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Body weight, g
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Fulton index/body weight ratio (x10 )
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PCNA+ cells/vessel, % +
Treg number (% CD4 cells) +
pSTAT3 cells (% Treg)
#
#,$ #,$
#,$ $ #,$
*=statistical significance compared to normoxic wildtype rats; #=statistical significance compared to normoxic ZDF rats; $=statistical significance compared to hypoxic wildtype rats; CO= cardiac output; mPAP= mean pulmonary arterial pressure; PCNA= proliferating cell nuclear antigen; PVR= pulmonary vascular resistance; pSTAT3=phosphorylated signal tranducer and activator of transcription 3; Treg= regulatory T lymphocytes.
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S0012-3692(16)00461-X
DOI:
10.1016/j.chest.2016.01.004
Reference:
CHEST 242
To appear in:
CHEST
Received Date: 6 October 2015 Revised Date:
27 November 2015
Accepted Date: 4 January 2016
Please cite this article as: Huertas A, Phan C, Bordenave J, Tu L, Thuillet R, Le Hiress M, Avouac J, Tamura Y, Allanore Y, Jovan R, Sitbon O, Guignabert C, Humbert M, Regulatory T cell dysfunction in idiopathic, heritable and connective tissue-associated pulmonary arterial hypertension, CHEST (2016), doi: 10.1016/j.chest.2016.01.004. 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.
Text word count: 2762
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Abstract word count: 242
Regulatory T cell dysfunction in idiopathic, heritable and connective tissue-associated pulmonary arterial hypertension Running title: Regulatory T lymphocytes in PAH
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Alice Huertas, MD, PhD1, 2, 3, Carole Phan, MSc1, 2, Jennifer Bordenave MSc1, 2, Ly Tu, PhD1, 2, Raphaël Thuillet1, 2, Morane Le Hiress, PhD1, 2, Jérôme Avouac, MD, PhD4, Yuichi Tamura, MD, PhD1, 2, Yannick Allanore, MD, PhD4, Roland Jovan, MD1, 2, 3, Olivier Sitbon, MD, PhD1, 2, 3, Christophe Guignabert, PhD1, 2 and Marc Humbert, MD, PhD1, 2, 3
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Affiliations: Inserm UMR_S 999, Hôpital Marie Lannelongue, Le Plessis Robinson, France; 2. Univ. Paris-Sud, Faculté de Médecine, Université Paris-Saclay, Le Kremlin Bicêtre, France; 3. AP-HP, Service de Pneumologie, Hôpital Bicêtre, Le Kremlin Bicêtre, France; 4. Cochin Hospital, Paris Descartes University, INSERM U1016 and CNRS UMR8104, AP-HP, Paris, France.
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1.
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Correspondence to: Dr Alice Huertas, MD, PhD INSERM UMR_S 999, Hôpital Marie Lannelongue, 133 Avenue de la Résistance, 92350 Le PlessisRobinson, FRANCE. E-mail: [email protected]
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Disclosures: AH, CP, JB, LT, RT, MLH, JA, YT, RJ and CG have no conflict of interest to declare. YA has/had consultancy relationship and/or has received research funding in relationship with the treatment of systemic sclerosis from Actelion, Bayer, Biogen Idec, Bristol-Myers Squibb, Genentech/ Roche, Inventiva, Medac, Pfizer, Sanofi/Genzyme, Servier and UCB. OS has relationships with pharmaceutical companies including Actelion Pharmaceuticals, Bayer HealthCare, GlaxoSmithKline, Pfizer and United Therapeutics. In addition to being an investigator in trials involving these companies, relationships include consultancy service and membership of scientific advisory boards. MH has relationships with drug companies including Actelion, Bayer Schering, GSK, Lilly, Novartis, Pfizer and United Therapeutics. In addition to being an investigator in trials involving these companies, relationships include consultancy service and membership of scientific advisory boards. Funding footnotes: This research was supported by grants from the French National Institute for Health and Medical Research (INSERM, the French National Agency for Research: grant no. ANR_12_JSV1_0004_01), the Association des Sclérodermiques de France (ASF) and in part by the Département Hospitalo-Universitaire (DHU) Thorax Innovation (TORINO) MLH was supported by the LabEx LERMIT (grant no ANR-10-LABX-0033-LERMIT). YT received a personal ERS/EU Marie Curie grant post-doctoral fellowship (RESPIRE2). CP is supported by the Fonds de Dotation "Recherche en Santé Respiratoire" - Fondation du Souffle.
ABSTRACT
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Pulmonary arterial hypertension (PAH) encompasses a group of conditions with distinct causes. Dysimmunity is a common feature of all forms of PAH and contributes to both disease susceptibility and/or progression. Regulatory T lymphocytes (Treg) are dysfunctional in idiopathic PAH (iPAH) patients in a
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leptin-dependent manner. However, it is not known whether these abnormalities are specific to iPAH. Hence, we hypothesized that (1) Treg dysfunction is also present in heritable (hPAH) and connective tissue disease-associated PAH (CTD-PAH); (2) defective leptin-dependent signaling is present in hPAH and CTD-
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PAH and could contribute to Treg dysfunction; (3) modulating leptin axis in vivo could protect against Treg dysfunction; (4) restoration of Treg activity could limit and/or reverse experimental chronic hypoxia (Hx)-
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induced pulmonary hypertension (PH) in vivo. We analysed 62 patients with PAH (30 iPAH, 18 hPAH and 14 CTD-PAH), 10 CTD patients without PAH and 20 healthy controls. Our results indicate that Treg are dysfunctional in all PAH forms tested, as well as in CTD patients without PAH. Importantly, leptin axis is crucial in Treg dysfunction in iPAH and CTD patients (with or without PAH), whereas in hPAH, Treg are
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altered in a leptin independent manner. Using leptin receptor (ObR)-deficient rats which develop less severe Hx-PH, we found that ObR-deficient rats are protected against decreased Treg function after Hx exposure. Taken altogether, our results suggest that Treg dysfunction is common to all forms of PAH and may
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contribute to the development and/or the progression of the disease.
Keywords: Pulmonary arterial hypertension; Leptin; Lymphocytes; Scleroderma
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INTRODUCTION Pulmonary arterial hypertension (PAH, Group 1, is a severe cardiopulmonary disorder characterized by
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chronic pre-capillary pulmonary hypertension (PH), defined by a mean pulmonary artery pressure at rest ≥ 25 mm Hg, in the presence of a normal pulmonary capillary wedge pressure (≤ 15 mm Hg)1,2. Despite rapid development of specific therapies, PAH carries a high mortality rate3,4. PAH may occur in a number of
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different clinical contexts: without an identifiable underlying cause (idiopathic or iPAH), or induced by drugs and toxins, or associated with other diseases, including connective tissue disease (CTD-PAH), portal
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hypertension, human immunodeficiency virus (HIV) infection2. When PAH occurs in a familial context, germline mutations in the Bone Morphogenetic Protein Receptor type 2 (BMPR2) gene are detected in at least 70% of cases, and around 20% of so-called idiopathic cases carry a BMPR2 mutation (heritable PAH or hPAH)5. Although different forms of PAH could reflect distinct physiopathological mechanisms, current
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evidence strongly suggests that inflammation and autoimmune disorders are common to all forms6,7. In particular, it is now widely accepted that immunological disorders contribute to both disease susceptibility and progression of vascular remodeling in PAH8-12. Nevertheless, the initiating triggers of dysimmunity and
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the exact mechanisms by which altered immune responses contribute to the disease development and/or progression of PAH remain unknown. Furthermore, to which extent immune disorders are implicated in the
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different forms of PAH is still an open question. In the immune system, CD4+ CD25+ Foxp3+ regulatory T lymphocytes (Treg) represent a key cell type: they are potent immunomodulators of the adaptive immune system, the proinflammatory cytokine responses and the activation of monocytes/macrophages13,14. Association between autoimmune disorders and PAH has been recognized for many years15-21. The absence of Treg cells in athymic nude rats leads to activated macrophage recruitment in lungs and exaggerated pulmonary vascular remodeling, following vascular injury with the vascular endothelial growth factor (VEGF) receptor inhibitor SU-541622,23. It has been
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demonstrated that Treg cells are dysfunctional in patients with iPAH24 and that this adaptive immune
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dysregulation may be partly explained by the excessive pulmonary endothelial cell (EC)-derived leptin present in these patients24, suggesting that leptin could represent a missing link between pulmonary endothelial dysfunction and immunological disorders in iPAH. The significance of this metabolic factor in other forms of PAH is still unexplored, in particular in patients who exhibit dysimmunity such as hPAH with
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BMPR2 mutations or systemic sclerosis (SSc), the most frequent autoimmune disease associated with PAH25. Indeed, recent publications underline a strong link between the presence of a BMPR2 mutation and an inappropriate inflammation within the context of PAH26-28. It has been shown that mice heterozygous for
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a null allele in Bmpr2 are more susceptible to develop PH under inflammatory stress when compared to control mice26 and that loss of endothelial Bmpr2 is sufficient to predispose to PH in association with
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perivascular inflammatory infiltration28. Finally, it has recently been demonstrated that this link is partly due to a p38-MK2 mechanism27.
In this study, we determined whether: 1) Treg dysfunction is identified in hPAH due to BMPR2 mutations and in CTD-PAH patients with SSc; 2) defective leptin-dependent signal transduction is present in hPAH
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and CTD-PAH and could contribute to Treg dysfunction as in iPAH; 3) modulating leptin axis in vivo could protect against Treg dysfunction; 4) the effective restoration of normal Treg activity could limit or even
Subjects
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METHODS
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reverse experimental chronic hypoxia-induced PH in vivo.
Adult PAH patients had a diagnosis confirmed by right-heart catheterization and a stable clinical and hemodynamic status for the last 3 months. Adult SSc patients had clinical stability for the last 3 months (with no need of chronic immunosuppressive and/or corticosteroid therapy) and exclusion of PAH by means of right-heart catheterization. Exclusion criteria for all patient groups were diabetes, metabolic syndrome and immunosuppressive and/or corticosteroid therapy in the last 3 months (Tables 1 and 2). Characteristics at diagnosis and follow-up were stored in the Registry of the French Network of PH in agreement with 4
French bioethics laws (Commission Nationale de l'Informatique et des Libertés). This study was approved
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by the local ethics committee (CPP Ile-de-France VII, Paris, France) and all patients signed written informed consent.
Animal models and hemodynamic measurements
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Zucker diabetic fatty (ZDF, Charles River Laboratories) male (4 weeks old) Sprague-Dawley rats (ZDF/Lepr fa/fa) and age matched lean control rats (ZDF/Lean Ctrl) were exposed to normoxia or normobaric hypoxia (FiO2 = 10 %) for 3 consecutive weeks. As previously described29, animals were
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anesthetized with isoflurane for catheterization. A polyvinyl catheter was introduced into the right jugular vein and pushed through the right ventricle into the pulmonary artery. Another polyethylene catheter was
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inserted into the right carotid artery. After measurement of hemodynamic parameters, blood was removed for peripheral blood mononuclear cell (PBMC) analyses. Animal studies were approved by the administrative panel on animal care from Université Paris-Sud (France).
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Flow cytometry analyses
After blood samples withdrawal, PBMCs from humans (patients and controls) or rats were obtained by standard Ficoll gradient centrifugation. The cells were carefully washed with PBS and resuspended in a
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staining buffer containing 10% of human serum. The cells were then fluorescently labeled with the following antibodies under non-permeabilized or permeabilized conditions (IntraPrep, following
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manufacturer’s instructions, Beckman Coulter), to enable surface or intracellular staining, respectively: in humans, fluorophore conjugated monoclonal anti-CD4, monoclonal anti-CD25 and monoclonal anti-CD127 (Treg cocktail), monoclonal anti-phosphorylated STAT3 (Y705 site) (Becton Dickinson) and fluorophore conjugated monoclonal anti-ObR (R&D Systems). In rats, fluorophore conjugated monoclonal anti-CD4, anti-CD25 and anti-FoxP3 (eBioscience) and monoclonal anti-phosphorylated STAT3 (Becton Dickinson). Flow cytometry gating conditions and the mean fluorescence intensity (MFI) were set and normalized, respectively, against isotype- and fluorophore-matched non-immune IgGs. Human Treg phenotype was defined as CD4+ CD25+ CD127low cells and rat Treg phenotype was defined as CD4+ CD25+ Foxp3+ cells 5
and was expressed as the percentage of total CD4+ cells. Flow cytometry data were acquired with a flow
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cytometer (MACSQuant) and analyzed by FlowJo software program (Tree Star, Inc). Statistical analyses Results are expressed as means ± SEM. A p<0.05 level of statistical significance was used for all analyses.
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All between-groups comparisons were assessed using One Way ANOVA in humans; post-hoc analysis of significant variables was performed using Bonferroni correction with all pairwise multiple comparisons. Differences between two selected groups (SSc-PAH and SSc patients) were compared using unpaired t-test.
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All between-groups comparisons in rats were assessed using non parametric Kruskal-Wallis analysis with Dunn’s correction. Pearson correlations were used to establish associations between the dependent variables
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and relevant independent variables. All statistical procedures were carried out using GraphPad Prism version 5.0 (GraphPad Software Inc.)
Patient populations
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RESULTS
Blood samples were collected from 62 patients with PAH, corresponding to iPAH (n=30), hPAH due to a
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BMPR2 mutation (n=18) and SSc-PAH (n=14) (Table 1). Seven SSc patients without PAH and 20 control subjects were also studied. Tables 1 and 2 summarize patient anthropometric, functional and hemodynamic
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characteristics.
Dysfunction of Treg cells in idiopathic, heritable and connective tissue disease-associated pulmonary arterial hypertension
To determine the functional status of Treg function, we measured Treg STAT3 phosphorylation (pSTAT3) as STAT3 represents an important signaling pathway for Treg activity30. We measured pSTAT3 levels in iPAH and hPAH patients carrying a BMPR2 mutation, SSc-PAH and SSc patients without PAH, as well as in control subjects. After Treg permeabilization, pSTAT3 quantification by flow cytometry revealed that the number of Treg-pSTAT3+ in the whole Treg population as well as the Treg-STAT3+ in the Treg 6
subpopulation expressing ObR were markedly decreased in all patients compared to controls. Interestingly,
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the number of Treg-pSTAT3 was similar among all groups of PAH as well as in SSc patients without PAH (Figure 1 and Table 3). Importantly, flow cytometry analyses revealed a normal Treg cell count in all groups, controls, PAH (iPAH, hPAH and SSc-PAH) and SSc patients, expressed as a percentage of CD4+ cells (Figure 2 and Table 3). These results are consistent with our previous work24, while others have
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suggested an increase in Treg cell number in iPAH patients31. Although influences of genetic and environmental factors cannot be excluded in these studies32, it is important to underlie that Ulrich et al. described an increase in FoxP3 in the total CD4+ cell population, whereas it is known that FoxP3 expression
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is no longer restricted to Treg and hence is not sufficient to identify Treg33,34. Interestingly, and consistently with our findings, Ulrich and coworkers did not find differences in FoxP3 expression in the Treg population
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between iPAH and controls31. Our present results indicate that Treg cell counts are normal but that Treg are substantially dysfunctional in all forms of PAH studied, to the same degree as autoimmune diseases, such as SSc.
Leptinemia in iPAH, hPAH and SSc-PAH compared with control and SSc patients
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Our recent work in iPAH patients has showed that dysfunctional pulmonary endothelium synthetizes abnormally high levels of leptin that contribute to altered functional status of circulating Treg24. However, the levels of leptinemia in hPAH and in SSc patients without PAH have not been investigated so far. First,
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we measured circulating levels of leptin in patients with iPAH, hPAH and SSc-PAH compared with control subjects and/or SSc patients. Of note, all patients and controls displayed similar body mass index (BMI). As
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previously published24, the leptinemia was higher in iPAH and SSc-PAH (with no difference between these groups) compared to controls. By contrast, hPAH patients and control subjects had normal leptinemia (Figure 3 and Table 1). We then compared SSc patients with or without PAH and there was no difference within these two groups (Figure 3 and Table 2). Since no correlation was found between serum leptin levels and BMI, disease severity and pro-inflammatory cytokine levels in our previous study, we did not perform these analyses. Our results indicate here that hPAH patients had a normal level of circulating leptin whereas in iPAH and SSc patients, with or without PAH, circulating leptin levels are highly increased compared to controls. 7
ObR expression on Treg cell membrane isolated from iPAH, hPAH and SSc-PAH compared with control
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subjects and SSc patients We next determined the expression level of leptin receptor ObR on Treg cell surface. We selected Treg cells among the fluorescently tagged PBMCs by flow cytometry analyses after peripheral venous blood withdrawal in controls and PAH patients. ObR expression on Treg membrane was markedly and similarly
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increased in iPAH and SSc-PAH patients, and even more in SSc patients without PAH. By contrast, hPAH patients displayed a normal ObR expression level on Treg with no difference compared to controls (Figure 4A-B and Table 3). Interestingly, the expression of ObR on Treg cell membrane did not correlate with
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serum leptin levels or BMI (Figure 4C-F and Table 3). Taken together, these findings suggest that leptin and its receptor expression on circulating Treg are not altered in hPAH as opposed to iPAH and SSc-PAH.
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Importantly, our findings indicate that the level of ObR expression is independent of the patients’ BMI or serum leptin levels.
Leptin axis modulates decrease Treg function in chronic hypoxia-induced PH in rats We then questioned to which extent leptin could play a role on Treg function in vivo and whether the
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presence of functional Treg could limit or even reverse experimental chronic hypoxia-induced PH. To this aim, we used rats lacking ObR, called Zucker Diabetic Fatty (ZDF) rats (ZDF/Lepr fa/fa) and exposed them to chronic hypoxia (FiO2 10%) or normoxia for three weeks and compared them to their wildtype littermates
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(ZDF/Lepr Crl), also exposed to chronic hypoxia or normoxia. Wildtype rats exposed to chronic hypoxia
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displayed decreased Treg function with decreased pSTAT3 level compared to wildtype normoxic animals. Interestingly, ZDF rats displayed a normal pSTAT3 level in normoxia and in hypoxia (Figure 5A and Table 4). Of note, Treg count was normal in all groups (Figure 5B). Overall, these results, together with our recent data indicating that leptin pathway contributes to chronic hypoxia-induced PH susceptibility35, suggest that decrease Treg function may contribute to chronic hypoxia-induced PH onset and/or progression.
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DISCUSSION
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To address if similar or distinct pathomechanisms contribute to Treg dysfunction in PAH, we analyzed Treg cell numbers and functional activity in iPAH, hPAH and SSc-PAH patients and compared our observations with those obtained in control and SSc patients with no evidence of PAH. We show, for the first time, that Treg cells are altered in all these PAH subgroups. In addition, our data highlight the
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differences in pathophysiological mechanisms leading to Treg dysfunction among iPAH, hPAH and SScPAH. Indeed, our results indicate that in iPAH and SSc-PAH patients, the leptin axis is crucial in Treg dysfunction whereas in hPAH, Treg cells are altered independently of the leptin signaling system. Consistent
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with these human findings, we are demonstrating here that ObR-deficient rats, which develop less severe PH35, are protected against decrease Treg function when exposed to chronic hypoxia. Taken together, our
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results support the concept that in the absence of appropriate Treg activity, pulmonary vascular injury may lead to progressive elevations of pulmonary artery pressure and PH.
Although it is now clear that PAH patients display inflammatory and immunologic disorders and that some inflammatory biomarkers predict mortality36-38, the causes of dysimmunity in the different forms of
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PAH development and/or progression are still not fully understood8,11,17,18,39-43. Our study presented here is the first to provide direct experimental evidence that Treg are equally dysfunctional regardless of the cause
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of PAH and that different pathomechanisms may explain this abnormality. Although STAT3 represents an important transcription factor for Treg activity30 and the main leptin-dependent signaling pathway44,45,
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further studies are needed to better characterize the phenotypic and functional status of human Treg cells in healthy individuals and PAH patients by in vitro suppression assay. Whereas it is well known that Treg control immune homeostasis by maintaining self-tolerance and suppressing a variety of physiological and pathological immune responses to non-self antigens13, the exact mechanisms by which they exert their function are not completely understood46,47. It has also been described that Treg are critical in limiting the extent of inflammation and in protecting effects against vascular remodeling13,22,23,32. We are indicating here new aspects of immune homeostasis as we are showing that leptin down-regulates Treg ability to control other immune cells in iPAH and SSc-PAH but not in hPAH due to BMPR2 mutations. While we suggest a 9
role for leptin in iPAH and SSc-PAH, Treg dysfunction in hPAH is not well understood. It needs to be noted
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that different biologic outcomes depend upon the influence from the local cytokine milieu32,48,49 thus we cannot exclude that our findings may be influenced by the local cytokine milieu and the differentiation status of the Treg. However, BMPRII-dependent signaling pathways might be implicated in Treg regulation acting on the level of STAT3 phosphorylation through classical Smad 1/5/8 signaling or p38 mitogen-activated
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protein kinase (MAPK) pathway, as it has been shown that hPAH patients display defective Smad signaling compensated for by an activation of p38MAPK signaling50. Further studies are needed to test these hypotheses and address this specific question. Interestingly, recent data indicate that BMPR-II deficiency
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promotes an exaggerated inflammatory response which can instigate PH development51.
Our recent study performed in iPAH patients and in rodent PH models indicate that increased
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leptinemia and leptin local production by dysfunctional pulmonary ECs contribute to vascular remodeling by acting on pulmonary ECs, pulmonary artery smooth muscle cell and perivascular monocyte/macrophage accumulation24,35. Despite these findings, the effect of pulmonary EC-derived leptin on systemic dysimmunity remains unclear. The present data highlight another role for pulmonary EC-derived leptin,
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suggesting a crosstalk between pulmonary dysfunctional ECs and Treg dysfunction21. Using ObR deficient rats, which are protected against PH development in chronic hypoxia35, we have shown a possible connection between pulmonary EC-derived leptin, dysimmunity and susceptibility of PH development.
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While pulmonary EC dysfunction may lead to altered Treg via excessive leptin secretion, the effects of
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dysfunctional Treg on pulmonary vascular remodeling is still unclear. Interestingly, it has been demonstrated that T cell-deficient athymic rats develop exaggerated PH and display excessive inflammation and leukotriene B4-secreting macrophage recruitment23,52,53. Furthermore, it has been reported that the pulmonary vasculature is sensitive to autoimmune stimuli, such as autoantibodies directed against pulmonary vasculature components, and that the vascular wall can respond by developing abnormal proliferative and apoptotic resistant phenotype16. Consistent with these notions, Treg dysfunction in PAH could contribute to disease onset and/or progression.
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However, several questions remain unanswered and in particular whether dysimmunity represents a
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cause or an effect of PAH onset is still unclear. In summary, our study indicates that hPAH patients display dysfunctional Treg regardless of the leptin level whereas in iPAH and SSc-PAH patients, Treg leptin-dependent dysfunction could represent a pivotal and central element in disease onset and/or progression, as a possible consequence of dysfunctional
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pulmonary endothelium.
Acknowledgments:
Author Contribution: Conception and design: AH, LT, CG and MH. Analysis and interpretation: all. Drafting manuscript: AH, CG and MH. Guarantor of the paper: AH.
The authors thank Prof. Florent Soubrier, Dr. Mélanie Eyries, Dr. Barbara Girerd and Dr David Montani for
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their help in the study.
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References:
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Figure legends
Figure 1. Regulatory T lymphocytes (Treg) are dysfunctional in patients with idiopathic, heritable and connective tissue disease-associated pulmonary arterial hypertension
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A: Group data of flow cytometry analysis of intracellular staining with anti-phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3) antibody in Treg called pSTAT3+ cells, expressed as percentage of the total Treg population, in control subjects (n=20), idiopathic PAH (iPAH, n=30), heritable
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PAH (hPAH, n=18) and SSc associated PAH (SSc-PAH, n=14) patients. B: Group data of flow cytometry analysis of pSTAT3+ cells, expressed as percentage of the total Treg population, in SSc-PAH (n=14) and
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SSc (n=7) patients. C: Group data of flow cytometry analysis of intracellular staining with antiphosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3) antibody in Treg expressing leptin receptor (ObR) called pSTAT3+ cells, expressed as percentage of the total Treg ObR+ population, in control subjects (n=20), idiopathic PAH (iPAH, n=30), heritable PAH (hPAH, n=18) and SSc associated
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PAH (SSc-PAH, n=14) patients. D: Group data of flow cytometry analysis of pSTAT3+ cells, expressed as percentage of the total Treg ObR+ population, in SSc-PAH (n=14) and SSc (n=7) patients. Values are expressed as mean ± sem. *: p<0.05. **: p<0.01. NS: non significant.
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Figure 2. Normal regulatory T lymphocytes (Treg) count in patients with idiopathic, heritable and
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connective tissue disease-associated pulmonary arterial hypertension A: Group data of circulating Treg cell count, expressed as percentage of the total CD4+ lymphocyte population, in control subjects (n=20), idiopathic PAH (iPAH, n=30), heritable PAH (hPAH, n=18) and SSc associated PAH (SSc-PAH, n=14) patients. B: Group data of circulating Treg cell count, expressed as percentage of the total CD4+ lymphocyte population, in SSc-PAH (n=14) and SSc (n=7) patients. Values are expressed as mean ± sem. NS: non significant.
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Figure 3. Circulating leptin is increased in idiopathic (i) and scleroderma (SSc) associated pulmonary
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arterial hypertension (PAH) patients but not in heritable PAH (hPAH) patients A: Group data of leptin level in serum from controls (n=20), iPAH (n=30), hPAH (n=18) and SSc-PAH (n=14) patients. B: Group data of leptin level in serum from SSc-PAH (n=14) and SSc (n=7) patients.
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Values are expressed as mean ± sem. *: p<0.05. **: p<0.01. NS: non significant. Figure 4. Leptin receptor (ObR) is differentially expressed in patients with pulmonary arterial hypertension (PAH) and scleroderma (SSc)
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A: Group data of ObR expression on circulating regulatory T lymphocytes (Treg), called ObR+ cells expressed as percentage of the total Treg population, in control subjects (n=20), idiopathic PAH (iPAH,
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n=30), heritable PAH (hPAH, n=18) and SSc associated PAH (SSc-PAH, n=14) patients. B: Group data of ObR expression on Treg, called ObR+ cells expressed as percentage of the total Treg population, in SScPAH (n=14) and SSc (n=7) patients. C-D: Correlation between ObR+ cells, expressed as percentage of the total Treg population and leptin in iPAH (n=30), hPAH (n=18), SSc-PAH (n=14) and SSc (n=7) patients (C)
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and in controls (n=20) (D). E-F: Correlation between ObR+ cells, expressed as percentage of the total Treg population and body mass index (BMI) in iPAH (n=30), hPAH (n=18), SSc-PAH (n=14) and SSc (n=7) patients (E) and in controls (n=20) (F). Values are expressed as mean ± sem. ***: p<0.001. NS: non
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significant.
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Figure 5. Leptin inhibition attenuates decrease regulatory T lymphocytes (Treg) function in chronic hypoxia-induced PH in rats
A: Group data of flow cytometry analysis of intracellular staining with anti-phosphorylated Signal Transducer and Activator of Transcription 3 (pSTAT3) antibody in Treg called pSTAT3+ cells, expressed as percentage of the total Treg population, in wildtype and transgenic rats lacking leptin receptor (ZDF), exposed to either exposed to normoxia or 3 weeks of chronic hypoxia (hypoxia). B: Group data of Treg cell count, expressed as percentage of the total CD4+ lymphocyte population, in wildtype and ZDF rats, exposed to either normoxia or hypoxia. Values are expressed as mean ± sem. * p<0.05. NS: non significant. 15
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Table 1. Characteristics of idiopathic and heritable pulmonary arterial hypertension patients and controls
hPAH n=18 51 ± 3.3* 6 / 12 (0.43)
25 ± 1
26 ± 1
25 ± 1
NA NA NA NA
10 2 11 2
0 2 6 3
2
2
NA NA
3 0
5 0
NA NA NA NA
3 18 9 0
6 8 4 0
NA NA NA NA NA NA
95 ± 24 461 ± 18 47.3 ± 2.5 5.8 ± 0.3 7.3 ± 0.6 8.4 ± 0.5
75 ± 29 484 ± 20 46.3 ± 2.6 5.1 ± 0.4 8.5 ± 1.2 7.9 ± 0.7
2
BMI, Kg/m
Specific PAH Therapy - ERA - PDE5i - ERA + PDE5i - ERA + PGI2 - PDE5i + PGI2 - ERA + PDE5i + PGI2 - No Treatment
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BNP, ng/L 6-MWD, m mPAP, mmHg CO, L/min PVR, Wood units PCWP, mmHg
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NYHA functional class, n class I class II class III class IV
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NA
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iPAH n=30 55 ± 2.5* 6 / 24 (0.25)
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Age, years Sex, M/F (ratio)
Controls n=20 47 ± 2.4 9 / 11 (0.82)
*=statistical significance compared to controls; 6-MWD=6-minute walk distance; BMI=body mass index; BNP=brain natriuretic peptide; CO=cardiac output; ERA=endothelin receptor antagonists; hPAH=heritable pulmonary arterial hypertension; iPAH=idiopathic pulmonary arterial hypertension; M/F=male/female; mPAP=mean pulmonary artery pressure; NA = not applicable; NYHA=New York Heart Association; PDE5i=Phophodiesterase 5 inhibitors; PCWP=pulmonary capillary wedge pressure; PGI2 =prostaglandin I2; PVR= pulmonary vascular resistance.
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Table 2. Characteristics of scleroderma associated pulmonary arterial hypertension patients, scleroderma patients ACCEPTED MANUSCRIPT and controls
Specific PAH Therapy - ERA - PDE5i - ERA + PDE5i - ERA + PGI2
25 ± 1
26 ± 1
24 ± 1
4 2 4 0
NA NA NA NA
1
NA
3 0 0
NA NA 0
NA NA NA NA
1 5 6 2
NA NA NA NA
NA NA NA NA NA NA
239 ± 76 320± 36 42.2 ± 3.0 5.7 ± 0.5 6.2 ± 0.9 10.1 ± 1.3
NA NA NA NA NA NA
NA NA NA NA
- PDE5i + PGI2
NA
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NYHA functional class, n class I class II class III class IV
NA NA NA
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- ERA + PDE5i + PGI2 - No Treatment Corticosteroids and/or immunosuppresants
BNP, ng/L 6-MWD, m mPAP, mmHg CO, L/min PVR, Wood units PCWP, mmHg
SSc n=7 64 ± 3.6* 0 / 7 (0.0)
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2
BMI, Kg/m
SSc-PAH n=14 71 ± 2.9* 2 / 12 (0.17)
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Age, years Sex, M/F (ratio)
Controls n=20 47 ± 2.4 9 / 11 (0.82)
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*=statistical significance compared to controls; 6-MWD=6-minute walk distance; BMI=body mass index; BNP= brain natriuretic peptide; CO=cardiac output; ERA=endothelin receptor antagonists; iPAH=idiopathic pulmonary arterial hypertension; M/F=male/female; mPAP=mean pulmonary artery pressure; NA = not applicable; NYHA= New York Heart Association; PDE5i=Phophodiesterase 5 inhibitors; PCWP=pulmonary capillary wedge pressure; PGI2=prostaglandin I2; PVR= pulmonary vascular resistance; SSc=scleroderma; SSc-PAH=scleroderma associated pulmonary arterial hypertension
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ACCEPTED MANUSCRIPT Table 3. Regulatory T lymphocyte data in idiopathic, heritable and scleroderma associated pulmonary arterial hypertension patients, scleroderma patients and controls
+
Treg number (% CD4 cells) +
Treg ObR cells (% Treg) +
pSTAT3 cells (% Treg) +
+
pSTAT3 cells (% Treg ObR )
iPAH n=30
hPAH n=18
SSc-PAH n=14
SSc n=7
5.0 ± 0.4
4.2 ± 0.4
5.2 ± 0.6
3.5 ± 0.3
4.3 ± 0.6
9.2 ± 2.2
37.6 ± 3.2*
8.5 ± 1.5
37.7 ± 6.9*
60.4 ± 1.7*
7.3 ± 1.1
3.5 ± 0.5*
4.2 ± 0.6*
2.9 ± 0.3*
4.0 ± 0.5*
11.7 ± 1.5
4.7 ± 0.6*
6.3 ± 0.6*
4.5 ± 0.9*
6.6 ± 0.7*
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Controls n=20
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*=statistical significance compared to controls; ObR= leptin receptor; pSTAT3=phosphorylated signal tranducer and activator of transcription 3; Treg= regulatory T lymphocytes
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Table 4. Characteristics of wildtype and ZDF rats in normoxia and hypoxia
Wildtype
ZDF
Normoxia n=4
Hypoxia n=4
Normoxia n=5
Hypoxia n=8
217 ± 4
213 ± 4
254 ± 4
252 ± 5
18.2 ± 0.6
32.8 ± 0.5 *
17.3 ± 0.2
27.7 ± 0.9
#,$
0.2 ± 0.02
0.6 ± 0.12 *
0.1 ± 0.01
0.4 ± 0.04
#,$
100 ± 6
49 ± 7 *
128 ± 2
79 ± 7
1.3 ± 0.2
2.2 ± 0.2 *
1.1 ± 0.1
1.7 ± 0.1
Muscularized pulmonary arteries, %
13 ± 4
71 ± 4 *
15 ± 5
48 ± 3
Wall thickness, %
27 ± 2
34 ± 2 *
19 ± 2
23 ± 2
1.9 ± 0.3
7.8 ± 0.5 *
2.4 ± 0.4
3.8 ± 0.3
5.7 ± 1.9
4.5 ± 1.5
4.8 ± 0.7
5.8 ± 0.9
8.3 ± 0.6
3.5 ± 0.2 *
5.3 ± 1.2
4.5 ± 0.3
mPAP, mmHg PVR, mmHg/mL/min CO, ml/min
TE D
Body weight, g
-3
EP
Fulton index/body weight ratio (x10 )
AC C
PCNA+ cells/vessel, % +
Treg number (% CD4 cells) +
pSTAT3 cells (% Treg)
#
#,$ #,$
#,$ $ #,$
*=statistical significance compared to normoxic wildtype rats; #=statistical significance compared to normoxic ZDF rats; $=statistical significance compared to hypoxic wildtype rats; CO= cardiac output; mPAP= mean pulmonary arterial pressure; PCNA= proliferating cell nuclear antigen; PVR= pulmonary vascular resistance; pSTAT3=phosphorylated signal tranducer and activator of transcription 3; Treg= regulatory T lymphocytes.
18
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT