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Original Research: Pulmonary Vascular Disease |

T-Helper 17 Cell Polarization in Pulmonary Arterial HypertensionT-Helper 17 Cells in Pulmonary Hypertension FREE TO VIEW

Aurélie Hautefort, MSc; Barbara Girerd, PhD; David Montani, MD, PhD; Sylvia Cohen-Kaminsky, PhD; Laura Price, MD, PhD; Bart N. Lambrecht, MD, PhD; Marc Humbert, MD, PhD; Frédéric Perros, PhD
Author and Funding Information

From the Faculté de médecine (Ms Hautefort and Drs Girerd, Montani, Cohen-Kaminsky, Humbert, and Perros), Université Paris-Sud, Le Kremlin-Bicêtre, France; AP-HP (Drs Girerd, Montani, and Humbert), DHU TORINO, Centre de Référence de l’Hypertension Pulmonaire Sévère, Service de Pneumologie et Réanimation Respiratoire, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; INSERM UMR-S 999 (Ms Hautefort and Drs Girerd, Montani, Cohen-Kaminsky, Humbert, and Perros), Labex LERMIT, Hypertension Artérielle Pulmonaire: Physiopathologie et Innovation Thérapeutique, Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France; Pulmonary Hypertension Service (Dr Price), Royal Brompton Hospital, London, England; and VIB Inflammation Research Center (Dr Lambrecht), University of Ghent, Gent, Belgium.

CORRESPONDENCE TO: Frédéric Perros, PhD, INSERM U999, Centre Chirurgical Marie Lannelongue, 133, Ave de la Résistance, F-92350 Le Plessis-Robinson, France; e-mail: frederic.perros@inserm.fr


FUNDING/SUPPORT: Ms Hautefort is supported by a PhD grant from Région Ile de France (CORDDIM). This study was supported by grants from the National Funding Agency for Research (ANR) [Grant ANR-13-JSV1-001] and from the Fondation pour la Recherche Médicale (FRM) [EQ20100318257].

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2015;147(6):1610-1620. doi:10.1378/chest.14-1678
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BACKGROUND:  Inflammation may contribute to the pathobiology of pulmonary arterial hypertension (PAH). Deciphering the PAH fingerprint on the inflammation orchestrated by dendritic cells (DCs) and T cells, key driver and effector cells, respectively, of the immune system, may allow the identification of immunopathologic approaches to PAH management.

METHODS:  Using flow cytometry, we performed immunophenotyping of monocyte-derived DCs (MoDCs) and circulating lymphocytes from patients with idiopathic PAH and control subjects. With the same technique, we performed cytokine profiling of both populations following stimulation, coculture, or both. We tested the immunomodulatory effects of a glucocorticoid (dexamethasone [Dex]) on this immunophenotype and cytokine profile. Using an epigenetic approach, we confirmed the immune polarization in blood DNA of patients with PAH.

RESULTS:  The profile of membrane costimulatory molecules of PAH MoDCs was similar to that of control subjects. However, PAH MoDCs retained higher levels of the T-cell activating molecules CD86 and CD40 after Dex pretreatment than did control MoDCs. This was associated with an increased expression of IL-12p40 and a reduced migration toward chemokine (C-C motif) ligand 21. Moreover, both with and without Dex, PAH MoDCs induced a higher activation and proliferation of CD4+ T cells, associated with a reduced expression of IL-4 (T helper 2 response) and a higher expression of IL-17 (T helper 17 response). Purified PAH CD4+ T cells expressed a higher level of IL-17 after activation than did those of control subjects. Lastly, there was significant hypomethylation of the IL-17 promoter in the PAH blood DNA as compared with the control blood.

CONCLUSIONS:  We have highlighted T helper 17 cell immune polarization in patients with PAH, as has been previously demonstrated in other chronic inflammatory and autoimmune conditions.

Figures in this Article

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance, leading to right ventricular failure and ultimately death.1 PAH has a complex and multifactorial pathogenesis in which excessive migration and proliferation of pulmonary vascular cells (ie, endothelial cells and smooth muscle cells) and dysregulated immune responses are critical contributors to inappropriate pulmonary vascular remodeling.2 Inflammation is a common denominator of the hallmark lesions of PAH35 and of its animal models.6 The heritable form of PAH (hPAH) is usually (> 80%) caused by mutations in the BMPR2.7 In animal models, it has been shown that greater endothelial injury and enhanced inflammatory response could be the underlying causes of the sensitivity and may work in concert with BMPR2 heterozygosity to promote the development of persistent pulmonary hypertension.8

Different studies have shown increased levels of cytokines in PAH, including the proinflammatory cytokines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12p70, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1,9,10 and the cytokine-like hormone leptin.11 Our group has demonstrated that IL-1α, IL-1β, IL-6, TNF-α, and IL-13 are linked to death in PAH.12 However, the specific role of these cytokines in the PAH pathogenesis remains elusive. They probably play a role in the self-perpetuating vicious cycle of endothelial cell activation13 and in the local adaptive immune response that occurs in the tertiary (ectopic) lymphoid follicles (tLTs) developing along the remodeled pulmonary vasculature in PAH.14 Glucocorticoids (GCs) are effective and clinically useful medicines for repressing inflammation in lung disease; however, except for a single successful case report,15 there is no evidence of GC efficacy in patients with idiopathic PAH (iPAH), even though cases of PAH associated with autoimmune diseases such as systemic lupus erythematosus or mixed connective tissue disease had an improvement in clinical and hemodynamic characteristics after GC treatment.16,17

To bridge innate and adaptive immunity, there is a specialized population of innate immune antigen-presenting cells, dendritic cells (DCs), that express all the receptors of the innate immune system and at the same time have the potential to take up antigen, process it into small peptides, and present it in the cleft of major histocompatibility complex (MHC)-I and MHC-II molecules to be recognized by T-cell receptors.18 T-lymphocyte differentiation is a highly organized process controlled by DCs, which secrete cytokines and induce polarized T helper (Th)1, Th2, or Th17 cell subsets that secrete interferon (IFN)-γ, IL-4/IL-5/IL-13, and IL-17, respectively. Under certain conditions, T cells may also differentiate into regulatory T cells, which suppress various aspects of inflammation and the activation of naive T cells. We have identified an intense recruitment of DCs into the wall of affected pulmonary arteries in severe pulmonary hypertension induced in rats by monocrotaline exposure and in human PAH lesions.19 Several findings suggest that disturbed DC function plays a critical role in the establishment of chronic inflammation and in the induction of autoimmunity as seen in a number of autoimmune disease states. In many autoimmune diseases, the number of DCs and their degree of immunostimulatory capacity for activating autoreactive T cells have been shown to be increased.2022

Here, we studied the phenotype and function of monocyte-derived DCs (MoDCs) from patients with PAH and control subjects, in terms of costimulatory molecule expression, cytokine profile, GC sensitivity, and crosstalk with Th cells in vitro. We then compared the in vitro DC-driven polarization of T cells with the Th profiles of the circulating lymphocytes in both groups of subjects.

Collection of Blood Samples

All patients and control subjects studied were part of the French Network on Pulmonary Hypertension, a program approved by our institutional ethics committee, and gave written informed consent:

  • • Protocol No: CO-08-003, ID Recherches et Collections Biologiques: 2008-A00485-50

  • • Principal investigator: Marc Humbert, MD, PhD

  • • CPP (Comité de protection des personnes) de Bicêtre (CPP IDF VII).

Subjects were > 18 years of age and patients with iPAH or hPAH (BMPR2 mutation carriers) had a diagnosis confirmed by right-sided heart catheterization. Blood samples were collected in patients with PAH during follow-up and in control subjects. Characteristics at diagnosis and follow-up were stored in the Registry of the French Network of Pulmonary Hypertension set up in agreement with French bioethics laws (French Commission Nationale de l’Informatique et des Libertés).23 Subject characteristics are described in Tables 14.

Table Graphic Jump Location
TABLE 1 ]  Clinicopathologic Features of Subjects Whose Blood Was Used for MoDC Generation, Characterization, and Cellular Assays

Data are mean ± unless otherwise indicated. 6MWD = 6-min walk distance; ERA = endothelin receptor antagonist; iPAH = idiopathic pulmonary arterial hypertension; MoDC = monocyte-derived dendritic cell; mPAP = mean pulmonary artery pressure; N/A = not applicable; NYHA = New York Heart Association; PAH = pulmonary arterial hypertension; PDE5 = phosphodiesterases type 5; PVR = pulmonary vascular resistance.

Table Graphic Jump Location
TABLE 2 ]  Clinicopathologic Features of Subjects Whose Blood Was Used for Study of Circulating Lymphocyte Subpopulations

Data are mean ± unless otherwise indicated. See Table 1 legend for expansion of abbreviations.

Table Graphic Jump Location
TABLE 3 ]  Clinicopathologic Features of Subjects Whose Blood Was Used for CD4+ T-Cell Isolation and Stimulation

Data are mean ± unless otherwise indicated. See Table 1 legend for expansion of abbreviations.

Table Graphic Jump Location
TABLE 4 ]  Clinicopathologic Features of Subjects Whose Blood Genomic DNA Was Used for Epigenetic Analysis

Data are mean ± unless otherwise indicated. PDE5-i = phosphodiesterases type 5 inhibitor. See Table 1 legend for expansion of other abbreviations.

Generation and Phenotypic Analysis of MoDCs from Peripheral Blood Mononuclear Cells

Monocytes isolated from peripheral blood mononuclear cells (PBMCs) by adherence were cultured with IL-4 and granulocyte macrophage colony stimulating factor (35 and 50 ng/mL, respectively) during 7 days. At day 7, immature MoDCs were isolated by positive selection (CD209 [Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin (DC-SIGN)] MicroBead Kit; Miltenyi Biotec) and primed with IFN-γ (10 ng/mL) with or without dexamethasone (Dex) (10−6 M, as described previously24) until day 8. Primed MoDCs were activated with lipopolysaccharide (1 μg/mL) for 1 day (± GolgiStop [Becton, Dickinson and Company] for intracellular cytokine staining according to the manufacturer’s instructions). Activated MoDCs from control subjects and patients with PAH were stained for flow cytometry or used in cellular assays.

Analysis of the Migratory Capacities of MoDCs

MoDCs were loaded onto the upper chambers of transwell plates and were allowed to migrate against the basal medium, chemokine (C-C motif) ligand (CCL) 19 or CCL21 (250 ng/mL/4 h), present in the lower chamber. Migrated cells were then counted with a flow cytometer. The chemotaxis indexes were calculated by dividing the number of migrated DCs in the presence of a chemotactic ligand by that in its absence (basal medium) and are expressed in percentages.

Heterologous Mixed Leukocyte Reaction Stimulated by MoDCs

Mixed leukocyte reactions (MLRs) were performed to assess the stimulatory capacity of the MoDCs. CD4+ T cells isolated by negative selection (CD4+ T Cell Isolation Kit; Miltenyi Biotec) from a healthy buffy coat for use as responders were cultured with stimulator cells (MoDCs from control subjects and patients with PAH) at a DC to T cell ratio of 1:100 for 4 days. The proliferative response of T lymphocytes was measured with the Click-iT EdU Cell Proliferation Assay (Life Technologies) with 5-ethynyl-2'-deoxyuridine solution (1 mM) added to the MLR at day 3. For intracellular detection of cytokines, GolgiStop solution was added to the MLRs at day 3. At day 4, T cells were processed for flow cytometry.

Isolation, Stimulation, and Phenotypic Analysis of Circulating CD4+ T Cells

CD4+ T cells were isolated from whole blood (control subjects and patients with iPAH) by negative selection with the RosetteSep Human CD4+ T Cell Enrichment Cocktail (STEMCELL Technologies Inc), activated for 24 h with the Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience), and then processed for flow cytometry.

Flow Cytometry

All primary antibodies used for flow cytometry are depicted in Table 5. Protocols are described in e-Appendix 1.

Table Graphic Jump Location
TABLE 5 ]  Characteristics of Antibodies Used in Flow Cytometry Analyses

IFN = interferon; MHC = major histocompatibility complex; MLR = mixed leukocyte reaction; TNF = tumor necrosis factor. See Table 1 legend for expansion of other abbreviations.

Epigenetic Assays for Th17 Immune Cell Monitoring in Blood

Epigenetic assays were performed by Epiontis on a service-based assay service (Berlin, Germany). Protocols are described in e-Appendix 1.

Statistical Evaluation

Quantitative variables are presented as mean ± SEM unless otherwise stated. We used Student t tests or Mann-Whitney tests to compare groups, depending on the normal or nonnormal nature of the distribution. We used the Kruskal-Wallis test to compare more than two groups. Analyses were performed with GraphPad Prism 6 software (GraphPad Software). P values < .05 were considered to indicate statistical significance.

Differences in iPAH MoDCs Sensitivity to Dex

After differentiation of monocytes into DCs, MoDCs were primed with IFN-γ (10 ng/mL) with or without Dex (10−6 M). The primed cells were then activated with lipopolysaccharide (1 μg/mL). The phenotype of MoDCs was characterized by the analysis of a range of DC markers by flow cytometry: CD80, CD86, CD40, MHC-II, CD11c, CD209, B7H1, B7H2, and ILT3. As expected, Dex decreased the expression of activation markers on MoDCs such as CD40, CD80, and CD86, and maintained a higher expression of markers for immature DCs such as CD209 (Fig 1A). In the absence of Dex treatment, there was no difference in the membrane expression of these DC markers between control subjects and patients with PAH (data not show). However, after Dex treatment, the expression of CD86 (P < .001), CD40, and B7H2 (both P < .05) remained higher in patients with PAH as compared with control subjects (Fig 1B). There were no modifications in the expression of the other costimulation molecules (data not shown).

Figure Jump LinkFigure 1 –  Phenotypic analysis by flow cytometry of monocyte-derived dendritic cells (MoDCs) from control subjects and patients with idiopathic PAH. A, Histograms of isotype control-stained and of CD40-, CD80-, CD86-, or CD209-stained interferon (IFN)-γ/lipopolysaccharide (LPS)-activated MoDCs (CD11c+major histocompatibility complex [MHC]-IIhigh) ± 10−6M Dex. B, Mean fluorescence intensity for CD40, CD86, and B7H2 staining of control and PAH Dex/IFN-γ/LPS-generated MoDCs, determined by flow cytometry; n = 5 in each condition. There was no detectable difference between control and PAH MoDCs without Dex (data not shown). Data are presented as MFI. CTRL = control; Dex = dexamethasone; MFI = mean fluorescent intensity; PAH = pulmonary artery hypertension.Grahic Jump Location

Maturation of DCs is associated with an increase in their migratory capacity to T-cell areas of the lymphoid organ through a chemoattracting gradient of CCL19 and CCL21. Interestingly, Dex treatment specifically decreased PAH MoDC migration toward CCL21 (P < .05) (Fig 2C).

Figure Jump LinkFigure 2 –  Migratory capacity of MoDCs measured by transwell migration assay. CTRL and PAH IFN-γ/LPS-generated MoDCs (CD11+MHC-IIhigh) untreated or treated with Dex were loaded onto the upper chambers of transwell plates and allowed to migrate against BM, CCL19 (250 ng/mL), or CCL21 (250 ng/mL) present in the lower chamber. A, BM. B, CCL19 (250 ng/mL). C, CCL21 (250 ng/mL). After 4 h of incubation, migrated cells were then counted with flow cytometer. The chemotaxis indexes were calculated by dividing the number of migrated dendritic cells in the presence of a chemotactic ligand by that in its absence (BM) and expressed in %; n = 5 in each condition. There was no detectable difference between control and PAH MoDCs without Dex (data not shown). BM = basal medium; CCL = chemokine (C-C motif) ligand. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

Finally, after priming and activation of MoDCs, we looked at their cytokine profile (TNF-α, IL-6, p40 IL-12/IL-23 subunit, and IL-10) production. After Dex treatment, p40 levels were higher in PAH MoDCs than in control MoDCs (P < .05) (Fig 3B). Expression of the other cytokines did not differ in control and PAH MoDCs (Figs 3A, 3B).

Figure Jump LinkFigure 3 –  Cytokine expression analysis in control and idiopathic PAH MoDCs with or without Dex treatment using flow cytometry. After fixation and permeabilization, CTRL and PAH IFN-γ/LPS-activated MoDCs (CD11+MHC-IIhigh) were stained for intracellular IL-6, IL-12p40, IL-10, and TNFα content. A, Untreated CTRL and PAH MoDCs. B, Dex-treated CTRL and PAH MoDCs. Data are presented as % of total CD11+MHC-IIhighMoDCs. n = 5 in each experimental condition. TNFα = tumor necrosis factor-α. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Enhanced Activation and Proliferation, and Specific Th17 Polarization of CD4 T Lymphocytes After Coculture With iPAH MoDCs

We analyzed the crosstalk between T lymphocytes and DCs in terms of the activation, proliferation, and polarization of CD4 T cells after coculture with primed (± Dex) and activated control and PAH MoDCs. After coculture with PAH MoDCs, a higher expression of the surface activation marker CD69 on CD3+CD4+ T cells was observed, as compared with T cells cocultured with control MoDCs, which remained higher when PAH MoDCs were pretreated with Dex (control subjects vs patients with PAH, P < .0001; and control subjects-Dex vs patients with PAH-Dex, P < .001) (Fig 4A). This was associated with a higher proliferation of these cells in both conditions (control subjects vs patients with PAH, P < .0001; and control subjects-Dex vs patients with PAH-Dex, P < .001) (Fig 4B). DC-driven T-cell polarization was assessed via the intracellular staining of cytokines in cocultured CD4 T lymphocyte for discriminated Th1 (IFN-γ), Th2 (IL-4), and Th17 (IL-17) responses (Figs 5AC, respectively) and intracellular staining cytokines, including IL-2, IL-10, and TNF-α, having a pleiotropic effects on lymphocyte activation, growth, and differentiation. (Fig 5D) There were higher IL-17 and IL-10 levels (both, P < .05) in CD4 T cells cocultured with PAH MoDCs than in T cells cultured with control MoDCs. Inversely, we observed a decrease in IL-4 levels in the same conditions, which was maintained when MoDCs were pretreated with Dex (P = .056). There was no significant difference in the other cytokines levels between the two groups (cocultured with control and PAH MoDCs ± Dex) (data not shown).

Figure Jump LinkFigure 4 –  MoDC-induced activation and proliferation of CD4 T cells in mixed leukocyte reactions (MLRs). CTRL and PAH IFN-γ/LPS-activated MoDCs (CD11+MHC-IIhigh) ± Dex were cocultured with CD3+CD4+T cells (ratio dendritic cell/T 1:100) during 4 d. A, CD69+CD3+CD4+ T cells (activated T cells). B, Proliferating CD3+CD4+ T cells. Data are presented as % of total CD3+CD4+ present in the MLR. n = 5 in each experimental condition. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 5 –  MoDC-driven T-cell polarization in MLR. Intracellular cytokine detection was carried out on CD3+CD4+ T cells from MLR. After 4 d of coculture of T cells with CTRL or PAH IFNγ/LPS-activated MoDCs (CD11+MHC-IIhigh) with or without Dex pretreatment. T-cell surfaces were stained for CD3 and CD4, fixed/permeabilized, and stained for intracellular cytokine. Measurements of IL-2, TNFα, and IL-10 were taken to assess their pleiotropic effects on lymphocyte activation, growth, and differentiation, as well as IFNγ (T helper [Th]1 response), IL-4 (Th2 response), and IL-17 (Th17 response). A, IFN-γ (Th1 response). B, IL-4 (Th2 response). C, IL-17 (Th17 response). D, IL-2, TNFα, and IL-10. Data are presented as % of total CD3+CD4+ present in the MLR; n = 5 in each experimental condition. See Figure 1, 3, and 4 legends for expansion of abbreviations.Grahic Jump Location
Specific Polarization of T Cells in Th17 Subpopulation in iPAH PBMC

To obtain an overview of the peripheral blood leukocyte composition, subpopulations of circulating lymphocytes were studied in PBMCs from patients with PAH and control subjects (Fig 6). The total CD3+ T-lymphocyte count was decreased in patients with PAH (P < .05) (Fig 6B). This resulted from the reduction in the number of the CD3+CD4+ Th cells in PAH (P < .05) (Fig 6C). There was no detectable alteration in the CD19+ B-lymphocyte and CD8+ T-lymphocyte counts in the PAH group. The polarization of isolated CD4+ T cells was determined in vitro after activation by phorbol 12-myristate 13-acetate/ionomycin according to their cytokine signature: Th1 (IL-2, IFN-γ), Th2 (IL-4, IL-13, IL-10), and Th17 (IL-17). We found an increase in the percentage of IL-17+ cells in iPAH Th cells as compared with control cells (P < .05) (Fig 7).

Figure Jump LinkFigure 6 –  Enumeration of circulating lymphocyte subpopulations. Subjects’ blood lymphocyte analysis was carried out with a No-Lyse No-Wash protocol, gating on Hoechst+ nucleated leukocytes (discrimination from Hoechst RBC counts). CD3+ (total T cells), CD3+CD4+ (Th cells), CD3+CD8+ (cytotoxic T cells), and CD19 (B cells) lymphocytes were counted by flow cytometry. A, CD19 (B cells) lymphocytes. B, CD3+ (total T cells). C, CD3+CD4+ (Th cells). D, CD3+CD8+ (cytotoxic T cells). Data are presented in 106 cells/mL. Enumeration was performed on control subjects (n = 20) and patients with idiopathic PAH (n = 17). See Figure 1 and 5 legends for expansion of abbreviations.Grahic Jump Location
Figure Jump LinkFigure 7 –  Control and idiopathic PAH T-cell polarization. CD3+CD4+ T cells were isolated from whole blood (control subjects and patients with PAH) by negative selection with a rosette and gradient technique, activated for 24 h with a cell stimulation cocktail (containing phorbol 12-myristate 13-acetate and ionomycin, plus protein transport inhibitors), then processed for staining of surface CD3 and CD4 antigens, and intracellular cytokines specific to T-cell subpopulations: A, B, IL-2 and IFNγ (Th1); C-E, IL-4, IL-13, and IL-10 (Th2); and F, IL-17 (Th17). Data are presented as % of total CD3+CD4+ cells. Cytokine profile was performed on CD3+CD4+ Th cells from control subjects (n = 16) and patients with idiopathic PAH (n = 8). See Figure 1 and 5 legends for expansion of abbreviations.Grahic Jump Location
Epigenetic Confirmation of the Th17 Response in PAH

We performed a methylation study of IL17A in genomic DNA from iPAH and hPAH and control PBMCs. Indeed, IL17A demethylation is the epigenetic signature of Th17 cells. There was no significant difference in terms of IL17A methylation between iPAH and hPAH (data not shown). Hence, we presented the results for IL17A methylation by comparing patients with PAH (iPAH + hPAH) with control subjects. There was an increase in the percentage of demethylated IL17A in PAH PBMC as compared with control subjects (P < .05) (hypomethylation) (Fig 8).

Figure Jump LinkFigure 8 –  Epigenetic assays for Th17 immune cell monitoring in blood. Methylation study of the IL17A gene was performed on genomic DNA from the blood of patients with PAH (n = 28; 19 with idiopathic PAH and nine with heritable PAH) and control subjects (n = 19). IL17A demethylation is the epigenetic signature of Th17 cells. See Figure 1 and 5 legends for expansion of abbreviations.Grahic Jump Location

We have demonstrated that under similar differentiation and activation conditions, iPAH MoDCs exhibit a profile of membrane costimulatory molecules similar to that of control MoDCs. As expected, GC (Dex) decreased the activation of MoDCs. However, PAH MoDCs retained a higher level of the T cell-activating molecules CD86 and CD40 after Dex pretreatment than did control MoDCs. This was associated with an increased expression of p40 (IL-12 and IL-23 subunit) and a reduced migration toward CCL21. Moreover, both with and without Dex, iPAH MoDCs induced a higher activation/proliferation of CD4+ T cells, associated with a reduced expression of IL-4 (Th2 response) and a higher expression of IL-17 (Th17 response), which may be related to the higher expression of p40 by PAH MoDCs. In PAH venous blood, we found a specific depletion of CD4+ T cells, maybe recruited, in the pulmonary tertiary lymphoid organs that we described in this disease.14 Purified iPAH CD4+ T cells expressed higher levels of IL-17 after activation than did control cells. Lastly, there was a significant increase in the percentage of demethylated DNA (hypomethylation) at the IL-17 promoter in the PAH blood DNA, as compared with the blood DNA of control subjects. This result strengthened the suspicion of a pathologic PAH-associated Th17 response.

Hence, we confirmed that PAH is associated with an unleashed inflammatory response that we highlighted by a reduced DC response to GC and an increased stimulation of T cells. This loss of immune control may favor chronic inflammation. We also demonstrated that PAH is associated with an increased Th17 cell prevalence. Th17 is a major issue in the field of autoimmunity25 and mixes ideally with the flavor of autoimmunity described in PAH.25 The argument for autoimmunity in PAH is supported by (1) the association of inflammatory, infectious, and autoimmune diseases with PAH26; (2) the presence of a female predominance in autoimmune diseases and in PAH27,28; (3) the high prevalence of autoantibodies in PAH2931; and (4) the pathophysiologic role of autoantibodies in vitro32 and in vivo in experimental models of PAH.33

Autoimmunity is also consistent with the lymphoid neogenesis reported previously in PAH.14,33 Indeed, tLTs emerge at various sites in response to persistent inflammation, as occurs in autoimmunity, and are often accompanied by tissue destruction, with progressive clinical symptoms.34 Similar to secondary lymphoid organs, tLTs harbor all immune cell subsets to conduct the key steps of primary immune responses. They are organized into distinct compartments, including separate T-cell areas, conventional DCs, B-cell follicles containing proliferating B centroblasts, and mantle zone B cells, as well as follicular DCs in activated germinal centers. Their formation is dependent on lymphorganogenic chemokines such as CCL20 and CXCL13 that are overexpressed in explanted iPAH lungs.14 Such structures are highly prevalent in the lungs of patients with the pulmonary complications of autoimmune diseases such as rheumatoid arthritis and Sjögren syndrome.35 It has been shown that IL17 may favor their neogenesis,36 which is consistent with the description of Th17 cells in PAH-associated tLTs.14

Moreover, the CD138+ plasma cells observed around pulmonary vascular lesions are consistent with locally produced autoantibodies targeting the pulmonary vascular components participating in the remodeling of the pulmonary vasculature in iPAH.14 Lastly, several lymphocyte populations dedicated to the inhibition of autoimmune reaction or to regulation of the T-cell response (regulatory T cells, natural killer, and natural killer T cells)3739 are decreased in number and in function in pulmonary hypertension.11,4042 Autoimmune diseases often result from the imbalance between regulatory T and Th17 cells producing IL-17, and this balance is impaired in nearly all clinical conditions, thus making the therapeutic target of Th17 signaling a promising treatment in chronic inflammation,25,43 and possibly in PAH, according to our results.

It is noteworthy that IL-12 and IL-23, two heterodimeric cytokines produced by DCs, are composed of a specific polypeptide, namely p35 for IL-12 and p19 for IL-23, a disulfide linked to a common p40 chain to form biologically active molecules. IL-23, rather than IL-12, was identified as the major factor responsible for lesions caused by chronic inflammation, acting through the induction of Th17 cells.44 By analyzing the IL-12p40 subunit, we cannot decipher whether iPAH is associated with IL-12, IL-23, or both IL-12 and IL-23 overproduction. The increased Th17 polarization of Th cells in coculture with iPAH MoDCs suggests that p40 overexpression could be linked to IL-23 overproduction. Control of IL-23 is broadly understood to involve the phosphatidylinositol-4,5-bisphosphate3-kinase/Akt, mitogen-activated protein kinase, and Syk-CARD9 intracellular signaling pathways45; however, we did not address the signaling leading to the pathologic phenotype of iPAH MoDCs. This certainly deserves future investigation.

In conclusion, the dysregulated immune response associated with iPAH or hPAH may be related in part to DC dysfunction and to increased Th17 immune polarization. Indeed, this polarization has been shown to be involved in several chronic inflammatory and autoimmune conditions, but not previously in PAH.

Author contributions: F. P. is the guarantor of this manuscript and takes responsibility for its content, including the data and analysis. A. H., B. G., D. M., S. C.-K., L. P., B. N. L., M. H., and F. P. had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects; and A. H., B. G., D. M., S. C.-K., L. P., B. N. L., M. H., and F. P. contributed to the study design, the data analysis and interpretation, and the writing of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Montani has received speaker fees or honoraria for consultations from Actelion Pharmaceuticals Ltd, Bayer, GlaxoSmithKline, Eli Lilly and Company, Novartis, Pfizer Inc, and United Therapeutics Corporation. Dr Humbert has received speaker fees or honoraria for consultations from Actelion Pharmaceuticals Ltd, Bayer, Bristol-Myers Squibb, GlaxoSmithKline, Eli Lilly and Company, Novartis, Pfizer Inc, and United Therapeutics Corporation. Ms Hautefort and Drs Girerd, Cohen-Kaminsky, Price, Lambrecht, and Perros have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript.

Other contributions: We thank M. Ali Alashkar, MSc, for technical assistance.

Additional information: The e-Appendix can be found in the Supplemental Materials section of the online article.

CCL

chemokine (C-C motif) ligand

DC

dendritic cell

Dex

dexamethasone

GC

glucocorticoid

hPAH

heritable pulmonary arterial hypertension

IFN

interferon

iPAH

idiopathic pulmonary arterial hypertension

MHC

major histocompatibility complex

MLR

mixed leukocyte reaction

MoDC

monocyte-derived dendritic cell

PAH

pulmonary arterial hypertension

PBMC

peripheral blood mononuclear cell

Th

T-helper

tLT

tertiary lymphoid follicle

TNF

tumor necrosis factor

Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med. 2004;351(14):1425-1436. [PubMed]
 
Huertas A, Perros F, Tu L, et al. Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: a complex interplay. Circulation. 2014;129(12):1332-1340. [CrossRef] [PubMed]
 
Dorfmüller P, Humbert M. Progress in pulmonary arterial hypertension pathology: relighting a torch inside the tunnel. Am J Respir Crit Care Med. 2012;186(3):210-212. [PubMed]
 
Price LC, Wort SJ, Perros F, et al. Inflammation in pulmonary arterial hypertension. Chest. 2012;141(1):210-221. [PubMed]
 
Kherbeck N, Tamby MC, Bussone G, et al. The role of inflammation and autoimmunity in the pathophysiology of pulmonary arterial hypertension. Clin Rev Allergy Immunol. 2013;44(1):31-38. [PubMed]
 
Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol. 2009;297(6):L1013-L1032. [PubMed]
 
West J, Austin E, Fessel JP, Loyd J, Hamid R. Rescuing the BMPR2 signaling axis in pulmonary arterial hypertension. Drug Discov Today. 2014;19(8):1241-1245. [PubMed]
 
Song Y, Coleman L, Shi J, et al. Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice. Am J Physiol Heart Circ Physiol. 2008;295(2):H677-H690. [PubMed]
 
Sanchez O, Marcos E, Perros F, et al. Role of endothelium-derived CC chemokine ligand 2 in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2007;176(10):1041-1047. [PubMed]
 
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(9):920-927. [CrossRef] [PubMed]
 
Huertas A, Tu L, Gambaryan N, et al. Leptin and regulatory T-lymphocytes in idiopathic pulmonary arterial hypertension. Eur Respir J. 2012;40(4):895-904. [PubMed]
 
Cracowski JL, Chabot F, Labarère J, et al. Proinflammatory cytokine levels are linked to death in pulmonary arterial hypertension. Eur Respir J. 2014;43(3):915-917. [PubMed]
 
Price LC, Caramori G, Perros F, et al. Nuclear factor κ-B is activated in the pulmonary vessels of patients with end-stage idiopathic pulmonary arterial hypertension. PLoS ONE. 2013;8(10):e75415. [CrossRef] [PubMed]
 
Perros F, Dorfmüller P, Montani D, et al. Pulmonary lymphoid neogenesis in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2012;185(3):311-321. [PubMed]
 
Ogawa A, Nakamura K, Mizoguchi H, et al. Prednisolone ameliorates idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2011;183(1):139-140. [PubMed]
 
Sanchez O, Sitbon O, Jaïs X, Simonneau G, Humbert M. Immunosuppressive therapy in connective tissue diseases-associated pulmonary arterial hypertension. Chest. 2006;130(1):182-189. [PubMed]
 
Cohen-Kaminsky S, Hautefort A, Price L, Humbert M, Perros F. Inflammation in pulmonary hypertension: what we know and what we could logically and safely target first. Drug Discov Today. 2014;19(8):1251-1256. [PubMed]
 
Lambrecht BN, Hammad H. The role of dendritic and epithelial cells as master regulators of allergic airway inflammation. Lancet. 2010;376(9743):835-843. [PubMed]
 
Perros F, Dorfmüller P, Souza R, et al. Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension. Eur Respir J. 2007;29(3):462-468. [PubMed]
 
Kim SJ, Diamond B. Modulation of tolerogenic dendritic cells and autoimmunity [published online ahead of print April 18, 2014]. Semin Cell Dev Biol.
 
Ganguly D, Haak S, Sisirak V, Reizis B. The role of dendritic cells in autoimmunity. Nat Rev Immunol. 2013;13(8):566-577. [PubMed]
 
Quintana FJ, Yeste A, Mascanfroni ID. Role and therapeutic value of dendritic cells in central nervous system autoimmunity. Cell Death Differ. 2015;22(2):215-224. [CrossRef] [PubMed]
 
Humbert M, Sitbon O, Chaouat A, et al. Pulmonary arterial hypertension in France: results from a national registry. Am J Respir Crit Care Med. 2006;173(9):1023-1030. [CrossRef] [PubMed]
 
Hu J, Kinn J, Zirakzadeh AA, et al. The effects of chemotherapeutic drugs on human monocyte-derived dendritic cell differentiation and antigen presentation. Clin Exp Immunol. 2013;172(3):490-499. [CrossRef] [PubMed]
 
Selmi C. Autoimmunity in 2013. Clin Rev Allergy Immunol. 2014;47(1):100-109. [CrossRef] [PubMed]
 
Perros F, Cohen-Kaminsky S, Dorfmüller P, et al;. Inflammation in pulmonary arterial hypertension.. In:Abraham D, Handler C, Dashwood M, Coghlan G., eds. Translational Vascular Medicine. London, England: Springer London; 2012:213-229.
 
Quintero OL, Amador-Patarroyo MJ, Montoya-Ortiz G, Rojas-Villarraga A, Anaya JM. Autoimmune disease and gender: plausible mechanisms for the female predominance of autoimmunity. J Autoimmun. 2012;38(2-3):J109-J119. [CrossRef] [PubMed]
 
Humbert M, Sitbon O, Chaouat A, et al. Survival in patients with idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension in the modern management era. Circulation. 2010;122(2):156-163. [CrossRef] [PubMed]
 
Dib H, Tamby MC, Bussone G, et al. Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma. Eur Respir J. 2012;39(6):1405-1414. [CrossRef] [PubMed]
 
Tamby MC, Chanseaud Y, Humbert M, et al. Anti-endothelial cell antibodies in idiopathic and systemic sclerosis associated pulmonary arterial hypertension. Thorax. 2005;60(9):765-772. [CrossRef] [PubMed]
 
Terrier B, Tamby MC, Camoin L, et al. Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2008;177(10):1128-1134. [CrossRef] [PubMed]
 
Bussone G, Tamby MC, Calzas C, et al. IgG from patients with pulmonary arterial hypertension and/or systemic sclerosis binds to vascular smooth muscle cells and induces cell contraction. Ann Rheum Dis. 2012;71(4):596-605. [CrossRef] [PubMed]
 
Colvin KL, Cripe PJ, Ivy DD, Stenmark KR, Yeager ME. Bronchus-associated lymphoid tissue in pulmonary hypertension produces pathologic autoantibodies. Am J Respir Crit Care Med. 2013;188(9):1126-1136. [CrossRef] [PubMed]
 
Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN. Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol. 2012;33(6):297-305. [CrossRef] [PubMed]
 
Rangel-Moreno J, Hartson L, Navarro C, Gaxiola M, Selman M, Randall TD. Inducible bronchus-associated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis. J Clin Invest. 2006;116(12):3183-3194. [CrossRef] [PubMed]
 
Rangel-Moreno J, Carragher DM, de la Luz Garcia-Hernandez M, et al. The development of inducible bronchus-associated lymphoid tissue depends on IL-17. Nat Immunol. 2011;12(7):639-646. [CrossRef] [PubMed]
 
Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol. 2011;11(2):119-130. [CrossRef] [PubMed]
 
Crome SQ, Lang PA, Lang KS, Ohashi PS. Natural killer cells regulate diverse T cell responses. Trends Immunol. 2013;34(7):342-349. [CrossRef] [PubMed]
 
Berzins SP, Smyth MJ, Baxter AG. Presumed guilty: natural killer T cell defects and human disease. Nat Rev Immunol. 2011;11(2):131-142. [CrossRef] [PubMed]
 
Perros F, Cohen-Kaminsky S, Humbert M. Understanding the role of CD4+CD25(high) (so-called regulatory) T cells in idiopathic pulmonary arterial hypertension. Respiration. 2008;75(3):253-256. [CrossRef] [PubMed]
 
Ormiston ML, Chang C, Long LL, et al. Impaired natural killer cell phenotype and function in idiopathic and heritable pulmonary arterial hypertension. Circulation. 2012;126(9):1099-1109. [CrossRef] [PubMed]
 
Perros F, Cohen-Kaminsky S, Gambaryan N, et al. Cytotoxic cells and granulysin in pulmonary arterial hypertension and pulmonary veno-occlusive disease. Am J Respir Crit Care Med. 2013;187(2):189-196. [CrossRef] [PubMed]
 
Isono F, Fujita-Sato S, Ito S. Inhibiting RORγt/Th17 axis for autoimmune disorders. Drug Discov Today. 2014;19(8):1205-1211. [CrossRef] [PubMed]
 
Gerosa F, Baldani-Guerra B, Lyakh LA, et al. Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells. J Exp Med. 2008;205(6):1447-1461. [CrossRef] [PubMed]
 
Wang Q, Franks HA, Porte J, et al. Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation. Immunology. 2011;134(1):60-72. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1 –  Phenotypic analysis by flow cytometry of monocyte-derived dendritic cells (MoDCs) from control subjects and patients with idiopathic PAH. A, Histograms of isotype control-stained and of CD40-, CD80-, CD86-, or CD209-stained interferon (IFN)-γ/lipopolysaccharide (LPS)-activated MoDCs (CD11c+major histocompatibility complex [MHC]-IIhigh) ± 10−6M Dex. B, Mean fluorescence intensity for CD40, CD86, and B7H2 staining of control and PAH Dex/IFN-γ/LPS-generated MoDCs, determined by flow cytometry; n = 5 in each condition. There was no detectable difference between control and PAH MoDCs without Dex (data not shown). Data are presented as MFI. CTRL = control; Dex = dexamethasone; MFI = mean fluorescent intensity; PAH = pulmonary artery hypertension.Grahic Jump Location
Figure Jump LinkFigure 2 –  Migratory capacity of MoDCs measured by transwell migration assay. CTRL and PAH IFN-γ/LPS-generated MoDCs (CD11+MHC-IIhigh) untreated or treated with Dex were loaded onto the upper chambers of transwell plates and allowed to migrate against BM, CCL19 (250 ng/mL), or CCL21 (250 ng/mL) present in the lower chamber. A, BM. B, CCL19 (250 ng/mL). C, CCL21 (250 ng/mL). After 4 h of incubation, migrated cells were then counted with flow cytometer. The chemotaxis indexes were calculated by dividing the number of migrated dendritic cells in the presence of a chemotactic ligand by that in its absence (BM) and expressed in %; n = 5 in each condition. There was no detectable difference between control and PAH MoDCs without Dex (data not shown). BM = basal medium; CCL = chemokine (C-C motif) ligand. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3 –  Cytokine expression analysis in control and idiopathic PAH MoDCs with or without Dex treatment using flow cytometry. After fixation and permeabilization, CTRL and PAH IFN-γ/LPS-activated MoDCs (CD11+MHC-IIhigh) were stained for intracellular IL-6, IL-12p40, IL-10, and TNFα content. A, Untreated CTRL and PAH MoDCs. B, Dex-treated CTRL and PAH MoDCs. Data are presented as % of total CD11+MHC-IIhighMoDCs. n = 5 in each experimental condition. TNFα = tumor necrosis factor-α. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 4 –  MoDC-induced activation and proliferation of CD4 T cells in mixed leukocyte reactions (MLRs). CTRL and PAH IFN-γ/LPS-activated MoDCs (CD11+MHC-IIhigh) ± Dex were cocultured with CD3+CD4+T cells (ratio dendritic cell/T 1:100) during 4 d. A, CD69+CD3+CD4+ T cells (activated T cells). B, Proliferating CD3+CD4+ T cells. Data are presented as % of total CD3+CD4+ present in the MLR. n = 5 in each experimental condition. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 5 –  MoDC-driven T-cell polarization in MLR. Intracellular cytokine detection was carried out on CD3+CD4+ T cells from MLR. After 4 d of coculture of T cells with CTRL or PAH IFNγ/LPS-activated MoDCs (CD11+MHC-IIhigh) with or without Dex pretreatment. T-cell surfaces were stained for CD3 and CD4, fixed/permeabilized, and stained for intracellular cytokine. Measurements of IL-2, TNFα, and IL-10 were taken to assess their pleiotropic effects on lymphocyte activation, growth, and differentiation, as well as IFNγ (T helper [Th]1 response), IL-4 (Th2 response), and IL-17 (Th17 response). A, IFN-γ (Th1 response). B, IL-4 (Th2 response). C, IL-17 (Th17 response). D, IL-2, TNFα, and IL-10. Data are presented as % of total CD3+CD4+ present in the MLR; n = 5 in each experimental condition. See Figure 1, 3, and 4 legends for expansion of abbreviations.Grahic Jump Location
Figure Jump LinkFigure 6 –  Enumeration of circulating lymphocyte subpopulations. Subjects’ blood lymphocyte analysis was carried out with a No-Lyse No-Wash protocol, gating on Hoechst+ nucleated leukocytes (discrimination from Hoechst RBC counts). CD3+ (total T cells), CD3+CD4+ (Th cells), CD3+CD8+ (cytotoxic T cells), and CD19 (B cells) lymphocytes were counted by flow cytometry. A, CD19 (B cells) lymphocytes. B, CD3+ (total T cells). C, CD3+CD4+ (Th cells). D, CD3+CD8+ (cytotoxic T cells). Data are presented in 106 cells/mL. Enumeration was performed on control subjects (n = 20) and patients with idiopathic PAH (n = 17). See Figure 1 and 5 legends for expansion of abbreviations.Grahic Jump Location
Figure Jump LinkFigure 7 –  Control and idiopathic PAH T-cell polarization. CD3+CD4+ T cells were isolated from whole blood (control subjects and patients with PAH) by negative selection with a rosette and gradient technique, activated for 24 h with a cell stimulation cocktail (containing phorbol 12-myristate 13-acetate and ionomycin, plus protein transport inhibitors), then processed for staining of surface CD3 and CD4 antigens, and intracellular cytokines specific to T-cell subpopulations: A, B, IL-2 and IFNγ (Th1); C-E, IL-4, IL-13, and IL-10 (Th2); and F, IL-17 (Th17). Data are presented as % of total CD3+CD4+ cells. Cytokine profile was performed on CD3+CD4+ Th cells from control subjects (n = 16) and patients with idiopathic PAH (n = 8). See Figure 1 and 5 legends for expansion of abbreviations.Grahic Jump Location
Figure Jump LinkFigure 8 –  Epigenetic assays for Th17 immune cell monitoring in blood. Methylation study of the IL17A gene was performed on genomic DNA from the blood of patients with PAH (n = 28; 19 with idiopathic PAH and nine with heritable PAH) and control subjects (n = 19). IL17A demethylation is the epigenetic signature of Th17 cells. See Figure 1 and 5 legends for expansion of abbreviations.Grahic Jump Location

Tables

Table Graphic Jump Location
TABLE 1 ]  Clinicopathologic Features of Subjects Whose Blood Was Used for MoDC Generation, Characterization, and Cellular Assays

Data are mean ± unless otherwise indicated. 6MWD = 6-min walk distance; ERA = endothelin receptor antagonist; iPAH = idiopathic pulmonary arterial hypertension; MoDC = monocyte-derived dendritic cell; mPAP = mean pulmonary artery pressure; N/A = not applicable; NYHA = New York Heart Association; PAH = pulmonary arterial hypertension; PDE5 = phosphodiesterases type 5; PVR = pulmonary vascular resistance.

Table Graphic Jump Location
TABLE 2 ]  Clinicopathologic Features of Subjects Whose Blood Was Used for Study of Circulating Lymphocyte Subpopulations

Data are mean ± unless otherwise indicated. See Table 1 legend for expansion of abbreviations.

Table Graphic Jump Location
TABLE 3 ]  Clinicopathologic Features of Subjects Whose Blood Was Used for CD4+ T-Cell Isolation and Stimulation

Data are mean ± unless otherwise indicated. See Table 1 legend for expansion of abbreviations.

Table Graphic Jump Location
TABLE 4 ]  Clinicopathologic Features of Subjects Whose Blood Genomic DNA Was Used for Epigenetic Analysis

Data are mean ± unless otherwise indicated. PDE5-i = phosphodiesterases type 5 inhibitor. See Table 1 legend for expansion of other abbreviations.

Table Graphic Jump Location
TABLE 5 ]  Characteristics of Antibodies Used in Flow Cytometry Analyses

IFN = interferon; MHC = major histocompatibility complex; MLR = mixed leukocyte reaction; TNF = tumor necrosis factor. See Table 1 legend for expansion of other abbreviations.

References

Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med. 2004;351(14):1425-1436. [PubMed]
 
Huertas A, Perros F, Tu L, et al. Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: a complex interplay. Circulation. 2014;129(12):1332-1340. [CrossRef] [PubMed]
 
Dorfmüller P, Humbert M. Progress in pulmonary arterial hypertension pathology: relighting a torch inside the tunnel. Am J Respir Crit Care Med. 2012;186(3):210-212. [PubMed]
 
Price LC, Wort SJ, Perros F, et al. Inflammation in pulmonary arterial hypertension. Chest. 2012;141(1):210-221. [PubMed]
 
Kherbeck N, Tamby MC, Bussone G, et al. The role of inflammation and autoimmunity in the pathophysiology of pulmonary arterial hypertension. Clin Rev Allergy Immunol. 2013;44(1):31-38. [PubMed]
 
Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol. 2009;297(6):L1013-L1032. [PubMed]
 
West J, Austin E, Fessel JP, Loyd J, Hamid R. Rescuing the BMPR2 signaling axis in pulmonary arterial hypertension. Drug Discov Today. 2014;19(8):1241-1245. [PubMed]
 
Song Y, Coleman L, Shi J, et al. Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice. Am J Physiol Heart Circ Physiol. 2008;295(2):H677-H690. [PubMed]
 
Sanchez O, Marcos E, Perros F, et al. Role of endothelium-derived CC chemokine ligand 2 in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2007;176(10):1041-1047. [PubMed]
 
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(9):920-927. [CrossRef] [PubMed]
 
Huertas A, Tu L, Gambaryan N, et al. Leptin and regulatory T-lymphocytes in idiopathic pulmonary arterial hypertension. Eur Respir J. 2012;40(4):895-904. [PubMed]
 
Cracowski JL, Chabot F, Labarère J, et al. Proinflammatory cytokine levels are linked to death in pulmonary arterial hypertension. Eur Respir J. 2014;43(3):915-917. [PubMed]
 
Price LC, Caramori G, Perros F, et al. Nuclear factor κ-B is activated in the pulmonary vessels of patients with end-stage idiopathic pulmonary arterial hypertension. PLoS ONE. 2013;8(10):e75415. [CrossRef] [PubMed]
 
Perros F, Dorfmüller P, Montani D, et al. Pulmonary lymphoid neogenesis in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2012;185(3):311-321. [PubMed]
 
Ogawa A, Nakamura K, Mizoguchi H, et al. Prednisolone ameliorates idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2011;183(1):139-140. [PubMed]
 
Sanchez O, Sitbon O, Jaïs X, Simonneau G, Humbert M. Immunosuppressive therapy in connective tissue diseases-associated pulmonary arterial hypertension. Chest. 2006;130(1):182-189. [PubMed]
 
Cohen-Kaminsky S, Hautefort A, Price L, Humbert M, Perros F. Inflammation in pulmonary hypertension: what we know and what we could logically and safely target first. Drug Discov Today. 2014;19(8):1251-1256. [PubMed]
 
Lambrecht BN, Hammad H. The role of dendritic and epithelial cells as master regulators of allergic airway inflammation. Lancet. 2010;376(9743):835-843. [PubMed]
 
Perros F, Dorfmüller P, Souza R, et al. Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension. Eur Respir J. 2007;29(3):462-468. [PubMed]
 
Kim SJ, Diamond B. Modulation of tolerogenic dendritic cells and autoimmunity [published online ahead of print April 18, 2014]. Semin Cell Dev Biol.
 
Ganguly D, Haak S, Sisirak V, Reizis B. The role of dendritic cells in autoimmunity. Nat Rev Immunol. 2013;13(8):566-577. [PubMed]
 
Quintana FJ, Yeste A, Mascanfroni ID. Role and therapeutic value of dendritic cells in central nervous system autoimmunity. Cell Death Differ. 2015;22(2):215-224. [CrossRef] [PubMed]
 
Humbert M, Sitbon O, Chaouat A, et al. Pulmonary arterial hypertension in France: results from a national registry. Am J Respir Crit Care Med. 2006;173(9):1023-1030. [CrossRef] [PubMed]
 
Hu J, Kinn J, Zirakzadeh AA, et al. The effects of chemotherapeutic drugs on human monocyte-derived dendritic cell differentiation and antigen presentation. Clin Exp Immunol. 2013;172(3):490-499. [CrossRef] [PubMed]
 
Selmi C. Autoimmunity in 2013. Clin Rev Allergy Immunol. 2014;47(1):100-109. [CrossRef] [PubMed]
 
Perros F, Cohen-Kaminsky S, Dorfmüller P, et al;. Inflammation in pulmonary arterial hypertension.. In:Abraham D, Handler C, Dashwood M, Coghlan G., eds. Translational Vascular Medicine. London, England: Springer London; 2012:213-229.
 
Quintero OL, Amador-Patarroyo MJ, Montoya-Ortiz G, Rojas-Villarraga A, Anaya JM. Autoimmune disease and gender: plausible mechanisms for the female predominance of autoimmunity. J Autoimmun. 2012;38(2-3):J109-J119. [CrossRef] [PubMed]
 
Humbert M, Sitbon O, Chaouat A, et al. Survival in patients with idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension in the modern management era. Circulation. 2010;122(2):156-163. [CrossRef] [PubMed]
 
Dib H, Tamby MC, Bussone G, et al. Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma. Eur Respir J. 2012;39(6):1405-1414. [CrossRef] [PubMed]
 
Tamby MC, Chanseaud Y, Humbert M, et al. Anti-endothelial cell antibodies in idiopathic and systemic sclerosis associated pulmonary arterial hypertension. Thorax. 2005;60(9):765-772. [CrossRef] [PubMed]
 
Terrier B, Tamby MC, Camoin L, et al. Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2008;177(10):1128-1134. [CrossRef] [PubMed]
 
Bussone G, Tamby MC, Calzas C, et al. IgG from patients with pulmonary arterial hypertension and/or systemic sclerosis binds to vascular smooth muscle cells and induces cell contraction. Ann Rheum Dis. 2012;71(4):596-605. [CrossRef] [PubMed]
 
Colvin KL, Cripe PJ, Ivy DD, Stenmark KR, Yeager ME. Bronchus-associated lymphoid tissue in pulmonary hypertension produces pathologic autoantibodies. Am J Respir Crit Care Med. 2013;188(9):1126-1136. [CrossRef] [PubMed]
 
Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN. Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol. 2012;33(6):297-305. [CrossRef] [PubMed]
 
Rangel-Moreno J, Hartson L, Navarro C, Gaxiola M, Selman M, Randall TD. Inducible bronchus-associated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis. J Clin Invest. 2006;116(12):3183-3194. [CrossRef] [PubMed]
 
Rangel-Moreno J, Carragher DM, de la Luz Garcia-Hernandez M, et al. The development of inducible bronchus-associated lymphoid tissue depends on IL-17. Nat Immunol. 2011;12(7):639-646. [CrossRef] [PubMed]
 
Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol. 2011;11(2):119-130. [CrossRef] [PubMed]
 
Crome SQ, Lang PA, Lang KS, Ohashi PS. Natural killer cells regulate diverse T cell responses. Trends Immunol. 2013;34(7):342-349. [CrossRef] [PubMed]
 
Berzins SP, Smyth MJ, Baxter AG. Presumed guilty: natural killer T cell defects and human disease. Nat Rev Immunol. 2011;11(2):131-142. [CrossRef] [PubMed]
 
Perros F, Cohen-Kaminsky S, Humbert M. Understanding the role of CD4+CD25(high) (so-called regulatory) T cells in idiopathic pulmonary arterial hypertension. Respiration. 2008;75(3):253-256. [CrossRef] [PubMed]
 
Ormiston ML, Chang C, Long LL, et al. Impaired natural killer cell phenotype and function in idiopathic and heritable pulmonary arterial hypertension. Circulation. 2012;126(9):1099-1109. [CrossRef] [PubMed]
 
Perros F, Cohen-Kaminsky S, Gambaryan N, et al. Cytotoxic cells and granulysin in pulmonary arterial hypertension and pulmonary veno-occlusive disease. Am J Respir Crit Care Med. 2013;187(2):189-196. [CrossRef] [PubMed]
 
Isono F, Fujita-Sato S, Ito S. Inhibiting RORγt/Th17 axis for autoimmune disorders. Drug Discov Today. 2014;19(8):1205-1211. [CrossRef] [PubMed]
 
Gerosa F, Baldani-Guerra B, Lyakh LA, et al. Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells. J Exp Med. 2008;205(6):1447-1461. [CrossRef] [PubMed]
 
Wang Q, Franks HA, Porte J, et al. Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation. Immunology. 2011;134(1):60-72. [CrossRef] [PubMed]
 
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