BackgroundData on cellular response and the decay of antibodies and T cells in time are scarce in lung transplant recipients (LTRs). Additionally, the development and durability of humoral and cellular immune responses have not been investigated in patients on the waitlist for lung transplantation (WLs). Here, we report our 6-month follow-up of humoral and cellular immune responses of LTRs and WLs, compared with controls.MethodsHumoral responses to two doses of the mRNA-1273 vaccination were assessed by determining spike (S)-specific IgG antibodies and neutralizing antibodies. Cellular responses were investigated by interferon gamma (IFN-γ) release assay (IGRA) and IFN-γ ELISpot assay at 28 days and 6 months after the second vaccination.ResultsIn LTRs, the level of antibodies and T-cell responses was significantly lower at 28 days after the second vaccination. Also, WLs had lower antibody titers and lower T-cell responses compared with controls. Six months after the second vaccination, all groups showed a decrease in antibody titers and T-cell responses. In WLs, the rate of decline of neutralizing antibodies and T-cell responses was significantly higher than in controls.ConclusionOur results show that humoral and cellular responses in LTRs, if they develop, decrease at rates comparable with controls. In contrast, the inferior cellular responses and the rapid decay of both humoral and cellular responses in the WL groups imply that WLs may not be protected adequately by two vaccinations and repeat boostering may be necessary to induce protection that lasts beyond the months immediately post-transplantation.

RATIONALE:Disease course in sarcoidosis is highly variable. Bronchoalveolar lavage fluid and mediastinal lymph nodes show accumulation of activated T cells with a T-helper (Th)17.1 signature, which correlates with non-resolving sarcoidosis. We hypothesize that the peripheral blood (PB) T cell phenotype may correlate with outcome. OBJECTIVES:To compare frequencies, phenotypes and function of circulating T cell populations in sarcoidosis patients with healthy controls (HCs) and correlate these parameters with outcome. METHODS:We used multi-color flow cytometry to quantify activation marker expression on PB T cell subsets in treatment-naïve patients and HCs. The disease course was determined after 2-year follow-up. Cytokine production was measured after T cell stimulation in vitro. MEASUREMENTS AND MAIN RESULTS:We observed significant differences between patients and HCs in several T cell populations, including CD8+ and CD4+ T cells, Th1/Th17 subsets, CD4+ T memory stem cells, regulatory T cells (Tregs) and γδ T cells. Decreased frequencies of CD4+ T cells and increased frequencies of Tregs and CD8+ γδ T cells correlated with worse outcome. Naïve CD4+ T cells displayed an activated phenotype with increased CD25 expression in patients with active chronic disease at 2-year follow-up. A distinctive Treg phenotype with increased expression of CD25, CTLA4, CD69, PD-1 and CD95 correlated with chronic sarcoidosis. Upon stimulation, both naïve and memory T cells displayed a different cytokine profile in sarcoidosis compared to HCs. CONCLUSIONS:Circulating T cell subpopulations of sarcoidosis patients display phenotypic abnormalities that correlate with disease outcome, supporting a critical role of aberrant T cell activation in sarcoidosis pathogenesis.

CD4+ T helper 2 (Th2) cells and group 2 innate lymphoid cells are considered the main producers of type-2 cytokines that fuel chronic airway inflammation in allergic asthma. However, CD8+ cytotoxic T (Tc) cells - critical for anti-viral defense - can also produce type-2 cytokines (referred to as 'Tc2' cells). The role of Tc cells in asthma and virus-induced disease exacerbations remains poorly understood, including which micro-environmental signals and cell types promote Tc2 cell formation. Here we show increased circulating Tc2 cell abundance in severe asthma patients, reaching peak levels during exacerbations and likely emerging from canonical IFNγ+ Tc cells through plasticity. Tc2 cell abundance is associated with increased disease burden, higher exacerbations rates and steroid insensitivity. Mouse models of asthma recapitulate the human disease by showing extensive type-2 skewing of lung Tc cells, which is controlled by conventional type-1 dendritic cells and IFNγ. Importantly, we demonstrate that the alarmin interleukin-33 (IL-33) critically promotes type-2 cytokine production by lung Tc cells in experimental allergic airway inflammation. Our data identify Tc cells as major producers of type-2 cytokines in severe asthma and during exacerbations that are remarkably sensitive to alterations in their inflammatory tissue micro-environment, with IL-33 emerging as an important regulator of Tc2 formation.

IntroductionPrevious studies have shown an increase of T cells and chemokines in vascular lesions of patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, detailed characterization of these T cells is still lacking, nor have treatment effects been evaluated.MethodsWe included 41 treatment-naive CTEPH patients at diagnosis, 22 patients at 1-year follow-up, and 17 healthy controls (HCs). Peripheral blood T cells were characterized by flow cytometry for subset distribution, cytokine expression and activation marker profile. We used multiplex immunofluorescence to identify CCR6+ T cells in endarterectomy tissue from 25 patients.ResultsAt diagnosis, proportions of CCR6+ CD4+ T cells were increased in CTEPH patients compared with HCs. Patients displayed a significantly reduced production capacity of several cytokines including TNFα, IFNγ, GM-CSF and IL-4 in CD4+ T cells, and TNFα and IFNγ in CD8+ T cells. CD4+ and CD8+ T cells showed increased expression of the immune checkpoint protein CTLA4. Multivariate analysis separated CTEPH patients from HCs, based on CCR6 and CTLA4 expression. At 1-year follow-up, proportions of CCR6+CD4+ T cells were further increased, IFNγ and IL-17 production capacity of CD4+ T cells was restored. In nearly all vascular lesions we found substantial numbers of CCR6+ T cells.ConclusionThe observed increase of CCR6+ T cells and modulation of the IFNγ and IL-17 production capacity of circulating CD4+ T cells at diagnosis and 1-year follow-up – together with the presence of CCR6+ T cells in vascular lesions - support the involvement of the Th17-associated CCR6+ T cell subset in CTEPH.

Pulmonary arterial hypertension (PAH) is rare disease that is categorized as idiopathic (IPAH) when no underlying cause can be identified. Lungs of most patients with IPAH contain increased numbers of T cells and dendritic cells (DCs), suggesting involvement of the immune system in its pathophysiology. However, our knowledge on circulating immune cells in IPAH is rather limited. We used flow cytometry to characterize peripheral blood DCs and T cells in treatment-naive IPAH patients, compared with connective-tissue disease-PAH (CTD-PAH) patients and healthy controls (HCs). At diagnosis, T-helper (Th) cells of IPAH patients were less capable of producing TNFα, IFNγ, IL-4 and IL-17 compared to HCs. IPAH patients showed a decreased frequency of Th2 cells and significantly enhanced expression of the CTLA4 checkpoint molecule in naive CD4+ T cells and both naive and memory CD8+ T cells. Frequencies and surface marker expression of circulating DCs and monocytes were essentially comparable between IPAH patients and HCs. Principal component analysis (PCA) separated IPAH patients—but not CTD-PAH patients—from HCs, based on T-cell cytokine profiles. At 1-year follow-up, the frequencies of IL-17+ production by memory CD4+ T cells were increased in IPAH patients and accompanied by increased proportions of Th17 and Tc17 cells, as well as decreased CTLA4 expression. Treatment-naive IPAH patients displayed a unique T-cell phenotype that was different from CTD-PAH patients and was characterized by reduced cytokine-producing capacity. These findings point to involvement of adaptive immune responses in IPAH, which may have an implication for the development of therapeutic interventions.

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old Tnfaip3DNGR1-KO mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 (Tnfaip3) gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of Tnfaip3DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3DNGR1-KO mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in Tnfaip3DNGR1-KO mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (Bmpr2), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the Bmpr2 gene.

INTRODUCTION:Autoreactivity against pulmonary vascular structures is thought to be involved in idiopathic pulmonary arterial hypertension (IPAH), but the underlying mechanisms remain poorly understood. We hypothesised that aberrant B-cell activation contributes to IPAH aetiology.METHODS:Mice with enhanced B-cell activation due to B-cell-specific overexpression of the B-cell receptor (BCR) signalling molecule Bruton's tyrosine kinase (BTK) were subjected to lung injury and examined for several pulmonary hypertension (PH) indices. Peripheral blood lymphocytes from patients with IPAH (n=13), connective tissue disease-associated PAH (CTD-PAH, n=9), congenital heart disease PAH (n=7), interstitial lung disease associated PH (n=17) and healthy controls (n=19) were characterised by 14-colour flow cytometry.RESULTS:Following pulmonary injury, BTK-overexpressing mice showed prolonged activation of B cells and CXCR5+ follicular T-helper (Tfh) cells, as well as features of PH development. Patients with CTD-PAH and CHD-PAH displayed reduced proportions of circulating non-switched-memory B cells (p=0.03, p=0.02, respectively). Interestingly, we observed increased BTK protein expression in naive (p=0.007) and memory B-cell subsets of patients with IPAH and CTD-PAH. BTK was particularly high in patients with IPAH with circulating autoantibodies (p=0.045). IPAH patients had low frequencies of circulating CXCR5+ Tfh cells (p=0.005). Hereby, the increased BTK protein expression in B cells was associated with high proportions of Tfh17 (p=0.018) and Tfh17.1 (p=0.007) cells within the circulating Tfh population.CONCLUSIONS:Our study shows that pulmonary injury in combination with enhanced B-cell activation is sufficient to induce PH symptoms in mice. In parallel, immune homeostasis in patients with IPAH is compromised, as evidenced by increased BCR signalling and cTfh17 polarisation, indicating that adaptive immune activation contributes to IPAH disease induction or progression.

Recent studies have provided evidence for an important contribution of the immune system in the pathophysiology of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). In this report, we investigated whether the inflammatory profile of pulmonary hypertension patients changes over time and correlates with patient WHO subgroups or survival. 50 PAH patients (16 idiopathic (I)PAH, 24 Connective Tissue Disease (CTD)-PAH and 10 Congenital Heart Disease (CHD)-PAH), 37 CTEPH patients and 18 healthy controls (HCs) were included in the study. Plasma inflammatory markers at baseline and after 1-year follow-up were measured using ELISAs. Subsequently, correlations with hemodynamic parameters and survival were explored and data sets were subjected to unbiased multivariate analyses. At diagnosis, we found that plasma levels of interleukin-6 (IL-6) and the chemokines (C-X3-C) motif legend CXCL9 and CXCL13 in CTD-PAH patients were significantly increased, compared with HCs. In idiopathic PAH patients the levels of tumor growth factor-β (TGFβ), IL-10 and CXCL9 were elevated, compared with HCs. The increased CXCL9 and IL-8 concentrations in CETPH patients correlated significantly with decreased survival, suggesting that CXCL9 and IL-8 may be prognostic markers. After one year of treatment, IL-10, CXCL13 and TGFβ levels changed significantly in the PAH subgroups and CTEPH patients. Unbiased multivariate analysis revealed clustering of PH patients based on inflammatory mediators and clinical parameters, but did not separate the WHO subgroups. Importantly, these multivariate analyses separated patients with < 3 years and > 3 years survival, in particular when inflammatory mediators were combined with clinical parameters. Our study revealed elevated plasma levels of inflammatory mediators in different PAH subgroups and CTEPH at baseline and at 1-year follow-up, whereby CXCL9 and IL-8 may prove to be prognostic markers for CTEPH patients. While this study is exploratory and hypothesis generating, our data indicate an important role for IL-8 and CXCL9 in CHD and CTEPH patients considering the increased plasma levels and the observed correlation with survival. In conclusion, our studies identified an inflammatory signature that clustered PH patients into WHO classification-independent subgroups that correlated with patient survival.

Background: Accumulating evidence shows involvement of the immune system in CTEPH pathogenesis. Previous studies have shown increased presence of T-cells and chemokines in CTEPH surgical PEA material. Currently, no data is present for further determination and classification of these T-cells. Moreover, no data are available regarding T-cell activation, cytokine production, and Th-subset division in treatment naïve CTEPH patients. Therefore, our aim was to more closely investigate peripheral blood T cells in CTEPH patients at diagnosis. Methods: Peripheral blood samples were collected from 43 CTEPH patients at baseline and 17 healthy controls (HC). Th-subset division, T-cell activation, and cytokine production were measured in CTEPH patients at diagnosis and compared to HC. Results: At baseline, we found increased proportions of CCR6+ CD4+ CD45RA- T-cells compared to HC. CD4+ CD45RA- T-cells of CTEPH patients showed decreased cytokine production. Strikingly, we found increased CTLA4 expression on CD4+ T cells, indicating a regulatory or immunomodulatory effect. After unsupervised clustering analysis for these T cell subsets and activation markers, CTEPH patients were clustered separately from HCs due to CTLA4 expression and CCR6+ cells. No specific changes were found for central vs peripheral thrombotic lesions at baseline within CTEPH patients. Conclusion: Taken together, we found evidence for CCR6+ CD4+ T cell-dependent changes in peripheral blood samples in CTEPH patients. We found decreased cytokine production in CD45RA- CD4+ T-cells, indicating suppression or exhaustion. Overall, our studies indicate a possible role for CCR6+ T cell-dependent immunopathology in CTEPH patients.

In the last decades it has become clear that dendritic cells (DCs) critically contribute to the development of T helper 2 (Th2)-mediated allergic diseases, such as asthma. Two main conventional DC (cDC) subsets, type 1 cDCs (cDC1s) and cDC2s, can be identified based on the expression of cell-surface molecules and transcription factors [1]. Using mouse models in which specific DC subsets are targeted or by adoptive transfer of DC subsets, it has been shown that upon activation in the airways cDC2s migrate towards the lung-draining lymph node where they induce allergen-specific Th2 cells, which subsequently promote eosinophilic airway inflammation [2]. The role of cDC1s in asthma is more controversial, although most recent reports indicate that cDC1s can efficiently suppress allergic airway inflammation [3], either by the induction of regulatory T cells [4] or through increased interleukin (IL)-12 production [5]. These functional properties of DC subsets were evaluated in mouse models, but to date it is unclear how these findings reflect the characteristics of cDC subsets in asthmatic patients with different disease severities. Therefore, we questioned whether DC subset frequencies and the surface expression of co-stimulatory and co-inhibitory molecules differ between healthy persons and asthmatic patients. To investigate this, we focused on the Th2-stimulatory molecules (CD86, OX40L) and Th2-inhibitory molecule PD-L1 [6–8]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Vroman reports grants from EAACI Young Investigatoru0027s Grant, during the conduct of the study. Conflict of interest: Dr. Tindemans has nothing to disclose. Conflict of interest: Dr. Lukkes has nothing to disclose. Conflict of interest: Dr. van Nimwegen has nothing to disclose. Conflict of interest: Dr. de Boer has nothing to disclose. Conflict of interest: Dr. Tramper reports grants from OM Pharma, grants from ESPID, grants from Stichting Coolsingel, during the conduct of the study; which were all paid directly to a research foundation Conflict of interest: Dr. Braunstahl reports grants from GSK, grants from Novartis, grants from AstraZeneca, grants from Chiesi, grants from Sanofi, outside the submitted work. Conflict of interest: Dr. Hendriks reports grants from Netherlands Lung Foundation, outside the submitted work. Conflict of interest: Dr. Kool reports grants from Netherlands Lung Foundation, during the conduct of the study.

ABSTRACT Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-kB ligand (RANKL), is used as a biomarker for assessing severity of liver fibrosis. However, its expression and role in pulmonary fibrosis are unknown. We hypothesized that OPG also has a role in pulmonary fibrosis. Human and mouse control and fibrotic lung tissue were used to examine OPG expression, and mouse precision-cut lung slices to study OPG regulation in pulmonary fibrosis. Serum from idiopathic pulmonary fibrosis (IPF) patients and controls was analysed to investigate whether OPG levels correlate with disease status as measured by lung function. OPG-protein levels were significantly higher in mouse and human fibrotic lung tissue compared to control. OPG-mRNA and protein production were induced in mouse precision-cut-lung slices upon TGFβ stimulation and could be inhibited with galunisertib, a TGFβ receptor kinase inhibitor. OPG-protein levels in fibrotic mouse lung tissue correlated with degree of fibrosis. Isolated lung fibroblasts from IPF patients had higher OPG-protein levels than control fibroblasts. Serum OPG levels in IPF patients, at first presentation, negatively correlated with diffusing capacity to carbon monoxide. Finally, serum OPG levels higher than 1234 pg/ml at first presentation were associated with progression of disease in IPF patients. In conclusion, OPG is produced in lung tissue, associates with fibrosis, and may be a potential prognostic biomarker for IPF disease progression. Validation in a larger cohort is warranted to further explore the role of OPG in pulmonary fibrosis and its potential for assessing the prognosis of fibrotic lung disease in individual patients. Take home message Osteoprotegerin is present in fibrotic lung tissue and high serum levels correlate with low lung function and IPF disease progression in this small study, indicating osteoprotegerin may have value as a biomarker to predict IPF progression

Pulmonale Hypertonie (PH) ist eine extrem belastende fortschreitende Erkrankung, die durch erhöhte pulmonal-arterielle Drücke gekennzeichnet ist. Diese führen zu rechtsventrikulärer (RV) Insuffizienz (Rechtsherzinsuffizienz), Herzinsuffizienz und letztlich zum Tod. Je nach Ätiologie werden Patienten mit PH in die folgenden 5 Gruppen eingeteilt: (1) Pulmonal-arterielle Hypertonie (PAH), (2) PH bei Erkrankungen des linken Herzens, (3) PH bei Lungenerkrankungen und/oder Hypoxie, (4) PH aufgrund chronischer Thromboembolien (CTEPH) und (5) PH mit unklaren multifaktoriellen Mechanismen. Derzeit ist die Prognose für Patienten mit PAH bzw. CTEPH, selbst mit PAH-spezifischer Pharmakotherapie, weiterhin schlecht. Die durchschnittliche 5-Jahres-Überlebensrate beträgt für PAH 57%–59% und für inoperable CTEPH 53%–69%. Daher ist ein besserer Einblick in die Pathogenese von PAH und CTEPH dringend erforderlich, um neue Behandlungsstrategien entwickeln zu können. Neuere Studien zeigen sowohl bei PAH- als auch bei CTEPH-Patienten ein erhöhtes Vorliegen und eine vermehrte Aktivierung angeborener und adaptiver Immunzellen. Darüber hinaus haben umfassende Forschungsarbeiten zu Biomarkern ergeben, dass zahlreiche Entzündungs- und Immunmarker bei PAH- und CTEPH-Patienten mit deren Hämodynamik und/oder Prognose korrelieren. Zunehmende Evidenzdaten über die pathologische Rolle von Immunzellen bei angeborener und adaptiver Immunität haben zu zahlreichen vielversprechenden präklinischen interventionellen Studien geführt. Diese ziehen ihrerseits innovative klinische Studien nach sich, die derzeit durchgeführt werden. Möglicherweise ist neben den aktuellen Behandlungen, die sich ausschließlich auf eine Vasodilatation stützen, eine Kombination immunmodulatorischer Therapien erforderlich, um eine wirksame Behandlung und Prävention gegen das Fortschreiten dieser Erkrankung zu etablieren. In dieser Übersichtsarbeit beschreiben wir die aktuellen Fortschritte bezüglich unserer Erkenntnisse über die Beteiligung der einzelnen Zelltypen des Immunsystems bei PH. Wir geben einen Überblick über den wachsenden Korpus an Evidenzdaten zu Entzündung und Immunität im Rahmen der Pathogenese von PH sowie über die Anwendung von inflammatorischen Biomarkern und immunmodulatorischen Therapien bei PAH und CTEPH.

In recent decades it has become clear that dendritic cells (DCs) critically contribute to the development of T helper 2 (Th2)-mediated allergic diseases, such as asthma. Two main conventional DC (cDC) subsets, type 1 cDCs (cDC1s) and cDC2s, can be identified based on the expression of cell-surface molecules and transcription factors [1]. Using mouse models in which specific DC subsets are targeted or by adoptive transfer of DC subsets, it has been shown that upon activation in the airways cDC2s migrate towards the lung-draining lymph node where they induce allergen-specific Th2 cells, which subsequently promote eosinophilic airway inflammation [2]. The role of cDC1s in asthma is more controversial, although most recent reports indicate that cDC1s can efficiently suppress allergic airway inflammation [3], either by the induction of regulatory T-cells [4] or through increased interleukin (IL)-12 production [5]. These functional properties of DC subsets were evaluated in mouse models, but to date it is unclear how these findings reflect the characteristics of cDC subsets in asthmatic patients with different disease severities. Therefore, we questioned whether DC subset frequencies and the surface expression of co-stimulatory and co-inhibitory molecules differ between healthy persons and asthma patients. To investigate this, we focused on the Th2-stimulatory molecules (CD86, OX40L) and Th2-inhibitory molecule PD-L1 [6–8]. Proportions of conventional dendritic cell subsets and the expression of co-stimulatory and co-inhibitory molecules in cDC2s are altered in asthma patients. These changes are most pronounced in asthmatics with frequent exacerbations. http://bit.ly/2wbb8UJ

Abstract Background Treatment of pulmonary sarcoidosis is recommended in case of significant symptoms, impaired or deteriorating lung function. Evidence-based treatment recommendations are limited and largely based on expert opinion. Prednisone is currently the first-choice therapy and leads to short-term improvement of lung function. Unfortunately, prednisone often has side-effects and may be associated with impaired quality of life. Methotrexate is presently considered second-line therapy, and appears to have fewer side-effects. Objective The primary objective of this trial is to investigate the effectiveness and tolerability of methotrexate as first-line therapy in patients with pulmonary sarcoidosis compared with prednisone. The primary endpoint of this study will be the change in hospital-measured Forced Vital Capacity (FVC) between baseline and 24 weeks. Secondary objectives are to gain more insights in response to therapy in individual patients by home spirometry and patient-reported outcomes. Blood biomarkers will be examined to find predictors of response to therapy, disease progression and chronicity, and to improve our understanding of the underlying disease mechanism. Methods/design In this prospective, randomized, non-blinded, multi-center, non-inferiority trial, we plan to randomize 138 treatment-naïve patients with pulmonary sarcoidosis who are about to start treatment. Patients will be randomized in a 1:1 ratio to receive either prednisone or methotrexate in a predefined schedule for 24 weeks, after which they will be followed up in regular care for up to 2 years. Regular hospital visits will include pulmonary function assessment, completion of patient-reported outcomes, and blood withdrawal. Additionally, patients will be asked to perform weekly home spirometry, and record symptoms and side-effects via a home monitoring application for 24 weeks. Discussion This study will be the first randomized controlled trial comparing first-line treatment of prednisone and methotrexate and provide valuable data on efficacy, safety, quality of life and biomarkers. If this study confirms the hypothesis that methotrexate is as effective as prednisone as first-line treatment for sarcoidosis but with fewer side-effects, this will lead to improvement in care and initiate a change in practice. Furthermore, insights into the immunological mechanisms underlying sarcoidosis pathology might reveal new therapeutic targets. Trial registration The study was registered on the 19th of March 2020 in the International Clinical Trial Registry, www.clinicaltrials.gov; ID NCT04314193.

BACKGROUND:Conventional type 1 dendritic cells (cDC1s) control anti-viral and anti-tumor immunity by inducing antigen-specific cytotoxic CD8+ T-cell responses. Controversy exists whether cDC1s also control CD4+ T helper 2 (Th2) cell responses, since suppressive and activating roles have been reported. DC activation status, controlled by the transcription factor NF-κB, might determine the precise outcome of Th-cell differentiation upon encounter with cDC1s. To investigate the role of activated cDC1s in Th2-driven immune responses, pulmonary cDC1s were activated by targeted deletion of A20/Tnfaip3, a negative regulator of NF-κB signaling.METHODS:To target pulmonary cDC1s, Cd207 (Langerin)-mediated excision of A20/Tnfaip3 was used, generating Tnfaip3fl/fl xCd207+/cre (Tnfaip3Lg-KO ) mice. Mice were exposed to house dust mite (HDM) to provoke Th2-mediated immune responses.RESULTS:Mice harboring Tnfaip3-deficient cDC1s did not develop Th2-driven eosinophilic airway inflammation upon HDM exposure, but rather showed elevated numbers of IFNγ-expressing CD8+ T cells. In addition, Tnfaip3Lg-KO mice harbored increased numbers of IL-12-expressing cDC1s and elevated PD-L1 expression in all pulmonary DC subsets. Blocking either IL-12 or IFNγ in Tnfaip3Lg-KO mice restored Th2 responses, whereas administration of recombinant IFNγ during HDM sensitization in C57Bl/6 mice blocked Th2 development.CONCLUSIONS:These findings indicate that the activation status of cDC1s, shown by their specific expression of co-inhibitory molecules and cytokines, critically contributes to the development of Th2 cell-mediated disorders, most likely by influencing IFNγ production in CD8+ T cells.

Chronic perivascular inflammation is a prominent feature in the lungs of idiopathic pulmonary arterial hypertension (IPAH). Although the proportions of conventional dendritic cells (cDCs) and plasmacytoid DCs are increased in IPAH lungs, it remains unknown whether activated cDCs play a pathogenic role. The Tnfaip3 gene encodes the ubiquitin-binding protein A20, which is a negative regulator of NF-κB, critically involved in DC activation. Targeting of Tnfaip3/A20 in cDCs was achieved by Clec9a (DNGR1)-Cre-mediated excision of the Tnfaip3 gene in Tnfaip3DNGR1-KO mice. Mice were evaluated for signs of pulmonary hypertension (PH) using right heart catheterization, echocardiography and measurement of the Fulton index. Inflammation was assessed by immunohistochemistry and flow cytometry. Pulmonary cDCs and mo-DCs from 31-week-old Tnfaip3DNGR1-KO mice showed modulated expression of cell surface activation markers compared to Tnfaip3DNGR1-WT mice. Tnfaip3DNGR1-KO mice developed elevated right ventricular (RV) systolic pressure and RV hypertrophy. The lungs of these mice displayed increased vascular remodeling and perivascular and peribronchial immune cell infiltration resembling tertiary lymphoid organs. Proportions of activated T cells and expression of IL-1β, IL-6 and IL-10 were enhanced in the lungs of Tnfaip3DNGR1-KO mice. Autoreactive IgA and IgG1 was detected in bronchoalveolar lavage and autoreactive IgA recognizing pulmonary endothelial antigens was present in the serum of Tnfaip3DNGR1-KO mice. All signs of PH were ameliorated in Tnfaip3DNGR1-KO mice by anti-IL-6 antibody treatment. These results indicate that activation of the NF-κB pathway in DCs, through deletion of A20/Tnfaip3, leads to experimental PH with accompanied pulmonary inflammation in an IL-6-dependent fashion.

Pulmonary hypertension (PH) is a debilitating progressive disease characterized by increased pulmonary arterial pressures, leading to right ventricular (RV) failure, heart failure and, eventually, death. Based on the underlying conditions, PH patients can be subdivided into the following five groups: (1) pulmonary arterial hypertension (PAH), (2) PH due to left heart disease, (3) PH due to lung disease, (4) chronic thromboembolic PH (CTEPH), and (5) PH with unclear and/or multifactorial mechanisms. Currently, even with PAH-specific drug treatment, prognosis for PAH and CTEPH patients remains poor, with mean five-year survival rates of 57%–59% and 53%–69% for PAH and inoperable CTEPH, respectively. Therefore, more insight into the pathogenesis of PAH and CTEPH is highly needed, so that new therapeutic strategies can be developed. Recent studies have shown increased presence and activation of innate and adaptive immune cells in both PAH and CTEPH patients. Moreover, extensive biomarker research revealed that many inflammatory and immune markers correlate with the hemodynamics and/or prognosis of PAH and CTEPH patients. Increased evidence of the pathological role of immune cells in innate and adaptive immunity has led to many promising pre-clinical interventional studies which, in turn, are leading to innovative clinical trials which are currently being performed. A combination of immunomodulatory therapies might be required besides current treatment based on vasodilatation alone, to establish an effective treatment and prevention of progression for this disease. In this review, we describe the recent progress on our understanding of the involvement of the individual cell types of the immune system in PH. We summarize the accumulating body of evidence for inflammation and immunity in the pathogenesis of PH, as well as the use of inflammatory biomarkers and immunomodulatory therapy in PAH and CTEPH.