Despite modern therapy, pulmonary arterial hypertension (PAH) remains a fatal condition. PAH is characterized by construction and remodelling of pulmonary arteries leading to increased pulmonary vascular resistance and right heart failure (1).

An increasing body of evidence points to inflammation as a central pathogenic factor in idiopathic PAH (iPAH) as well as PAH secondary to other conditions (2–8). Perivascular inflammation and lymphoid tissue are found in lungs of PAH patients, and concordantly in murine models of pulmonary hypertension (2, 9–12). Elevated numbers of mast cells and T-helper type 2 (Th2) lymphocytes as well as increased expression of Th2 cytokines were repeatedly found in patients with pulmonary hypertension (2, 8, 13–16). Analogously, preclinical studies indicate an important underlying role of Th2-mediated immune signaling in the induction of morphological and functional changes found in PAH (2, 16–23).

The concept that the endothelium-derived peptide endothelin-1 (ET-1) serves as a major driver of PAH pathobiology is broadly accepted (1, 24–26). Prepro-endothelin-1 is the precursor of big-ET-1, which is converted to mature, bioactive ET-1 (24, 26, 27). Patients with pulmonary hypertension show elevated pulmonary vascular expression of ET-1 as well as increased levels of circulating ET-1 (25, 28).

ET-1 is a potent vasoconstrictor in the pulmonary circulation through activation of the G protein-coupled receptors endothelin A receptor (ET_A) and endothelin B receptor (ET_B) expressed on pulmonary arterial smooth muscle cells (PASMCs) (26, 27). The vasoconstrictive response induced by ET-1 involves thromboxane A₂ (TXA₂) release and consecutive TXA₂ receptor activation (29, 30). Downstream signalling of ET-1-evoked vasoconstriction critically depends on protein kinase C isozyme alpha (PKCα) (31). Contrariwise, activation of ET_B located on endothelial cells induces vasodilation via release of nitric oxide (NO) and prostaglandins (26, 27), partially dampening the vasoconstrictive effects of ET-1.

Besides its vasomotor actions, ET-1 promotes immune cell trafficking (32), such as via release of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 from monocytes and macrophages (33), or via ET_A-dependent release of IL-6 from vascular smooth muscle cells (34).

Endothelin receptor antagonists (ERAs) are routinely used for the treatment of PAH. However, whether ET_A-selective targeting or dual inhibition of ET_A/ET_B is superior to the other is controversially discussed.

In systemic sclerosis (SSc), PAH is a common vascular complication, and an important driver of mortality (24). The prognosis of PAH secondary to SSc (SSc-PAH) has been critically linked to the presence of anti-ET_A autoantibodies (AAb) (35). Autoimmunity is believed to play a significant role in PAH pathobiology (2, 5, 6, 35–40), and blood plasmablast levels were shown to be elevated in PAH patients (36). However, the potential involvement of anti-ET_B AAb in PAH is currently unknown and the immunomodulatory role of ET_B in the context of pulmonary hypertension remains largely elusive.

In this study, anti-ET_B AAb levels were assessed in PAH patients for the first time. To better characterize the immunomodulatory functions of ET_B, we additionally studied the effects of rescued ET_B deficiency in mice on pulmonary vascular disease, independent of and dependent on pulmonary Th2 inflammation.

Circulating ET-1 is cleared from the blood via ET-1/ET_B complex internalization (27, 41–43) and ET_B deficiency results in increased ET-1 plasma levels (44–46). Thus, effects on PAH-associated cardiovascular pathologies were studied in parallel in mice overexpressing prepro-ET-1 (_preET^tg) to allow differentiation between ET-1- and ET_B-mediated effects.

We hypothesized that ET_B plays an anti-inflammatory role, alleviating Th2-evoked pathologies of the cardiovascular system.

In our study, we evaluated anti-ET_B AAb in PAH patients for the first time and found increased levels in SSc-PAH patients. Furthermore, we characterized the immunomodulatory role of ET_B in the context of PAH using a mouse model of PAH due to rescued ET_B deficiency. Our data point to an important role of ET_B in immune homeostasis, with functional ET_B deficiency unleashing PAH development under inflammatory conditions.

ET_B deficiency is associated with defective ET-1 clearance (27, 41–43), and increased levels of plasma ET-1 (44–46). In order to distinguish ET-1- and ET_B-dependent effects in ET_B ^-/- mice, we studied a second transgenic mouse model in parallel, namely, prepro-endothelin-1 overexpressing (_preET^tg) mice.

Here, we show that pulmonary hypertension, pulmonary vascular hyperresponsiveness, and right ventricular hypertrophy were present in _preET^tg as well as in ET_B ^-/- mice, arguing for ET-1-specific effects. In _preET^tg mice, both pulmonary hypertension and right ventricular hypertrophy, however, were exclusively detected in highly aged (≥16 months old) mice, possibly as a result of decreasing NO-mediated compensatory effects with increasing age (61). In contrast, in ET_B ^-/- mice, PAH-associated alterations were generally observed at a younger age than in _preET^tg mice, which may be the result of synergistic unfavorable effects of ET_B deficiency and consecutive defective clearance of ET-1. Yet, as opposed to the characteristic findings in PAH patients, relevant pulmonary arterial remodeling was absent in both transgenic mouse lines, suggesting that the observed pulmonary hypertensive phenotypes are primarily driven by an increased vascular tone due to an imbalance of vasoconstrictive and vasodilatory mechanisms, rather than by extensive vascular remodeling in the pulmonary circulation.

Increased numbers of lymphocytes were found in BAL of both transgenic mouse models, again pointing to ET-1 as causative trigger. This finding is in line with previously described chronic lymphocytic lung inflammation as a result of prepro-ET-1 overexpression (58).

Of note, pronounced peripheral perivascular cluster of lymphoid infiltrates were exclusively found in ET_B ^-/- lungs, arguing for a dysregulation of the immune system due to the loss of ET_B. The perivascular space is a unique lung compartment, which might have been underestimated (62). Capillaries as well as lymphatic vessels from the periphery are found in the perivascular space, predominantly around the pulmonary arteries. This compartment is rather inactive in healthy lungs, but gains major significance in many types of lung inflammation. It is hypothesized that in certain conditions, inflammatory mediators induce extravasation of fluid and leukocytes from the periarterial capillaries, leading to thick cellular cuffs around the pulmonary arteries (63, 64). This defense mechanism may contribute to secondary lesions or processes such as the development of tertiary lymphoid tissue.

The data presented here give a strong hint that ET_B is involved in the regulation of perivascular infiltration of pulmonary arteries. This finding is of specific interest as perivascular infiltrates and lymphoid tissue are commonly found in lungs of PAH patients and in preclinical models of pulmonary hypertension (2, 9–12).

As previously shown by us and others, induction of pulmonary Th2 inflammation in mice induces relevant PAH-associated features such as perivascular inflammation, hyperresponsiveness of the pulmonary vasculature to vasoconstrictive stimuli, complex pulmonary arterial remodeling, and increase in right ventricular systolic pressure (2, 16–23). Importantly, a key role of Th2 inflammation in the pathogenesis of pulmonary hypertension has been demonstrated in lung-specific IL-13-overexpressing mice, which develop spontaneous pulmonary hypertension, pulmonary arterial remodeling, and right ventricular hypertrophy (22). Interestingly, also in Fra-2 transgenic mice, a well-described model of SSc-PAH and interstitial lung disease, a strong underlying Th2 phenotype is present (56, 65).

Here, we analyzed the effects of pulmonary Th2 inflammation as a second hit in ET_B ^-/- mice. Notably, both pulmonary vascular hyperresponsiveness and right ventricular hypertrophy were aggravated secondary to Th2 inflammation in ET_B ^-/- mice. Moreover, in ET_B ^-/- lungs, pulmonary perivascular inflammation and collagen deposition were increased.

On the cytokine level, IL-12 subunit p40 (IL-12p40) levels were largely increased in BAL of ET_B ^-/- mice, whereas Th2 cytokines were similar in ET_B ^-/- and WT mice. Increased IL-12p40 levels are notable with respect to the exaggerated PAH phenotype in ET_B ^-/- mice as well as the increased pulmonary collagen deposition, since PAH patients show elevated levels of circulating IL-12p40 (12). Analogously, IL-12p40 serum levels are increased in mice deficient for chemokine receptor CCR7, which develop pulmonary hypertension, pulmonary arterial remodeling, and perivascular lymphoid infiltrates in the lung (12). Moreover, IL-12p40 has been identified as a central profibrotic mediator in murine lung fibrosis (60).

Pulmonary vascular hyperresponsiveness to the stimuli ET-1 and TXA₂ analog U46619 was shown to be aggravated in ET_B ^-/- lungs compared to the WT lungs. Interestingly, pulmonary vascular hyperresponsiveness to serotonin was unchanged in ET_B ^-/- lungs, arguing for stimulus-specific alterations of the here assessed vasomotor responses. Mechanistically, ET-1-evoked hyperresponsiveness was most likely based on increased TXA₂ release following vascular ET-1 application in ET_B ^-/- mice, as indicated here by the elevated TXB₂ perfusate levels.

Increased responsiveness as a result of ET_A and/or TXA₂ receptor upregulation, however, was ruled out in this study. In fact, ET_A was downregulated in ET_B ^-/- lungs following induction of Th2 inflammation, possibly as a counter-response to the increase in local ET-1 expression in ET_B ^-/- lungs. Taken together, both the upregulation of ET-1 expression and the increased release of IL-12p40 may have contributed to the here observed exaggeration of the PAH phenotype in ET_B ^-/- mice following induction of pulmonary Th2 inflammation.

It can be assumed that the anti-inflammatory effects of ET_B signaling described in this study are primarily mediated via ET_B receptor activation on vascular and inflammatory cells. In contrast, secondary immunomodulatory effects in response to ET_B-regulated sodium and water reabsorption in the kidney are unlikely to underlie the detected anti-inflammatory properties of ET_B in the lung. Specifically, ET_B exerts natriuretic functions (66), and collecting duct-specific deficiency of ET_B accordingly causes systemic hypertension with decreased urinary aldosterone excretion and plasma renin activity (67). Reduced activation of the renin–angiotensin–aldosterone system is, however, characteristically associated with mitigated inflammation (68). The same holds true for natriuretic peptides, which are abundantly released upon volume expansion (69) and have protective immunomodulatory properties (70). These anti-inflammatory effects of renal ET_B deficiency stand in contrast to the pro-inflammatory effects in the lung detected in our study. Therefore, the anti-inflammatory role of ET_B in the lung seems unrelated to its natriuretic function.

Our finding that Th2 inflammation as a second hit augments hallmarks of PAH is in line with previous findings in mice expressing a hypomorphic bone morphogenetic protein receptor type 2 (BMPR2) transgene, which showed an increase in right ventricular systolic pressure following induction of a Th2 immune response (23). As Th2-mediated aggravation of PAH phenotypes has been repeatedly shown, it is tempting to speculate that mediators of Th2 signaling may serve as potential targets in PAH. This needs to be evaluated in further studies.

PAH may occur in SSc patients with or without accompanying interstitial lung disease and/or digital ulcers (71, 72). The endothelin system is believed to play a central role in SSc-PAH, and SSc-PAH patients benefit from ERA treatment (24, 71, 72). ERAs are further indicated to treat SSc-related digital ulcers (72). Additional SSc-associated complications seem to involve the endothelin system. Alveolitis is frequently observed in SSc patients (73, 74). Intriguingly, alveolitis was also experimentally induced in mice treated with anti-ET_A AAb and anti-angiotensin II type 1 receptor (AT₁R) AAb-positive IgG derived from SSc patients (75). Moreover, expression of type I collagen in fibroblasts following treatment with IgG from SSc patients correlated with anti-ET_A AAb levels, suggesting an underlying role of the endothelin system in SSc-associated fibrosis (75), as also discussed elsewhere (76).

The immunomodulatory actions of ET_B shown here may be of relevance for the early phase of PAH, in which inflammation, endothelial dysfunction, and hyperresponsiveness of the pulmonary vasculature are believed to play relevant mechanistic roles. In the chronic disease state, reflected by profound remodeling of the pulmonary arteries, ET_B, however, may play a less prominent role, as indicated by the fact that ET_A-selective blockers do not appear to be superior to dual ET_A/ET_B therapy in established PAH (77, 78). Prospective clinical trials comparing selective ERA (inhibition of ET_A) head-to-head against dual ERA (combined inhibition of ET_A/ET_B) therapy at early disease time points may be required to identify a potential advantage of selective ERA therapy in PAH.

In conclusion, our data show an anti-inflammatory role of ET_B. ET_B deficiency as a single hit is associated with spontaneous formation of marked lymphoid infiltrates in the perivascular space of the lung, in addition to pulmonary hypertension, pulmonary vascular hyperresponsiveness, and right ventricular hypertrophy. Th2 inflammation as a second hit aggravates PAH-associated pathologies in ET_B ^-/- mice. The pathogenic role of anti-ET_B AAb in SSc-PAH needs to be evaluated in further studies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the ethics committee (Charité - Universitätsmedizin Berlin, Germany; EA1/179/17). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by institutional authorities of the Charité – Universitätsmedizin Berlin, Germany, and the Local State Office of Health and Social Affairs Berlin (LAGeSo; Berlin, Germany).

CT and MW conceived and designed the research. CT, CG, TT, OK, BG, JN, and JHe performed experiments. CT, CG, JL, JHö, TT, OK, BG, JN, BO, AG, HH, ES, and MW analyzed data. All authors interpreted the results of the experiments. CT, CG, JL, JHö, TT, OK, and ES prepared figures. CT and JL drafted the manuscript. All authors edited and revised the manuscript and approved the final version of the manuscript.

This study received funding from the German Research Foundation (SFB-TR84, C6/C9 to MW and Z01b to AG) and Actelion Pharmaceuticals Germany GmbH. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

CT received funding for research from Deutsche Gesellschaft für Pneumologie, Bayer HealthCare, Boehringer Ingelheim, and for lectures from Actelion Pharmaceuticals, Boehringer Ingelheim. HH is CEO of CellTrend GmbH, Luckenwalde, Germany. MW received funding for research from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Deutsche Gesellschaft für Pneumologie, European Respiratory Society, Marie Curie Foundation, Else Kröner Fresenius Stiftung, CAPNETZ STIFTUNG, International Max Planck Research School, Actelion, Bayer Health Care, Biotest AG, Boehringer Ingelheim, NOXXON Pharma, Pantherna, Quark Pharma, Silence Therapeutics, Vaxxilon, and for lectures and advisory from Actelion, Alexion, Aptarion, Astra Zeneca, Bayer Health Care, Berlin Chemie, Biotest, Boehringer Ingelheim, Chiesi, Glaxo Smith Kline, Insmed, Novartis, Teva and Vaxxilon.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.