Circulating Interleukin-7 in Human Pulmonary Arterial Hypertension

Objectives: Interleukin-7 (IL-7) secures B cell maturation, regulatory T and natural killer (NK) cell survival, and homeostasis, all of which are important for beneficial immunomodulation in pulmonary arterial hypertension (PAH). However, the role and potential impact of IL-7, VEGF-C and the vascular injury markers ICAM-1, and VCAM-1 on the pathobiology and severity of PAH is unknown. Methods: EDTA blood was collected during cardiac catheterization from the superior vena cava (SVC), pulmonary artery (PA), and ascending aorta (AAO) in children with pulmonary hypertension (PH) [n = 10; 9.1 (3.9–18.5) years] and non-PH controls [n = 10; 10.5 (2.0–17.3) years]. Compartment-specific plasma concentrations of IL-7, VEGF-C, aldosterone, ICAM-1, and VCAM-1 were determined using Meso Scale Discovery's multi array technology and the LIAISON Aldosterone Assay. Results: Children with PH had approximately 50% lower IL-7 (p < 0.01) and 59% lower VEGF-C plasma levels (p < 0.001) in the SVC, PA, and AAO versus non-PH controls. IL-7 and VEGF-C concentrations negatively correlated with the pulmonary vascular resistance (PVR)/systemic vascular resistance (SVR) ratio (rho = −0.51 and r = −0.62, respectively). Central-venous IL-7 strongly positively correlated with VEGF-C (r = 0.81). Most patients had a step down in ICAM-1 and VCAM-1 plasma concentrations across the pulmonary circulation and both ICAM-1 and VCAM-1 transpulmonary gradients negatively correlated with invasive hemodynamics. Conclusion: This manuscript is the first report on decreased circulating IL-7 and VEGF-C plasma concentrations in human PAH and their inverse correlations with invasive surrogates of PAH severity. Additional and larger studies are needed to explore the role of the immune-modulatory IL-7 and VEGF-C in pediatric and adult PAH.


INTRODUCTION
The inflammatory response and its resolution, delivered by the innate and adaptive immune systems, are important for development, disease severity, and responses to therapy, in many patients with pulmonary arterial hypertension (PAH) (1,2). The pro-inflammatory phenotype of pulmonary endothelial cells in PAH is characterized by increased surface expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, and accompanied by an excessive release of cytokines and chemokines (3). The sequelae include impairment of angiogenesis and cardiovascular repair mechanisms, and promotion of pulmonary vascular remodeling. Emerging evidence suggests that natural killer (NK) cells (4)(5)(6) and regulatory T cells (Tregs, CD4+) (7) play major, protective immune-modulatory roles in both preclinical and human PAH, and associated right ventricular dysfunction (RVD). Additional protective factors include hepatocyte growth factor (HGF) that reduces PAH severity and inflammation by attenuation of NF-kB signaling (8), and suppresses vascular medial hyperplasia and extracellular matrix accumulation (9) in the monocrotaline PAH rat model. Interleukin-7 (IL-7) forms a heterodimer with HGF and secures B cell maturation, T and NK cell survival, and homeostasis (10). However, in contrast to HGF, the role and potential impact of IL-7 on the pathobiology and severity of PAH is unknown. In addition, VEGF-C, aldosterone, and the aforementioned vascular injury markers ICAM-1 and VCAM-1 have not been studied in children with PAH, and have not been correlated with invasive hemodynamics.

Study Population
10 PH patients and 10 non-PH controls underwent combined right and left heart catheterization. Non-PH controls had left ventricular outflow tract obstruction (LVOTO, n = 9) or s/p reconstruction of a double aortic arch (n = 1). EDTA blood samples, pressure recordings, and blood gas analysis (SpO2) were obtained near-simultaneously at three sites: superior vena cava (SVC), pulmonary artery (PA), and ascending aorta (AAO). To ensure compartment-specific analysis, none of the PH patients, and non-PH controls had intra-or extracardiac shunts. The legal caregivers of each study subject gave written informed consent. Inclusion criteria were defined according to the World Symposium on Pulmonary Hypertension in Nice (2018): mean pulmonary artery pressure (mPAP) > 20 mmHg (14,15). Detailed information on PH-patients and non-PH controls including demographic data, functional status, and hemodynamics can be found in Table 1. The study has been approved by Hannover Medical School (#2200).

Biomarker Assays
EDTA whole blood samples obtained during cardiac catheterization were immediately centrifuged for 10 min at 1300g. Aldosterone plasma concentrations were measured using the LIAISON Aldosterone Assay (REF 310450, DiaSorin Inc., Stillwater, MN, USA), a chemiluminescent immunoassay (CLIA) in routine clinical use. One sample had to be diluted 1:10 because the initial measured aldosterone concentration was above the detection limit of 1000 pg/ml. IL

Statistical Analysis
Statistical analyses were performed with the GraphPad Prism 6 software and R. Data are expressed as mean ± standard error (SEM) and were tested for normal distribution with D'Agostino-
In order to test whether pro-angiogenic factors are decreased in pediatric PAH, we measured plasma VEGF-C concentrations, and found -similarly to IL-7-approximately 59% lower circulating VEGF-C levels in pediatric PAH vs. controls (SVC, PA, AAO; p < 0.001) that correlated negatively with the PVR/SVR ratio (r = −0.62, p = 0.0034; Figure 1B). Central-venous IL-7 strongly positively correlated with VEGF-C (r = 0.81, p < 0.0001; Figure 2), suggesting a possible shared upstream pathway negatively affecting IL-7 mediated immunomodulation and VEGF-C induced angiogenesis in pediatric PAH. In addition, there was a trend toward higher aldosterone levels in PAH vs. controls in children, but there was no correlation between plasma aldosterone and PAH severity (PVR/SVR ratio; Figure 1C right panel). We had previously studied 41 adult patients with IPAH, and 8 age-matched, unrelated controls (16). Aldosterone plasma concentrations were similar in the PAH children under study (SVC, mean 283.6 ± 73.0 pg/ml; Figure 1C) and those of adults with IPAH (mean 248.5 ± 38.8 pg/ml) (16).

Transpulmonary Decrease in the Vascular Injury Markers ICAM-1 and VCAM-1 in Pediatric PH Patients vs. Non-PH Controls
Next, we investigated the vascular injury markers ICAM-1 and VCAM-1 in pediatric PH. Most patients had a step down in ICAM-1 ( Figure 3A) and VCAM-1 (Figure 3B

DISCUSSION
Here, we report for the first time invasive, compartmentspecific levels of circulating IL-7, VEGF-C, aldosterone, ICAM-1 and VCAM-1 in children with moderate PH. We found a very pronounced decrease in plasma IL-7 concentrations in pediatric PH patients vs. non-PH controls, which inversely correlated with precapillary PH severity, as assessed by the ratio of PVR/SVR. Similarly to IL-7, we found approximately 59% lower circulating VEGF-C levels in pediatric PAH vs. controls that correlated negatively with the PVR/SVR ratio. Central-venous IL-7 strongly positively correlated with VEGF-C. Additionally, there was a trend toward higher aldosterone levels in pediatric PH patients vs. controls, with levels similar to those previously measured in adults (16). Transpulmonary gradient analysis revealed that most patients had a step down in ICAM-1 and VCAM-1 plasma concentrations across the pulmonary circulation, indicating either intrapulmonary uptake or increased intrapulmonary degradation of these vascular injury markers.
The rationale to investigate circulating IL-7 in (pediatric) PAH was its aforementioned role in NK and Treg cell survival (4)(5)(6)(7), both of which are thought to be crucial for the adaptations necessary in PAH and RVD. NK cells are the innate immune equivalent of cytotoxic T cells that spot stressed or damaged cells and induce targeted cell death. NK cells are mainly circulating in the blood stream, but resident populations have also been found in lung, liver, and lymph nodes. NK cells are reduced in number and function in peripheral blood of adults with PAH, compared to control subjects, releasing less cytotoxic granules (4). Dysregulated immunity resulting from deficient Treg activity in athymic rats contributes to increased inflammation, leading to macrophage activation and rapid progressive PAH and RV failure (7).
Consistent with our finding of the greatly decreased IL-7 concentrations in the systemic and pulmonary circulation of children with PAH (Figure 1), others had demonstrated reduced expression of IL-7 receptor (IL-7R) in circulating PBMC and CD4+ T-cells from adult scleroderma (SSc) patients with PAH vs. those without PAH (17).
According to the aforementioned SSc-PAH study and our current findings, we postulate that blood cell IL-7/IL7R signaling is decreased in adult and pediatric PAH. However, increased immunoreactivity for IL-7 was found in tertiary (ectopic) lymphoid tissues, i.e., highly organized perivascular follicles, in adult idiopathic PAH (IPAH) lungs, suggesting specific immuneadaptive mechanisms in the pathophysiology of the disease, at least in adults (18). So far, it is unclear whether intrapulmonary IL-7 signaling is a detrimental driver or counterregulatory protector in pulmonary lymphoid neogenesis (18), and other hallmarks of PAH pathobiology.
Additionally, we revealed for the first time decreased levels of VEGF-C in pediatric PAH patients. VEGF-C primarily signals through vascular endothelial growth factor receptor 3 (VEGFR3) which is expressed in endothelial cells and regulates cardiovascular development and lymphangiogenesis (19,20). VEGFR3 has been found to be a positive regulator of bone morphogenetic protein receptor 2 (BMPR2) signaling in PAH and knockout of VEGFR3 in endothelial cells caused exacerbation of hypoxia-induced PH in mice (21). Consistent with our finding of significantly decreased VEGF-C plasma levels in PH children, the expression of VEGFR3 (both VEGFR3 mRNA and protein) was found to be decreased in pulmonary arterial endothelial cells isolated from adult PAH patients vs. controls (21).
In our previous human biomarker study, ICAM-1 was increased in adult IPAH and CTD-PAH vs. controls, whereas VCAM-1 and pro-inflammatory, anti-angiogenic interleukin 12 (IL-12) were significantly elevated in CTD-PAH only (16). In the same study, we showed for the first time that a clinically relevant aldosterone/galectin-3 axis exists in adult PAH and associates with clinical symptoms (WHO functional class) (16).

CONCLUSION
The limitations of our study are common in biomarker studies on a rare disease, and include small sample size (children), non-healthy controls (invasive diagnostics), and a wide age range. Nevertheless, this article is the first report on decreased circulating IL-7 and VEGF-C plasma concentrations in human PAH and their inverse correlations with invasive surrogates of PAH severity. Additional and larger studies are warranted to explore the role of the immune-modulatory IL-7 and VEGF-C at multiple locations, cells and tissues (circulation, pulmonary vascular/lymph vessels, RV myocardium) in pediatric and adult PAH.

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

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Hannover Medical School (#2200). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
FD provided clinical data, performed experiments and statistical analysis, produced display items, and wrote parts of the manuscript. EL supervised, performed experiments and statistical analysis, and produced display items. PC performed advanced data analysis and produced display items. HB, CH, and GH collected blood samples during pediatric cardiac catheterizations. GH conceptualized, designed and supervised the study, performed experiments, and wrote the manuscript. All authors reviewed and revised the manuscript for important intellectual content.

FUNDING
This study was supported by the German Research Foundation (DFG; HA4348/2-2 and KFO311 HA4348/6-2 to GH) and the European Pediatric Pulmonary Vascular Disease Network (www.pvdnetwork.org). GH receives additional funding from the Federal Ministry of Education and Research (BMBF ViP+ program 03VP08053; BMBF 01KC2001B).