Elevated Interleukin 6 Levels Predict Clinical Worsening in Pediatric Pulmonary Arterial Hypertension

Abstract

OBJECTIVE

To assess whether circulating interleukin 6 (IL-6) is associated with measures of disease severity and clinical worsening in pediatric pulmonary arterial hypertension (PAH).

STUDY DESIGN

IL-6 was measured by ELISA in serum samples from a cross-sectional cohort from the NHLBI PAH Biobank (PAHB) (n=175) and a longitudinal cohort from Children’s Hospital Colorado (CHC) (n=61). Associations between IL-6, disease severity, and outcomes were studied with regression and Kaplan-Meier analysis.

RESULTS

In analyses adjusted for age and sex, each log-unit higher IL-6 was significantly associated in the PAHB cohort with higher pulmonary vascular resistance indices, lower odds of having idiopathic PAH or treatment with prostacyclin, and higher odds of having PAH associated with a repaired congenital shunt. In the CHC cohort, each log-unit higher IL-6 was significantly associated with higher mean pulmonary arterial pressure (mPAP) over time. Kaplan-Meier analysis in the CHC cohort revealed that IL-6 was significantly associated with clinical worsening (a composite score of mortality, transplant, or palliative surgery) (p=0.037).

CONCLUSIONS

IL-6 was significantly associated with worse hemodynamics at baseline and over time and may be associated with clinical worsening. IL-6 may provide a less invasive method for disease monitoring and prognosis in pediatric PAH as well as a potential therapeutic target.

Pulmonary arterial hypertension (PAH) is a progressive disease in both children and adults that results in significant vascular remodeling due to increased resistance in the pulmonary circulation (1). If left untreated, PAH can lead to right heart failure and death. Similar to the criteria for adults, PAH in children is defined as having) a mean pulmonary arterial pressure (mPAP) higher than 20 mmHg,) a pulmonary vascular resistance index (PVRI) of 3 Wood units*m² or greater, and a pulmonary capillary wedge pressure (PCWP) < 15 mmHg at rest, according to the proceedings of the 6th World Symposium on Pulmonary Hypertension (2,3). Definitive diagnosis of PAH is made by right heart catheterization (RHC), a procedure which is very invasive for children and carries a non-negligible risk of death, cardiac arrest, and mechanical circulatory support (1,4). Further, monitoring disease severity by measuring 6-minute walk distance (6MWD) is not feasible in small children (5). Echocardiography, therefore, is the mainstay for monitoring disease progression in pediatric PAH (3) but often lacks the sensitivity for making therapeutic decisions.

Pediatric PAH shares similarities with adult PAH, and adult therapies are leveraged for use in pediatric patients (6,7). Children, however, are distinct in that their lungs and pulmonary vascular bed are immature and still developing within a rapidly changing immunological milieu (8). The causes of pediatric PAH are also different from those in adults, with a larger proportion of cases due to congenital heart disease and fewer cases of idiopathic PAH (IPAH) or cases due to connective tissue disease, drug exposure, or infection (9). This difference in etiology presents greater promise for the reversal of the disease in children if the underlying mechanisms of PAH are better understood.

In addition to being released in response to tissue damage and upregulated during infection and active autoimmune disease, interleukin 6 (IL-6), a pro-inflammatory cytokine, has been increasingly recognized as a major contributor to the pathobiology of PAH (10,11). In small single center studies, IL-6 was elevated in adults with PAH and associated with right ventricular dysfunction and decreased survival (12–14). In a large adult cohort of diverse PAH subtypes and severities, IL-6 was associated with disease-related forms of PAH, worse hemodynamics and function, and decreased survival, including in less severe PAH (15). IL-6 has not been studied as widely in children, though in one small study of pediatric PAH using a broad cytokine panel and univariate analysis, IL-6 was associated with adverse outcomes, suggesting the possible applicability of IL-6 as a prognostic marker among pediatric patients (16). This study examined the association between IL-6 levels and hemodynamics, functional variables, and outcomes in pediatric PAH in a longitudinal cohort to investigate the relationship between IL-6 and disease progression over time.

METHODS

Samples and clinical data were obtained from two pediatric cohorts: a cross-sectional cohort from the National Institutes of Health and National Heart, Lung, and Blood Institute Pulmonary Arterial Hypertension Biobank (PAHB) (n=175) and a longitudinal cohort from Children’s Hospital Colorado (CHC) (n=61). Subjects were age 21 or younger in both cohorts. The PAH Biobank includes clinical data and serum samples of subjects with WHO Group 1 PAH, collected from 5 enrollment centers across North America. Specimen collection was approved by the Institutional Review Boards at each site, and an informed consent from parents or legal guardians of all subjects under age 18 was obtained, in addition to an assent from subjects ages 7–18. De-identified patient data was managed by the PAH Biobank, and whole blood specimens procured via venipuncture at the time of enrollment were stored as serum in secure freezers at the University of Cincinnati. Within the PAHB cohort, a subset of 42 subjects who had a right heart catheterization (RHC) within 12 months of enrollment was studied to evaluate relationships between hemodynamics and IL-6 levels. The CHC cohort includes subjects who were seen at least twice and were followed for pulmonary hypertension with RHC, echocardiography, and serum sampling at each visit. A subset of the PAH Biobank cohort was also enrolled in the CHC cohort for longitudinal evaluation (n=41). Thus, the CHC cohort was only used for longitudinal analysis. This study was conducted in accordance with the Declaration of Helsinki and approved by the Johns Hopkins University Institutional Review Board (NA_00069663, Baltimore, MD).

The electrochemiluminescent sandwich immunosorbent assay used in this study to measure serum IL-6 levels was previously described in Simpson et al. (15)

Statistical Analyses

Continuous variables are presented as means with standard deviations or medians with interquartile range as appropriate, based on normality of the data. Comparisons were made between variables using t-tests, Kruskall-Wallis tests, and Mann-Whitney tests as appropriate. IL-6 concentrations were log transformed for regression due to non-normality. Associations between IL-6 levels and clinical variables were analyzed using regression models adjusted for the PAH clinically relevant variables of age and sex (17,18). A longitudinal regression accounting for the nested nature of the data (i.e. multiple samples per subject, using mixed linear and logistic models) was then performed on the CHC cohort. Relationships between IL-6 levels and time to mortality, transplant, or palliative shunt (Pott’s shunt or atrial septectomy) were studied in the CHC cohort using Kaplan-Meier analysis with dichotomization of subjects based on the median IL-6 level of the cohort and were explored further using Cox proportional hazard models adjusted for age and sex. A P value equal to or less than 0.05 was considered statistically significant. All statistical analyses were conducted with Stata (Version 15.1, StataCorp, College Station, TX).

RESULTS

Subjects from the PAHB cohort (n=175) were mostly female (59%) with a median age of 13 (interquartile range (IQR) 8–17) (Table I). Most subjects had IPAH (48%) or disease-associated PAH (APAH), especially congenital heart disease-associated PAH (APAH-CHD) (46%). The PAHB cohort had a median mPAP of 52 mmHg, PVRI of 11 Wood units*m², PCWP of 9 mmHg, and cardiac index of 3.5 L/min/m². Most subjects were on multiple medications, including calcium channel blockers (CCBs), phosphodiesterase (PDE5) inhibitors, endothelin receptor antagonists (ERAs), and IV or subcutaneous prostacyclin or prostacyclin analogs (IV/SQ PCA).

Table 1.

Demographics and Clinical Characteristics of the PAH Biobank and Children’s Hospital Colorado Cohorts

	PAH Biobank	Children’s Hospital Colorado	p value
Demographics
Subjects, n	175	61
Age, years	13 (8–17)	5 (3–8)	<0.001
Sex, n female (%)	104 (59%)	37 (60%)	0.900
Height, cm	117 (92–150)	104 (84–123)	0.087
Weight, kg	19.85 (12.10–44.50)	15.90 (11.65–26.18)	0.007
BSA, m2	0.74 (0.52–1.26)	0.69 (0.51–0.99)	0.120
NYHA FC, n I/II/III/IV (% missing)	19/54/49/11 (28%)	17/9/4/2 (47%)	0.960
6MWD, median m (% missing)	442 (43%)	427 (70%)	0.510
Etiology, n IPAH/FPAH/APAH	84/11/80	23/5/33
Visits, n	1	2 (1–4)
IL-6 Levels
IL-6, pg/mL	1.42 (0.89–2.48)	1.16 (0.57–2.69)	0.250
Hemodynamics
RAP, mmHg	7 (5–10)	6 (5–8)	0.120
mPAP, mmHg	52.0 (39.5–60.8)	38.5 (28.8–52.3)	0.005
PCWP, mmHg	9 (7–11)	8 (6–9)	0.060
PVRI, Wood units*m2	11.0 (5.5–22.7)	7.5 (4.7–13.4)	0.290
Cardiac output, L/min	3.2 (2.3–4.3)	2.8 (1.8–3.6)	0.750
Cardiac index, L/min/m2	3.5 (2.7–4.3)	3.6 (2.8–4.8)	0.100

All data presented as median (IQR) unless otherwise specified.

Definition of abbreviations: BSA: body surface area; NYHA FC: New York Heart Association Functional Class; 6MWD: six-minute walk distance; IPAH: idiopathic pulmonary arterial hypertension; FPAH: familial pulmonary arterial hypertension; APAH: disease-associated pulmonary arterial hypertension; RAP: right atrial pressure; mPAP: mean pulmonary arterial pressure; PCWP: pulmonary capillary wedge pressure; PVRI: pulmonary vascular resistance index. Hemodynamic data is from subjects with a right heart catheterization within 12 months of PAH Biobank enrollment (n=42) or at enrollment in the Children’s Hospital Colorado cohort.

Subjects from the CHC cohort (n=61) were mostly female (60%) and younger than the PAHB cohort, with a median age of 5 (IQR 3–8) (Table 1). Most subjects had IPAH (38%) or APAH-CHD (48%). The CHC cohort had a median mPAP of 39 mmHg, PVRI of 8 Wood units*m², PCWP of 8 mmHg, and cardiac index of 3.6 L/min/m². CHC subjects were treated with PAH therapies similar to those in the PAHB cohort.

Subjects in the PAHB cohort had a median IL-6 value of 1.42 pg/mL (IQR 0.89–2.48) (Table 1). In the PAHB cohort, subjects with APAH had significantly higher IL-6 levels than other subtypes (1.65, IQR 1.05–2.92 pg/mL, p<0.001) (Figure 1). In particular, APAH-CHD subjects with a repaired congenital shunt had the highest IL-6 levels (2.32, IQR 1.08–3.44 pg/mL, p=0.006) compared with other APAH subgroups, which also included PAH associated with connective tissue disease, portopulmonary hypertension, and CHD without a shunt repair. Ultimately, each log-unit higher IL-6 was significantly associated with lower odds of having IPAH (OR 0.56, 95% CI 0.37–0.83, p=0.004) but higher odds of having APAH (OR 1.86, 95% CI 1.24–2.79, p=0.003), particularly APAH-CHD with a congenital shunt repair (OR 1.60, 95% CI 1.04–2.45, p=0.033) (Table 2).

Figure 1.

Comparison of IL-6 levels among PAH subtypes in the PAH Biobank cohort: idiopathic PAH (IPAH), familial PAH (FPAH), and disease-associated PAH (APAH). 25th percentile, median, and 75th percentile values are presented for each subtype.

Table 2.

IL-6 Adjusted Associations with Clinical Variables at Baseline in the PAH Biobank Cohort and Longitudinally in the Children’s Hospital Colorado Cohort

	PAH Biobank	Children’s Hospital Colorado
	Baseline Regression Coefficient (95% CI, p value)	Longitudinal Regression Coefficient (95% CI, p value)
RAP, mmHg	1.53 (−0.44–3.49, 0.123)	0.34 (−0.05–0.72, 0.085)
mPAP, mmHg	9.06 (−0.01–18.12, 0.050)	2.92 (0.83–5.01, 0.006)
PCWP, mmHg	0.11 (−1.55–1.76, 0.897)	0.31 (−0.06–0.68, 0.101)
PVRI, Wood units*m2	7.56 (1.29–13.82, 0.020)	0.66 (−0.17–1.49, 0.118)
Cardiac output, L/min	0.21 (−0.65–1.06, 0.625)	0.18 (0.02–0.33, 0.025)
Cardiac index, L/min/m2	0.15 (−0.68–0.97, 0.718)	0.17 (−0.02–0.4, 0.085)
Stroke volume, mL	0.00 (−0.01–0.02, 0.750)	---
PA compliance, mL/mm Hg	−0.24 (−0.66–0.18, 0.254)	---
Heart rate, beats/min	5.17 (−5.96–16.29, 0.352)	---
6MWD, m	−19.54 (−51.25–12.18, 0.224)	−9.28 (−26.88–8.32, 0.301)
	Odds Ratio (95% CI, p value)
IPAH	0.56 (0.37–0.83, 0.004)	---
APAH	1.86 (1.24–2.79, 0.003)	---
APAH-CHD with shunt repair	1.60 (1.04–2.45, 0.033)	---
IV/SQ prostacyclin	0.65 (0.43–0.97, 0.036)	---

Definition of abbreviations: PA: pulmonary arterial; CHD: congenital heart disease; IV/SQ: intravenous or subcutaneous. See Table 1 for other abbreviations. Hemodynamic data is from subjects with a right heart catheterization within 12 months of PAH Biobank enrollment (n=42). All regression coefficients and odds ratios were adjusted for age and sex.

In an age and sex adjusted analysis of subjects with a right heart catheterization within 12 months of enrollment in the PAHB cohort (n=42), each log-unit higher IL-6 was significantly associated with a 9.06 mmHg higher mPAP (95% CI −0.01–18.12, p=0.050) and a 7.56 Wood units*m² higher PVRI (95% CI 1.29–13.82, p=0.020) (Table 2).

In an age and sex adjusted, longitudinal analysis of the CHC cohort, each log-unit higher IL-6 was significantly associated over time with a higher 2.92 mmHg mPAP (95% CI 0.83–5.01 mmHg, p=0.006) and a 0.18 L/min higher cardiac output (95% CI 0.02–0.33 L/min, p=0.025) (Table 2).

Treatment with IV/SQ prostacyclin analogs was used as another measure of clinical severity. In an age and sex adjusted analysis of the PAHB cohort, the odds of being treated with IV/SQ prostacyclin analogs was decreased with increasing IL-6 (OR 0.65, 95% CI 0.43–0.97, p=0.036) (Table 2). There was no statistical significance, however, between IL-6 levels and treatment type over time in the CHC cohort and no statistical significance between IL-6 levels and functional metrics, such as 6MWD and New York Heart Association Functional Class, in either cohort.

In unadjusted Kaplan Meier analysis, IL-6 was significantly associated with time to death, transplant, or palliative shunt in PAH (p=0.037) (Figure 2). However, in Cox proportional hazard regression, the positive relationship between IL-6 levels and mortality, transplant, or palliative surgery was no longer statistically significant after adjustment for age and sex (HR 3.61, 95% CI 0.78–16.73, p=0.101).

Figure 2.

Kaplan-Meier analysis of clinical worsening (mortality, transplant, or palliative surgery) among PAH subjects with IL-6 levels above vs. below the median (log rank p=0.037).

DISCUSSION

IL-6 is a pro-inflammatory cytokine that has been implicated in the endothelial dysfunction and resultant remodeling of the pulmonary vasculature that characterizes PAH (10,11). IL-6 has been studied in greater depth in adults as an early, pathobiologic biomarker of disease development as well as a possible therapeutic target. Currently, a clinical trial is underway in adults investigating the efficacy of tocilizumab, an IL-6 receptor inhibitor, in PAH (NCT02676947). IL-6, however, has not been comprehensively evaluated in a pediatric-specific population in which congenital anomalies, developmental changes of both the lung and pulmonary vasculature, and a changing inflammatory response are critical factors in the development of the disease.

This study examines the association between IL-6, hemodynamics, and clinical variables in diverse pediatric PAH subtypes and provides novel longitudinal data on IL-6 levels and outcomes in this population. Our results show that APAH-CHD subjects had the highest IL-6 levels among PAH subtypes. Further, we demonstrate a clinically significant increase in mPAP and PVRI with increasing IL-6. Moreover, IL-6 levels were associated with an increase in mPAP over time and with time to death, transplant, or palliative shunt. The relationship between IL-6 and clinical worsening, however, was attenuated in Cox proportional hazard modeling adjusted for age and sex, which may have been in part due to the small size of the cohort and the low number of adverse events. Nevertheless, taken together, these findings suggest that IL-6 may provide key insights into PAH progression in pediatric patients and indicate when an escalation in care may be warranted, particularly in the disease-associated subtypes of PAH.

Our findings show decreased odds of being treated with IV/SQ prostacyclin analogs with each log-unit increase in IL-6, which may be a result of the anti-inflammatory properties of prostacyclins, which have been shown to reduce circulating IL-6 levels (19). Unfortunately, we were not able to specifically evaluate the cause of the association in this study. It is also possible that other therapeutic combinations of CCBs, PDE5 inhibitors, and ERAs may have been used instead of IV/SQ prostacyclin analogs in subjects with moderate disease, leading to the observed association between increased IL-6 levels and decreased odds of prostacyclin use. Our results also showed an unexpected increase in cardiac output for each log-unit increase in IL-6 over time, despite an increase in mPAP, which may be due to the inherent inaccuracies of measuring cardiac output by Fick in children and in the setting of a shunt (20,21).

Overall, the results of preclinical animal and tissue culture studies support the involvement of enhanced inflammation in the pathogenesis of PAH (10,12,22,23). Only a few studies to date have examined plasma biomarkers in pediatric PAH, which include growth differentiation factor-15, connective tissue growth factor, and N-terminal pro-B-type natriuretic peptide (NT-proBNP) (24–26). Unlike IL-6, NT-proBNP points to more downstream effects of PAH, such as latent heart failure or excess cardiomyocyte stretch (27–29). Animal models of PAH, on the other hand, have shown that perivascular inflammation precedes vascular remodeling of the pulmonary circuit (30). IL-6 thus offers an upstream marker of disease development and may aid in the earlier diagnosis and treatment of PAH in children. In particular, PAH patients with poor outcomes and the highest IL-6 levels, such as those with APAH-CHD, may be more likely to benefit from novel anti-IL-6 therapies like tocilizumab to halt the progression of the disease or even reverse its course. Measuring IL-6, therefore, would be useful for monitoring the therapeutic effect of such therapies. Despite a number of promising IL-6 studies among adults with PAH, IL-6 to date has not been evaluated extensively in children as a potential mediator of pediatric PAH pathobiology or as a prospective therapeutic target. Our work suggests that despite the varying etiologies for PAH in children, there may be a similar background of inflammation and endothelial dysfunction that underlies the pathobiology of the disease in children, meriting further investigation of anti-IL-6 therapies in this population.

A strength of this research is its study of a well-phenotyped, multicenter pediatric PAH cohort with detailed hemodynamic and functional data across a number of PAH subtypes. Further, this study includes a longitudinal cohort which enabled an investigation of the relationship between IL-6 and disease progression.

Limitations of this study include the small sample sizes of the cohorts and the time difference between the hemodynamic measures and serum samples in the PAHB cohort, in which only a quarter of the subjects had a right heart catheterization within 12 months of enrollment. Further, some outcomes were limited, particularly 6MWD which was not available for all subjects. There was also a combination of incident and prevalent subjects which may have affected overall IL-6 levels. Additionally, most subjects were being treated for PAH at the time of enrollment, which may have affected actual IL-6 levels in the serum from the disease (19,31). Because IL-6 is also upregulated in infectious and autoimmune states, further study is needed to determine if IL-6 remains a specific marker for pulmonary vascular dysfunction when other co-morbidities are present.

In conclusion, IL-6 offers a more upstream, mechanistic marker of disease development than those studied to date in children with PAH and may aid in earlier diagnosis and initiation of treatment. Because IL-6 is associated with unfavorable hemodynamic changes and clinical worsening in PAH, measuring circulating IL-6 may provide a less invasive method for monitoring the progression of the disease in children. Finally, the association of IL-6 with pediatric PAH, mirroring that of adult PAH, suggests that IL-6 could be studied as a potential therapeutic target for children, in conjunction with adult studies.

Abbreviations List:

IL-6

Interleukin 6

PAH

Pulmonary Arterial Hypertension

CHC

Children’s Hospital Colorado

PAHB

PAH Biobank

IPAH

Idiopathic Pulmonary Arterial Hypertension

APAH

Disease-Associated Pulmonary Arterial Hypertension

APAH-CHD

Congenital Heart Disease-Associated Pulmonary Arterial Hypertension

6MWD

6-Minute Walk Distance

ELISA

Enzyme-Linked Immunosorbent Assay

IQR

Interquartile Range

RHC

Right Heart Catheterization

RAP

Right Atrial Pressure

mPAP

Mean Pulmonary Arterial Pressure

PCWP

Pulmonary Capillary Wedge Pressure

PVRI

Pulmonary Vascular Resistance Index

IV/SQ PCA

Continuous infusion (IV or subcutaneous) or inhalation prostacyclin analog