Cytokine Signatures Differentiate Systemic Sclerosis Patients at High Versus Low Risk for Pulmonary Arterial Hypertension

Kathleen D. Kolstad; Avani Khatri; Michele Donato; Sarah E. Chang; Shufeng Li; Virginia D. Steen; Paul J. Utz; Purvesh Khatri; Lorinda Chung


Arthritis Res Ther. 2022;24(39) 

In This Article

Materials and Methods

Patient Population

Clinical data and serum samples for PAH and high-risk PAH patients are from the Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma (PHAROS) study, a US-based, multi-center registry of SSc patients at high risk for (high-risk PAH) or with incident PAH confirmed by RHC within 6 months of enrollment. Clinical data and serum samples were collected from 2006 to 2016. For this study, the Institutional Review Board at 22 participating US centers approved the PHAROS protocol and patients provided written informed consent prior to enrollment.

Incident PAH patients had a RHC within 6 months of enrollment demonstrating mean pulmonary artery pressure (mPAP) ≥25 mmHg, pulmonary capillary wedge pressure (PCWP) ≤15 mmHg, and no significant interstitial lung disease as determined by chest imaging and/or a forced vital capacity (FVC) >60% predicted. High-risk PAH patients were considered to be at risk for development of definitive PAH and were defined as having any one of the following features: (1) diffusing capacity of carbon monoxide (DLCO) <55% predicted with a FVC of >70% predicted, (2) FVC/DLCO ratio >1.6, or (3) estimated right ventricular systolic pressure (RVSP) ≥40 mmHg on echocardiography at the time of enrollment. There were 81 incident PAH patients and 71 high-risk PAH patients included in the current study.

Patients with SSc considered to be low-risk for SSc-PAH had a DLCO ≥ 80%, FVC ≥ 80%, and RVSP ≤35mmHg or normal echocardiogram if no measurable tricuspid regurgitant jet was observed at the time of enrollment (n=10). This patient group was enrolled at the Stanford Rheumatology Clinic. Healthy controls (HC) (n=20) were enrolled through the Stanford rheumatology-dermatology clinic and they had no known autoimmune disease.

Baseline demographics and clinical characteristics were compared between the four patient groups, including age, gender, race, cutaneous subtype (limited versus diffuse), autoantibody subtype, disease duration, pulmonary function test findings (FVC% predicted, DLCO % predicted), and RVSP on echocardiogram. All serum samples were drawn at the time of disease risk classification, at patient enrollment or in the case of incident SSc-PAH patients, within 6 months of diagnostic RHC.

Cytokine Expression Profiling and Analysis

Serum samples were obtained from the above-described patient groups. Sample collection and storage was standardized across study sites. Blood samples were collected in red top tubes and centrifuged at the end of the clotting time (30–60 min) for 20 min at 1100–13,000g at room temperature. Serum was stored in aliquots at 80 °C. Cytokines chosen for analysis were based on cytokines assessed in early feasibility studies using core facility commercial arrays (Supplementary Table 1). The cytokines assessed in these early studies were limited by assay availability at the facility. A custom 14-plex array and the Immune Monitoring 65-Plex Human ProcartaPlex™ Panel array from Invitrogen were used for cytokine analysis to include all cytokines of interest. Samples were diluted 1:100 in assay buffer and run in duplicate. Diluted samples were incubated with magnetic beads for 1 hour. Beads were washed twice and incubated with detection antibody for 30 min, followed by two washes and incubation with Streptavidin-PE for 30 mins. Beads were washed twice, resuspended in reading buffer, and analyzed on the Luminex Flexmap 3D system. Samples were normalized by background correction and removed if the correlation between technical replicates was <0.8. Mean values and standard deviations for each cytokine by the patient group can be found in Supplementary Table 2. Cytokine expression was compared between patient groups using Tukey's test. A multiple hypotheses corrected p value <0.05 was considered significant. The complex heatmap shows the magnitude of the fold changes between conditions and their statistical significance for each comparison. Significance was based on the false discovery rate (FDR) threshold of 5%.