Autoimmunity Is a Significant Feature of Idiopathic Pulmonary Arterial Hypertension

Abstract

Rationale: Autoimmunity is believed to play a role in idiopathic pulmonary arterial hypertension (IPAH). It is not clear whether this is causative or a bystander of disease and if it carries any prognostic or treatment significance. Objectives: To study autoimmunity in IPAH using a large cross-sectional cohort. Methods: Assessment of the circulating immune cell phenotype was undertaken using flow cytometry, and the profile of serum immunoglobulins was generated using a standardized multiplex array of 19 clinically validated autoantibodies in 473 cases and 946 control subjects. Additional glutathione S-transferase fusion array and ELISA data were used to identify a serum autoantibody to BMPR2 (bone morphogenetic protein receptor type 2). Clustering analyses and clinical correlations were used to determine associations between immunogenicity and clinical outcomes. Measurements and Main Results: Flow cytometric immune profiling demonstrates that IPAH is associated with an altered humoral immune response in addition to raised IgG3. Multiplexed autoantibodies were significantly raised in IPAH, and clustering demonstrated three distinct clusters: "high autoantibody," "low autoantibody," and a small "intermediate" cluster exhibiting high concentrations of ribonucleic protein complex. The high-autoantibody cluster had worse hemodynamics but improved survival. A small subset of patients demonstrated immunoglobulin reactivity to BMPR2. Conclusions: This study establishes aberrant immune regulation and presence of autoantibodies as key features in the profile of a significant proportion of patients with IPAH and is associated with clinical outcomes.

Keywords: BMPR2; IPAH; autoantibodies; autoimmune; pulmonary arterial hypertension.

Figure 1.

The peripheral immune profile in idiopathic pulmonary arterial hypertension (IPAH) is one of an activated immune response. Circulating leukocytes from peripheral blood mononuclear cell fractions of peripheral whole blood were analyzed in subjects with IPAH (n = 26) and healthy donor control subjects (n = 29) using flow cytometry. (A) Sunburst plots showing average distribution of B-cell, T follicular helper (T_FH) cell, and regulatory T (Treg) cell populations as a percentage of parent population from their respective panels of CD19⁺ B cells, T helper/T_FH cells, and CD4⁺ T cells in healthy donors and subjects with IPAH. (B) Abundance of cell populations for plasmablasts (CD3⁻ CD19⁺ CD38⁺ IgD⁻), double-negative B cells (CD3⁻ CD19⁺ CD38⁻ CD27⁻ IgD⁻), NSMs (CD3⁻ CD19⁺ CD27⁺ IgD⁺), switched memory B cells (CD3⁻ CD19⁺ CD38⁺ CD27⁺ IgD⁻), “circulating” T_FH cells (CD3⁺ CD4⁺ CXCR5⁺ CD45RA⁻ PD-1⁺), Treg cells (CD3⁺ CD4⁺ CD25⁺ CD127⁻), and CCR4⁺-primed Treg cells (CD3⁺ CD4⁺ CD25⁺ CD27⁻ CCR4⁺). Plots show median with interquartile range except for Treg cells (mean with SD). To control for multiple hypothesis testing, false discovery rates were estimated using the Benjamini-Hochberg procedure on a per-panel basis, and resultant q values are presented. We report here as significant tests q < 0.05. CD = cluster of differentiation; cTFH = circulating T follicular helper; CXCR5 = C-X-C chemokine receptor type 5; NSM = nonswitched memory B cell; PD1 = programmed cell death protein 1.

Figure 2.

Circulating immunoglobulin and IL-21 concentrations in subjects with idiopathic pulmonary arterial hypertension (IPAH). (A and B) Nephelometry of peripheral immunoglobulin classes in subjects with IPAH (n = 10) and healthy donor control subjects (n = 27) for (A) major immunoglobulin classes (data shown as mean with SD for IgG and IgM and as median with interquartile range for IgA) and (B) IgG subclasses (data shown as mean with SD for IgG1 and IgG3 and as median with interquartile range for IgG2 and IgG4). Patients with IPAH were selected according to the availability of plasma samples from the immunophenotyping cohort. To control for multiple hypothesis testing, false discovery rates were estimated using the Benjamini-Hochberg procedure for a total of four tests, and resultant q values are presented. We report here as significant tests q < 0.05. (C) IL-21 concentrations in subjects with IPAH (n = 17) and healthy donor control subjects (n = 60). Data are shown as median with interquartile range with Mann-Whitney test. ns = not significant.

Figure 3.

Strategy for the detection of autoantibody biomarkers in subjects with pulmonary arterial hypertension (PAH) and healthy donor control subjects and subsequent cluster analysis for the evaluation of clinical characteristics in subjects with PAH on the basis of autoantibody status. HPAH = heritable PAH; IPAH = idiopathic PAH; PCH = pulmonary capillary hemangiomatosis; PVOD = pulmonary venoocclusive disease.

Figure 4.

Autoantibody analysis in pulmonary arterial hypertension (PAH). Circulating autoantibodies were assayed using the ProtoPlex Autoimmune Panel in 473 patients with PAH and 946 age- and sex-matched healthy control subjects. (A) Autoantibody concentrations were compared in both healthy control subjects and patients with PAH (left-side graphs) and among clusters of patients with PAH (right-side graphs). Positivity was defined as 0.75Q + 2IQR of the control population (shown as a dashed line set at 1 for the normalized autoantibody concentration). Box plots show the median and IQR (25%, 75%); whiskers represent the end of the box plot ± 1.5 × IQR. The y-axis represents the scaled autoantibody concentration, which is normalized to have a median of 0 in the control population and a positivity threshold of 1. (B) Heat map showing autoantibody positivity prevalence as a percentage in cases with PAH and control subjects. FDR-adjusted q values were calculated across 19 tests and are indicated using asterisks (*q < 0.05, **q < 0.005, and ***q < 0.0005). (C) Heat map showing autoantibody positivity prevalence among clusters of cases with PAH: high, low, and intermediate. FDR-adjusted q values were calculated across 19 tests and are indicated using asterisks and indicate which autoantibodies are driving the clustering. Note that the null hypotheses of no difference among clusters are not sensible to test, because clusters were defined on the basis of observed values. B and C summarize the significance testing results shown in detail in A. FDR = false discovery rate; IQR = interquartile range; La/SS-B = La antigen; RNP = ribonucleic protein; Ro/SS-A = Ro antigen; SCL = scleroderma antigen; U1snRNP-68 = U1 small nuclear ribonucleic protein 68.

Figure 5.

Clinical comparison of clusters in pulmonary arterial hypertension (PAH). (A–G) Box plot comparison of stratification of clinical outcomes in the PAH cohort as determined using autoantibody cluster analysis for (A) PVR, (B) cardiac output, (C) age at diagnosis, (D) PAWP, (E) BMI, (F) thyroid-stimulating hormone (TSH) concentration, and (G) mPAP. Box plots represent the median with IQR (25th–75th percentile); whiskers represent the end of the box plot ± 1.5 × IQR. Statistical analysis was performed on all available values using two-tailed ANOVA with false discovery rate–adjusted q values calculated across 178 tests. TSH concentrations were log transformed before the ANOVA. (H) Kaplan-Meier survival analysis for patients up to 20 years after diagnosis (n = 462). Statistical analysis was performed using pairwise log-rank tests with a global log-rank test on 2 degrees of freedom. BMI = body mass index; IQR = interquartile range; mPAP = mean pulmonary arterial pressure; PAWP = pulmonary arterial wedge pressure; PVR = pulmonary vascular resistance.

Figure 6.

Putative antibodies to BMPR2 (bone morphogenetic protein receptor 2) are present in sera of patients with pulmonary arterial hypertension (PAH). (A) Heat map showing reactivity after a glutathione S-transferase fusion human proteomic screen to identify sera reactivity to proteins in the BMPR pathway. Sera were obtained from patients with idiopathic PAH (IPAH) (n = 5) and with comparator autoimmune diseases (T1 DM, n = 3; AAV, n = 3; SLE, n = 2; SSc, n = 3). Heat map shows mean reactivity (arbitrary units). (B) Quantitative analysis of IgG reactivity against a recombinant peptide of the BMPR2 extracellular domain (ECD) in sera from patients with IPAH/heritable PAH (n = 350) and healthy donors (n = 55). The Mann-Whitney test was performed between control subjects and PAH groups. (C) Quenching of identified seropositive samples (n = 5) with free ECD before incubation on ELISA effectively quenches binding at >1,000 ng. Data are shown as mean with SD and expressed as a percentage of nonquenched serum. (D and E) Effect of serum preincubation from control subjects (n = 5) or patients with PAH with seropositivity to BMPR2 as shown by ELISA (n = 5) on pulmonary arterial smooth muscle cells for 1 hour, followed by stimulation with 10 ng/ml BMP4 for 1 hour. Relative quantification of downstream ID1 (D) and ID3 (E) by quantitative PCR and normalized to HPRT and B2M. AAB = autoantibody; AAV = antineutrophil cytoplasmic antibody–associated vasculitis; ACVRL1 = activin A receptor like type 1; AU = arbitrary units; B2M = β-2-microglobulin; BMP = bone morphogenetic protein; ENG = endoglin; GDF2 = growth differentiation factor 2; HPRT = hypoxanthine phosphoribosyltransferase 1; ID = inhibitor of DNA binding protein; SLE = systemic lupus erythematosus; SSc = systemic sclerosis; T1 DM = type 1 diabetes mellitus.