Identification of serum exosome proteins in systemic sclerosis with interstitial lung disease by aptamer proteomics

Abstract

Objective: A major unmet need for Systemic Sclerosis (SSc) clinical management is the absence of well validated biomarkers for early diagnosis of SSc-associated interstitial lung disease (SSc-ILD). The objective of this study was to identify proteins contained within serum exosomes that may serve as potential biomarkers to differentiate patients with Diffuse SSc without SSc-ILD from patients with Diffuse SSc with SSc-ILD employing aptamer-based proteomics.

Methods: Serum exosomes were isolated from two cohorts of patients. The first cohort included 15 patients with Diffuse SSc without SSc-ILD and 14 patients with Diffuse SSc with SSc-ILD and the second cohort included 12 patients with Diffuse SSc with SSc-ILD and 12 patients with Diffuse SSc without SSc-ILD. SOMAscan aptamer proteomics was performed with the first cohort and quantified the concentration levels of 1,305 proteins. Significant associations of differentially elevated or reduced proteins (p < 0.05 |FC|>1.2) discriminating between the two SSc clinical subsets were assessed. Validation of the results obtained from the proteomics analysis of the first cohort was performed with the second cohort.

Results: The aptamer proteomic analysis identified 29 proteins increased and 9 proteins decreased in SSc with SSc-ILD as compared to SSc without SSc-ILD. Principal component analysis using the 20 most significantly differentially expressed proteins resulted in excellent separation of the two SSc clinical subsets. Most of the differentially increased proteins converged around enhanced inflammatory responses, immune cell activation, cell death, and abnormal vascular functions and several of them displayed a highly significant correlation with the CO Diffusion Capacity Levels.

Conclusion: Aptamer proteomic analysis of circulating exosomes from patients with Diffuse SSc with and without SSc-ILD identified several biologically plausible biomarkers that may be of value to differentiate these two SSc clinical subsets.

Keywords: Aptamer proteomics; Interstitial lung disease; Serum exosomes; Systemic sclerosis.

Fig. 1

(A) Principal Component Analysis using the top 20 proteins (p < 0.05/FC>|1.2|) contained in serum exosomes from patients with Diffuse SSc with SSc-ILD compared to serum exosomes from patients with Diffuse SSc without ILD. Red circles, Diffuse SSc with SSc-ILD patients; and Green circles, Diffuse SSc without ILD. (B) Box Whisker plots for differentially expressed proteins in patients with Diffuse SSc with SSc-ILD and Diffuse SSc without ILD. Expression pattern using relative fluorescence units of 6 representative proteins among the top dysregulated proteins (p < 0.05), derived from SOMAscan^®. Data are shown as Box Whisker plots with mean expression depicted as a red + sign and median expression indicated by a horizontal line

Fig. 2

Systems Biology analysis of the significantly differentially expressed proteins was conducted using Ingenuity Pathway Analysis, Bio Function Analysis and analysis of Upstream Regulators of Diffuse SSc with SSc-ILD compared with Diffuse SSc without ILD. A. Functional Category; B. Inflammatory Response; C. Vasculogenesis; D. Upstream Regulator Analysis that best correlates with the observed expression changes in Diffuse SSc with SSc-ILD. E. Downstream targets of TNFα; F. Downstream targets of NFκB; G. Downstream targets of TGFβ1

Fig. 3

Network and cluster analysis using the STRING database of functional and physical protein associations

Fig. 4

Correlation of Elafin, TNR 4, FCN 2, and thrombospondin 4 with DLCO

Fig. 5

A. Validation of Elafin increase in SSc with ILD by ELISA analysis. B.Validation of thrombospondin 4 reduction in SSc with ILD by Western blot analysis. Example of Western blot and commassie blue stained gel (CB). Quantification of thrombospondin 4 in serum exosomes samples from SSc-ILD patients (ILD) and from SSc patients without ILD (No ILD)