Targeted proteomics in extracellular vesicles identifies biomarkers predictive for therapeutic response in sarcoidosis

Abstract

Background: ∼30% of patients with sarcoidosis, a systemic granulomatous disease of unknown cause, need treatment to alleviate symptoms or prevent organ damage. Prednisone and methotrexate (MTX) are the most commonly used drugs; however, success of treatment varies from patient to patient. In this study, we search for biomarkers and pathways that predict response to treatment with prednisone or MTX in extracellular vesicles (EVs).

Methods: A targeted proteomics approach (OLINK Bioscience) was used in which 92 proteins were measured in two baseline EV fractions in 32 patients treated for pulmonary sarcoidosis (eight responders and eight non-responders each for prednisone and MTX). The top three proteins were replicated in 62 prednisone- and 76 MTX-treated patients.

Results: We identified 11 differentially expressed proteins (DEPs) between responders and non-responders to prednisone treatment, and 16 DEPs for patients treated with MTX. Reactome pathway analysis showed DEPs in prednisone to be involved in nuclear factor kappa B and interleukin signalling pathways. The DEPs in MTX were involved in transduction of GPI-anchored proteins and MAPK signalling pathway. CHI3L1 for prednisone and CPA1 for MTX were replicated as significant predictors of response.

Conclusion: This study is the first to show that in pulmonary sarcoidosis the response to treatment with prednisone or MTX can be predicted at baseline by different EV proteins active in different pathways. Using these markers and associated pathways to identify patients with a high probability of response to therapy will aid personalised treatment choice and improve treatment outcome.

FIGURE 1

Schematic overview of workflow. First an OLINK proteomics approach was performed on a discovery cohort (n=32) to search for potential predictive biomarkers for response to treatment with either prednisone or methotrexate (MTX). The top three predictive proteins for each treatment group were replicated in a second cohort (n=138) by ELISA. ILD: interstitial lung disease.

FIGURE 2

The bubble plot of Reactome pathway analysis of proteins differently expressed between responders and non-responders in the discovery cohort treated with a) prednisone and b) methotrexate. GM-CSF: granulocyte-macrophage colony-stimulating factor; GPI: glycosylphosphatidylinositol-anchored glycoproteins; IGFBP: insulin-like growth factor binding protein; MAPK: mitogen-activated protein kinase kinase; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B-cells; NFE2L2: nuclear factor erythroid-derived 2-like 2; VLDLR: very-low-density-lipoprotein receptor.

FIGURE 3

Box-plots of protein concentrations in the replication cohort of sarcoidosis patients treated with a–c) prednisone and d–f) methotrexate (MTX). Proteins measured are a) CHI3L1 in LDL, b) CD163 in HDL, c) CXCL16 in LDL, d) MMP2 in LDL, e) CPA1 in HDL and f) uPA in LDL. Concentration is shown in log2 scale with boxes representing interquartile range and whiskers the minimum and maximum for non-responders (NON) and responders (RES) to treatment. Concentrations of CHI3L1 and CPA1 are significantly different between responders and non-responders treated with prednisone and methotrexate. LDL: low-density lipid particles; HDL: high-density lipid particles. **: p<0.01.