Sarcoidosis exosomes stimulate monocytes to produce pro-inflammatory cytokines and CCL2

Casper J E Wahlund,Ahmed Ibrahim,Susanna Kullberg,Susanne Gabrielsson,Elga Bandeira,Ana Lukic,Anna Smed-Sörensen,Loïc Steiner,Johan Grunewald,Gozde Gucluler Akpinar,Anders Eklund,Rico Lepzien

doi:10.1038/s41598-020-72067-7

Abstract

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities, but cell sources and mechanisms have not been identified. In the current study, we found that BALF exosomes from sarcoidosis patients, but not from healthy individuals, induced a dose-dependent elevation of intracellular IL-1β in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1β, IL-6 and TNF from both PBMCs and enriched monocytes, suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients, and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast, a cysteinyl leukotriene receptor antagonist. Further, reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation, and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis.

Highlights

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment
We found that the ultracentrifuged preparations from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients and healthy volunteers contained exosomes of similar size distributions (Fig. 1A) and mode diameters (131 nm and 130 nm for patients and healthy controls respectively, Fig. 1B)
Corroborating our earlier findings where we found more exosomal protein in patient compared to healthy control B ALF4, exosomes were more abundant in the BALF from patients compared to healthy controls (2.8 × 106 vesicles/ml, Fig. 1C)

Summary

Introduction

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment. Reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation, and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis. In a separate validation cohort, the blocking assay was repeated and showed that Montelukast decreased CCL2 levels for approximately half of the 30 patient exosome stimulations. This opens for further investigations of exosome effects on sarcoid inflammation, and of Montelukast as an inhibitor of exosome-mediated inflammation in certain sarcoidosis patients

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Results

Conclusion