RNY1 partitions into extracellular vesicles and ribonucleoprotein particles during airway inflammation to regulate macrophage programming

Abstract YRNAs are small noncoding RNAs that are abundant in both cells and biofluids. Prior research has shown that the secretion of extracellular YRNAs (exYRNAs) changes in response to inflammatory stimuli. However, the mechanisms by which exYRNA packaging and dynamics in biofluids regulate inflammation remain poorly understood. In this study, we found that one YRNA species, RNY1, increased in airway fluid during allergen-induced lung inflammation and correlated with neutrophil infiltration. Using RNase sensitivity assays and size exclusion chromatography, we determined that RNY1 was present in airway fluid extracellular vesicles (EVs) and ribonucleoproteins (RNPs), while another YRNA species, RNY3, was present only in EVs. Both the EV and RNP-containing fractions of airway fluid had a unique ability to program cellular inflammation. Airway fluid EVs increased expression of an alternative activation program in macrophages including Arg1, Ym1, Il10, and Il6, while RNPs induced gene expression more consistent with a classic pro-inflammatory phenotype. RNY1 contributed to the programming of macrophages by airway EVs, as macrophages treated with EVs isolated from RNY1-/- mice demonstrated lower induction of Arg1 and Ym1. Together, these results define the form and function of exYRNAs in lung biofluid and support their role in communicating signals during inflammation. Competing Interest Statement The authors have declared no competing interest. Footnotes Ethics approval statement: All mouse experiments were performed with approval from the Vanderbilt Institutional Animal Care and Use Committee. All material in this manuscript is original and has not been published elsewhere. All work in this manuscript upholds principles of rigorous and reproducible research in science. Abbreviations - EV - Extracellular vesicle - EP - extracellular particle - RNP - Ribonucleoprotein particle - OVA - ovalbumin - HDM - House dust mite - BALF - Bronchoalveolar lavage fluid - UF100 - ultrafiltrated extracellular material >100 kDa - UF10 - ultrafiltrated extracellular material 10 kDa - Tx - Triton X-100 - PK - Proteinase K - SEC - size exclusion chromatography