Small Extracellular Vesicles Containing miR-381-3p from Keratinocytes Promote T Helper Type 1 and T Helper Type 17 Polarization in Psoriasis

T helper cells are crucial for psoriasis pathogenesis. Communication between and psoriatic keratinocytes (KCs) helps drive the Th1 Th17 response, but underlying mechanism is not well-understood. Small extracellular vesicles (sEVs) emerging mediators of intercellular communication. Here, we investigated role KC-derived sEVs in response psoriasis. We isolated characterized from KCs under normal (untreated) (cytokine-treated) conditions. both conditions exhibited a cup-shaped morphology expressed markers CD63 CD81. cytokine-treated can be taken up by CD4+T cells, leading to induction polarization. RNA sequencing revealed that miR-381-3p was significantly increased patients with Moreover, sEVs-containing responsible sEVs-induced further found targeted 3? untranslated region E3 ubiquitin-ligase UBR5 stabilized ROR?t protein expression. It also FOXO1, associated activated T-bet transcription. Taken together, propose transfer through sEVs, inducing polarization promoting development. Our findings motivate future studies or their specific cargoes as therapeutic candidates Psoriasis chronic inflammatory skin disorder formation silvery scales because complex interplay activation immune (Hawkes et al., 2017Hawkes J.E. Chan T.C. Krueger J.G. pathogenesis development novel therapies.J Allergy Clin Immunol. 2017; 140: 645-653Abstract Full Text PDF PubMed Scopus (357) Google Scholar). Multiple factors contribute psoriasis, including genetic, environmental, immunologic triggers 2018Hawkes Adalsteinsson J.A. Gudjonsson Ward N.L. Research techniques made simple: murine models human psoriasis.J Invest Dermatol. 2018; 138: e1-e8Abstract (39) Exaggerated T-cell responses critical (Tamehiro 2019Tamehiro N. Nishida K. Sugita Y. Hayakawa Oda H. Nitta T. al.Ras homolog gene family H (RhoH) deficiency induces psoriasis-like dermatitis TH17 cell polarization.J 2019; 143: 1878-1891Abstract (6) Targeting cytokines has resulted effective treatment. Among subsets, IFN-?–producing (Th) 1 IL-17A–producing recognized progression (Cheung 2016Cheung K.L. Jarrett R. Subramaniam S. Salimi M. Gutowska-Owsiak D. Chen Y.L. al.Psoriatic recognize neolipid antigens generated mast phospholipase delivered exosomes presented CD1a.J Exp Med. 2016; 213: 2399-2412Crossref Scholar; Hawkes However, precise regulation disease remains unclear. main constituents epidermis involved lesions. now generally realized actively affect epidermal microenvironment orchestrate (Boehncke Schön, 2015Boehncke W.H. Schön M.P. Psoriasis.Lancet. 2015; 386: 983-994Abstract (1110) Although increasing emphasis been placed on communication 30–200 nm derived all kinds (Beuzelin Kaeffer, 2018Beuzelin Kaeffer B. Exosomes miRNA-loaded biomimetic nanovehicles, focus potentials preventing type-2 diabetes linked metabolic syndrome.Front 9: 2711Crossref (25) Scholar, Théry 2018Théry C. Witwer K.W. Aikawa E. Alcaraz M.J. Anderson J.D. 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Here sought identify whether might influence thus lead studied showed micro (miRNA) composition small had treated mimic molecular sEVs-mediated modulation illustrate potential opening possibility sEVs?T-cell interaction harnessed platform innovative strategies diseases. To investigate function healthy conditions, were left untreated cytokine cocktails (10 ng/ml IL-17A, 10 IL-22, IFN-?, 25 TNF-?) (Lowes 2014Lowes M.A. Suárez-Fariñas Immunology psoriasis.Annu Rev 2014; 32: 227-255Crossref (844) Yang 2018Yang Jin Ke X. Zhang al.E3 ligase Trim21 ubiquitylates stabilizes keratin 17 STAT3 2568-2577Abstract (27) Scholar) culture medium 24 hours. purified (noncytokine-treated sEVs) (cytokine-treated (Jiang 2019Jiang Fang Shao Dang Qiao P. al.Keratinocyte activate neutrophils enhance inflammation psoriasis.FASEB 33: 13241-13253Crossref (30) Purified typical structure, which confirmed transmission electron microscopy (Figure 1a). The average size 128.5 (from KCs) 131.5 KCs), analyzed nanoparticle-tracking analysis. most collected less than 200 1b). Furthermore, CD81 identified these flow cytometry 1c) western blot analysis 1d). GM130 absent, ruling out contamination Golgi Given contribution possible cells. First, labeled Dil incubated them peripheral blood donors fluorescence detected cultured those without 2a), supporting then stimulation anti-CD3 anti-CD28 mAbs presence KCs. After KCs, mRNA levels IL-17F, IL-23, IL-36, TNF-?, T-bet, upregulated compared IL-4, Gata3, Foxp3 no significant change 2b). frequency CD4+IFN-?+ CD4+IL-17A+ when There difference CD4+IL-4+ CD4+CD25+Foxp3+ groups 2c d). Similar changes observed ELISA 2e). Together, data indicate miRNA expression profiles remarkable differences 3a Supplementary Table S1), miRNAs 114 downregulated 3b). four miRNAs—miR-381-3p, miR-365-5p, miR-4488, miR-619-5p—significantly 3c These markedly 3e). them, induced 3f) well IFN-? IL-17A 3g). amount positively correlated PASI score 3h). may regulate directly assess regulates Th-cell polarization, transfected mimics inhibitors 4a). Whereas levels, suppressed 4b). 4c). Increased promoted Th1- Th17-cell 4d). In contrast, CD4+IL-17+ reduced 4e). Next, examine involvement exosome-induced effects, could transferred Cy3-labeled mimics, Cy3 indicating into 4f). negative control stimulated 4g). relative 4h) 4i). promotes functions gain insight combined bioinformatics tools, TargetScan, CLIP-seq starBase version 2.0 database [Li 2014Li J.H. Fau L.S. Zhou Z.H. Qu L-H. Lh al.starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA protein-RNA networks large-scale CLIP-Seq data.Nucleic Acids Res. 42: D92-D97Crossref (2306) Scholar]), miRDB, miRanDa, predict putative targets 5a b). Two target genes, function, selected this study. an demonstrated ubiquitylation cause proteasomal degradation ROR?t, transcription (Wu 2015Wu Z. Kuchroo V.K. DUBA-UBR5 axis: transactivation.Cell 25: 273-274Crossref (1) FOXO1 direct suppresser several receptors (Ichiyama 2016Ichiyama Gonzalez-Martin Kim B.S. H.Y. al.The microRNA-183-96-182 cluster pathogenicity negatively regulating Foxo1 expression.Immunity. 44: 1284-1298Abstract (103) (Lainé 2015Lainé Martin Luka Mir Auffray Lucas al.Foxo1 cell-intrinsic inhibitor ROR?t-Th17 program.J 195: 1791-1803Crossref (53) serving regulator program. validate wild type luciferase reporter constructs included genes mutant constructs, contained mutant-binding sequences 5c). Compared group, decreased inhibition lost transfection mutant-type construct 5d). next evaluated influenced endogenous levels. inhibitors. Notably, after corresponding controls 5e). phosphorylation kinase B miR-381-3 (Supplementary Figure S1a). Immunofluorescence staining cytoplasmic transposition S1b). S1c). 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