The Role Of Keratinocyte Exosomes In The Inflammatory Dysfunction In Psoriasis

BackgroundPsoriasis is a persistent inflammatory skin disease,characterized by activation of keratinocytes and infiltration of immune cells.It severely affects patients’physical and psychologic wellbeing and has been considered as a major global health problem.Although the pathogenesis of paoriasis is complex,the immunological disorder is considered to be a key reason.Th1 and Th17 have been proved to be important members in the pathogenesis of psoriasis.In the recent years,increasing evidences show that neutrophils play a nonnegligible role in the disease.Keratinocytes,the main cellular component of the epidermis,are also essential to disease progression.It is generally accepted that the complex communication between activated keratinocytes and infiltrated immune cells such as T cells and neutrophils,leading to the disease development.Exosomes have been reported recently as important mediators of intercellular communication recently.Exosomes are a specific population of nanometer-sized(30–150nm)extracellular vesicles that are products of the endolysosomal pathway.They harbor and transfer diverse cargoes of proteins,RNAs,and lipids,providing new implications for the pathology ofmany diseases.Keratinocytes are the main constituents of epidermis and contribute to the formation of psoriatic lesions.They also have the ability to secrete exosomes.However,the role of keratinocyte exosomes in mediating the communication between keratinocytes and immune cells is yet to be explored.Objective1.To determine the characteristics of keratinocyte exosomes under normal and psoriatic conditions.2.To explore the role of keratinocyte exosomes in the pathogensis of psoriasis.3.To study the mechanism by which keratinocyte exosomes participate in the pathogenesis of psoriasisMethods1.Keratinocyte exosomes were extracted by ultra-high speed centrifugation.Their morphology and surface markers were detected and identified by transmission electron microscopy,nanoparticle tracking analysis and Western blot.2.Neutrophils and CD4~+T cells were stimulated separately by keratinocyte exosomes under normal and psoriatic conditions.The possible role of keratinocyte exosomes was observed.The effect of keratinocyte exosomes in the pathogensis in vivo was investigated by using imiquimod(IMQ)–induced psoriasis-like mice.3.Mass spectrometry and high-throughput sequencing were used to identify the protein/micro RNA components of psoriasis-like keratinocyte exosomes.The characteristics and possible functions of these components were also analyzed.4.The possible molecular mechanisms of their regulatory effects were further analyzed,according to the functions of psoriatic keratinocytes exosomes on neutrophils and CD4~+T cells,as wells as their specific protein/micro RNA profiles.Results1.Keratinocyte exosomes were isolated.These isolated particles presented characteristics of exosomes with a size of less than 150 nm,which was confirmedby transmission electron microscopy and NTA.Exosomal markers,including CD9,CD63,CD81,and HSP70,were identified in exosomes by Western blot,compared with the whole cell lysate.And GM130 was absent,thus excluding contamination with Golgi.2.Psoriasis-like keratinocyte exosomes were taken up by neutrophils and triggered expressions of proinflammatory factors in neutrophils.These exosomes also promote polarization of Th1 and Th17 cells.The proinflammatory role of psoriasis-like keratinocyte exosomes in vivo was also verified in imiquimod(IMQ)–induced psoriasis-like mice.3.Compared with non–cytokine-treated keratinocyte exosomes,psoriasis-like keratinocyte exosomes exhibited 72(10.9%)proteins up-regulated and 96(14.5%)proteins down-regulated.GO analyze showed these proteins participate in pathways which regulate proinflammatory factors in neutrophils.Small RNA sequencing analysis showed remarkable differences in the mi RNA profiles in psoriasis-like keratinocyte exosomes,with 28 mi RNAs upregulated and 114 downregulated.4.Psoriasis-like keratinocyte exosomes led to activation of the NF-κB and p38signaling pathways and the subsequent expressions of proinflammatory factors.Exosomal mi R-381-3p is responsible for exosomes-induced polarization of Th1 and Th17 cells.UBR5 and Foxo1 are downstream targets of mi R-381-3p and are possible mediators of mi R-381-3p-induced effects.ConclusionOur findings demonstrated the role of psoriasis-like keratinocyte exosomes on neutrophils and T cells,promoting psoriasis development.We also verified the role of keratinocyte exosomes in the pathogenesis of the disease in imiquimod-induced mouse psoriasis model.We highlight the release of exosomes as a communication strategy between keratinocytes and immune cells in inflammatory diseases such as psoriasis.Our findings motivate future studies of keratinocyte exosomes,or their specific cargoes,as therapeutic candidates for psoriasis.