Effect And Mechanism Of Keratinocytes Acting On Dendritic Cells Through Exosomes On Psoriasis

Background and purpose of the resesrch: Psoriasis is an immune-mediated chronic inflammatory skin disease.The global prevalence rate is about 3%,and about 0.47% in China.It is characterized by excessive proliferation,abnormal differentiation of keratinocytes and infiltration of inflammatory cells.Clinically,it is characterized by erythema and plaques covered with silvery lamellar scales,which are common in scalp,back and extensional side of limbs.The pathogenesis of psoriasis is still unclear.At present,it is generally believed that it is the result of the joint action of genetic,environmental and immune factors.Among the immune factors,the interaction between activated keratinocytes and infiltrated inflammatory cells is an important reason for the occurrence and development of the disease.For patients with mild to moderate psoriasis,local treatment is the main method.Phototherapy,oral drugs or injection of biological agents are important means to treat patients with severe psoriasis.Dendritic cells(DCs)are powerful antigen-presenting cells,which play an important role in autoimmune diseases and the induction of immune tolerance.Previous studies have shown that DCs are involved in the occurrence and development of psoriasis.DCs originated from CD34 + bone marrow multifunctional hematopoietic stem cells,distributed in nonlymphoid tissues and parenchymal organs with blood,and developed into immature DCs.They mature under the stimulation and action of inflammatory mediators,and then migrate to lymph nodes through lymphatic vessels,secreting chemokines and cytokines to activate T lymphocytes and produce specific immune response.DCs in the skin include LCs(Langham’s cells),p DCs(plasmacytoid dendritic cells)and m DCs(myeloid dendritic cells),and in the dermis are mainly m DCs.DCs were detected in psoriatic lesions,and it was found that the expression of costimulatory molecules CD83 and CD86 increased.In addition,studies have shown that damaged keratinocytes in psoriatic lesions will release their own nucleotides and form LL-37-own nucleotide complex with LL-37,stimulating p DCs to produce type I interferon,leading to the maturation and activation of m DCs.Mature dendritic cells can produce IL-12 and IL-23,activating and stimulating Th1 / Th17 cells to form DC-T cell clusters,and finally drive the activation of T cells.The important feature of psoriasis is that T cells produce effector cytokines and induce the proliferation and abnormal differentiation of keratinocytes.In conclusion,exploring the interaction between DCs and keratinocytes is an important research direction at home and abroad.In recent years,many studies have shown that exosomes are important mediators of intercellular communication.They are nano-sized(30-150nm)extracapsular vesicles and the product of endolysosomal pathway.They can carry and transfer kinds of proteins,RNA and lipids,and provide a new direction for the study of the mechanism of many diseases.In inflammatory diseases,there is increasing evidence that exosomes may come from a variety of cell types,such as epithelial cells,macrophages,dendritic cells and lymphocytes.Keratinocytes are the main component of epidermis.They participate in the formation of psoriasis lesions,and affect the microenvironment of epidermis and the function of immune cells in diseases.Previous studies have shown that keratinocytes can also secrete exosomes and promote the occurrence and development of psoriasis by promoting the proliferation of neutrophils and affecting Th1 / Th2 polarization.This research intends to explore the effect of keratinocyte derived exosomes on the function of dendritic cells and its related mechanism,so as to lay a certain foundation for the study of the role and mechanism of exosomes in the occurrence and development of psoriasis,so as to provide a research foundation for the new direction of psoriasis treatment.This topic mainly includes the following three parts:Section I: The effect of exosomes derived by Keratinocytes on the function of Dendritic CellsMethod: 1.HaCaT keratinocyte lines were used,and the cells were treated with cytokines to simulate the state of keratinocytes in psoriasis.Exosomes were isolated and purified from the supernatants of cell cultures of the two groups by ultracentrifugation,and identified respectively.2.Collect peripheral blood of healthy people,then extract PBMC from collected blood and dendritic cells were induced and cultured.3.Cytokine-treated and untreated HaCaT keratinocyte derived exosomes were coincubated with immature dendritic cells.The expression of costimulatory molecules on dendritic cells was measured,and the expression of cytokines IL-12 and IL-23 in cell supernatant was measured by Enzyme-Linked Immunosorbent Assay(ELISA).Result:1.The two groups of vesicles isolated and purified from cytokine-treated and untreated HaCaT keratinocyte culture supernatants were consistent with the diameter and morphological characteristics of exosomes,and there was no significant difference in the secretion of exosomes between the two groups.2.After the exosomes of the two groups were co-incubated with immature dendritic cells,the expression of costimulatory molecules on dendritic cells in the cytokine-treated group increased,and the serection of cytokines IL-12 and IL-23 in the supernatant increased.Section II: The mechanism of exosomes derived by Keratinocytes affecting the function of Dendritic CellsMethod: 1.RNA was extracted from exosomes derived from cytokine-treated and untreated HaCaT keratinocyte respectively.RNA-seq and q RT-PCR were used to detect and verify the miRNA highly expressed in the exosomes of cytokine-treated group.2.Through the bioinformatics website——http://starbase.sysu.edu.cn/index.php/,we predict the downstream of highly expressed miRNA,screen and search the literature,and select specific molecules as the target of miRNA for further research.3.Exosomes derived from cytokine-treated and untreated HaCaT keratinocytes were co-incubated with immature dendritic cells.The effect of miRNA was verified by Wstern Blot and double Luciferase reporter gene detection.Result: 1.RNA from exosomes derived from keratinocytes treated and untreated with cytokines was extracted and sequenced.The five most significantly differentially expressed were: mi R-11615,mi R-378,mi R-7054,mi R-6650 and mi R-1692.q RT PCR was used to detect the expression of these five miRNAs in HaCaT keratinocytes in cytokine-treated and untreated group,keratinocyte derived exosomes in two groups,and dendritic cells coincubated with two groups of exosomes.It was found that mi R-378 was highly expressed and had statistical significance.2.Through the bioinformatics website,we predicted the downstream of mi R-378 and screened out the targets supported by more than two websites.Through searching and reading the literatures,we finally took RORA as the molecular of follow-up research.3.The expression level of RORA in dendritic cells incubated with keratinocytes,keratinocytes and exosomes of the two groups was detected by Western Blot.We found that the expression level of RORA in dendritic cells treated with cytokines decreased significantly,while overexpression of mi R-378 could reduce the expression level of RORA and the increase of RORA expression level after inhibiting mi R-378.Section III: The effect and mechanism of mouse Keratinocytes on psoriasis mice model through exosomesMethod: 1.BALB / C female mice aged 6-8 weeks were selected,and 5% Imiquimod cream(IMQ)50mg / d was applied to the ear skin,and Vaseline 50 mg in the control group.After the mice were killed,the ears were taken and the epidermis was isolated,then the keratinocytes in the epidermis were isolated and cultured.After 24 hours,the exosomes in the keratinocyte supernatant of the control group and IMQ group were isolated and purified.2.Keratinocytes in the ear epidermis of mice in the control group and IMQ group were isolated and cultured.The concentration of mi R-378 in keratinocytes of mice in the IMQ group was knocked down and mi R-378 in keratinocytes of mice in the overexpression control group was overexpressed.The exosomes in the cell supernatants of the four groups were isolated and purified respectively.BALB / C female mice aged 6-8 weeks were selected,and 5% imiquimod cream(IMQ)50mg / day was applied to the ear skin to construct the mouse ear psoriasis model.At the same time,mice were subcutaneously injected with PBS,control exosomes,IMQ exosomes,knockdown mi R-378 exosomes in control group,and overexpressed mi R-378 exosomes in IMQ group at 20μg,and the mice were killed 7 days later.The severity of skin lesions was evaluated by HE staining and immunohistochemical staining.The expression of costimulatory molecules on the surface of dermal dendritic cells was detected by flow cytometry.Result: On the 8th day,the mice were killed by cervical dislocation,and the ear skin samples were taken.The severity of skin inflammation was evaluated by HE staining and immunohistochemical staining(Ki67).It was found that compared with normal KC group,psoriasis KC group and normal KC overexpression mi R-378 group,the skin inflammation was aggravated,the scales increased,the epidermis thickened,and the infiltrating cells in the dermis increased,which was higher than that in psoriasis KC group,Psoriasis KC inhibited mi R-378 group,the inflammation decreased and the epidermal thickness became thinner.After the epidermis and dermis of skin lesions was isolated,the cells in dermis were extracted,and the expression of DC characteristic molecules was detected by flow cytometry.It was found that compared with normal KC group,psoriatic KC group and normal KC overexpression mi R-378 group,the expression of CD80,CD86 and MHC II increased,while psoriatic KC inhibited the expression of CD80,CD86 and MHC II in mi R-378 group.Conclusion: 1.There was no significant difference in the secretion of exosomes between cytokine treated and untreated HaCaT keratinocyte culture supernatants,After coincubation with immature dendritic cells,the expression of costimulatory molecules on the surface of dendritic cells in the cytokine treated HaCaT keratinocyte derived exosomes group increased,and the expression of cytokines IL-12 and IL-23 in the supernatant increased.2.Keratinocytes treated by cytokines mimicking psoriasis highly express mi R-378,and WB results showed that the level of RORA downstream gene of mi R-378 in dendritic cells treated with cytokines decreased significantly,while overexpression of mi R-378 could reduce the expression level of RORA,and the expression level of RORA increased after inhibition of mi R-378.3.Exosomes derived from keratinocytes in psoriasis mice model aggravate the inflammation of ear psoriasis lesions(erythema,scale and skin thickness),promote the expression of DC cell surface molecules in dermis,while the inflammation was lighter after inhibiting mi R-378 in keratinocytes in psoriasis model mice.After overexpression of mi R-378 in normal mouse keratinocytes,the inflammation was worse than that injected with normal mouse keratinocyte derived exosomes.