Human Umbilical Cord Mesenchymal Stem Cells-derived Exosomes Alleviate Psoriasis-like Skin Inflammation

Background:Psoriasis(Psoriasis)is a common chronic inflammatory disease related to immunity in dermatology.The etiology and pathogenesis of psoriasis are still unclear.According to the current research progress,scholars agree that IL-23/Th17 axis carry a big weight in the occurrence and development of psoriasis.Interleukin(IL)-23 is a pro inflammatory factor,which is mainly secreted by dendritic cells(DCS).T helper cell 17(Th17)is differentiated by helper T cells and can release factors such as IL-17 which play a multi-effect role in cell raising and activation.IL-23/IL-17 signal axis inhibitor is effective in the treatment of psoriasis,indicating that IL-23/Th17 axis is a good target for the treatment of psoriasis.At present,the main methods to treat psoriasis clinically include external drug therapy,optical therapy,traditional systematic drug therapy,biological agents and small molecule targeted drug therapy.However,some patients still can not achieve very satisfactory treatment results after the above treatment.Therefore,exploring new treatment methods is a research hotspot in the field of psoriasis.Mesenchymal stem cells(MSCs)have been used in clinic for more than 10 years.They have strong immune regulation and good therapeutic effect on immune related diseases.Previous clinical reports have shown that allogeneic MSCs can reduce the clinical symptoms of patients with psoriasis..Previous preclinical studies have shown that allogeneic MSCs have a therapeutic effect on psoriasis.Human umbilical cord mesenchymal stem cells(hucMSCs)are typical adult stem cells.Compared with stem cells from other sources,they have superiority of low immunogenicity,non-invasive acquisition,and easy in vitro expansion.Therefore,hucMSCs are a promising cell in cell therapy.With the improvement of the understanding level of exosomes,it is gradually recognized that MSCs derived exosomes go hand in hand with their therapeutic effects.Exosomes are nanovesicles(50-200 nm)derived from most living cells and are thought to play an important role in transmitting information between cells.MSCs derived exosomes(MSCs-Exo)can basically include the functions of MSCs.It has been reported that MSCs-Exo has therapeutic effects on atopic dermatitis(AD)and inflammatory bowel disease(IBD).Psoriasis,AD and IBD are all immune-mediated inflammatory diseases.However,the mechanism of action of MSCs-Exo in psoriasis is still largely unclear.The purpose of this research is to extract hucMSCs-Exo,identify hucMSCs and hucMSCs-Exo;To explore the mechanism of hucMSCs-Exo in inhibiting psoriasis-like inflammation;meanwhile,a psoriasis-like mouse model was constructed to observe whether human umbilical cord mesenchymal stem cells exosomes(hucMSCs-Exo)could reduce the inflammatory responses in psoriasis-like mice.Part 1:Identification of human umbilical cord mesenchymal stem cells and their extraction,identification and intake of exosomesTo ensure the accuracy and consistency of the experimental results,the hucMSCs used in our entire experiment were the third generation hucMSCs,and the extracted hucMSCs-Exo were obtained from the third generation hucMSCs.Objective:1.Identify hucMSCs to determine whether hucMSCs have the characteristics of stem cells.2.Extracting hucMSCs-Exo,identify and determine the concentration of the prepared hucMSCs-Exo,to determine whether the hucMSCs-Exo we obtained has the characteristics of exosomes,so as to ensure that the subsequent experiments can be carried out smoothly.3.To observe whether hucMSCs-Exo can be ingested by human immortalized keratinocytes(HaCat),so as to provide a basis for subsequent cell and animal experiments.Method:1.Identification of HucMSCs:1)We use flow cytometry to check specific surface signs of hucMSCs,such as positive markers CD29,CD44,CD 105 and negative marker HLA-DR;2)hucMSCs were induced to osteogenic for 21 days and adipogenic for 14 days.The differentiation was identified by Oil Red O staining and alizarin red staining.2.Identification of HucMSCs-Exo:1)The supernatant of hucMSCs with excellent growth state was collected and hucMSCs-Exo was extracted by ultra-high speed ionization method.2)Observe the shape and size of hucMSCs-Exo by transmission electron microscope(TEM).3)Detect the specific markers of hucMSC-Exo by protein Western Blotting,such as positive markers CD9,CD63,CD81,TSG101 and negative marker GAPDH;4)Test the diameter and concentration of hucMSCs-Exo through Nanoparticle tracking analysis(NTA);5)Measure concentration of hucMSCS-Exo by BCA protein concentration assay kit.3.Intake of HucMSCs-Exo:After co-culturing the prepared labeled hucMSCs-Exo with HaCat cells for 12 hours,the uptake of hucMSCs-Exo was observed under a confocal laser scanning microscope(CLSM).Result:1.HucMSCs:1)Morphology:Observed under an inverted microscope,hucMSCs are uniform in size and shape,arranged in a fusiform,swirled,and adherent to the wall;2)Dechoring flow cytometry found that the positive surface markers such as CD29,CD44,CD 105 were highly expressed in HUCMSCS cells,while HLA-DR negative surface markers were low;3)The results of induced differentiation showed that:at the end of osteogenic induction experiment,orange nodules with clear boundary were observed between HUCMSCS cells by alizarin red staining experiment;At the end of the lipogenic induction experiment,Oil Red O staining displayed that there were many clustered oils among HUCMSCS cells.2.HucMSCs-Exo:1)Morphology:observed under TEM,hucMSCs-Exo were round or cup-shaped with intact membrane;2)Western Blotting showed that HucMSCs-Exo significantly expressed four transmembrane proteins CD9,CD63,CD81,TSG101,and HUCMSCS cells did not see it;GAPDH was not expressed in hucMSCs-Exo,but highly expressed in hucMSCs cells;3)The diameter of hucMSCs-Exo was mainly concentrated between 55 and 167 nm by NTA detection,the particle concentration was 2.3*1011 Particles/mL;4)The protein concentration of hucMSCs-Exo determined by BCA kit was about 0.3～0.5μg/μl.3.Intake of HucMSCs-Exo:After co-culturing the prepared labeled hucMSCs-Exo with HaCat cells for 12 hours,more hUCMSCs-Exo aggregated in HaCat cells and around the nucleus were observed under CLSM.Conclusion:1.HucMSCs cells showed slender spindle and vortex adherent state;high expression of CD29,CD44,CD105,low expression of HLA-DR;potential to differentiate into osteoblasts and adipocytes.2.HucMSCs-Exo are round or cup-shaped,with intact cell membrane;high expression of CD9,CD63,CD81,TSG101,low expression of GAPDH;the diameter is mainly between 55 and 167 nm,and the particle concentration is about 2.3*1011 Particles/mL;The protein concentration is about 0.3 to 0.5 μg/μl.3.HaCat cells can successfully take up hucMSCs-Exo.Part 2:The mechanism of HucMSCs-Exo inhibiting the maturation and activation of dendritic cells and the secretion of inflammatory factors in keratinocytesObjective:1.Explore the optimal concentration of hucMSCs-Exo co-culture with DCs and HaCat cells.2.To explore whether hucMSCs-Exo can inhibit the maturation,activation and secretion of pro-inflammatory factor IL-23 of DCs.3.To investigate whether hucMSCs-Exo can inhibit the expression of transcription factor Stat3/p-Stat3 and the secretion of inflammatory factor IL-23 and chemokine CCL20 in HaCat cells.Method:1.Set the concentration of hucMSCs-Exo to several gradients of 0.5μg/ml,1μg/ml,2.5μg/ml,5μg/ml,10μg/ml,and co-culture with DCs and HaCat cells respectively.According to the changes of CD11c,MHCII,CD86 expression in DCs and the changes of IL-23 and CCL20 secreted by HaCat cells,the optimal co-culture concentration was selected.2.DCs were extracted from the bone marrow of 4-6 weeks old C57BL/6 mice and cultured.The DCs were divided into three groups:imDC,mDC+PBS,mDC+hucMSCs-Exo,and hucMSCs-Exo and DCs were co-cultured for 24 hours.1)The cells were collected,and flow cytometry was used to detect the specific markers CD11c,MHCII,and CD86 of DCs,to observe the changes in the ratio of CD11c,MHCII,and CD86 of DCs in each group,and to explore the effect of hucMSCs-Exo on the maturation and activation of DCs;2)The supernatant of the cells in each group was collected,the expression of IL-23 was detected by enzyme linked immunosorbent assay(ELISA),and the effect of hucMSCs-Exo on the secretion of cytokines by DCs was observed.3.HaCat cells were stimulated with IL-17A to create a psoriasis-like inflammatory environment.The cells were divided into IL-17A+PBS group and IL-17A+hucMSCs-Exo,and hucMSCs-Exo was co-cultured with HaCat cells for 24 hours.1)Collect cells from each group,and detect the expression of transcription factor Stat3/p-Stat3 by Western Blotting;2)Collect cell supernatant from each group,detect the expression of inflammatory factor IL-23 and chemokine CCL20 by ELISA,and observe the impacts of hucMSCs-Exo on the secretes cytokines and chemokines of HaCat cells.Result:1.By setting different concentration gradients,it was found that the hucMSCs-Exo concentration of 2.5μg/ml was the best concentration for co-culture with DCs and HaCat cells.2.DCs:Compared with the PBS group adding the same dose,the expression of CD11c+MHCⅡ+,CD11c+CD86+ and CD11c+MHCII+CD86+in the cells decreased significantly in the DCs added to the hucMSCs-Exo group,and the content of IL-23 in the cell supernatant also decreased.3.HaCat cells:Compared with the PBS group adding the same dose,hucMSCs-Exo can significantly inhibit the expression of Stat3/p-Stat3 in HaCat cells,and the contents of IL-23 and CCL20 in the cell supernatant also decreased.Conclusion:1.HucMSCs-Exo can inhibit the maturation and,activation of DCs and secretion of inflammatory factor IL-23.2.HucMSCs-Exo can inhibit the expression of transcription factor Stat3/p-Stat3 and the secretion of pro-inflammatory factor IL-23 and chemokine CCL20 in HaCat cells.Part 3:The mechanism of HucMSCs-Exo alleviating skin inflammation in psoriasis-like miceObjective:1.To explore the optimal dose of hucMSCs-Exo injected subcutaneously into the skin of psoriasis-like mice.2.To construct a psoriasis-like mouse model to observe whether hucMSCs-Exo can alleviate the inflammation response in psoriasis-like mice.3.To explore the mechanism of hucMSCs-Exo inhibiting skin inflammation in psoriasis-like mice.Method:1.Exploring the dose of subcutaneous injection of hucMSCs-Exo:We set up dose gradients of 10μg/mice,30μg/mice,50μg/mice,100μg/mice,and 400μg/mice,and the hucMSCs-Exo was subcutaneously injected into the back skin of the mice at multiple points on the 0th,2th,and 4th days of mouse modeling,and the changes of the back skin of the mice were observed.According to the changes of the back skin erythema,scale and skin thickness of the mice at different doses,the final dose of subcutaneous injection of hucMSCs-Exo was selected..2.Modeling:The back skin and hair of 8-week-old C57BL/6 mice were shaved and smeared with 5%imiquimod(IMQ)cream(62.5mg/each)every day for 6 days.Twenty-four mice were randomly divided into four groups,i.e,control group(externally coated with Vaseline ointment),IMQ group(externally coated with imiquimod cream),IMQ+PBS group,and IMQ+hucMSCs-Exo group.The hucMSCs-Exo(50ug/each)was subcutaneously injected into the back skin of mice at multiple points on the 0th,2th,and 4th daysof mouse modeling,and the PBS group was injected with PBS solution(50ul/each)to observe the changes of the back skin of mice in each group.3.The back skin of the mice in each group was collected,and one part was subjected to histopathological sections to observe the histopathological changes of the back skin of the mice in each group;the other part was detected by Western Blotting to detect the changes of the transcription factors Stat3/p-Stat3,inflammatory factors IL-17,IL-23 and chemokine CCL20 in the back skin of the mice in each group.Result:1.By observing the relief of erythema,scaling and skin thickness on the back of the mice,we found that the skin symptoms of mice that was injected subcutaneously of hucMSCs-Exo 50μg/mice significantly relieved.And the skin thickness was not significantly relieved compared with 50μg/mice,so we chose 50μg/mice for the dose of subcutaneous injection of hucMSCs-Exo in our subsequent experiments.2.Modeling:1)The back skin of the mice gradually thickened,erythema and white scales appeared,and some fissures were seen;2)The histopathology of the mice skin showed thickening of the epidermis,hyperkeratosis and parakeratosis,and granular,the epidermis was significantly reduced,the spinous layer was thickened,the epidermal process was extended,and lymphocyte infiltration was seen around;3)Western Blotting detected the significant expressions of Stat3/p-Stat3,IL-17,IL-23,and CCL20 in the skin of psoriasis-like mice.3.Changes of symptoms on the back skin of mice:1)Compared with the subcutaneous injection of PBS group,subcutaneous injection of hucMSCs-Exo can significantly relieve the erythema,scale and skin thickness of the back skin of the mice,and the PASI score was significantly reduced;2)The histopathology of the mice skin showed that,compared with the subcutaneous injection of PBS group,subcutaneous injection of hucMSCs-Exo can significantly reduce the degree of epidermal thickening.4.Detection of related factors in the back skin of mice:Compared with the subcutaneous injection of PBS group,subcutaneous injection of hucMSCs-Exo can significantly inhibit the expression of transcription factors Stat3/p-Stat3,inflammatory factors IL-17A,IL-23 and chemokine CCL20.Conclusion:1.HucMSCs-Exo may inhibit the overactivation of IL-23/Th17 axis by inhibiting the signaling pathway STAT3/p-STAT3,and reduce the release of inflammatory factor IL-17,pro-inflammatory factor IL-23 and chemokine CCL20.2.HucMSCs-Exo can significantly reduce the inflammatory response in psoriasis-like mice.