Function And Regulation Of PD-L1 In Keratinocytes And Intervention Of Indirubin

Psoriasis is an immune-related chronic inflammatory disease.Due to its refractory and high recurrence,it causes extreme discomfort to the patient's body and puts great pressure on the economy and spirit.Although there has been a history over one hundred years of the drug treatment,current drugs do not completely cure psoriasis.And the pathogenesis of psoriasis has not been clarified so far,so medical researchers need to continue to explore the mechanism of psoriasis,in order to find proper drugs to treat the disease.We previously analyzed the differential genes between lesional skins from 3 psoriatic patients and normal skins from 3 healthy volunteers based on the technology of microarray.And many core genes related to psoriasis were found.With the development of sequencing technology,high-throughput transcriptome sequencing has replaced microarray as a better technology in the field of bioanalysis.In this study,high-throughput transcriptome sequencing technology was used to find that PD-L1(the ligand of immune checkpoint PD-1)was differenctly expressed in psoriatic skins and healthy skins.Furthermore,the function and regulation of PD-L1 in the keratinocytes were studied,and the therapeutic effect of PD-L1-related drug indirubin in psoriasis was explored and verified preliminary.Chapter ? Transcriptome sequencing of psoriasis vulgaris Objective: To investigate the pathogenesis of psoriasis vulgaris in Chinese population and to find core target genes in the disease.Methods: Psoriatic skins and normal skins were collected and totle RNA was extracted for transcriptome sequencing.Differential genes were screened and compared with the reported differential genes.Bioinformatics analysis of differentially expressed genes such as GO,pathway,and co-expression networks was conducted to find key target genes in the pathogenesis of psoriasis.Immunohistochemistry on the target genes was performed in psoriatic skins and normal skins.Results: A total of 2597 differential genes were found,including 2139 coding genes and 458 non-coding genes.Compared with the reported differential genes,1676 new differential genes were identified,including HACL1,ACAA,TECR,GADD45 G,SH2D1A,ITGAV,SH2D1 A,et al.There were 1335 up-regulated genes and 804 down-regulated genes in the coding differential gene.GO terms related to up-regulated genes mainly included cell cycle,immune response,inflammatory reaction and keratinocyte differentiation,etc.Down-regulaed GO terms included fatty acid synthesis and metabolism,lipid metabolism and viral transcription,et al.Pathway analysis revealed that cytokine-cytokine interaction pathway,metabolic pathway,NF?B signaling pathway,PPAR signaling pathway,NOD-like receptor signaling pathway,RIG-I-like receptor signaling pathway,chemokine signaling pathway,cell cycle,apoptosis,fatty acid degradation pathway,peroxidase,T cell receptor signaling pathway,tight junction,JAK-STAT signaling pathway,and Toll-like receptor signaling pathway played important roles in psoriasis.Co-expression network analysis revealed 49 core genes,including IL-17 A,IL-26,PD-L1,et al.In this study,PD-L1 was selected as a target gene for further study.The results of immunohistochemistry showed that PD-L1 was normally expressed in healthy human skin keratinocytes,but significantly decreased in in psoriatic lesions.Conclusions: The differential genes based on Chinese psoriasis transcriptome sequencing are different from those in the reported literatures.Transcriptome sequencing can provide significant advantages in exploring the pathogenesis of psoriasis.The decreased PD-L1 in psoriatic skin keratinocytes may contributed to the pathogenesis of psoriasis.Chapter ? Construction of PD-L1 knockout keratinocytes model Objective: To construct a stable PD-L1 knockout human epidermal keratinocytes model in order to study the role of PD-L1 in psoriatic keratinocytes or other skin diseases with the decreased PD-L1 expression.Methods: Three pairs of gRNA primers,designed based on the PD-L1's sequence,were ligated with the lentiCRISPRv2 vector to construct recombinant plasmids.The lentivirus was packaged with the recombinant plasmid and infected the human immortalized keratinocyte(HaCaT).The puromycin was applied for the screening of mixed clones.PD-L1 gene sequences of mixed clones were used to detect the mutation effect,and western blot experiments were performed to verify the knockout effect.The monoclonal cells from the selected mixed clones were screened by limiting dilution,and the knockout effect of PD-L1 was verified by western blot experiments.Finally,the method of TA cloning was used to detect the mutation locus in PD-L1 knockout keratinocytes.Results: Three lentiCRISPRv2-gRNA recombinant plasmids were successfully constructed.The sequencing results of the mixed clones selected by puromycin showed that only the groups of Cas1 and Cas2 obtained the positive results,and the Cas3 group negative.Therefore,the two positive groups were chosen for the subsequent experiments.Western blot experiments showed that Cas1 group showed better PD-L1 knockout effect than Cas2 group.Therefore,Cas1 group mixed clones were used to the screening of monoclonal cells.Western blot experiments showed that the three screened monoclonal cells didn't express PD-L1 protein,which means that we obtained three groups of PD-L1 knockout positive monoclonal cells.The results of TA cloning showed that there were different deletions or insertions on the two chromosomes of the knockout cells compared with the wild-type cells.Frameshift mutation resulted in the termination of protein translation,and short chain polypeptide with only 32 amino acids was obtained.Conclusions: We successfully constructed a PD-L1 knockout immortalized human keratinocyte model.Chapter ? Effect of PD-L1 Knockout on the secretion of cell regulatory factors in the keratinocytes Objective: To investigate the changes of the regulatory factors(IL-1?,CXCL1,CXCL9,CCL20,S100A8,S100A9,IL-6 and CXCL2)in PD-L1 knockout keratinocytes,in order to clarify the role of PD-L1 in epidermal keratinocytes.Methods: Real-time PCR(polymerase chain reaction)and ELISA(enzyme linked immunosorbent assay)experiments were performed to compare the secretion of pro-inflammatory factor(IL-1? and IL-6),chemokines(CXCL1,CXCL2,CXCL9 and CCL20)and antimicrobial peptides(S100A8 and S100A9)in PD-L1 knockout human immortalized keratinocytes and negative control keratinocytes.At the same time,the IL-1?,CCL20 and S100A8 levels in the two cells with or without 100 ng/ml IL-17 A stimulation for 24 h were detected by real-time PCR experiment.Results: Compared with the control keratinocytes,the levels of IL-1?,CXCL1,CXCL9,CCL20,S100A8 and S100A9 in PD-L1 knockout keratinocytes were significantly up-regulated,while IL-6 and CXCL2 levels were not significantly different.The expression levels of IL-1?,CCL20 and S100A8 in the control keratinocytes were significantly increased under the stimulation of IL-17 A for 24 h.However,the effect of IL-17 A in PD-L1 knockout cells was more obvious.There were higher levels of IL-1?,CCL20 and S100A8 in PD-L1 knockout keratinocytes.Conclusions: PD-L1 not only directly inhibits inflammation in human epidermal keratinocytes,but also inhibits IL-17A-mediated inflammatory response.Chapter ? Regulation of PD-L1 in keratinocytes Objective: To understand the regulatory factors of PD-L1 in human epidermal keratinocytes in order to find the cause of down-regulated PD-L1 in psoriatic keratinocytes.Methods: IFN-?(25 ng/ml),IL-17A(100 ng/ml),IL-17F(100 ng/ml),IL-1?(20 ng/ml),IL-2(10 ng/ml)),IL-22(100 ng/ml),IL-10(20 ng/ml),and TNF-?(20 ng/ml)stimulate human primary epidermal keratinocytes for 24 h.PD-L1 levels were detected by real-time PCR and flow cytometry.The effects of IFN-? concentration(25,50,100 ng/ml)and time(0,3,6,12,24,48 h)were examined.Six candidate miRNAs were predicted by the method of network database and four miRNA(miR-16-5P,miR-15a-5P,miR-195-5P and miR-15b-5P)mimics were chosen to stimulat keratinocytes for overexpression studies.PD-L1 levels were detected by real-time PCR and western blot.In situ hybridization was used to detect the expression of miR-15a-5P and miR-195-5P in psoriatic lesions and normal skin.Results: TNF-? and IFN-? up-regulated the expression of PD-L1 in human keratinocytes,and they had synergistic effects.Other cytokines had no significant effect.The effect of IFN-? was the most significant,and increased with time.The expression of PD-L1 was up-regulated after IFN-? stimulated keratinocytes for 3 h,and reached its peak after 48 h.The expression of PD-L1 didn't upregulate following the increased concentration of IFN-?.There were same PD-L1 expression levels under the stimulation of 25ng/ml IFN-? and 100 ng/ml IFN-?.The inhibitory cytokine IL-10 had no regulatory effect on the expression of PD-L1.In vitro,miR-15a-5P mimic and miR-195-5P mimic,not miR-16-5P mimic and miR-15b-5P mimic,downregulated the expression of PD-L1 in human keratinocytes.The results of in situ hybridization showed that the expression of miR-15a-5P in psoriatic keratinocytes was higher than that in normal skin,while the expression of miR-195-5P in keratinocytes showed no significant difference.Conclusions: Down-regulated PD-L1 in psoriatic keratinocytes is not associated with inflammatory factor stimulation and may be associated with overexpression of miR-15a-5P in lesional keratinocytes.Chapter ? Indirubin relieves the symptoms and inflammatory response of psoriatic mice in a PD-L1-dependent manner Objective: Previous co-expression network analysis revealed that indirubin was negatively correlated with PD-L1 in epidermal keratinocytes.In the study,we aimed to investigate whether the treatment effect of indirubin in psoriasis was related to PD-L1.Methods: 1.Eight-week-old BALB/c mice were randomly divided into three groups: control,imiquimod(IMQ)-induced psoriasis-like mouse model,IMQ+ indirubin.The expression of PD-L1 in the epidermal keratinocytes was observed by immunofluorescence costaining.2.BALB/c mice were randomly divided into four groups: control,IMQ,IMQ + indirubin,IMQ + indirubin + PD-L1 antibody.Skin photoes were taken.HE(hematoxylin-eosin)staining and real-time PCR were used to investigate the morphological changes of skin tissue and the secretion of inflammatory factors(IL-1?,IL-23 A,IL-17 A,IL-22).3.BALB/c mice were randomly divided into three groups: IMQ,IMQ + indirubin,and IMQ + PD-L1 Fc.Skin photoes were taken.HE(hematoxylin-eosin)staining was used to investigate the morphological changes of skin tissue.IL-17 A expression and ??T cell number were examined by real-time PCR and immunofluorescence staining.4.The primary epidermal keratinocytes were used as in vitro cell models,and the cells were divided into 4 groups: control,miR-15a-5P,indirubin,miR-15a-5P+ indirubin.PD-L1 mRNA expression were investigated by the method of real-time PCR.Results: 1.PD-L1 expression in the epidermal keratinocytes of IMQ-induced psoriasis-like mice was significantly lower than that of control mice.The expression of PD-L1 in psoriatic keratinocytes was significantly up-regulated after the treatment of indirubin.2.Indirubin relieved erythema,hyperplasia and other psoriatic symptoms,and reduced epidermal thickness,inflammatory cell infiltration,as well as dowregulating the expression levels of inflammatory cytokines(IL-1?,IL-23 A,IL-17 A and IL-22).However,therapeutic effect of indirubin was weakened after the treatment of PD-L1 antibody and the symptoms of skin lesions in mice were aggravated,with the increasing epidermis thickness and the infiltration of inflammatory cells.The expression levels of IL-1?,IL-23 A,IL-17 A and IL-22 also increased.3.Indirubin had a better therapeutic effect than PD-L1 Fc in alleviating IMQ-induced mouse psoriasis-like symptoms and inflammatory response.4.MiR-15a-5P mimic down-regulated the expression of PD-L1 in primary epidermal keratinocytes.Howerver,the down-regulation of miR-15a-5P mimic disappeared when indirubin was added.Conclusions: Indirubin is a multi-targeted drug that can alleviate IMQ-induced mouse psoriasis-like symptoms and inflammatory responses by regulating PD-L1 expression in epidermal keratinocytes.And the regulation was carried by affecting the function of miR-15a-5P.