Mechanism Of BET And New Inhibitor NHWD-870 In Regulating The Progression Of Psoriasis By ScRNA-seq

Psoriasis is a complex chronic inflammatory disease.It is currently considered that psoriasis not only affects skin and joints,but also is associated with a variety of systemic diseases,and is closely related to metabolic abnormalities and cardiovascular risk events.Epigenetics plays an important role in the pathogenesis of psoriasis.Bromodomains（BRDs）family is one of the most representative epigenetic readers.BET family are widely distributed and play an important role in various tissues.Small molecule inhibitors developed against BET protein have shown good therapeutic effects on inflammation,metabolic abnormalities,tumors and infectious diseases.We have originally developed a novel and powerful small molecule inhibitor of BET,NHWD-870.In this study,based on single-cell transcriptome sequencing technology,we explored the mechanism of NHWD-870 regulating psoriatic skin inflammation and peripheral blood metabolism,providing a new idea for basic research of psoriasis and exploring a new therapeutic strategy for clinical management of patients with psoriasis which have potential value of clinical transformation and application.BET and its inhibitor NHWD-870 regulate keratinocyte inflammation in psoriasisObjective（1）To clarify the therapeutic effect of NHWD-870 on psoriatic dermatitis in mice,and explore more suitable administration methods and drug preparations.（2）To explore the specific mechanism of BET and its inhibitor NHWD-870 regulating psoriatic skin inflammation,thus providing theoretical basis for the clinical transformation and application of NHWD-870.Methods（1）Psoriatic dermatitis in mice was induced by IMQ and IL-23,then treated by systemic oral or local administration of BET inhibitor NHWD-870.Skin inflammation,spleen size and body weight were compared before and after treatment.（2）In vitro transdermal experiments were performed using bamapig,measuring drug concentrations in the receiving medium at4hh,8,12h and 24h,respectively.Dermis and epidermis were separated to detect the concentration of drug retention in skin,calculating accumulative permeability,average permeability and drug retention in skin within a certain period of time.（3）NHWD-870 hydrogels were used to treat IMQ-induced psoriatic skin lesions on the back of mice.RNA-seq was taken to analyze and verify differentially expressed genes and conduct KEGG enrichment analysis.（4）Skin lesions from normal and psoriatic patients were collected for single-cell transcriptome sequencing,and the single cell landscape of psoriatic skin was described.The expression of BET protein family genes was examined in different cell populations,and verified by tissue immunofluorescence and qPCR.（5）qPCR and Western Blot were used to detect the changes of IL-17R family genes in HaCaT cells,which were treated with NHWD-870 at different concentrations and time points,or knockdown of BRD2/3/4.（6）Psoriatic keratinocyte inflammation was induced by IL-17A and IL-36α/γ,and the changes of keratinocyte inflammation related genes（CXCl2/3,S100A9,DEFB4A and LCN2）were compared before and after NHWD-870 treatment,or BRD2/3/4 knockdown.（7）Chromatin immunoprecipitation sequencing（ChIP-seq）was used to search for transcription factors that might be regulated by NHWD-870 in inhibiting keratinocyte inflammation and verified by ChIP-PCR.（8）qPCR and Western Blot were used to detect the changes of NFKBIZ in HaCaT cells induced by inflammation before and after NHWD-870 treatment or BRD2/3/4 knockdown.（9）The expression of NFKBIZ in epidermal cell populations of normal subjects and patients with psoriasis was detected by single cell sequencing.Results（1）Both systemic oral and local administration of NHWD-870could effectively inhibit psoriatic dermatitis in mice.The specific manifestations were as follows:the inflammation of back skin in IMQ+NHWD-870 treatment group was significantly alleviated,epidermal thickness and PASI score was significantly reduced;NHWD-870 could significantly alleviate spleen enlargement induced by IMQ and IL-23-induced psoriatic ear inflammation in mice.（2）NHWD-870 could achieve an appropriate inhibitory effect on psoriatic dermatitis at a dose of 0.01%,with slighter effect of the external drug on the overall state and body weight of mice.（3）In vitro transdermal test showed that the average penetration rates of NHWD-870 hydrochloride and free base were 0.7544ng·cm^-2h^-1and 0.7368ng·cm^-2h^-1,respectively.There was no significant difference between the two vectors,manifesting about 90%of NHWD-870 drug remained in local skin and about 10%of the drug entered the medium through the skin.（4）Transcriptome sequencing of mouse skin tissue suggested that,the expression levels of 1845 genes in NHWD-870+hydrogel group were more than 2 times compared with IMQ+hydrogel group,including1130 up-regulated genes and 715 down-regulated genes,Enriched by KEGG function among 715 down-regulated genes suggested cytokine-cytokine receptor interaction and IL-17 signaling Pathway were down-regulated most obviously after treatment.（5）After NHWD-870+hydrogel treatment,The expression of Il-17,Il-23,Il1f8,Il1f9,Il-12,Il-17r,Cxcl1,Cxcl2,Cxcl3,Ccl4,Defb4S100a9 and Lcn2 genes decreased in mouse skin lesions.（6）Compared with the control group,single cell sequencing and in vitro immunofluorescence showed that BRD2 and BRD4 were significantly upregulated in the basal and upper keratinocytes（KCs）of psoriatic lesions and para-psoriatic tissues.BRD2/4 showed no significant changes in other cells.The expression of BRD3 in various groups of skin cells was low,and no significant difference between normal subjects and patients with psoriasis.（7）m RNA levels of IL-17RA,IL-17RC and IL-17RE were significantly down-regulated in HaCaT cells treated with NHWD-870 for48H.The protein level of NHWD-870 decreased significantly after 72H treatment.In HaCaT cells,simultaneous knockdown of BRD2,BRD3 and BRD4 with siRNA received consistent results.（8）m RNA levels of CXCL2/3,S100A9,DEFB4A and LCN2 were down-regulated in HaCaT cells treated with NHWD-870.The results were consistent after knocking down BRD2/3/4 in HaCaT cells.（9）The expression of CXCL2/3,DEFB4A,S100A9 and LCN2 in HaCaT cells was significantly down-regulated after NHWD-870treatment in vitro by IL-17A and IL36α/γinduced psoriatic keratinocytes inflammation.CXCL2/3,DEFB4A,S100A9 and LCN2 were also significantly down-regulated by IL-17A and IL36α/γstimulation to knock down BRD2/3/4 HaCaT compared with siNC.（10）ChIP-seq results suggested that BRD4 could bind to the promoter region of NFKBIZ,and the peak disappeared after NHWD-870treatment.In vitro verification suggested that both NHWD-870 treatment and BRD2/3/4 knockdown could inhibit the expression of NFKBIZ.（11）The results of sc RNA-seq analysis showed that the expression level of NFKBIZ in psoriatic KCs was higher than melanocytes,myeloid cells and T lymphocytes,and the expression level of NFKBIZ in psoriatic skin KCs was higher than that in psoriatic and normal KCs.Conclusions（1）BET inhibitor NHWD-870 can effectively inhibit skin inflammation in psoriatic mice.（2）In vitro transdermal experiments showed that 90%of NHWD-870 remained in the skin while only 10%entered the receiving medium through the skin,which proved that NHWD-870 is beneficial for the external treatment of psoriasis.（3）The expression of BRD2/4 in psoriatic lesions KCs was increased.（4）BET and its inhibitor NHWD-870 regulate psoriatic inflammation by directly inhibiting keratinocyte IL-17R and transcription factor IκBζ.The mechanism of BET and its inhibitor NHWD-870 in regulating the lipid metabolism of psoriatic peripheral blood monocytesObjectives（1）To show the landscape of the peripheral blood of psoriatic patients by ScRNA-seq and analyze the metabolic state of each cell cluster.（2）To explore the specific mechanism of BET and its inhibitor NHWD-870 in regulating the lipid metabolism of peripheral blood monocytes in psoriasis patients,and to provide a theoretical basis for the clinical transformation of NHWD-870.Methods（1）Peripheral blood samples from normal subjects,patients with psoriasis and 12 weeks after treatment were collected,and mononuclear cells were isolated.Then,single cell capture,cDNA library construction,high-throughput sequencing and raw data processing were performed.（2）Unsupervised clustering and classical cell marker genes were combined to determine the peripheral blood cell population and analyze the changes in the proportion of each cell population between groups.（3）All peripheral blood cells were reclustered using KEGG metabolic pathway genes,and metabolizing-related genes were used as a gene set for AUCell score to analyze the metabolic pathway changes of monocytes,T cells,B cells and NK cells in peripheral blood.（4）Combined with unsupervised clustering of peripheral blood monocytes and classical marker genes of monocytes,subpopulation analysis of monocytes was conducted to determine the changes in the proportion of each subpopulation between groups.（5）Three monocyte subpopulations were evaluated by the character of stem cell score,pseudotime analysis,and differential gene analysis.The expression of CD36 in peripheral blood monocytes was verified by flow cytometry.（6）KEGG metabolic pathway genes were used to recluster monocytes,and metabolization-related genes were used as a gene set for AUCell score to analyze the metabolic pathway changes of monocytes in three subgroups.（7）Correlation analysis was conducted between PASI score and gene expression of S-Mono subsets of monocytes,and the top 400 genes with the most correlation were screened.Upstream transcription factors were screened through the database,and functional enrichment analysis was performed.（8）Using the http://bioinfo.life.hust.edu.cn/h TFtarget database website to screen the gene which can regulate the CD36.（9）CD36 expression was detected by flow cytometry at 0H,12H,24H and 36H after THP-1 cells were treated with different concentrations of NHWD-870.The expression of CD36 was detected by flow cytometry after BRD4 knockdown in THP-1 cells.Results（1）26 peripheral blood samples were collected,including peripheral blood samples from 11 patients with psoriasis before treatment,peripheral blood samples from 11 patients with psoriasis after treatment for 12 weeks,and peripheral blood samples from 4 normal controls as controls.Mononuclear cells were isolated from peripheral blood to detect cell viability,which all met the standard of single cell sequencing.（2）After data preprocessing,remove double cells and red blood cells,unsupervised clustering method is applied to determine the group of31 cells,for a total of 67990 immune cells,mark 13 kind of cells,including B cells,basophils,Cycling cells,DCs cells,hematopoietic stem cells,monocytes,neutrophils,NK cells,NPC precursor cells.There was no difference in the proportion of p DCs cells,plasmoid cells,platelets and T cells in normal subjects,patients with psoriasis and after treatment.（3）In the visualization of peripheral blood single-cell metabolism clustering,it can be roughly divided into the upper left part and the lower right part.The upper left part is mainly composed of monocytes,while the lower right part is mainly composed of T cells,B cells,NK cells and other cells.The scores of metabolic pathways in monocytes were all high,and the scores of amino acid metabolism,carbon metabolism and lipid metabolism were higher in the monocytes of psoriasis patients,and the scores of coenzyme factor,vitamin metabolism and nucleic acid metabolism were lower.（4）Peripheral blood monocytes are divided into three subgroups,namely Classical Monocyte(C-Mono,CD14^hiCD16^low),Nonclassical monocytes(N-Mono,CD14^lowCD16^hi)and Special monocytes(S-Mono,CD14^midCD16^low).The proportion of S-Mono subgroup was more specific in patients with psoriasis.（5）The S-Mono subpopulation had higher stem cell characteristic score,and the analysis of pseudo sequence locus of monocytes suggested that the S-Mono subpopulation was in the early stage of differentiation.（6）The total metabolism,lipid metabolism,coenzyme factor and vitamin metabolism,amino acid metabolism and nucleic acid metabolism pathway scores of S-Mono subgroup monocytes were higher than those of C-Mono and N-Mono subgroups.Carbon metabolism in the three groups of cells in the middle level.The lipid metabolism of S-Mono subsets of monocytes increased in patients with psoriasis.（7）There were 124 upstream regulatory transcription factors of the top400 genes associated with PASI score in S-Mono subsets,which were mainly enriched in myeloid cell differentiation and histone modification.（8）BRD4 is a potential transcription factor regulating CD36,and BET and its inhibitor NHWD-870 can regulate the expression level of CD36 in monocytes.Conclusions（1）The metabolic level of peripheral blood monocytes was more active in patients with psoriasis.（2）The specificity of S-Mono subgroup monocytes increased in peripheral blood of patients with psoriasis.（3）The monocytes of S-Mono subgroup have the following characteristics:high degree of stem cell characteristics,early stage of differentiation,and active lipid metabolism.（4）BRD4 and its inhibitor NHWD-870 regulate lipid metabolism in patients with psoriasis by directly inhibiting CD36 expression in peripheral blood monocytes.Figures 35;Tables 29;Reference 136.