Next Generation Sequencing Facilitates Quantitative Analysis of dermal Transcriptomes of Imiquimod (IMQ)-Induced Psoriasis-Like Dermatitis Treated by Xiao-Yin-Fang (XYF) Therapy
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ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare dermal transcriptomes of control group (wild-type IMQ-untreated C57/BL6 mice), model group (wild-type IMQ-treated C57/BL6 mice) and XYF group (model group mice treated by XYF). Methods: Dermal mRNA profiles of control group, model group and XYF group mice were generated by deep sequencing. Total RNA samples were prepared from intact dermal ear sheets of mouse ear skin from two mice. Double-strand cDNA was generated from equal amounts of total RNA by following TruSeq8 RNA Library Prep Kit v2 (#RS-122-2001/2002, Illumina). The cDNA libraries were sequenced using Illumina Hi-seq2500. STAR software was utilized for sequence alignment between the preprocessing sequence and reference genome sequence of mice downloaded from the Ensembl database(Mus_musculus.GRCm38.90,ftp://ftp.ensembl.org/pub/release-90/gtf/mus_musculus/ Mus_musculus.GRCm38.90.chr.gtf.gz). Transcript assembly of mRNA sequencing data was performed by StringTie software. The expression levels for each of the genes were normalized to FPKM (fragments per kilobase of transcript per million fragments mapped). DESeq 2 was applied to conduct the analysis of differentially expressed genes (DEG). The cutoffs of DEG method were determined as the P value < = 0.05, the FDR value < = 0.05 and the Fold Change (FC) value > = 2. Results: Heatmap analysis of differentially expressed genes (DEG) revealed distinct transcriptomes between different groups, which displayed a partial reversal of pathological gene-expressing modifications by the treatment of XYF. Detailed comparison identified shared DEGs of opposite deviations between control group versus model group and model group versus XYF group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered the dermis from disease-model group was greatly disturbed in cytokine-cytokine receptor interaction, chemokine signaling pathways, IL-17 signaling pathway, TNF signaling pathway, Wnt signaling pathway and PI3K-Akt signaling pathway. As for XYF therapy, several metabolic pathways, Calcium signaling, cAMP signaling pathway and cGMP-PKG signaling pathway were affected within the dermis. Further Gene Set Enrichment Analysis (GSEA) exposed multiple metabolic pathways, which involved ether lipid metabolism, alpha linolenic acid metabolism, arachidonic acid metabolism, linoleic acid metabolism, glycerophospholipid metabolism, glycosaminoglycan biosynthesis heparan sulfate, along with VEGF signaling and cell cycle were considerably suppressed in the dermal tissue when given XYF. Conclusions: XYF might regulate various inflammatory signaling, metabolic processes and microvascular formation to influence γδT cell biology.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare epidermal transcriptomes of control group (wild-type IMQ-untreated C57/BL6 mice), model group (wild-type IMQ-treated C57/BL6 mice) and XYF group (model group mice treated by XYF). Methods: Epidermal mRNA profiles of control group, model group and XYF group mice were generated by deep sequencing. Total RNA samples were prepared from intact epidermal ear sheets of mouse ear skin from two mice. Double-strand cDNA was generated from equal amounts of total RNA by following TruSeq8 RNA Library Prep Kit v2 (#RS-122-2001/2002, Illumina). The cDNA libraries were sequenced using Illumina Hi-seq2500. STAR software was utilized for sequence alignment between the preprocessing sequence and reference genome sequence of mice downloaded from the Ensembl database(Mus_musculus.GRCm38.90,ftp://ftp.ensembl.org/pub/release-90/gtf/mus_musculus/ Mus_musculus.GRCm38.90.chr.gtf.gz). Transcript assembly of mRNA sequencing data was performed by StringTie software. The expression levels for each of the genes were normalized to FPKM (fragments per kilobase of transcript per million fragments mapped). DESeq 2 was applied to conduct the analysis of differentially expressed genes (DEG). The cutoffs of DEG method were determined as the P value < = 0.05, the FDR value < = 0.05 and the Fold Change (FC) value > = 2. Results: Heatmap analysis of differentially expressed genes (DEG) revealed distinct transcriptomes between different groups, which displayed a partial reversal of pathological gene-expressing modifications by the treatment of XYF. Detailed comparison identified shared DEGs of opposite deviations between control group versus model group and model group versus XYF group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered the epidermis from disease-model group was highly enriched in multiple inflammatory signaling, involving cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, IL-17 signaling pathway and cGMP-PKG signaling pathway. As for XYF therapy, PI3K-Akt signaling pathway, Calcium signaling, Hippo signaling pathway and cGMP-PKG signaling pathway were modulated in the epidermis. Further Gene Set Enrichment Analysis (GSEA) exposed downregulations in focal adhesion, ECM receptor interaction, TGF-β signaling pathway and glyoxylate and dicarboxylate metabolism in XYF-treated psoriasis-like epidermis. Conclusions: XYF might regulate various inflammatory signaling, metabolic processes and microvascular formation to influence γδT cell biology.
Project description:Purpose: To compare epidermal LC transcriptomes of control group (wild-type IMQ-untreated C57/BL6 mice) and IMQ group (wild-type IMQ-treated C57/BL6 mice). Results: KEGG pathway analysis implied that lesional LCs were highly enriched in several inflammatory signalling pathways, including cytokine-cytokine receptor interactions, Th1, Th2 and Th17 cell differentiation, tumor necrosis factor signalling and IL-17 signalling pathways, complement and coagulation cascades, and NOD-like receptor signalling and PI3K-Akt signalling pathways. Gene set enrichment analysis plots revealed enrichment of cytokine activity, chemoattractant activity, protein lipid complex binding, and low-density lipoprotein particle binding gene sets in LCs from IMQ+ mice compared with IMQ- mice. Conclusions: These results confirmed a proinflammatory role for epidermal LCs in the pathogenesis of psoriasis.
Project description:Psoriasis is one of the most common dermatological disorders, characterized by increased epidermal hyperplasia and immune cell infiltration. Psychological stress has been reported to contribute to the severity, aggravation, and relapse of psoriasis. We developed a chronic restrain stress (CRS)-imiquimod (IMQ)-induced psoriasis-like mouse model and performed a comprehensive comparative transcriptomic and metabolic analysis with control mice, CRS-treated mice, and IMQ-treated mice to investigate how psychological stress affects psoriasis. We found that CRS-IMQ-induced psoriasis-like mice showed significant exacerbation of psoriasis-like skin inflammation compared with mice treated with IMQ only. Mice of the CRS+IMQ group showed increased expression of keratinocyte proliferation and differentiation genes, differential regulation of cytokines, and promotion of the linoleic acid metabolism. Our study provides new insights into the effects of psychological stress on psoriasis pathogenesis and the mechanisms involved, which provides clues for development of therapeutics or biomarkers.
Project description:We then performed gene expression profiling analysis using data obtained from RNA-seq of 9 skin tissues including 3 per group in vaseline-treated group, imq model group and bergapten-gel-treated imq model group.
Project description:Psoriasis is a chronic inflammatory skin disorder that is predominantly characterized by sharply demarcated chronic erythematous plaques. Although its etiological mechanisms are largely unknown, recent evidence suggests that the topical application of imiquimod (IMQ) cream causes psoriasis-like skin inflammation in humans and mice. Skin examined 4 hours after IMQ cream treatment. Results provide insight into the role of each knockout mice phenotype in the response to IMQ-induced psoriasis model.
Project description:B16-BL6 mouse melanoma that had been maintained in C57BL/6J mice were used to evaluate the 5-aminolevulinic acid (ALA) effects on radiotherapy. Mice were divided into 4 groups after implantation of B16-BL6 cells; (1) control group; (2) ALA treatment; (3) Xray treatment; (4) ALA and Xray treatment.
Project description:CaMK4 has an important function in autoimmune diseases, and the contribution of CaMK4 in psoriasis remains obscure. Here, we show that CaMK4 expression is significantly increased in psoriatic lesional skin from psoriasis patients compared to healthy human skin as well as inflamed skin from an imiquimod (IMQ)-induced mouse model of psoriasis compared to healthy mouse skin. Camk4-deficient (Camk4−/−) mice treated with IMQ exhibit reduced severity of psoriasis compared to wild-type (WT) mice. There are more macrophages and fewer IL-17A+γδ TCR+ cells in the skin of IMQ-treated Camk4−/− mice compared to IMQ-treated WT mice. CaMK4 inhibits IL-10 production by macrophages, thus allowing excessive psoriatic inflammation. Deletion of Camk4 in macrophages alleviates IMQ-induced psoriatic inflammation in mice. In keratinocytes, CaMK4 inhibits apoptosis as well as promotes cell proliferation and the expression of pro-inflammatory genes such as S100A8 and CAMP. Taken together, these data indicate that CaMK4 regulates IMQ-induced psoriasis by sustaining inflammation and provides a potential target for psoriasis treatment.
Project description:Segmental instillation of lipopolysaccharide (LPS) by bronchoscopy can be used as a model to safely induce transient airway inflammation in the human lung. The LPS challenge model enables investigation of cellular mechanisms involved in pulmonary inflammatory processes as well as pharmacodynamic analysis of investigational drugs for the treatment of respiratory diseases. Aim of this work was to describe the transcriptomic profile of the human segmental LPS challenge model with contextualization to major respiratory diseases. Pre-challenge bronchoalveolar lavage fluid (BAL) and biopsies were sampled from twenty-eight smoking, healthy subjects, followed by segmental LPS challenge and saline control challenge. Twenty-four hours post instillation, BAL and biopsies were collected from the challenged lung segments. Total RNA of cells from BAL and biopsy samples were sequenced for subsequent data analysis. Differential gene expression analysis resulted in 6316 upregulated differentially expressed genes (DEGs) and 241 downregulated DEG in BAL, but only one downregulated DEG in biopsy samples after LPS challenge compared to saline challenge. Upregulated DEG in BAL were related to molecular functions such as “Inflammatory response” or “antimicrobial humoral immune response mediated by antimicrobial peptide”, enriched biological processes such as “chemokine receptor activity”, and upregulated pro-inflammatory pathways such as “Wnt signaling pathway”, “Ras signaling pathway” or “JAK-STAT signaling pathway”. Furthermore, the segmental LPS challenge model resembled aspects of the five most prevalent respiratory diseases chronic obstructive pulmonary disease (COPD), asthma, pneumonia, tuberculosis and lung cancer and featured similarities with acute exacerbations in COPD (AECOPD) and community-acquired pneumonia (CAP). Overall, our study provides extensive information about the transcriptomic profile from BAL cells and mucosal biopsies following LPS challenge in healthy smokers. It expands the knowledge about the LPS challenge model providing potential overlap with respiratory diseases in general and infection-triggered respiratory insults such as AECOPD in particular.
Project description:Background: Imiquimod (IMQ) produces a cutaneous phenotype in mice frequently studied as an acute model of human psoriasis. Whether this phenotype depends on strain or sex has never been systematically investigated on a large scale. Such effects, however, could lead to conflicts among studies, while further impacting study outcomes and efforts to translate research findings. Methods: RNA-seq was used to evaluate the psoriasiform phenotype elicited by IMQ in both sexes of 7 mouse strains (C57BL/6J, BALB/cJ, CD1, DBA/1J, FVB/NJ, 129X1/SvJ and MOLF/EiJ). Results: In most strains, IMQ altered gene expression in a manner consistent with human psoriasis, partly due to innate immune activation and decreased homeostatic gene expression. The IMQ response of MOLF males was aberrant, however, with decreased expression of differentiation-associated genes (elevated in other strains). Key aspects of the IMQ response differed between the two most commonly studied strains (BALB/c and C57BL/6). Compared with BALB/c, the C57BL/6 phenotype showed increased expression of genes associated with DNA replication, IL-17A activation and CD8+ T cells, but decreased expression of genes associated with interferon signaling and CD4+ T cells. Surprisingly, although IMQ-induced expression shifts mirrored psoriasis, correspondence was similar or better for other human skin diseases (e.g., eschars, acne, atopic dermatitis). For BALB/c, MOLF, and 129X1 strains, genes altered by IMQ corresponded better to those altered in human skin infections or wounds compared with those altered in psoriasis lesions. Conclusions: These findings demonstrate strain-dependent aspects of IMQ dermatitis that warrant consideration in planning and interpreting experimental studies. We have further shown that IMQ does not uniquely model psoriasis but in fact triggers a core set of pathways active in diverse skin diseases. These observations challenge the view of IMQ dermatitis as a mouse phenotype uniquely appropriate for studying psoriasis as opposed to other human skin conditions.
Project description:Aim: To study the effect of electroacupuncture (EA) on circRNA expression of plasma Exosomes and signal pathway of type 2 diabetic mice (T2DM). Methods: 10 mice were randomly selected as normal group. 20 mice were for T2DM model preparation and then were randomly divided into model and model + EA group. Mice in model + EA group were given EA treatment. The changes of fasting blood glucose (FBG) and the Islet structure were evaluated. Plasma exosomes of mice were subjected to RNA sequencing. Bioinformatics analysis on differentially expressed circRNA was performed. 12 differentially expressed circRNA were confirmed for real-time quantitative PCR (qPCR). Results: EA treatment reduced the FBG, preserved islets structure and reduced the islet β cell apoptotic rate of T2DM mice. RNA-sequencing analysis showed EA treatment lead to significant change of 165 circRNA expressions. GO and KEGG revealed that the effects of EA on T2DM mainly focus on regulating the functions and pathways related to multi-link metabolism, cell growth and organ development. Especially, the thyroid hormone signaling pathway was actively regulated by EA. circRNA/miRNA interactions analysis revealed that that mmu-mir-7092-3p was closely combined with circINPP4B, suggesting phosphatidylinositol signaling pathway was affected by EA. 12 circRNAs were identified to have significant differences by qRT-PCR. Conclusion: EA intervention can significantly protect islet function and improve FBG in T2DM, which may be related to the regulation on thyroid hormone and Phosphatidylinositol signaling pathway.