m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder in which excessive CD4+ T-cell activation and imbalanced effector T-cell differentiation play critical roles. Recent studies have implied a potential association between posttranscriptional N6-methyladenosine (m6A) modification and CD4+ T-cell-mediated humoral immunity. However, how this biological process contributes to lupus is not well understood. In this work, we investigated the role of the m6A methyltransferase like 3 (METTL3) in CD4+ T-cell activation, differentiation, and SLE pathogenesis both in vitro and in vivo. METHODS: The expression of METTL3 was knocked down and METTL3 enzyme activity was inhibited using siRNA and catalytic inhibitor, respectively. In vivo evaluation of METTL3 inhibition on CD4+ T-cell activation, effector T-cell differentiation, and SLE pathogenesis was achieved using a sheep red blood cell (SRBC)-immunized mouse model and a chronic graft versus host disease (cGVHD) mouse model. RNA-seq was performed to identify pathways and gene signatures targeted by METTL3. m6A RNA-immunoprecipitation qPCR was applied to confirm the m6A modification of METTL3 targets. RESULTS: METTL3 was defective in the CD4+ T cells of SLE patients. METTL3 expression varied following CD4+ T-cell activation and effector T-cell differentiation in vitro. Pharmacological inhibition of METTL3 promoted the activation of CD4+ T cells and influenced the differentiation of effector T cells, predominantly Treg cells, in vivo. Moreover, METTL3 inhibition increased antibody production and aggravated the lupus-like phenotype in cGVHD mice. Further investigation revealed that catalytic inhibition of METTL3 reduced Foxp3 expression by enhancing Foxp3 mRNA decay in a m6A-dependent manner, hence suppressing Treg cell differentiation. CONCLUSION: In summary, our findings demonstrated that METTL3 was required for stabilizing Foxp3 mRNA via m6A modification to maintain the Treg differentiation program. METTL3 inhibition contributed to the pathogenesis of SLE by participating in the activation of CD4+ T cells and imbalance of effector T-cell differentiation, which could serve as a potential target for therapeutic intervention in SLE.

Integration of metabolomics and lipidomics reveals serum biomarkers for systemic lupus erythematosus with different organs involvement.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects various organs or systems. We performed metabolomic and lipidomic profiles analyses of 133 SLE patients and 30 HCs. Differential metabolites and lipids were integrated, and then the biomarker panel was identified using binary logistic regression. We found that a combination of four metabolites or lipids could distinguish SLE from HC with an AUC of 0.998. Three lipids were combined to differentiate inactive SLE and active SLE. The AUC was 0.767. In addition, we also identified the biomarkers for different organ phenotypes of SLE. The AUCs for diagnosing SLE patients with only kidney involvement, skin involvement, blood system involvement, and multisystem involvement were 0.766, 0.718, 0.951, and 0.909, respectively. Our study succeeded in identifying biomarkers associated with different clinical phenotypes in SLE patients, which could facilitate a more precise diagnosis and assessment of disease progression in SLE.

Single-cell transcriptome reveals immunopathological cell composition of skin lesions in subacute cutaneous lupus erythematosus.

Subacute cutaneous lupus erythematosus (SCLE) is a clinical subtype of cutaneous lupus erythematosus with psoriatic-like or annular papules with scaly erythemas, the pathological mechanism of which is poorly understood. To investigate the immune pathogenesis of SCLE, we performed single-cell RNA sequencing (scRNA-seq) of SCLE skin lesions and integrated the scRNA-seq data from skin tissues of healthy controls (HCs). Our results identified expanded fibroblasts and keratinocytes subtypes, abnormally activated lymphocyte and inflammatory M1 macrophages in SCLE. In SCLE, stromal cells, such as keratinocytes and fibroblasts, showed enhanced chemotactic functions for recruiting immune cells. Importantly, interferon-related genes were identified as key intermediate genes in the potential trajectory of fibroblasts, keratinocytes, and B cells from HCs to SCLE. Our investigation provides a comprehensive description of cell composition in SCLE and highlights several important clues for understanding the pathogenesis of SCLE.

Celecoxib-induced drug fever: A rare case report and literature review.

WHAT IS KNOWN AND OBJECTIVE: Drug fever is frequently misdiagnosed, especially during concurrent infection. Celecoxib causes various adverse effects; however, celecoxib-induced drug fever is rarely reported. CASE SUMMARY: A 32-year-old man presented with pyrexia after 17 days of celecoxib therapy, which was reintroduced following 3-day total drug cessation. His fever recurred after this unsuspected rechallenge, which aided in the ultimate identification of the offending drug. A Naranjo Score of 8 led us to infer that drug fever was "probably" caused by celecoxib. WHAT IS NEW AND CONCLUSION: This is the first report of celecoxib-induced drug fever, aimed at assisting its diagnosis, particularly with rarely suspected causative drugs.

Tanshinone â ¡A inhibits homocysteine-induced proliferation of vascular smooth muscle cells via miR-145/CD40 signaling.

Tanshinone IIA (Tan IIA), isolated from the traditional Chinese herb Danshen, exhibits broad cardiovascular protective effects. However, the effect of Tan IIA on Homocysteine (Hcy)-induced proliferation of vascular smooth muscle cells (VSMCs) remains unknown. We herein determined whether Tan IIA exerted anti-proliferative effect in Hcy-treating VSMCs, and further investigated the underlying mechanism (miR-145/CD40 signaling). The results showed that Tan IIA significantly inhibited VSMCs proliferation induced by Hcy in a dose-dependent manner, and reversed the VSMCs injury as indicated by decreased KLF4 and increased Calponin expression. In view of the key role of miR-145 in VSMCs, we further explored the role of miR-145 on the protective effect of Tan IIA against Hcy-induced VSMCs proliferation. The miR-145 expression was down-regulated and its targeted gene CD40 was up-regulated in Hcy-treating VSMCs, while the Tan IIA reversed the effect of Hcy, suggesting the miR-145/CD40 may be involve in the protective effect of Tan IIA. To determine the speculation, miR-145 inhibitor was used to inhibit miR-145 expression. The results indicated that miR-145 inhibitor can suppress the protective effects of Tan IIA against Hcy-induced VSMCs proliferation. Collectively, present study demonstrates that Tan IIA inhibits Hcy-induced proliferation of VSMCs via miR-145/CD40 signaling.

Downregulation of BDH2 modulates iron homeostasis and promotes DNA demethylation in CD4+ T cells of systemic lupus erythematosus.

DNA hypomethylation plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Here we investigated whether 3-hydroxy butyrate dehydrogenase 2 (BDH2), a modulator of intracellular iron homeostasis, was involved in regulating DNA hypomethylation and hyper-hydroxymethylation in lupus CD4+ T cells. Our results showed that BDH2 expression was decreased, intracellular iron was increased, global DNA hydroxymethylation level was elevated, while methylation level was reduced in lupus CD4+ T cells compared with healthy controls. The decreased BDH2 contributed to DNA hyper-hydroxymethylation and hypomethylation via increasing intracellular iron in CD4+ T cells, which led to overexpression of immune related genes. Moreover, we showed that BDH2 was the target gene of miR-21. miR-21 promoted DNA demethylation in CD4+ T cells through inhibiting BDH2 expression. Our data demonstrated that the dysregulation of iron homeostasis in CD4+ T cells induced by BDH2 deficiency contributes to DNA demethylation and self-reactive T cells in SLE.

Pharmacodynamic properties and bioequivalence of dalteparin sodium subcutaneous injection in healthy Chinese male subjects.

1. Dalteparin sodium (DS) is a low molecular weight heparin that is widely used in the treatment of thromboembolism. The purpose of this study was to compare the pharmacodynamic properties and bioequivalence of the two formulations of DS with subcutaneous injection in healthy Chinese male subjects. 2. In this randomized, open-label, two-period crossover study, a total of 24 male subjects were recruited to receive single subcutaneous doses of test and reference DS injection in two different sequences (12 subjects each) with a seven-day washout period. Plasma samples were obtained at different time points after administration of the injection and measured by chromogenic substrate assay. The pharmacodynamic parameters including Emax, AUEC0-T, AUEC0-∞ and Tmax were analyzed to evaluate the bioequivalence of two DS formulations. 3. The relative bioequivalence was 107.7 ± 15.5 and 106.6 ± 29.8 for Anti-Xa and Anti-IIa, two major active metabolites of DS, respectively. The 90% confidence intervals (CIs) of the geometric mean ratio (test/reference) of Emax, AUEC0-T and AUEC0-∞ were 98.71-104.40%, 101.95-112.13% and 102.38-112.10% for Anti-Xa, and 100.88-110.42%, 95.76-112.62% and 92.24-111.32% for Anti-IIa, respectively, and all of the 90% CIs were within 80-125%. The T1/2 of reference and test were 2.88 ± 1.21 h and 2.76 ± 0.97 h for Anti-Xa, 1.87 ± 0.62 h and 1.96 ± 1.52 h for Anti-IIa. 4. Based on the pharmacodynamic parameters and FDA Guidance on DS and regulatory criteria for bioequivalence, the test and reference formulations were bioequivalent in healthy Chinese male subjects.

Aberrant histone modifications of global histone and MCP-1 promoter in CD14+ monocytes from patients with coronary artery disease.

OBJECTIVES: To investigate whether there are aberrant acetylation modifications in global histone and monocyte chemoattractant protein-1 (MCP-1) promoter in monocytes from patients with coronary artery disease (CAD) and demonstrate the potential mechanisms. METHODS: CD14+ monocytes were isolated from 13 patients with CAD and 18 confirmed non-CAD controls using magnetic beads. Global histone H3/H4 acetylation and H3K4/H3K27 tri-methylation levels were measured with enzyme-linked immunosorbent assay. Quantitative real time-PCR was performed to detect the mRNA expression levels of MCP-1 and enzymes involved in histone modification processes. Histone modification levels in MCP-1 promoter were assessed by ChIP-qPCR assay. RESULTS: Our results showed a markedly lower global histone H3 acetylation level in monocytes from CAD patients. Global H3K27 tri-methylation level was significantly increased in monocytes from CAD patients. Furthermore, the mRNA expression levels of epigenetic modification enzymes HDAC3, SIRT1, P300, JMJD3 and SUV39H1 were decreased significantly in monocytes from CAD patients, while HDAC7 mRNA expression level was markedly increased. MCP-1 mRNA expression level was increased histone H3/H4 acetylation levels in MCP-1 promoter were markedly increased in monocytes of CAD patients. CONCLUSION: Aberrant histone modifications, including acetylation and tri-methylation, were found both in global histone and specific MCP-1 gene locos in monocytes from patients with CAD. Aberrant epigenetic modification enzymes expressions may be the regulatory mechanism responsible for aberrant histone modifications.

Tacrolimus decreases insulin sensitivity without reducing fasting insulin concentration: a 2-year follow-up study in kidney transplant recipients.

New Onset Diabetes after Transplantation (NODAT) is defined as sustained hyperglycemia developing in patients without diabetes history before transplantation. A cohort study was performed to access the effects of tacrolimus on insulin secretion and insulin sensitivity and consequently in the development of NODAT in kidney transplant recipients. Then, we further investigated the association between NODAT and single-nucleotide polymorphisms of IRS-1 and IRS-2 in renal allograft recipients. One hundred and fifty-eight kidney transplant patients, receiving tacrolimus as the base immunosuppressant, were divided into two groups: with or without NODAT. Plasma levels of fasting insulin concentration (FINS) and C-peptide were determined by enhanced chemiluminescence immunoassay and ADVIA Centaur C peptide assay, respectively. The genotypes of Gly1057Asp in IRS-2 and Gly972Arg in IRS-1 were detected through polymerase chain reaction fragment length polymorphism in NODAT and non-NODAT patients. It was found that the concentrations of fasting plasma insulin and C-peptide in NODAT and non-NODAT patients treated with tacrolimus were higher than that in healthy volunteers (p < 0.05). Fasting plasma insulin concentration in NODAT was significantly elevated compared with than that in non-NODAT group (p < 0.05). But there are no statistical differences in fasting plasma C-peptide concentrations between NODAT and non-NODAT groups. The allele and genotype frequencies of IRS-2 Gly1057Asp and IRS-1 Gly972Arg in NODAT patients were not significantly different from non-NODAT patients (p > 0.05). In conclusion, insulin resistance is the primary cause of tacrolimus-induced NODAT. The IRS-2 Gly1057Asp and IRS-1 Gly972Arg genotypes are not related to NODAT.

Hypermethylation of DDAH2 promoter contributes to the dysfunction of endothelial progenitor cells in coronary artery disease patients.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) may be a biomarker for vascular function and cardiovascular risk in patients with coronary artery disease (CAD). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates the function of EPCs. This study aimed to examine whether hypermethylation of DDAH2 promoter contributes to impaired function of EPCs in CAD patients. METHODS: Peripheral blood mono-nuclear cells from 25 CAD patients and 15 healthy volunteers were collected and differentiated into EPCs. EPCs were tested for their adhesive capability. DDAH2 mRNA expression was analyzed by real-time PCR, and the methylation of DDAH2 promoter was detected by bisulfite genomic sequencing. RESULTS: DDAH2 promoter in EPCs from CAD patients was hypermethylated and the methylation level was negatively correlated to DDAH2 mRNA level and adhesion function of EPCs. Homocysteine impaired the adhesion function of EPCs, accompanied by lower DDAH2 expression and higher methylation level of DDAH2 promoter, compared to controls. These effects of homocysteine were reversed by pretreatment with Aza, an inhibitor of DNA methyltransferase. CONCLUSION: Hypermethylation in DDAH2 promoter is positively correlated to the dysfunction of EPCs in CAD patients. Homocysteine disrupts EPCs function via inducing the hypermethylation of DDAH2 promoter, suggesting a key role of epigenetic mechanism in the progression of atherosclerosis.

Fibroblast: A Novel Target for Autoimmune and Inflammatory Skin Diseases Therapeutics.

Fibroblasts are crucial components of the skin structure. They were traditionally believed to maintain the skin's structure by producing extracellular matrix and other elements. Recent research illuminated that fibroblasts can respond to external stimuli and exhibit diverse functions, such as the secretion of pro-inflammatory factors, adipogenesis, and antigen presentation, exhibiting remarkable heterogeneity and plasticity. This revelation positions fibroblasts as active contributors to the pathogenesis of skin diseases, challenging the traditional perspective that views fibroblasts solely as structural entities. Based on their diverse functions, fibroblasts can be categorized into six subtypes: pro-inflammatory fibroblasts, myofibroblasts, adipogenic fibroblasts, angiogenic fibroblasts, mesenchymal fibroblasts, and antigen-presenting fibroblasts. Cytokines, metabolism, and epigenetics regulate functional abnormalities in fibroblasts. The dynamic changes fibroblasts exhibit in different diseases and disease states warrant a comprehensive discussion. We focus on dermal fibroblasts' aberrant manifestations and pivotal roles in inflammatory and autoimmune skin diseases, including psoriasis, vitiligo, lupus erythematosus, scleroderma, and atopic dermatitis, and propose targeting aberrantly activated fibroblasts as a potential therapeutic strategy for inflammatory and autoimmune skin diseases.

The impact of psoriasis on idiopathic pulmonary fibrosis: a two-sample Mendelian randomization study.

BACKGROUND: The association between psoriasis and pulmonary fibrosis has been reported in observational studies. However, the association is vulnerable to bias from using immunosuppressants such as methotrexate, which can cause fibrosis in multiple organs. The present study is to investigate whether psoriasis could independently increase the risk of idiopathic pulmonary fibrosis (IPF). METHODS: We conducted a two-sample Mendelian randomization (MR) study using summary statistics from genome-wide association studies. The random-effects inverse variance weighted analysis was used as the primary method. Some secondary analyses were further performed, including a sensitivity analysis using a genetic instrument that excluded extended single nucleotide polymorphisms (SNPs) in the major histocompatibility complex (MHC) gene region. RESULTS: Our study included 9267 cases and 364,071 controls for psoriasis, 2018 cases, and 373,064 controls for IPF of European ancestry, respectively. Genetically predicted psoriasis was associated with a higher risk of IPF (odds ratio (OR), 1.14; 95% confidence interval (CI), 1.08-1.22; P < 0.001). Sensitivity analyses did not uncover any statistically significant evidence of bias resulting from pleiotropy or the genetic instruments, including SNPs in the MHC gene region. CONCLUSIONS: These MR analyses support that genetically predicted psoriasis was associated with the risk of IPF, implying that pulmonary fibrosis in patients with psoriasis should not be neglected, even if they are not receiving immunosuppressant therapy.

Gut-tropic T cells and extra-intestinal autoimmune diseases.

Gut-tropic T cells primarily originate from gut-associated lymphoid tissue (GALT), and gut-tropic integrins mediate the trafficking of the T cells to the gastrointestinal tract, where their interplay with local hormones dictates the residence of the immune cells in both normal and compromised gastrointestinal tissues. Targeting gut-tropic integrins is an effective therapy for inflammatory bowel disease (IBD). Gut-tropic T cells are further capable of entering the peripheral circulatory system and relocating to multiple organs. There is mounting evidence indicating a correlation between gut-tropic T cells and extra-intestinal autoimmune disorders. This review aims to systematically discuss the origin, migration, and residence of gut-tropic T cells and their association with extra-intestinal autoimmune-related diseases. These discoveries are expected to offer new understandings into the development of a range of autoimmune disorders, as well as innovative approaches for preventing and treating the diseases.

B-Cell Receptor Repertoire: Recent Advances in Autoimmune Diseases.

In the field of contemporary medicine, autoimmune diseases (AIDs) are a prevalent and debilitating group of illnesses. However, they present extensive and profound challenges in terms of etiology, pathogenesis, and treatment. A major reason for this is the elusive pathophysiological mechanisms driving disease onset. Increasing evidence suggests the indispensable role of B cells in the pathogenesis of autoimmune diseases. Interestingly, B-cell receptor (BCR) repertoires in autoimmune diseases display a distinct skewing that can provide insights into disease pathogenesis. Over the past few years, advances in high-throughput sequencing have provided powerful tools for analyzing B-cell repertoire to understand the mechanisms during the period of B-cell immune response. In this paper, we have provided an overview of the mechanisms and analytical methods for generating BCR repertoire diversity and summarize the latest research progress on BCR repertoire in autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjögren's syndrome (pSS), multiple sclerosis (MS), and type 1 diabetes (T1D). Overall, B-cell repertoire analysis is a potent tool to understand the involvement of B cells in autoimmune diseases, facilitating the creation of innovative therapeutic strategies targeting specific B-cell clones or subsets.

Modified Lichong decoction intervenes in colorectal cancer by modulating the intestinal flora and the Wnt/ß-catenin signaling pathway.

BACKGROUND: The pathogenesis and treatment of colorectal cancer (CRC) continue to be areas of ongoing research, especially the benefits of traditional Chinese medicine (TCM) in slowing the progression of CRC. This study was conducted to investigate the effectiveness and mechanism of action of modified Lichong decoction (MLCD) in inhibiting CRC progression. METHODS: We established CRC animal models using azoxymethane/dextran sodium sulfate (AOM/DSS) and administered high, medium, or low doses of MLCD or mesalazine (MS) for 9 weeks to observe MLCD alleviation of CRC. The optimal MLCD dose group was then subjected to metagenomic and RNA sequencing (RNA-seq) to explore the differentially abundant flora and genes in the control, model and MLCD groups. Finally, the mechanism of action was verified using WB, qRT‒PCR, immunohistochemistry and TUNEL staining. RESULTS: MLCD inhibited the progression of CRC, and the optimal effect was observed at high doses. MLCD regulated the structure and function of the intestinal flora by decreasing the abundance of harmful bacteria and increasing that of beneficial bacteria. The differentially expressed genes were mainly associated with the Wnt/ß-catenin pathway and the cell cycle. Molecular biology analysis indicated that MLCD suppressed the Wnt/ß-catenin pathway and the epithelial-mesenchymal transition (EMT), inhibited abnormal cell proliferation and promoted intestinal epithelial cell apoptosis. CONCLUSION: MLCD mitigated the abnormal growth of intestinal epithelial cells and promoted apoptosis, thereby inhibiting the progression of CRC. This inhibition was accomplished by modifying the intestinal microbiota and disrupting the Wnt/ß-catenin pathway and the EMT. Therefore, MLCD could serve as a potential component of TCM prescriptions for CRC treatment.

Aberrant H3K4me3 modification of immune response genes in CD4+ T cells of patients with systemic lupus erythematosus.

BACKGROUND: Increasing evidence has highlighted the significant role of histone modifications in pathogenesis of systemic lupus erythematosus (SLE). However, few studies have comprehensively analyzed trimethylation of histone H3 lysine 4 (H3K4me3) features at specific immune gene loci in SLE patients. METHODS: We conducted H3K4me3 chromatin immunoprecipitation sequencing (ChIP-seq) on CD4+ T cells from SLE patients and healthy controls (HC). Differential H3K4me3 peaks were identified, followed by enrichment analysis. We integrated online RNA-seq and DNA methylation datasets to explore the relationship between H3K4me3 modification, DNA methylation and gene expression. We validated several upregulated peak regions by ChIP-qPCR and confirmed their impact on gene expression using RT-qPCR. Finally, we investigated the impact of H3K4 methyltransferases KMT2A on the expression of immune response genes. RESULTS: we identified 147 downregulated and 2701 upregulated H3K4me3 peaks in CD4+ T cells of SLE. The upregulated peaks primarily classified as gained peaks and enriched in immune response genes such as FCGR2A, C5AR1, SERPING1 and OASL. Genes with upregulated H3K4me3 and downregulated DNA methylations in the promoter were highly expressed in SLE patients. These genes, including OAS1, IFI27 and IFI44L, were enriched in immune response pathways. The IFI44L locus also showed increased H3K27ac modification, chromatin accessibility and chromatin interactions in SLE. Moreover, knockdown of KMT2A can downregulate the expression of immune response genes in T cells. CONCLUSION: Our study uncovers dysregulated H3K4me3 modification patterns in immune response genes loci, which also exhibit downregulated DNA methylation and higher mRNA expression in CD4+ T cells of SLE patients.

RFX1 regulates foam cell formation and atherosclerosis by mediating CD36 expression.

BACKGROUND AND AIMS: Atherosclerosis (AS) is a continuously low-grade inflammatory disease, and monocyte-derived macrophages play a vital role in AS pathogenesis. Regulatory factor X1 (RFX1) has been reported to participate in differentiation of various cells. Our previous report showed that RFX1 expression in CD14+ monocytes from AS patients was decreased and closely related to AS development. Macrophages mostly derive from monocytes and play an important role in AS plaque formation and stability. However, the functions of RFX1 in the formation of macrophage-derived foam cells and consequent AS development are unclear. METHODS: We explored the effects of RFX1 on oxidation low lipoprotein (ox-LDL)-stimulated foam cell formation and CD36 expression by increasing or silencing Rfx1 expression in mouse peritoneal macrophages (PMAs). The ApoE-/-Rfx1f/f or ApoE-/-Rfx1f/f Lyz2-Cre mice fed a high-fat diet for 24 weeks were used to further examine the effect of RFX1 on AS pathogenesis. We then performed dual luciferase reporter assays to study the regulation of RFX1 for CD36 transcription. RESULTS: Our results demonstrate that RFX1 expression was significantly reduced in ox-LDL induced foam cells and negatively correlated with lipid uptake in macrophages. Besides, Rfx1 deficiency in myeloid cells aggravated atherosclerotic lesions in ApoE-/- mice. Mechanistically, RFX1 inhibited CD36 expression by directly regulating CD36 transcription in macrophages. CONCLUSIONS: The reduction of RFX1 expression in macrophages is a vital determinant for foam cell formation and the initiation of AS, proving a potential novel approach for the treatment of AS disease.

Keratinocyte-to-macrophage communication exacerbate psoriasiform dermatitis via LRG1-enriched extracellular vesicles.

Rationale: Macrophage-associated inflammation and keratinocytes excessive proliferation and inflammatory cytokines secretion induced by stimulation play an important role in the progression of psoriasiform dermatitis. However, how these two types of cells communicate remains obscure. Methods: We induced a mouse model with experimental psoriasiform dermatitis by Imiquimod (IMQ). To investigate whether damaged keratinocytes promote macrophage polarization and accelerate skin lesions by releasing extracellular vesicle (EV), purified EV were isolated from the primary epidermis of 5-day IMQ-induced psoriasiform dermatitis model mice, and then fluorescence-labeled the EV with PKH67. The EV was injected into the skin of mice treated with IMQ or vehicle 2 days in situ. In addition, we established a co-culture system of the human monocytic cell line (THP-1) and HaCaT, and THP-1/HaCaT conditioned media culture model in vitro respectively. Subsequently, we evaluated the effect of Leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EV on macrophage activation. Results: We demonstrated macrophages can significantly promote keratinocyte inflammation and macrophage polarization may be mediated by intercellular communication with keratinocytes. Interestingly, IMQ-induced 5-day, keratinocyte-derived EV recruited macrophage and enhanced the progression of skin lesions. Similar to results in vivo, EV released from M5-treated HaCaT significantly promotes Interleukin 1ß (IL-1ß) and Tumor necrosis factor α (TNF-α) expression of THP-1 cells. Importantly, we found that LRG1-enriched EV regulates macrophages via TGF beta Receptor 1 (TGFßR1) dependent process. Conclusion: Our findings indicated a novel mechanism for promoting psoriasiform dermatitis, which could be a potential therapeutic target.

Epigallocatechin-3-gallate inhibits homocysteine-induced apoptosis of endothelial cells by demethylation of the DDAH2 gene.

Our previous study showed that hypermethylation of dimethylarginine dimethylaminohydrolase 2 contributes to homocysteine-induced apoptosis of human umbilical vein endothelial cells. Epigallocatechin-3-gallate is a green tea-derived phenol which has been proved beneficial on atherosclerosis. It was demonstrated that epigallocatechin-3-gallate inhibits DNA methyltransferase activity and reactivates methylation-silenced genes in cancer cells. The aim of this study was to address whether epigallocatechin-3-gallate could induce DNA demethylation of the dimethylarginine dimethylaminohydrolase 2 gene, contributing to prevent endothelial cells from apoptosis induced by homocysteine. Human umbilical vein endothelial cells (ATCC, CRL-2480) were treated with homocysteine (1 mM) for 48 hours with or without epigallocatechin-3-gallate (20 µM) or 5-Aza (DNA methyltransferase inhibitor, 5 µM). Apoptosis rate of human umbilical vein endothelial cells was assayed by flow cytometry with an annexin V-FITC apoptosis detection kit. The mRNA and protein expression level of dimethylarginine dimethylaminohydrolase 2 and DNA methyltransferase 1 were detected by real-time PCR and Western blot, respectively. DNA methylation level of dimethylarginine dimethylaminohydrolase 2 was assayed by methylation specific PCR. The binding level of DNA methyltransferase 1 in the promoter of dimethylarginine dimethylaminohydrolase 2 was determined by chromatin immunoprecipitation-quantitative real-time PCR. It was shown that the apoptosis rate was decreased significantly in human umbilical vein endothelial cells treated with homocysteine compared with the control. Furthermore, the mRNA and protein level of dimethylarginine dimethylaminohydrolase 2 were downregulated, the dimethylarginine dimethylaminohydrolase 2 gene promoter was hypermethylated, and the DNA methyltransferase 1 mRNA and protein level were increased in human umbilical vein endothelial cells treated with homocysteine. Chromatin immunoprecipitation-quantitative real-time PCR revealed that homocysteine-induced binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter was increased. Pretreatment with epigallocatechin-3-gallate or 5-Aza inhibited such effects of homocysteine. In conclusion, epigallocatechin-3-gallate exerted protective effects on homocysteine-induced apoptosis in human umbilical vein endothelial cells by inhibiting promoter hypermethylation of the dimethylarginine dimethylaminohydrolase 2 gene and inducing dimethylarginine dimethylaminohydrolase 2 expression. These effects may be due to the decreased DNA methyltransferase 1 expression and binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter induced by epigallocatechin-3-gallate. This research suggests that modulating the epigenetic processes might be a novel plausible way for treatment of atherosclerosis.

Homocysteine-induced hypermethylation of DDAH2 promoter contributes to apoptosis of endothelial cells.

Homocysteine (Hcy) could induce apoptosis of endothelial cells (ECs). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) is recognized as a protective factor to improve the endothelial function. Defect of DDAH2 has been confirmed to be involved in the Hcy-induced dysfunction of endothelial NO system. This study was to determine whether Hcy could inhibit DDAH2 expression and induce apoptosis of ECs via increasing DNA methylation level of DDAH2 promoter and whether DNA methylation inhibitor 5-azacytidine (5-aza) could attenuate the effect of Hcy on ECs. Firstly, human umbilical vein endothelial cells (HUVECs) were treated by Hcy with or without 5-aza for 48 h. MTT assay showed that Hcy reduced the viability of HUVECs in a dose-dependent manner. The level of asymmetric dimethylarginine (ADMA) and the apoptosis rate of HUVECs treated with Hcy at 1.0 mM were increased significantly compared with that of control. Moreover, we found that mRNA level of DDAH2 was down-regulated and DNA methylation level of DDAH2 promoter was increased significantly in HUVECs treated with Hcy, in concomitance with up-regulated protein level of DNA methyltransferase 1 (DNMT1). Furthermore, we also found that 5-aza could neutralize the effect of Hcy on ECs through up-regulating mRNA level of DDAH2 and inducing demethylation of DDAH2 promoter. In conclusion, hypermethylation of DDAH2 contributes to Hcy induced apoptosis of ECs. Modulating DNA methylation status of DDAH2 promoter may be a potential strategy for treatment of endothelial dysfunction.