Serine deficiency exacerbates psoriatic skin inflammation by regulating S-adenosyl methionine-dependent DNA methylation and NF-κB signalling activation in keratinocytes.

BACKGROUND: Serine metabolism is crucial for tumour oncogenesis and immune responses. S-adenosyl methionine (SAM), a methyl donor, is typically derived from serine-driven one-carbon metabolism. However, the involvement of serine metabolism in psoriatic skin inflammation remains unclear. OBJECTIVES: To investigate the association between serine metabolism and psoriatic skin inflammation. METHODS: Clinical samples were collected from patients with psoriasis and the expression of serine biosynthesis enzymes was evaluated. The HaCaT human keratinocyte cell line was transfected with small interfering RNA (siRNA) of key enzyme or treated with inhibitors. RNA sequencing and DNA methylation assays were performed to elucidate the mechanisms underlying serine metabolism-regulated psoriatic keratinocyte inflammation. An imiquimod (IMQ)-induced psoriasis mouse model was established to determine the effect of the SAM administration on psoriatic skin inflammation. RESULTS: The expression of serine synthesis pathway enzymes, including the first rate-limiting enzyme in serine biosynthesis, phosphoglycerate dehydrogenase (PHGDH), was downregulated in the epidermal lesions of patients with psoriasis compared with that in healthy controls. Suppressing PHGDH in keratinocytes promoted the production of proinflammatory cytokines and enrichment of psoriatic-related signalling pathways, including the tumour necrosis factor-alpha (TNF-α) signalling pathway, interleukin (IL)-17 signalling pathway and NF-κB signalling pathway. In particular, PHGDH inhibition markedly promoted the secretion of IL-6 in keratinocytes with or without IL-17A, IL-22, IL-1α, oncostatin M and TNF-α (mix) stimulation. Mechanistically, PHGDH inhibition upregulated the expression of IL-6 by inhibiting SAM-dependent DNA methylation at the promoter and increasing the binding of myocyte enhancer factor 2A. Furthermore, PHGDH inhibition increased the secretion of IL-6 by increasing the activation of NF-κB via SAM inhibition. SAM treatment effectively alleviated IMQ-induced psoriasis-like skin inflammation in mice. CONCLUSIONS: Our study revealed the crucial role of PHGDH in antagonising psoriatic skin inflammation and indicated that targeting serine metabolism may represent a novel therapeutic strategy for treating psoriasis.

Assessment of relationships between bullous pemphigoid and neurological diseases: A bidirectional two-sample Mendelian randomization study.

Bullous pemphigoid (BP) is the most prevalent autoimmune vesiculobullous skin illness that tends to affect the elderly. Growing evidence has hinted a correlation between BP and neurological diseases. However, existing observational studies contained inconsistent results, and the causality and direction of their relationship remain poorly understood. To assess the causal relationship between BP and neurological disorders, including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD), and stroke. A bidirectional two-sample Mendelian randomization (MR) adopted independent top genetic variants as instruments from the largest accessible genome-wide association studies (GWASs), with BP (n = 218 348), PD (n = 482 730), AD (n = 63 926), stroke (n = 446 696), and MS (n = 115 803). Inverse variance weighted (IVW), MR-Egger, weighted mode methods, weighted median, and simple mode were performed to explore the causal association. Multiple sensitivity analyses, MR-Pleiotropy Residual Sum and Outlier (PRESSO) was used to evaluate horizontal pleiotropy and remove outliers. With close-to-zero effect estimates, no causal impact of BP on the risk of the four neurological diseases was discovered. However, we found that MS was positively correlated with higher odds of BP (OR = 1.220, 95% CI: 1.058-1.408, p = 0.006), while no causal associations were observed between PD (OR = 0.821, 95% CI: 0.616-1.093, p = 0.176), AD (OR = 1.066, 95% CI: 0.873-1.358, p = 0.603), stroke (OR = 0.911, 95% CI: 0.485-1.713, p = 0.773) and odds of BP. In summary, no causal impact of BP on the risk of PD, AD, MS and stroke was detected in our MR analysis. However, reverse MR analysis identified that only MS was positively correlated with higher odds of BP, but not PD, AD and stroke.

Formylpeptide receptor 1 contributes to epidermal barrier dysfunction-induced skin inflammation through NOD-like receptor C4-dependent keratinocyte activation.

BACKGROUND: Skin barrier dysfunction may both initiate and aggravate skin inflammation. However, the mechanisms involved in the inflammation process remain largely unknown. OBJECTIVES: We sought to determine how skin barrier dysfunction enhances skin inflammation and molecular mechanisms. METHODS: Skin barrier defect mice were established by tape stripping or topical use of acetone on wildtype mice, or filaggrin deficiency. RNA-Seq was employed to analyse the differentially expressed genes in mice with skin barrier defects. Primary human keratinocytes were transfected with formylpeptide receptor (FPR)1 or protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) small interfering RNA to examine the effects of these gene targets. The expressions of inflammasome NOD-like receptor (NLR)C4, epidermal barrier genes and inflammatory mediators were evaluated. RESULTS: Mechanical (tape stripping), chemical (acetone) or genetic (filaggrin deficiency) barrier disruption in mice amplified the expression of proinflammatory genes, with transcriptomic profiling revealing overexpression of formylpeptide receptor (Fpr1) in the epidermis. Treatment with the FPR1 agonist fMLP in keratinocytes upregulated the expression of the NLRC4 inflammasome and increased interleukin-1ß secretion through modulation of ER stress via the PERK-eIF2α-C/EBP homologous protein pathway. The activation of the FPR1-NLRC4 axis was also observed in skin specimens from old healthy individuals with skin barrier defect or elderly mice. Conversely, topical administration with a FPR1 antagonist, or Nlrc4 silencing, led to the normalization of barrier dysfunction and alleviation of inflammatory skin responses in vivo. CONCLUSIONS: In summary, our findings show that the FPR1-NLRC4 inflammasome axis is activated upon skin barrier disruption and may explain exaggerated inflammatory responses that are observed in disease states characterized by epidermal dysfunction. Pharmacological inhibition of FPR1 or NLRC4 represents a potential therapeutic target.

CREB1-driven CXCR4hi neutrophils promote skin inflammation in mouse models and human patients.

Neutrophils have a pathogenic function in inflammation via releasing pro-inflammatory mediators or neutrophil extracellular traps (NETs). However, their heterogeneity and pro-inflammatory mechanisms remain unclear. Here, we demonstrate that CXCR4hi neutrophils accumulate in the blood and inflamed skin in human psoriasis, and correlate with disease severity. Compared to CXCR4lo neutrophils, CXCR4hi neutrophils have enhanced NETs formation, phagocytic function, neutrophil degranulation, and overexpression of pro-inflammatory cytokines and chemokines in vitro. This is accompanied by a metabolic shift in CXCR4hi neutrophils toward glycolysis and lactate release, thereby promoting vascular permeability and remodeling. CXCR4 expression in neutrophils is dependent on CREB1, a transcription factor activated by TNF and CXCL12, and regulated by de novo synthesis. In vivo, CXCR4hi neutrophil infiltration amplifies skin inflammation, whereas blockade of CXCR4hi neutrophils through CXCR4 or CXCL12 inhibition leads to suppression of immune responses. In this work, our study identifies CREB1 as a critical regulator of CXCR4hi neutrophil development and characterizes the contribution of CXCR4hi neutrophils to vascular remodeling and inflammatory responses in skin.

Cross-disease characterization of fibroblast heterogeneities and their pathogenic roles in skin inflammation.

Fibroblasts are critical pro-inflammatory regulators in chronic inflammatory and fibrotic skin diseases. However, fibroblast heterogeneity and the absence of a unified cross-disease taxonomy have hindered our understanding of the shared/distinct pathways in non-communicable skin inflammation. By integrating 10× single-cell data from 75 skin samples, we constructed a single-cell atlas across inflammatory and fibrotic skin diseases and identified 9 distinct subsets of skin fibroblasts. We found a shared subset of CCL19+ fibroblasts across these diseases, potentially attracting and educating immune cells. Moreover, COL6A5+ fibroblasts were a distinct subset implicated in the initiation and relapse of psoriasis, which tended to differentiate into CXCL1+ fibroblasts, inducing neutrophil chemotaxis and infiltration; while CXCL1+ fibroblasts exhibited a more heterogeneous response to certain inflammatory conditions. Differentiation trajectory and regulatory factors of these fibroblast subsets were also revealed. Therefore, our study presents a comprehensive atlas of skin fibroblasts and highlights pathogenic fibroblast subsets in skin disorders.

Keratin 17 covalently binds to alpha-enolase and exacerbates proliferation of keratinocytes in psoriasis.

Dysregulated glucose metabolism is an important characteristic of psoriasis. Cytoskeletal protein keratin 17 (K17) is highly expressed in the psoriatic epidermis and contributes to psoriasis pathogenesis. However, whether K17 is involved in the dysregulated glucose metabolism of keratinocytes (KCs) in psoriasis remains unclear. In the present study, loss- and gain-of-function studies showed that elevated K17 expression was critically involved in glycolytic pathway activation in psoriatic KCs. The level of α-enolase (ENO1), a novel potent interaction partner of K17, was also elevated in psoriatic KCs. Knockdown of ENO1 by siRNA or inhibition of ENO1 activity by the inhibitor ENOBlock remarkably suppressed KCs glycolysis and proliferation. Moreover, ENO1 directly interacted with K17 and maintained K17-Ser44 phosphorylation to promote the nuclear translocation of K17, which promoted the transcription of the key glycolysis enzyme lactic dehydrogenase A (LDHA) and resulted in enhanced KCs glycolysis and proliferation in vitro. Finally, either inhibiting the expression and activation of ENO1 or repressing K17-Ser44 phosphorylation significantly alleviated the IMQ-induced psoriasis-like phenotype in vivo. These findings provide new insights into the metabolic profile of psoriatic KCs and suggest that modulation of the ENO1-K17-LDHA axis is a potentially innovative therapeutic approach to psoriasis.

CD36-SREBP1 Axis Mediates TSLP Production in Obesity-Exacerbated Atopic Dermatitis.

Obesity is associated with an increased risk of atopic dermatitis (AD) and may accelerate its development. Keratinocyte dysfunction has been observed in obesity-related skin diseases, including psoriasis and acanthosis nigricans, but is not fully understood in AD. In this study, we found that high-fat diet-induced obesity exacerbated AD-like dermatitis in mice, with elevated inflammatory molecules and increased CD36-SREBP1-related fatty acid accumulation in the lesional skin. Blocking CD36 or SREBP1 with chemical inhibitors effectively alleviated AD-like inflammation, decreased fatty acid accumulation, and downregulated TSLP expression in obese calcipotriol (MC903)-treated mice. Moreover, palmitic acid treatment induced TSLP overexpression in keratinocytes through the activation of the CD36-SREBP1 signaling pathway. The chromatin immunoprecipitation assay further revealed increased binding of SREBP1 to the TSLP promoter region. Our findings provide compelling evidence that obesity triggers the activation of the CD36-SREBP1-TSLP axis in keratinocytes, leading to epidermal lipid disorders and the aggravation of AD-like inflammation. By targeting CD36 or SREBP1, future combination therapies or modified treatment strategies could be developed to help manage patients with both obesity and AD.

An IGFBP7hi endothelial cell subset drives T cell extravasation in psoriasis via endothelial glycocalyx degradation.

Dysfunction of vascular endothelial cells (ECs) facilitates imbalanced immune responses and tissue hyperinflammation. However, the heterogeneous functions of skin ECs and their underlying mechanism in dermatoses remain to be determined. Here, focusing on the pathogenic role of skin ECs in psoriasis, we characterized the molecular and functional heterogeneity of skin ECs from healthy individuals and psoriasis patients at the single-cell level. We found that endothelial glycocalyx destruction, a major feature of EC dysfunction in psoriasis, was a driving force during the process of T cell extravasation. Interestingly, we identified a skin EC subset, IGFBP7hi ECs, in psoriasis. This subset actively responded to psoriatic-related cytokine signaling, secreted IGFBP7, damaged the endothelial glycocalyx, exposed the adhesion molecules underneath, and prepared the endothelium for immune-cell adhesion and transmigration, thus aggravating skin inflammation. More importantly, we provided evidence in a psoriasis-like mouse model that anti-IGFBP7 treatment showed promising therapeutic effects for restoring the endothelial glycocalyx and alleviating skin inflammation. Taken together, our results depict the distinct functions of EC clusters in healthy and psoriatic skin, identify IGFBP7hi ECs as an active subset modulating vascular function and cutaneous inflammation, and indicate that targeting IGFBP7 is a potential therapeutic strategy in psoriasis.

Neutrophil extracellular traps promote keratinocyte inflammation via AIM2 inflammasome and AIM2-XIAP in psoriasis.

The infiltration of neutrophils in the epidermis and the release of neutrophil extracellular traps (NETs) are important events in the pathogenesis of psoriasis, but the regulatory roles and internal mechanism of NETs in psoriasis are largely unknown. Here, we demonstrate that NETs can activate the absent-in-melanoma-2 (AIM2) inflammasome in keratinocytes through the p38-MAPK signalling pathway, and targeting NETs with CI-amidine in vivo reduces AIM2 expression and ameliorates imiquimod-induced psoriasis-like phenotype in mice. Notably, NETs-activated AIM2 in keratinocytes not only promotes IL-1ß production through the classical inflammasome pathway but also promotes IFN-γ production via X-linked inhibitor of apoptosis protein (XIAP), thereby mediating the immune responses of keratinocytes. In conclusion, our study demonstrates that the NETs-AIM2 axis exerts multiple pro-inflammatory effects on keratinocytes and may serve as a potential target for psoriasis therapy.

CXCL12/CXCR4 Axis Drives the Chemotaxis and Differentiation of B Cells in Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune bullous skin disease characterized by autoantibodies against the hemidesmosomal proteins in the skin and mucous membranes. The efficiency of B-cell‒targeting biologics in BP indicates the important role of B cells in its pathogenesis. However, abnormal B-cell migration and differentiation in BP require further elucidation. We showed that the number of antibody-secreting cells increased in the circulation and skin lesions of patients with BP and was correlated with disease severity. Bulk RNA sequencing of the peripheral B cells identified 171 upregulated and 408 downregulated genes in patients with BP compared with those in healthy controls, among which CXCR4 was significantly upregulated. Notably, CXCR4+ B cells were enriched in BP skin lesions and exhibited antibody-secreting cell characteristics. Correspondingly, an elevated level of CXCL12, the CXCR4 ligand, was detected in the blister fluid and serum of patients with BP, mediating the chemotaxis and accumulation of CXCR4+ B cells to BP skin lesions. Moreover, CXCL12 activated the transcription factor c-Myc, thus promoting B-cell differentiation into antibody-secreting cells and facilitating autoantibody production, which was blocked by CXCR4 inhibitor in vitro. Collectively, our study reveals that the CXCL12/CXCR4 axis plays a pathogenic role in modulating B-cell trafficking and differentiation, thus targeting CXCR4 represents a potential strategy for treating BP and other autoimmune diseases.

Nuclear-targeted EGF receptor enhances proliferation and migration of human anaplastic thyroid cancer cells.

INTRODUCTION: Epidermal growth factor (EGF) has various important physiological functions, which it exerts by binding to the epidermal growth factor receptor (EGFR). Reports show that EGF expression is strongly correlated with the occurrence and development of many types of tumour. To date, however, the relationship between EGF/EGFR and the occurrence and development of thyroid carcinoma remains unclear. MATERIAL AND METHODS: In the current study, we investigated this phenomenon using human anaplastic thyroid carcinoma cell lines (SUN-80). RESULTS: The results indicated that EGF triggered the EGFR-mediated intracellular signalling pathway, including signal transducers and activators of transcription 1/3/5 (STAT1/3/5) and protein kinase B (AKT) in a time- and dose-dependent manner. In addition, results from EGF-induced EGFR internalization and co-localization analyses showed that clathrin, Rab5/7, and EEA1 play critical roles in the intracellular trafficking of EGF/EGFR. Interestingly, EGF triggered EGFR translocation into the nucleus, while nuclear-localized EGFR affected cell cycle distribution, thereby significantly promoting the ration of S phase. Overall, these findings indicated that nuclear EGFR exerts biological activity and physiological functions, including changing cell cycle, which in turn promotes proliferation and migration of SUN-80 cells. CONCLUSION: These findings lay a foundation for further explorations seeking to understand the biological effects of the EGF/EGFR system on the occurrence and development of thyroid cancer.

Quinolinic Acid, a Tryptophan Metabolite of the Skin Microbiota, Negatively Regulates NLRP3 Inflammasome through AhR in Psoriasis.

Psoriasis is a chronic inflammatory skin disease whose pathogenesis involves skin microbiota dysbiosis. Multiple studies have revealed the changes in microbiota abundances between psoriatic lesions and healthy skin. However, the metabolic pathways of skin microbiota (especially tryptophan metabolism, which is closely related to immunosuppression) are far less understood. In this study, we first detected the major microbial metabolites of tryptophan on the skin surfaces, finding that the quinolinic acid was significantly lower in the lesional skin of patients with psoriasis than in that of healthy subjects and correlated negatively with the severity of psoriasis. In vitro and in vivo, applying quinolinic acid significantly alleviated skin inflammation in an AhR-dependent manner, resulting in inhibition of the NLRP3 inflammasome activation. Furthermore, in mice with imiquimod-induced psoriasis-like dermatitis, topical application of Ahr-targeted small interfering RNA substantially exacerbated the disease severity, with increased NLRP3 inflammasome activation. Collectively, our data suggest that quinolinic acid, a skin microbiota-derived metabolite, negatively regulates aryl hydrocarbon receptor-NLRP3 inflammasome signaling activation in patients with psoriasis, providing an insight into the correlation between microbiota metabolism and the host skin in individuals with psoriasis.

LCN2 Mediates Skin Inflammation in Psoriasis through the SREBP2‒NLRC4 Axis.

Lipocalins are a family of secreted adipokines that regulate cell lipid metabolism and immune responses. Although we have previously revealed that LCN2 modulates neutrophil activation in psoriasis, the other roles of LCN2 in psoriatic local inflammation have remained elusive. In this study, we found that 24p3R, the well-known specific receptor of LCN2, was highly expressed in the lesional epidermis of patients with psoriasis. Silencing 24p3R (also known as slc22a17) alleviated hyperkeratosis, inflammatory cell infiltration, and overexpression of inflammatory mediators in an imiquimod-induced psoriasis-like mouse model. In vitro, LCN2 enhanced the expression of proinflammatory factors in primary keratinocytes, such as IL-1ß, IL-23, CXCL1, and CXCL10, which was paralleled by enforced cholesterol biosynthetic signaling. Importantly, taking in vivo and in vitro approaches, we discovered the SREBP2, a vital transcriptional factor in cholesterol synthesis pathway, as the critical mediator of LCN2-induced keratinocyte activation, which bound to the promoter region of NLRC4. Suppressing SREBP2 in mice attenuated NLRC4 signaling and psoriasis-like dermatitis. Taken together, this study identifies the critical role of LCN2‒SREBP2‒NLRC4 axis in the pathogenesis of psoriasis and proposes 24p3R or SREBP2 as a potential therapeutic target for psoriasis.

Keratin 17 Promotes T Cell Response in Allergic Contact Dermatitis by Upregulating C-C Motif Chemokine Ligand 20.

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity response to skin contact allergens in which keratinocytes are critical in the initiation of early responses. Keratin 17 (K17) is a cytoskeletal protein inducible under stressful conditions and regulates multiple cellular processes, especially in skin inflammatory diseases; however, knowledge regarding its contribution to ACD pathogenesis remains ill defined. In the present study, we clarified the proinflammatory role of K17 in an oxazolone (OXA)-induced contact hypersensitivity (CHS) murine model and identified the underlying molecular mechanisms. Our results showed that K17 was highly expressed in the lesional skin of ACD patients and OXA-induced CHS mice. Mice lacking K17 exhibited alleviated OXA-induced skin inflammation, including milder ear swelling, a reduced frequency of T cell infiltration, and decreased inflammatory cytokine levels. In vitro, K17 stimulated and activated human keratinocytes to produce plenty of proinflammatory mediators, especially the chemokine CCL20, and promoted keratinocyte-mediated T cell trafficking. The neutralization of CCL20 with a CCL20-neutralizing monoclonal antibody significantly alleviated OXA-induced skin inflammation in vivo. Moreover, K17 could translocate into the nucleus of activated keratinocytes through a process dependent on the nuclear-localization signal (NLS) and nuclear-export signal (NES) sequences, thus facilitating the activation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3), further promoting the production of CCL20 and T cell trafficking to the lesional skin. Taken together, these results highlight the novel roles of K17 in driving allergen-induced skin inflammation and suggest targeting K17 as a potential strategy for ACD.

Phospholipase A2 enzymes represent a shared pathogenic pathway in psoriasis and pityriasis rubra pilaris.

Altered epidermal differentiation along with increased keratinocyte proliferation is a characteristic feature of psoriasis and pityriasis rubra pilaris (PRP). However, despite this large degree of overlapping clinical and histologic features, the molecular signatures these skin disorders share are unknown. Using global transcriptomic profiling, we demonstrate that plaque psoriasis and PRP skin lesions have high overlap, with all differentially expressed genes in PRP relative to normal skin having complete overlap with those in psoriasis. The major common pathway shared between psoriasis and PRP involves the phospholipases PLA2G2F, PLA2G4D, and PLA2G4E, which were found to be primarily expressed in the epidermis. Gene silencing each of the 3 PLA2s led to reduction in immune responses and epidermal thickness both in vitro and in vivo in a mouse model of psoriasis, establishing their proinflammatory roles. Lipidomic analyses demonstrated that PLA2s affect mobilization of a phospholipid-eicosanoid pool, which is altered in psoriatic lesions and functions to promote immune responses in keratinocytes. Taken together, our results highlight the important role of PLA2s as regulators of epidermal barrier homeostasis and inflammation, identify PLA2s as a shared pathogenic mechanism between PRP and psoriasis, and as potential therapeutic targets for both diseases.

Cyclin-Dependent Kinase 7 Promotes Th17/Th1 Cell Differentiation in Psoriasis by Modulating Glycolytic Metabolism.

Excessive activation of CD4+ T cells and T helper type (Th) 17/Th1 cell differentiation are critical events in psoriasis pathogenesis, but the associated molecular mechanism is still unclear. Here, using quantitative proteomics analysis, we found that cyclin-dependent kinase 7 (CDK7) expression was markedly increased in CD4+ T cells from patients with psoriasis compared with healthy controls and was positively correlated with psoriasis severity. Meanwhile, genetic or pharmacological inhibition of CDK7 ameliorated the severity of psoriasis in the imiquimod-induced psoriasis-like mouse model and suppressed CD4+ T-cell activation as well as Th17/Th1 cell differentiation in vivo and in vitro. Furthermore, the CDK7 inhibitor also reduced the enhanced glycolysis of CD4+ T cells from patients with psoriasis. Proinflammatory cytokine IL-23 induced increased CDK7 expression in CD4+ T cells and activated the protein kinase B/mTOR/HIF-1α signaling pathway, enhancing glycolytic metabolism. Correspondingly, CDK7 inhibition significantly impaired IL-23-induced glycolysis via the protein kinase B/mTOR/HIF-1α pathway. In summary, this study shows that CDK7 promotes CD4+ T-cell activation and Th17/Th1 cell differentiation by regulating glycolysis, thus contributing to the pathogenesis of psoriasis. Targeting CDK7 might be a promising immunosuppressive strategy to control skin inflammation mediated by IL-23.

IRAK2 Has a Critical Role in Promoting Feed-Forward Amplification of Epidermal Inflammatory Responses.

Many inflammatory skin diseases are characterized by altered epidermal differentiation. Whether this altered differentiation promotes inflammatory responses has been unknown. Here, we show that IRAK2, a member of the signaling complex downstream of IL-1 and IL-36, correlates positively with disease severity in both atopic dermatitis and psoriasis. Inhibition of epidermal IRAK2 normalizes differentiation and inflammation in two mouse models of psoriasis- and atopic dermatitis-like inflammation. Specifically, we demonstrate that IRAK2 ties together proinflammatory and differentiation-dependent responses and show that this function of IRAK2 is specific to keratinocytes and acts through the differentiation-associated transcription factor ZNF750. Taken together, our findings suggest that IRAK2 has a critical role in promoting feed-forward amplification of inflammatory responses in skin through modulation of differentiation pathways and inflammatory responses.

Synthesis and anti-inflammatory activity of novel steroidal chalcones with 3ß-pregnenolone ester derivatives in RAW 264.7 cells in vitro.

To identify new potential anti-inflammatory agents, we herein report the synthesis of novel steroidal chalcones with 3ß-pregnenolone esters of cinnamic acid derivatives using pregnenolone as the starting material. The structures of the newly synthesised compounds were confirmed by 1H NMR, 13C NMR, HRMS and infrared imaging. All the derivatives were examined to determine their in vitro anti-inflammatory profiles against LPS-induced inflammation in RAW 264.7 cells; the derivates were evaluated by the quantification of the pro-inflammatory mediator nitric oxide (NO) in the cell culture supernatant based on the Griess reaction, which measures nitrite levels, followed by an in vitro cytotoxicity study. Among these novel derivatives, compound 11e [3ß-3-phenyl acrylate-pregn-5-en-17ß-yl-3' -(p-fluoro)-phenylprop-2'-en-1'-one] was identified as the most potent anti-inflammatory agent, which showed significant anti-inflammatory activity by inhibiting the LPS-induced pro-inflammatory mediator NO in a dose-dependent manner without any cytotoxicity. Moreover, compound 11e markedly inhibited the expression of pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2), in LPS-induced RAW 264.7 cells. Further studies confirmed that compound 11e significantly suppressed the transcriptional activity of NF-κB in activated RAW 264.7 cells. Molecular docking study revealed the strong binding affinity of compound 11e to the active site of the pro-inflammatory proteins, which confirmed that compound 11e acted as an anti-inflammatory mediator. These results indicated that steroidal chalcones with 3ß-pregnenolone esters of cinnamic acid derivatives might be considered for further research in the design of anti-inflammatory drugs, and compound 11e might be a promising therapeutic anti-inflammatory drug candidate.

Neutrophils Enhance Cutaneous Vascular Dilation and Permeability to Aggravate Psoriasis by Releasing Matrix Metallopeptidase 9.

Neutrophil infiltration and papillary vessel dilation are hallmarks of the initiation phase of psoriatic lesions. However, how neutrophils aggravate psoriasis development during transendothelial migration and the interaction between neutrophils and cutaneous vascular endothelial cells are less well-understood. In this study, we reported that neutrophils and cutaneous vascular endothelial cells activated each other when neutrophils migrated through the cutaneous endothelial barrier. In addition, neutrophil infiltration into skin lesions caused vascular remodeling including cutaneous vasodilation and enhanced vascular permeability in vivo and in vitro. Microarray gene profile data showed that matrix metallopeptidase (MMP)-9 was overexpressed in psoriatic neutrophils, and zymography assay further validated the bioactivity of MMP-9 secreted by psoriatic neutrophils. Moreover, MMP-9 activated vascular endothelial cells through the extracellular signal‒regulated kinase 1/2 and p38-MAPK signaling pathways, enhancing CD4+ T-cell transmigration in vitro. Correspondingly, an MMP-9 inhibitor significantly reduced cutaneous vasodilation, vascular permeability, and psoriatic symptoms in an imiquimod- or IL-23‒induced psoriasiform mouse model. Overall, our study demonstrates that neutrophil-derived MMP-9 induces cutaneous vasodilation and hyperpermeability by activating cutaneous vascular endothelial cells, thus facilitating psoriatic lesion development, which increases our knowledge on the role of neutrophils in the pathogenesis of psoriasis.

Aryl Hydrocarbon Receptor in Cutaneous Vascular Endothelial Cells Restricts Psoriasis Development by Negatively Regulating Neutrophil Recruitment.

Vascular endothelial cells (VECs) that line the interiors of blood vessels participate in physiological and inflammatory processes. All skin cell types express the aryl hydrocarbon receptor (AhR), which is involved in the pathogenesis of psoriasis. However, the role of the cutaneous VEC AhR in the pathogenesis of psoriasis remains elusive. In the present study, we found that AhR protein expression and activation were downregulated in psoriatic VECs. Furthermore, cutaneous VEC-specific AhR-knockout (AhRcVECs-KO) mice were established. Using imiquimod and IL-23-induced psoriasis models, we found that skin inflammation was exacerbated with excessive neutrophil recruitment in AhRcVECs-KO mice. Furthermore, neutrophil neutralization alleviates exacerbated inflammation in imiquimod-treated AhRcVECs-KO mice. In addition, cutaneous VECs in AhRcVECs-KO mice exhibited increased dilation and activation compared with those in control mice. Finally, AhR-deficient microvascular endothelial cells stimulated by proinflammatory cytokines showed increased ICAM-1 expression in vivo and in vitro, which may have facilitated neutrophil recruitment. In summary, our study demonstrates that AhR in dermal VECs restricts psoriasis development by negatively regulating neutrophil recruitment, thereby providing insight into the pathogenesis of psoriasis.