Association of TNF-Î± polymorphisms (âˆ’857, âˆ’863 and âˆ’1031), TNF-Î± serum level and lipid profile with acne vulgaris
Background: Acne is an inflammatory condition principally affected by genetic and dietary factors. Investigation into functional polymorphisms of TNF-Î± gene and their association with acne vulgaris will be helpful in exploring genetic influence on skin immune mediated inflammatory events. In the present study, we analyzed association of TNF-Î± gene polymorphisms, its expression levels and lipid profiles in a large cohort of acne patients and controls. Methods: We used PCR-RFLP to study association of TNF-Î± polymorphisms at âˆ’857C/T, âˆ’863C/A and âˆ’1031 T/C sites with acne vulgaris. Lipid profiles were measured using enzymatic end-point method. The serum levels of TNF-Î± and apolipoprotein a were measured using ELISA. NIH, LDlink was used to investigate patterns of linkage disequilibrium across south Asian reference genome (Punjabi from Lahore Pakistan). Results: We found that TNF-Î± âˆ’863 polymorphism is strongly associated with acne in overall population as well as in gender and severity based groups of acne patients. Polymorphisms at âˆ’863 and âˆ’1031 position were in linkage disequilibrium. Importantly, TNF-Î± serum level was significantly increased in acne patients with severe disease symptoms. Furthermore, levels of total cholesterol (TC) and triglycerides (TG) were significantly increased, whereas high density lipoprotein cholesterol (HDL-C) level was significantly decreased in acne patients. The levels of apolipoprotein a varied widely in studied populations and no significant difference was found in the analyzed groups. Conclusion: In conclusion, we found that TNF-Î± expression increases in acne patients affected by TNF-Î± polymorphisms, and that the lipid profile is specifically disrupted in acne patients.
Skinomics, transcriptional profiling approaches to molecular and structural biology of epidermis
Skinomics is a field of bioinformatics applied specifically to skin biology and, by extension, to dermatology. Skinomics has been expanding into extensive genome-wide association studies, eg, of psoriasis, proteomics, lipidomics, metabolomics, metagenomics, and the studies of the microbiome. Here, the current state of the field of transcriptomics is reviewed, including the studies of the gene expression in human skin under several healthy and disease conditions. Specifically, transcriptional studies of epidermal differentiation, skin aging, effects of cytokines, inflammation with emphases on psoriasis and atopic dermatitis, and wound healing are reviewed. The transition from microarrays to NextGen sequencing is noted and potential future directions suggested.
Cardiac Fibroblast Transcriptome Analyses Support a Role for Interferogenic, Profibrotic and Inflammatory Genes in Anti-SSA/Ro-Associated Congenital Heart Block
The signature lesion of SSA/Ro autoantibody-associated congenital heart block (CHB) is fibrosis and a macrophage infiltrate, supporting an experimental focus on cues influencing the fibroblast component. The transcriptomes of human fetal cardiac fibroblasts were analyzed using two complementary approaches. Cardiac injury conditions were simulated in vitro by incubating human fetal cardiac fibroblasts with supernatants from macrophages transfected with the SSA/Ro-associated hY3. The top ten upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response (e.g. IFI44L, MX1, MX2, and Rsad2). Within the fibrotic pathway, transcript levels of EDN1, PDE4D, CXCL2 and CXCL3 were upregulated, while others, including ADM, RAPGEF3, TIMP1, TIMP3 and DUSP1, were downregulated. Agnostic DAVID analysis revealed a significant increase in inflammatory genes, including C3AR1, the complement C3A receptor, F2RL3, and NCF2. In addition, the stimulated fibroblasts expressed high levels of phospho-MEF2C (a substrate of ERK5), which was inhibited by BIX 02189, a specific inhibitor of ERK5. Translation to human disease leveraged an unprecedented opportunity to interrogate the transcriptome of fibroblasts freshly isolated and cell sorted without stimulation from a fetal heart with CHB and a matched healthy heart. Consistent with the in vitro data, five IFN response genes were among the top ten most highly expressed transcripts in the CHB fibroblasts. In addition, the expression of matrix-related genes reflected fibrosis. These data support the novel finding that cardiac injury in CHB may occur secondary to abnormal remodeling due in part to upregulation of type 1 IFN response genes.
Embryonic AP1 Transcription Factor Deficiency Causes a Collodion Baby-Like Phenotype
AP1 transcription factors are important controllers of gene expression in the epidermis, and altered AP1 factor function can perturb keratinocyte proliferation and differentiation. However, our understanding of how AP1 signaling changes may underlie or exacerbate skin disease is limited. We have shown that inhibiting AP1 factor function in suprabasal adult epidermis leads to reduced filaggrin levels and to a phenotype that resembles the genetic disorder, ichthyosis vulgaris. We now show that inhibiting AP1 factor function during development in embryonic epidermis produces marked phenotypic changes including reduced filaggrin mRNA and protein levels, compromised barrier function, marked ultrastructural change and enhanced dehydration susceptibility that resembles the phenotype observed in the flaky tail mouse, a model for ichthyosis vulgaris. In addition, the AP1 factor-deficient newborn mice also display a collodion membrane phenotype which is not observed in flaky tail mice or in newborn individuals with ichthyosis vulgaris, but is present in other forms of ichthyosis. This mixed phenotype suggests that a need for a better understanding of the possible role of filaggrin loss and AP1 transcription factor deficiency in ichthyoses and collodion membrane formation.
Role of activated platelets in severe acne scarring and adaptive immunity activation [Letter]
A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers
Chronic nonhealing venous leg ulcers (VLUs) are widespread and debilitating, with high morbidity and associated costs; about $15 billion is spent annually on the care of VLUs in the United States. Despite this, there is a paucity of treatments for VLUs because of the lack of pathophysiologic insight into ulcer development as well as the lack of knowledge regarding biologic actions of existing VLU-targeted therapies. The bioengineered bilayered living cellular construct (BLCC) skin substitute is a U.S. Food and Drug Administration-approved biologic treatment for healing VLUs. To elucidate the mechanisms through which the BLCC promotes healing of chronic VLUs, we conducted a clinical trial (NCT01327937) in which patients with nonhealing VLUs were treated with either standard of care (compression therapy) or the BLCC together with standard of care. Tissue was collected from the VLU edge before and 1 week after treatment, and the samples underwent comprehensive microarray mRNA and protein analyses. Ulcers treated with the BLCC skin substitute displayed three distinct transcriptomic patterns, suggesting that BLCC induced a shift from a nonhealing to a healing tissue response, involving modulation of inflammatory and growth factor signaling, keratinocyte activation, and attenuation of Wnt/beta-catenin signaling. In these ways, BLCC application orchestrated a shift from the chronic nonhealing ulcer microenvironment to a distinctive healing milieu resembling that of an acute, healing wound. Our findings provide in vivo evidence in VLU patients of pathways that can be targeted in the design of new therapies to promote healing of chronic VLUs.
Cyclic amp, ERK5, and transdifferentiation of cardiac fibroblasts in the pathogenesis of autoimmune congenital heart block [Meeting Abstract]
Background/Purpose: Maternal autoantibodies (Ab) reactive with the Ro/La ribonucleoprotein complex are associated with the development of cardiac injury in a fetus passively exposed to these Ab. This study evaluated the irreversible scarring phenotype characteristic of heart block involving the mitogen activated protein kinase (MAPK) pathway and its regulation by cAMP. Methods: The aorta from a healthy 2nd trimester fetal heart was cannulated using a Langendorff preparation with the addition of proteolytic enzymes to yield a single cell suspension of primary human fetal cardiac fibroblasts. Cultured cells were treated with secreted products generated from activated macrophages with or without BIX 02189 (10uM, a specific MEK5/ERK5 inhibitor) and forskolin (10uM to raise cAMP). After RNA isolation and cDNA library preparation, RNA-Seq, transcriptome analysis (Data as log2 transcripts per million) and qPCR were performed. Results: Incubation of fibroblasts with supernatants from macrophages transfected with hY3 (ssRNA associated with Ro60), shown to induce a pro-fibrotic phenotype, resulted in the increased expression of 3836 genes. Based on DAVID functional annotation, the top clusters represented were Actin Binding, Cytoskeletal Protein Binding, Cell Adhesion, Signal Peptide, and Contractile Fiber, all processes considered typical of the myofibroblast phenotype. In addition, RAPGEF3, an endogenous ERK5 inhibitor, and Adrenomedullin, which increases cAMP, were downregulated while PDE4D, an inhibitor of cAMP generation, was upregulated (Table 1). These data are consistent with previous literature supporting the association of lowered intracellular cAMP and upregulation of pro-fibrotic genes. Given that cAMP attenuates the activity of ERK5, BIX 02189 was used to evaluate the transcriptome. Of the 3836 genes upregulated by hY3 macrophage supernatants, 617 were reversed by the subsequent addition of BIX. Among the upregulated genes were pro-fibrosing genes such as EDN1 and TGFbeta2 and among those downregulated were genes that resist fibrosis including CLU, RAPGEF3, and ADM. The latter two are associated with a cAMP dependent inhibition of ERK5 and increased cAMP, respectively. The pro-fibrosing EDN1 result was confirmed by qPCR and as expected was attenuated by forskolin. Conclusion: These data support that the link of anti-Ro immune complex activated macrophages and the pathogenic fibroblast phenotype may relate to a decrease in cAMP levels. These results highlight potential novel targets for therapy and solidify the role of ERK5 in the transdifferentiation of fetal fibroblasts in the context of congenital heart block. (Table Presented)
Transcriptome profile of cells isolated from a CHB heart support an exuberant inflammatory/pro-fibrotic cascade [Meeting Abstract]
Background/Purpose: Histologic correlates of anti-Ro associated congenital heart block (CHB) are apoptosis and calcification of cardiomyocytes with fibrosis of the AV node surrounded by infiltrating macrophages and giant cells. This study leveraged an unprecedented opportunity to interrogate the transcriptome of different cell types in a fetal heart dying with CHB. Methods: Aortas from a 19 wk CHB fetal heart and a healthy 22 wk heart were cannulated using a Langendorff preparation with proteolytic enzymes to yield a single cell suspension. DAPI negative cells were isolated by flow using antibodies to CD14/CD45, CD31, and podoplanin, to yield leukocytes, endothelial cells, and fibroblasts, respectively. After RNA isolation and cDNA library preparation, RNA-Seq and transcriptome analysis were performed. Expression of lineage markers was consistent with the isolates based on flow markers. Data are expressed as log2 transcripts per million. Results: Transcriptomes of the two hearts for each isolated fraction were compared. For leukocytes, in CHB vs healthy there were 5000 genes greater than a threshold ratio of 0.78 (expressed as log2 difference). Based on the DAVID annotation, data were organized into clusters of closely related genes. Within the term inflammatory response (p=1.66E-4) for the ratio of CHB/healthy, the genes were IL8 (4.13), IL6 (6.72), TNFa (0.78), and EDN1 (5.17). In addition, leukocyte gene expression of FCGR3A, TLR7 and IRF5 was higher in CHB vs control (2.6,1.37 and 0.91, respectively), a result supporting that the requisite machinery is upregulated to effect anti-RohYRNA ligation and activate macrophages via TLR signaling. For endothelial cells, 7000 genes exceeded a ratio of 0.78 for CHB/healthy. Within the term inflammatory response (p=1E-5), FOS, FOSB, NFKB1A, and NFKBIZ were expressed with ratios of 1.47, 2.46, 1.03, and 2.75. Within the categories IMMUNE (p=4E-2) and cell adhesion (p=1.42E-06) higher expressed genes included CTGF (2.06), PTGS2 (2.53), SOCS3 (1.56), and OAS3 (2.52) along with ICAM1 (2.12), CCL2 (2.75), IL32 (2.81), VCAM1 (3.85), and SELE (2.4). Likewise within the death category (2.62E- 08), PPP1R15A (1.39), XAF1 (3.77), GADD45B (1.94), and TNFAIP3 (3.06) were increased in CHB. These data support endothelial cells in leukocyte recruitment. For the fibroblasts, 4500 genes were above the CHB/healthy threshold (Table 1). CHB fibroblasts showed increased expression of pro-fibrotic genes while attenuating anti-fibrotic genes. The cardiomyopathy marker, XIRP2 (3.15), as well as genes resisting and promoting vascular stiffness (ELN [-2.23] and TTN [2.12], respectively) were also identified. Conclusion: These data support that autoimmune CHB is a complex disease with contributions from death pathways, inflammation and fibrosis. Scarring likely results from a multi pronged pro-fibrotic environment whereby fibrosis promoting genes undermine genes that forestall fibrosis. (Table Presented)
The homeoprotein DLX3 and tumor suppressor p53 co-regulate cell cycle progression and squamous tumor growth
Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and transcriptomic approaches, we demonstrate that DLX3 operates through a p53-regulated network. DLX3 and p53 physically interact on the p21 promoter to enhance p21 expression. Elevating DLX3 in keratinocytes produces a G1-S blockade associated with p53 signature transcriptional profiles. In contrast, DLX3 loss promotes a mitogenic phenotype associated with constitutive activation of ERK. DLX3 expression is lost in human skin cancers and is extinguished during progression of experimentally induced mouse squamous cell carcinoma (SCC). Reinstatement of DLX3 function is sufficient to attenuate the migration of SCC cells, leading to decreased wound closure. Our data establish the DLX3-p53 interplay as a major regulatory axis in epidermal differentiation and suggest that DLX3 is a modulator of skin carcinogenesis.Oncogene advance online publication, 2 November 2015; doi:10.1038/onc.2015.380.
Keratinocyte p38delta loss inhibits Ras-induced tumor formation, while systemic p38delta loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin
p38delta expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38delta in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38delta exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38delta gene ablation inhibited the growth of tumors generated from v-rasHa -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38delta is required for Ras-induced tumorigenesis. Gene expression profiling of v-rasHa -transformed p38delta-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38delta-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1beta, IL-6, IL-17, and TNFalpha. Additionally, p38delta-null skin and p38delta-null keratinocytes exhibited increased p38alpha activation and signaling in response to acute inflammatory challenges, suggesting a role for p38alpha in stimulating the elevated inflammatory response in p38delta-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38delta+/+ skin. Altogether, our results indicate that p38delta signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment. (c) 2015 Wiley Periodicals, Inc.