Dermatology faculty, researchers, and medical students attended the Society for Investigative Dermatology Annual Meeting in Portland, OR April 26-29, 2017. They presided over symposia, presented abstracts, and attended talks. Their activities are listed below:
Dr. Amanda MacLeod (Duke University) along with Dr. Niroshana Anadasabapthy (Harvard University) presided over the Adaptive and Autoimmunity Mini-Symposium.
Dr. Rambi Cardones, as a member of the MDS Board of Directors, presided over the Medical Dermatology Society at the SID panel along with Dr. Daniela Kroshinsky (MA General Hospital) and Dr. Janet Fairley (University of Iowa).
Dr. Jennifer Zhang presented her research (abstract 868) at the Skin and Hair Development Biology Mini-Symposium.
Jeff Smith from Dr. Rajagopal’s lab presented his work (abstract 678) at a Plenary Session.
Kwaby Badu-Nkansah from Dr. Lechler’s lab presented his work (abstract 558) at the Growth Factors, Cell Adhesion and Matrix Biology Mini-Symposium.
Abstracts
Abstract 116
F Li, X Liu and C Li. Oncogenic reprogramming of human primary melanocytes into potent tumor initiating cells.
Cancer stem cells have been implicated in melanoma development and treatment. However, their origin and biological properties are still not well understood. Here we report that a cocktail of four genetic factors – myc, ras, dominant negative p53, and Oct-4 – could induce oncogenic transformation of primary human melanocytes rapidly and effectively. The transformed cells showed unlimited self-renewal ability with robust expression of telomerase activities. In addition, they expressed typical cancer stem cell markers such as CD133, CD15, and ALDH. They also exhibited with extraordinary strong tumor-initiating ability – transplantation of as few as 100 of the transformed cells into nude mice induced xenograft tumor formation. We believe the induced melanoma stem cells will be useful in studying molecular mechanisms involved in melanomagenesis and melanoma treatment.
Abstract 558
KA Badu-Nkansah, J Underwood and T Lechler. Proteomic evaluation of desmosomes reveals novel components essential for maintaining epidermal integrity.
Desmosomes are cell-cell adhesion structures that provide mechanical robustness to the epidermis and heart. Perturbation of desmosomes occurs in genetic, autoimmune and infectious disease and can lead to blistering of the skin. While the core components of the desmosomes were identified decades ago, more recent work has identified additional desmosome-associated proteins that have either regulatory and/or non-canonical functions. Here we used proximity biotinylation and quantitative mass spectrometry to identify novel desmosome-associated proteins. We performed separate analyses to enrich for proteins at the outer and inner dense plaques and identified many candidates. Remarkably, we would a significant overlap with proteins associated with adherens junctions. We show that this is due, in part, to promiscuity in the association of some proteins with adherens junctions and desmosomes in different tissues. In addition, we demonstrate that a subset of the identified proteins colocalize with core desmosomal proteins and require these proteins for their localization. Finally, we performed functional evaluation of the role of Crk and Crkl, two homologous proteins that were identified in our analysis. Loss of Crk/Crkl in cultured keratinocytes and in the mouse epidermis resulted in defects in keratin organization, impaired adhesion, and neonatal lethality. These data position Crk/Crkl as crucial regulators of the desmosome:keratin interaction which is essential for mechanical integrity of the skin and highlights the utility of the approach to identify novel regulators of the desmosome.
Abstract 573
J Suwanpradid, M Shih, B YE Guttman-Yassky, L Que, R Tighe and AS MacLeod. Arginase1/Nos2 imbalance in macrophages mediates cutaneous contact hypersensitivity responses.
Allergic contact dermatitis (ACD) is a common inflammatory skin disease. Macrophages (MAC) play relevant roles in orchestrating DC-T cell clusters relevant to ACD pathogenesis. MAC are reciprocally regulated by Arginase1 (Arg1) and inducible nitric oxide synthase (Nos2). Arg1 performs anti-inflammatory, whereas Nos2 performs pro-inflammatory functions. Arg1/Nos2 imbalance in MAC potentially mediates many inflammatory diseases. However, their roles in ACD remain unknown. We found that human ACD lesions and allergic contact hypersensitivity (CHS) mice contained high levels of Nos2+ monocytes/MAC (p0.05). In vitro stimulation of bone marrow-derived MAC with a hapten allergen suppressed Arg1, but induced Nos2 expression, which was normalized by dexamethasone, a common ACD treatment (p0.05). To confirm the importance of Nos2 to CHS, Nos2-/- mice were exposed to DNFB allergen and noted with decreased ear thickness compared to wildtype mice (p0.05). To determine how Nos2 was regulated in this response, we explored the role of Arg1. Using mice with myeloid cell specific deletion in Arginase-1 (Argfl/fl;LysMcre+/-) in the in vivo CHS model , we found that these mice have increased ear thickness along with high Nos2-expressing MAC (all p0.05) compared to control mice following DNFB challenge. The functional relevance of increased Nos2 in Arg1-deficient MAC is further supported by the finding that DNFB-elicited Argfl/fl;LysMcre+/- mice treated with a selective Nos2 inhibitor exhibited significantly decreased ear thickening compared to vehicle-treated CHS mice and wild type controls (p0.05). Together, our findings reveal a key role for Arg1 in suppressing skin CHS through dampening Nos2 and Arg1/Nos2 imbalance contributes to skin inflammation in this context. Therefore, restoring this imbalance may represent a potential therapeutic avenue in treating ACD.
Abstract 576
B Yang, J Suwanpradid, RS Lagunes, H Choi, P Hoang, D Wang, S Abraham and AS MacLeod. IL-27 promotes skin wound repair through stimulation of keratinocyte proliferation and host immunity.
Skin wound repair requires a coordinated program of epithelial cell proliferation and differentiation as well as resistance to invading microbes. However, the factors that trigger epithelial cell proliferation in this inflammatory process are incompletely understood. Recent studies from our lab and others demonstrate that IL-27 is produced by activated antigenpresenting cells in the skin upon skin damage and is shown to exert both pro-inflammatory and anti-inflammatory effects. The potential functional role of IL-27 in wound repair is currently unknown. Here, we demonstrate that IL-27 is rapidly and transiently produced by CD301b+ monocyte-derived macrophages and DC in the skin following injury. The functional role of IL-27 and CD301b+ cells is demonstrated by the finding that CD301b-depleted mice exhibit delayed wound closure in vivo which could be rescued by topical IL-27 treatment. Furthermore, genetic ablation of IL-27 receptor (Il27Ra-/-) attenuates wound healing, suggesting an essential role for IL-27 signaling in skin regeneration in vivo. Mechanistically, IL-27 feeds back on keratinocytes to stimulate cell proliferation and re-epithelialization in the skin, whereas IL-27 leads to suppression of keratinocyte terminal differentiation. Finally, we identify that IL-27 potently increases expression of the anti-viral oligoadenylate synthase 2 (OAS2), however does not affect expression of anti-bacterial human beta defensin 2 (HBD2) or regenerating islet-derived protein 3-alpha (REGIIIa). Together, our data suggest a previously unrecognized role for IL-27 in regulating epithelial cell proliferation and anti-viral host defense during the normal wound healing response. Our results may have major implications in our understanding of how keratinocyte proliferation and protective antiviral immunity is regulated during wound repair.
Abstract 594
J Kwock, B Yang, J Maycock, M McFadden, J Suwanpradid, L Pontius, P Hoang, S Horner and AS MacLeod. IL-27 promotes innate antiviral competence in wounds.
Following skin injury, multiple innate immune signaling pathways are activated in order to re-establish the antimicrobial barrier. While the induction of skin antibacterial molecules is well defined, regulation of antiviral proteins in the skin remains largely unknown. Here, we show that antiviral proteins are potently induced following skin injury and are highly expressed by keratinocytes and macrophages in the skin. Because we previously reported that interleukin-27 (IL-27) is produced upon skin injury to promote wound repair, we next tested whether rhIL-27 can stimulate expression of the antiviral peptides oligoadenylate synthetase 1, (OAS1), OAS2, OASL, Mx1 and other antiviral proteins in human keratinocytes and macrophages. All antiviral proteins tested were significantly stimulated by rhIL-27 (p < 0.001), whereas rhIL-27 failed to induce the antibacterial peptide human beta defensin 2 (HBD2). Virus envelope protein immunostaining and qPCR on human macrophages and keratinocytes treated with IL-27 and infected with various viruses demonstrated that rhIL-27 significantly suppresses viral infection in a cell-intrinsic manner. Together, our data suggest that IL-27 provides antiviral competence in skin wounds through activation of antiviral proteins. Our results help elucidate mechanistic insights into antiviral immunity in wound healing.
Abstract 609
L Pontius, J Suwanpradid, J Kwock, B Yang, J Maycock, R Kedl, G Hammer and AS MacLeod. IL-27 signaling is essential for IL-15 production and mediates contact hypersensitivity.
Contact hypersensitivity (CHS) is mediated by Ag-specific skin-resident memory T cells (TRM). Recent studies have shown that optimal memory T cell responses are dependent upon IL-27 signaling. Testing the functional in vivo relevance of IL-27 signaling in CHS, we find that IL27Ra-/- mice have a significantly reduced CHS response compared to wild type mice (p < 0.001). Persistence and maintenance of skin TRM is dependent on the common g-chain cytokines IL-7 and IL-15 produced by skin epithelial and innate immune cells. However, the factors that regulate the transcription of IL-7 and IL-15 are not known. We have made the unexpected observation that IL-27 significantly stimulates IL-7 and IL-15 expression in human epithelial keratinocytes and macrophages (p < 0.05). IL-27-mediated induction of IL-7 and IL- 15 in human keratinocytes was dependent on STAT1, but independent of type I interferons, as demonstrated in siRNA experiments. Here, siSTAT1, but not siIFNAR1, resulted in significant reduction of IL-27 mediated IL-7 and IL-15 production (p < 0.05). This result was further supported by our findings that P-STAT1 translocated to the nucleus from the cytoplasm following IL-27 stimulation of human keratinocytes. Additionally, IL27Ra-/- mice had significantly lower levels of IL-7 (up to 12-fold decrease, p < 0.001) and IL-15 (up to 239-fold decrease, p < 0.001) in the epidermis at baseline and upon hapten-induced elicitation, whereas such changes were much less pronounced in skin-draining lymph nodes (IL-7 with up to 2.59-fold decrease, p < 0.001; IL-15 with up to 1.57-fold decrease, p < 0.001). Collectively, our data demonstrate that IL-27 is critical for CHS and stimulates IL-7 and IL-15. Our results suggest that the IL-27 pathway may be a potential therapeutic target.
Abstract 678
JS Smith, L Nicholson, J Suwanpradid, R Glenn, J Gundry, P Alagesan, N Knape, AR Atwater, MD Gunn, AS MacLeod, RJ Lefkowitz and S Rajagopal. Biased CXCR3 ligands differentially alter allergic contact hypersensitivity and chemotaxis.
Allergic contact dermatitis (ACD) is a disease with few targeted therapies. Chemokines play an important role in ACD through the recruitment of T-cells that express the chemokine receptor (CKR) CXCR3. Chemokines signal through CKRs, a subgroup of the G proteincoupled receptor (GPCR) family, which are targeted in >30% of drugs. However, few drugs target CKRs. Classically, GPCRs were thought to act as simple switches turned on by agonists and off by antagonists. We now appreciate that GPCRs adopt multiple conformations that link to distinct signaling pathways, such as G-proteins and ß-arrestins (ßarrs). These pathways can be selectively activated by a novel class of receptor ligands, termed biased agonists, which signal through some pathways while blocking signaling through others. The purpose of this study was to determine the roles that G-proteins and ßarrs play in ACD by selectively targeting signaling with CXCR3 biased agonists. Mouse and human cell chemotaxis was determined through transwell migration, and the effects of CXCR3 ligands on ACD were assessed in the DNFB allergic contact hypersensitivity (CHS) mouse model. Patient biopsies of patch tested skin were analyzed. Our results show that ßarr signaling through CXCR3 is necessary for full efficacy chemotaxis of both mouse and human T-cells. A topically applied ßarr-biased ligand doubled (ph0.05) the CHS inflammatory response in WT, but not in ßarr2 KO or CXCR3 KO, mice. Flow cytometry of mouse skin demonstrated increased T-cells following ßarr-biased drug treatment. We conclude that CXCR3 ßarr-mediated signaling is critical for effector T-cell recruitment that underlies the inflammatory response in CHS. These findings suggest that biased ligands could be utilized to selectively target CKRs for therapeutic benefit.
Abstract 795
A Dikshit, JY Jin, J Hwang, S Degan, Y Deng, C Li and JY Zhang. K63-Ubiquitin enzyme UBE2N and its variant UBE2V2 play crucial roles in melanoma cell growth and survival.
Recent advances in oncotherapies and immunotherapies have markedly expanded melanoma treatment options. Nevertheless, the benefits of these treatments are often short-lived due to the development of resistance. We have previously established the onco-protective effect of the K63-ubiquitin (K63-Ub) deubiquitinating enzyme CYLD. In this study, we studied the role of the K63-Ub ubiquitin enzyme UBE2N and its functionally essential partners, UBE2V1 and UBE2V2, in melanoma growth and progression. We found that UBE2N and UBE2V2 were expressed and activated at elevated levels in malignant melanoma cell lines compared to normal melanocytes. Inhibition of UBE2N either pharmacologically or genetically via CRIPSR-mediated gene deletion and shRNA-mediated gene-silencing markedly decreased melanoma cell growth and sensitized cells to decarbonize and radiation treatments in vitro. In addition, we found that knockdown of UBE2V2 but not UBE2V1 significantly increased melanoma cell apoptosis and inhibited cell growth and migration. Surprisingly, gene silencing of UBE2N and UBE2V2 increased activation of the AKT and JNK pathways, and sensitized A375 melanoma cells to AKT and JNK inhibitors. In agreement with the in vitro data, subcutaneous tumor growth analysis showed that A375 melanoma cells with UBE2N and UBE2V2 knockdowns displayed significantly reduced tumor growth compared with cells with UBE2V1 knockdown or non-silencing control. Moreover, tumors with UBE2N or UBE2V2 knockdown expressed markedly elevated levels of E-cadherin and cleaved caspase 3. Further examination of the lung tissues revealed that UBE2N or UBE2V2 knockdown abolished pulmonary metastasis. These results indicate that UBE2N and UBE2V2 are crucial for melanoma growth, progression, and survival. We are currently using proteomic approaches to understand the molecular mechanisms mediating these effects.
Abstract 868
JY Jin, T Lechler and JY Zhang. BRAF activation induces sweat gland neogenesis in a WNT signaling dependent manner.
Sweat glands (SGs) are the most abundant skin appendages of our body. They are crucial for thermoregulation, wound healing and detoxification. SG deficiency is a major clinical issue for severely burned victims, people with congenital hypohidrotic ectodermal dysplasia, and subsets of diabetic and GVHD patients. However, methods to treat these deficiencies are limited by our lack of knowledge of if and how SG can be generated in adults. It is believed that, like hair follicles, SG budding from epidermis is triggered by signals derived from dermal cells, which in mouse are restricted to embryonic foot pads. Here we demonstrate that epidermal expression of the mutant Braf600E induced robust development of SG-duct like structures in adult mouse back and ear skins, as well as tongues. These structures showed openings to the skin surface, and expressed the luminal protein marker K18. RNA and protein analyses revealed that Braf600E markedly increased Wnt5a and Wnt10a, and downregulated Eda and Shh signaling pathways that are known to be critical for SG maturation. Topical treatments with the Wnt-inhibitor ICG001 diminished the ductal formation, indicating that Wnt signaling is required for Braf600E-induced SG neogenesis. This work offers both new insights into signaling mechanisms underlying SG specification and morphogenesis and suggests novel treatments for disorders associated with SG loss. Currently, we are investigating whether forced Eda and Shh pathway activation can enhance the SG ductal development and maturation into acini, and whether the animal data can be recapitulated in regenerated human skin.
Abstract LB949
S Mallam, R Streilein, H Suga, T Tedder, RP Hall. In vitro expansion of desmoglein specific peripheral blood B cells of patients with pemphigus.
B cells are known to play a major role in the pathogenesis of autoimmune diseases as precursors of autoantibody producing plasma cells. Pemphigus vulgaris (PV) and foliaceus (PF) are severe blistering diseases of the skin caused by autoantibodies directed against desmoglein-1 (DSG1) and desmoglein-3 (DSG3). The purpose of this study is to utilize in vitro expansion and differentiation of B cells from patients with PV and PF to help better understand the frequency of DSG1/DSG3 specific B cells in patients with active skin disease. B cells were isolated from peripheral blood (PB) samples from patients with clinically active PV (n=2), PF (n=2), and healthy controls (HC) (n=2). B cells were expanded in vitro using a cell culture system that supports B cell proliferation and differentiation into immunoglobulin-secreting plasma cells. PB B cells (10/well) were cultured in 96 well plates for 12 days. Supernatants were tested for the presence of DSG1 or DSG3 antibodies by ELISA. The frequency of wells with ELISA values outside of 3x the interquartile range (IQR) were calculated for each subject. Supernatants from PV/PF patients showed a higher frequency of wells producing IgG anti DSG1 or DSG3 than controls (mean frequency wells>3x IQR: PV/PF=4.87%; HC=1.89%, p=0.03). This study demonstrates that in vitro stimulation of PB B cells from patients with PV/PF produce an increased frequency of IgG anti DSG1/DSG3 detected by ELISA compared to PB B cells from normal subjects. Utilizing these techniques, we will be able to analyze the change in antigen-specific B cell frequency in patients after treatment with rituximab compared to those treated with conventional therapy and increase the understanding of the mechanism of rituximab treatment failure and recurrences.