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LB811 Arginase1 in myeloid cells controls allergic contact hypersensitivity through tempering NOS2-associated inflammation
| ABSTRACTS LB810
LB811
Characterization of cell-type specific expression of CXCR3 ligands in morphea YB Rainwater, H Jacobe and GA Hosler Dermatology, UT Southwestern Medical Center, Dallas, TX Morphea is a disease of dermal inflammation and resultant sclerosis with considerable functional and cosmetic consequences. Cellular and molecular events in pathogenesis of morphea remain poorly elucidated. Preliminary studies using genome-wide gene expression analysis support the role of gamma interferon mediated pathways in the early pathogenesis of morphea. Specifically, CXCL-9 and 10, known ligands of CXCR3 receptor, were shown to be strongly upregulated in morphea. The purpose of this study is to characterize the inflammatory infiltrate in morphea and investigate cell-type specific expression of CXCR3 ligands in morphea using immunohistochemistry (IHC). Our data show that the cellular infiltrate of early inflammatory morphea lesions consists of predominantly T lymphocytes, with significantly increased CD4 positive cells and CD4:CD8 ratio of 1.8. The immune cell infiltrate of early morphea was enriched in CXCR3+ immune cells. CXCR3 co-localizes with CD4+ cells, but not CD8+ lymphocytes. We demonstrate that CXCL9 is expressed by a small subset of cells of varying morphologies within perivascular, periadnexal, and interstitial infiltrates. CXCL9 expression co-localized with CD68, but not CD20, CD34, CD123, or SMA, suggesting that dermal macrophages may secrete CXCL9 ligand. Moreover, CXCL9 expression was detected in close proximity to CXCR3 expressing immune cells. Based on our findings, we suggest that macrophage expression of CXCR3 ligands, specifically CXCL9, in morphea may enhance recruitment of CXCR-3 expressing activated T lymphocytes into the lesional skin and propagate early stages of inflammation.
Arginase1 in myeloid cells controls allergic contact hypersensitivity through tempering NOS2-associated inflammation J Suwanpradid1, M Shih1, T Phillips1, B Yang1, A Birukova2, L Que2, R Tighe2 and A MacLeod1,3 1 Dermatology, Duke University, Durham, NC, 2 Department of Medicine, Duke University, Durham, NC and 3 Immunology, Duke, Durham, NC Arginase1 (Arg1) and inducible nitric oxide synthase (iNOS or NOS2) regulate inflammation in a reciprocal manner. Arg1 is expressed at highest levels in dermal macrophages of human and murine skin and is associated with anti-inflammatory functions. However, roles of Arg1 in regulating cutaneous inflammation in allergic contact dermatitis (ACD) are unknown. Notably, macrophages are critical to contact hypersensitivity (CHS), a murine model of ACD, but whether Arg1 in myeloid cells can functionally regulate CHS has not been studied. Using the CHS model with the hapten DNFB, we show that Arg1-/flox;LysMcre and Arg1flox/flox;Tie2cre mice have an increase in ear thickness, IL-6 and Nos2 expression compared to control mice (all p0.05). The relevant role of increased Nos2 in conditional Arg1 deficient mice during CHS is further substantiated by the finding that mice lacking iNOS showed reduced ear swelling (p0.05) compared to WT mice. Furthermore, in vivo treatment with an iNOS inhibitor prior to elicitation with DNFB suppresses ear thickening upon DNFBchallenge in conditional Arg1 knockout mice, but not in control mice (p0.05). Stimulation of bone marrow derived macrophages with DNBS in vitro induced Nos2 and IL-6 expression in macrophages and this response could be normalized by dexamethasone (p0.05). Finally, topical dexamethasone treatment of mouse ears in vivo reduced ear swelling in DNFBchallenged ears in control mice (p0.05), but this effect was absent in conditional Arg1 knockout mice, suggesting that dexamethasone-mediated suppression of CHS requires Arg1 in myeloid cells. Together, our data demonstrate that loss of Arg1 increases CHS through increase in Nos2 and that the immunosuppressive function of dexamethasone is entailed through an increase of Arg1 expression in myeloid cells. Together, our data suggest that targeting the Arg1/iNOS pathway may reveal a novel therapeutic avenue for ACD patients.
LB812
LB813
LB814
LB815
Interdependency of oxidative stress and UPR signaling underlying cutaneous inflammation and tissue damage caused by a prototype of arsenical, phenylarsine oxide RK Srivastava, C Li, Z Weng, A Agarwal, CA Elmets, F Afaq and M Athar University of Alabama at Birmingham, Birmingham, AL The skin vesicating chemicals e.g. arsenicals were developed as chemical war agents. Their mechanism of action remains unclear. Phenylarsine oxide (PAO), a strong oxidant is a prototype of arsenicals and could be used as a surrogate to define the mechanism by which arsenicals cause sever skin lesions. Among various murine models tested Ptch1+/-/SKH1 was highly sensitive to PAO. We show that topical application of PAO on the skin of these animals induced erythema, edema and microvesication. These changes were associated with the enhanced production of pro-inflammatory cytokines and reactive oxygen species (ROS) leading to the massive death of epidermal keratinocytes of the skin. The molecular mechanism underlying cutaneous injury involves activation of unfolded protein response (UPR) signaling. Similar to observations in murine skin, treatment of human skin keratinocytes, NHEK and HaCaT, with PAO upregulated inflammatory and UPR signaling pathways doseand time-dependently. Specifically, PERK-regulated UPR proteins GRP78, p-PERK, p-eIF2a, ATF4 and CHOP were induced in these keratinocytes. Although this signaling was triggered at an early time-point, the cell death was mainly observed at later time-points. Treatment of keratinocytes with chemical chaperone, 4-phenylbutyric acid (4-PBA) or antioxidant, N-acetylcysteine (NAC) significantly attenuated PAO-induced inflammatory signaling, UPR activation and apoptosis. Employing siRNAebased approaches, CHOP was found to be a key regulator of apoptosis and pro-inflammatory responses. To validate these observations, we tested the efficacy of 4-PBA and NAC in murine skin. A significant reduction in PAO-induced alterations in the pathobiology of murine skin was observed following treatment with these agents. These data suggest that, PAO has potent inflammatory, vesicant and cell death inducing properties in the skin and can serve as surrogate arsenical to develop the molecular target-based antidotes against arsenicals.
Acne, atopic dermatitis, and psoriasis e Integrating clinical transcriptomics studies to discover novel therapeutic targets and indication expansion opportunities JM Freudenberg and DK Rajpal Target Sciences Computational Biology, GlaxoSmithKline, King of Prussia, PA Acne, atopic dermatitis, and psoriasis all are chronic inflammatory skin diseases and although quite different in their signs and symptoms they are known to share a number of pathways that are dys-regulated in the disease state. We hypothesized that human clinical transcriptomics signatures could be used to a) determine unique and/or shared mechanisms, and b) combined with genetic evidence could offer novel targets, and biomarkers. We reviewed and selected 25 independent human transcriptome-wide datasets from the NCBI Gene Expression Omnibus that were derived from human skin biopsy samples from published clinical trials. The resulting combined dataset set comprises a total of 1,376 human patient samples from either lesional, non-lesional, or healthy control skin. By conducting a meta-analysis we identified 3 disease signatures, each comprising a set of genes significantly changed in skin disease lesions compared to either non-lesion or normal control samples. The signatures revealed common patterns across all three diseases but also subsets of genes that were dysregulated in a disease-specific fashion. We then investigated the underlying molecular mechanisms by pathway analysis and identified core mechanisms associated with disease. This work leads to the recognition that seemingly very different immune mediated dermatological diseases be compared and differentiated at the transcriptional level, providing clues to the underlying biology and pathogenesis. As demonstrated in our meta-analysis, clinical transcriptomics can further provide opportunities to expand a portfolio of drug’s indication space, help design validation approaches for new targets and to develop new biomarkers.
Palmitoyl-KVK-L-ascorbic acid increases type I procollagen expression by inhibition of DNA methyltrasferase activity J Lim, H An, J Kim, S Moon and S Chang Celltrion, Incheon, Korea (the Republic of) Ultraviolet (UV) irradiation reduces production of type I procollagen, the major structural protein in human cutaneous connective tissue. Also, UV irradiation induces hypermethylation of CpGs in the promoter of the COL1A1 and COL1A2 genes for type I procollagen. In this study, we showed the effects of Palmitoyl-KVK-L-ascorbic acid on the enzymatic activity of DNA methyltransferase (DNMT) and the type I procollagen expression. Our results showed that Palmitoyl-KVK-L-ascorbic acid inhibits DNMT activity in a dose dependent manner. Using procollagen Type I C-Peptide EIA assays, we observed that Palmitoyl-KVK-L-ascorbic acid increased the secretion of type I procollagen from human dermal fibroblasts into the culture medium. Also, analysis of PCR amplification showed that Palmitoyl-KVK-L-ascorbic acid induced the COL1A1 and COL1A2 genes expression. These results indicate that Palmitoyl-KVK-L-ascorbic acid might be a promising candidate for the treatment of photoaged skin.
Validation of EpiScreenTM as a predictive in vitro cell-based assay for screening dermatological drugs by high content approaches J Young1, M Fernandes1, V Chapuis1, E Duchemin-Pelletier1, P Girling2 and P Poydenot1 1 CYTOO, Grenoble, France and 2 CELLnTEC, Bern, Switzerland Reconstructed epidermis are the gold standard in vitro model for compound and skin care product testing. Analysis by high content imaging is challenging however with sample preparation requiring sectioning and immunohistochemistry. To go beyond viability and IL-1 release tests for screening, we developed automated 96-well cultures of stratified microepidermis (ME) adapted to high throughput and high content (HC) readouts. Each well contains 250 miniature epidermis that express terminal differentiation markers, loricrin and hornerin. To clarify the relevance of the model with respect to in vivo predictability, we analyzed ME response to two retinoids (ATRA and retinol). Retinoids are well-known inducers of epidermal dedifferentiation in vitro but stimulate proliferation and differentiation in vivo, while enhancing production of hyaluronate (HA) in both systems. Using HC confocal microscopy and customized image analysis methods, optically sectioned ME were analyzed for abundance and distribution of six differentiation markers as well as morphology and proliferation. In parallel, supernatants were collected for HA ELISA measurements. Results from triplicate well averages show that both retinoids at pharmacological dose (10-6 M) induce hyperproliferation of basal layer keratinocytes and reduce tranglutaminase levels by 2-fold. ATRA was more effective than retinol for stimulation of HA secretion. Interestingly, both retinoids provoke a premature and sustained higher expression of involucrin, extending the suprabasal zone. The local change in differentiation profile did not affect ME height as confirmed by 3D image reconstruction. Finally, addition of a cytokine mix to ME is shown to induce a phenotype typical of psoriasis that can be antagonized with retinoids, including a decrease in expression of antimicrobial peptides. In conclusion, EpiScreenTM in healthy and disease model states can provide quantitative in vitro data that simulate the pro-differentiation and anti-inflammatory activity of retinoids in vivo.
www.jidonline.org B11
LB811
Characterization of cell-type specific expression of CXCR3 ligands in morphea YB Rainwater, H Jacobe and GA Hosler Dermatology, UT Southwestern Medical Center, Dallas, TX Morphea is a disease of dermal inflammation and resultant sclerosis with considerable functional and cosmetic consequences. Cellular and molecular events in pathogenesis of morphea remain poorly elucidated. Preliminary studies using genome-wide gene expression analysis support the role of gamma interferon mediated pathways in the early pathogenesis of morphea. Specifically, CXCL-9 and 10, known ligands of CXCR3 receptor, were shown to be strongly upregulated in morphea. The purpose of this study is to characterize the inflammatory infiltrate in morphea and investigate cell-type specific expression of CXCR3 ligands in morphea using immunohistochemistry (IHC). Our data show that the cellular infiltrate of early inflammatory morphea lesions consists of predominantly T lymphocytes, with significantly increased CD4 positive cells and CD4:CD8 ratio of 1.8. The immune cell infiltrate of early morphea was enriched in CXCR3+ immune cells. CXCR3 co-localizes with CD4+ cells, but not CD8+ lymphocytes. We demonstrate that CXCL9 is expressed by a small subset of cells of varying morphologies within perivascular, periadnexal, and interstitial infiltrates. CXCL9 expression co-localized with CD68, but not CD20, CD34, CD123, or SMA, suggesting that dermal macrophages may secrete CXCL9 ligand. Moreover, CXCL9 expression was detected in close proximity to CXCR3 expressing immune cells. Based on our findings, we suggest that macrophage expression of CXCR3 ligands, specifically CXCL9, in morphea may enhance recruitment of CXCR-3 expressing activated T lymphocytes into the lesional skin and propagate early stages of inflammation.
Arginase1 in myeloid cells controls allergic contact hypersensitivity through tempering NOS2-associated inflammation J Suwanpradid1, M Shih1, T Phillips1, B Yang1, A Birukova2, L Que2, R Tighe2 and A MacLeod1,3 1 Dermatology, Duke University, Durham, NC, 2 Department of Medicine, Duke University, Durham, NC and 3 Immunology, Duke, Durham, NC Arginase1 (Arg1) and inducible nitric oxide synthase (iNOS or NOS2) regulate inflammation in a reciprocal manner. Arg1 is expressed at highest levels in dermal macrophages of human and murine skin and is associated with anti-inflammatory functions. However, roles of Arg1 in regulating cutaneous inflammation in allergic contact dermatitis (ACD) are unknown. Notably, macrophages are critical to contact hypersensitivity (CHS), a murine model of ACD, but whether Arg1 in myeloid cells can functionally regulate CHS has not been studied. Using the CHS model with the hapten DNFB, we show that Arg1-/flox;LysMcre and Arg1flox/flox;Tie2cre mice have an increase in ear thickness, IL-6 and Nos2 expression compared to control mice (all p0.05). The relevant role of increased Nos2 in conditional Arg1 deficient mice during CHS is further substantiated by the finding that mice lacking iNOS showed reduced ear swelling (p0.05) compared to WT mice. Furthermore, in vivo treatment with an iNOS inhibitor prior to elicitation with DNFB suppresses ear thickening upon DNFBchallenge in conditional Arg1 knockout mice, but not in control mice (p0.05). Stimulation of bone marrow derived macrophages with DNBS in vitro induced Nos2 and IL-6 expression in macrophages and this response could be normalized by dexamethasone (p0.05). Finally, topical dexamethasone treatment of mouse ears in vivo reduced ear swelling in DNFBchallenged ears in control mice (p0.05), but this effect was absent in conditional Arg1 knockout mice, suggesting that dexamethasone-mediated suppression of CHS requires Arg1 in myeloid cells. Together, our data demonstrate that loss of Arg1 increases CHS through increase in Nos2 and that the immunosuppressive function of dexamethasone is entailed through an increase of Arg1 expression in myeloid cells. Together, our data suggest that targeting the Arg1/iNOS pathway may reveal a novel therapeutic avenue for ACD patients.
LB812
LB813
LB814
LB815
Interdependency of oxidative stress and UPR signaling underlying cutaneous inflammation and tissue damage caused by a prototype of arsenical, phenylarsine oxide RK Srivastava, C Li, Z Weng, A Agarwal, CA Elmets, F Afaq and M Athar University of Alabama at Birmingham, Birmingham, AL The skin vesicating chemicals e.g. arsenicals were developed as chemical war agents. Their mechanism of action remains unclear. Phenylarsine oxide (PAO), a strong oxidant is a prototype of arsenicals and could be used as a surrogate to define the mechanism by which arsenicals cause sever skin lesions. Among various murine models tested Ptch1+/-/SKH1 was highly sensitive to PAO. We show that topical application of PAO on the skin of these animals induced erythema, edema and microvesication. These changes were associated with the enhanced production of pro-inflammatory cytokines and reactive oxygen species (ROS) leading to the massive death of epidermal keratinocytes of the skin. The molecular mechanism underlying cutaneous injury involves activation of unfolded protein response (UPR) signaling. Similar to observations in murine skin, treatment of human skin keratinocytes, NHEK and HaCaT, with PAO upregulated inflammatory and UPR signaling pathways doseand time-dependently. Specifically, PERK-regulated UPR proteins GRP78, p-PERK, p-eIF2a, ATF4 and CHOP were induced in these keratinocytes. Although this signaling was triggered at an early time-point, the cell death was mainly observed at later time-points. Treatment of keratinocytes with chemical chaperone, 4-phenylbutyric acid (4-PBA) or antioxidant, N-acetylcysteine (NAC) significantly attenuated PAO-induced inflammatory signaling, UPR activation and apoptosis. Employing siRNAebased approaches, CHOP was found to be a key regulator of apoptosis and pro-inflammatory responses. To validate these observations, we tested the efficacy of 4-PBA and NAC in murine skin. A significant reduction in PAO-induced alterations in the pathobiology of murine skin was observed following treatment with these agents. These data suggest that, PAO has potent inflammatory, vesicant and cell death inducing properties in the skin and can serve as surrogate arsenical to develop the molecular target-based antidotes against arsenicals.
Acne, atopic dermatitis, and psoriasis e Integrating clinical transcriptomics studies to discover novel therapeutic targets and indication expansion opportunities JM Freudenberg and DK Rajpal Target Sciences Computational Biology, GlaxoSmithKline, King of Prussia, PA Acne, atopic dermatitis, and psoriasis all are chronic inflammatory skin diseases and although quite different in their signs and symptoms they are known to share a number of pathways that are dys-regulated in the disease state. We hypothesized that human clinical transcriptomics signatures could be used to a) determine unique and/or shared mechanisms, and b) combined with genetic evidence could offer novel targets, and biomarkers. We reviewed and selected 25 independent human transcriptome-wide datasets from the NCBI Gene Expression Omnibus that were derived from human skin biopsy samples from published clinical trials. The resulting combined dataset set comprises a total of 1,376 human patient samples from either lesional, non-lesional, or healthy control skin. By conducting a meta-analysis we identified 3 disease signatures, each comprising a set of genes significantly changed in skin disease lesions compared to either non-lesion or normal control samples. The signatures revealed common patterns across all three diseases but also subsets of genes that were dysregulated in a disease-specific fashion. We then investigated the underlying molecular mechanisms by pathway analysis and identified core mechanisms associated with disease. This work leads to the recognition that seemingly very different immune mediated dermatological diseases be compared and differentiated at the transcriptional level, providing clues to the underlying biology and pathogenesis. As demonstrated in our meta-analysis, clinical transcriptomics can further provide opportunities to expand a portfolio of drug’s indication space, help design validation approaches for new targets and to develop new biomarkers.
Palmitoyl-KVK-L-ascorbic acid increases type I procollagen expression by inhibition of DNA methyltrasferase activity J Lim, H An, J Kim, S Moon and S Chang Celltrion, Incheon, Korea (the Republic of) Ultraviolet (UV) irradiation reduces production of type I procollagen, the major structural protein in human cutaneous connective tissue. Also, UV irradiation induces hypermethylation of CpGs in the promoter of the COL1A1 and COL1A2 genes for type I procollagen. In this study, we showed the effects of Palmitoyl-KVK-L-ascorbic acid on the enzymatic activity of DNA methyltransferase (DNMT) and the type I procollagen expression. Our results showed that Palmitoyl-KVK-L-ascorbic acid inhibits DNMT activity in a dose dependent manner. Using procollagen Type I C-Peptide EIA assays, we observed that Palmitoyl-KVK-L-ascorbic acid increased the secretion of type I procollagen from human dermal fibroblasts into the culture medium. Also, analysis of PCR amplification showed that Palmitoyl-KVK-L-ascorbic acid induced the COL1A1 and COL1A2 genes expression. These results indicate that Palmitoyl-KVK-L-ascorbic acid might be a promising candidate for the treatment of photoaged skin.
Validation of EpiScreenTM as a predictive in vitro cell-based assay for screening dermatological drugs by high content approaches J Young1, M Fernandes1, V Chapuis1, E Duchemin-Pelletier1, P Girling2 and P Poydenot1 1 CYTOO, Grenoble, France and 2 CELLnTEC, Bern, Switzerland Reconstructed epidermis are the gold standard in vitro model for compound and skin care product testing. Analysis by high content imaging is challenging however with sample preparation requiring sectioning and immunohistochemistry. To go beyond viability and IL-1 release tests for screening, we developed automated 96-well cultures of stratified microepidermis (ME) adapted to high throughput and high content (HC) readouts. Each well contains 250 miniature epidermis that express terminal differentiation markers, loricrin and hornerin. To clarify the relevance of the model with respect to in vivo predictability, we analyzed ME response to two retinoids (ATRA and retinol). Retinoids are well-known inducers of epidermal dedifferentiation in vitro but stimulate proliferation and differentiation in vivo, while enhancing production of hyaluronate (HA) in both systems. Using HC confocal microscopy and customized image analysis methods, optically sectioned ME were analyzed for abundance and distribution of six differentiation markers as well as morphology and proliferation. In parallel, supernatants were collected for HA ELISA measurements. Results from triplicate well averages show that both retinoids at pharmacological dose (10-6 M) induce hyperproliferation of basal layer keratinocytes and reduce tranglutaminase levels by 2-fold. ATRA was more effective than retinol for stimulation of HA secretion. Interestingly, both retinoids provoke a premature and sustained higher expression of involucrin, extending the suprabasal zone. The local change in differentiation profile did not affect ME height as confirmed by 3D image reconstruction. Finally, addition of a cytokine mix to ME is shown to induce a phenotype typical of psoriasis that can be antagonized with retinoids, including a decrease in expression of antimicrobial peptides. In conclusion, EpiScreenTM in healthy and disease model states can provide quantitative in vitro data that simulate the pro-differentiation and anti-inflammatory activity of retinoids in vivo.
www.jidonline.org B11