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264 Patients with pemphigus foliaceus may retain antibody reactivity against calcium dependent epitopes of desmoglein 1 in remission
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Corticotropin-releasing hormone receptor blocker suppresses cytokine expression induced by cathelicidine peptides H Lee and S Lee CHA Bundang Medical Center, CHA University, Seongnam-si, Korea (the Republic of) Rosacea is a chronic inflammatory skin condition that manifests unique inflammatory responses to normal environmental stimuli such as ultraviolet light radiation, heat and stress. The pathophysiology of rosacea appears to be defined by consistently abnormal innate immune activity, abnormal barrier function, and neurogenic dysregulation. The corticotropin releasing hormone (CRH), a neuropeptide originally isolated from the hypothalamus, is also produced by the skin, where it can act as a regulatory element of local neuroendocrine interactions. CRH has been reported to play a role in the inflammation by stimulating release of proinflammatory cytokines, such as IL-1b, IL-6, IL-8 and vascular endothelial growth factor. However, there have been no reports regarding the functional role of CRH in rosacea. We investigated if CRH receptor blocker could influence on inflammation induced by cathelicidin, which had a major role in rosacea. We cultured normal human keratinocytes in low Ca2+ condition (0.06mM) and high Ca2+ condition (1.6mM) and treated them with CRH and cathelicidine peptides (3.2mM) or CRH alone. Then we added CRHR-1 inhibitor to both groups. Supernatants were collected and IL-8 and TNF-a production were analyzed by ELISA. IL-8 and TNF-a release were increased in keratinocytes co-stimulated with CRH and cathelicidine compared to the cells treated with cathelicidine alone or CRH alone, especially in cells cultured in high Ca2+ condition. IL-8 and TNF-a release were suppressed by CRHR-1 inhibitors. This observation demonstrated possible immunomodulatory role of CRH in rosacea.
Hyaluronic acid oligosaccharides suppress cytokine expression induced by cathelicidine peptides H Lee and S Lee Department of Dermatology, CHA Bundang Medical Center, CHA University, Seongnam-si, Korea (the Republic of) Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. The pathophysiology of rosacea appears to be defined by consistently abnormal innate immune activity such as increased expression of cathelicidin, kallikrein 5 and Toll-like receptor (TLR) 2, abnormal barrier function, and neurogenic dysregulation. Hyaluronic acid (HA) is a high molecular weight linear glycosaminoglycan composed of repeating disaccharide subunits of D-glucuronic acid and N-acetyl glucosamine. The function of HA was initially thought to be mainly structural, but this paradigm changed when receptors to HA and its ability to regulate multiple functions were found. HA binds several different proteins that are able to influence immune function such as CD44, TLR2 and TLR4. Recently, it has been reported that HA oligosaccharides (oligo-HA) could suppress TLRdependent cytokine expression. In this study we investigated if oligo-HA could influence on inflammation induced by cathelicidin, which had a major role in rosacea. We cultured normal human keratinocytes in low Ca2+ condition (0.06mM) and high Ca2+ condition (1.6mM) and treated them with the cathelicidine peptides (3.2mM) and oligo-HA (10mg/ml) or the cathelicidine peptides alone. Supernatants were collected and IL-8 and TNF-a production were analyzed by ELISA. IL-8 and TNF-a release were suppressed in keratinocytes co-stimulated with oligo-HA and cathelicidine peptides compared to the cells treated with cathelicidine peptides alone, especially in cells cultured in high Ca2+ condition. This observation demonstrated possible immunomodulatory role of oligo-HA in rosacea.
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Patients with pemphigus foliaceus may retain antibody reactivity against calcium dependent epitopes of desmoglein 1 in remission K Kamiya1, Y Aoyama2, J Yamagami3, O Yamasaki4, Y Tokura5 and K Iwatsuki4 1 Department of Dermatology, Jichi Medical University, Shimotsuke, Japan, 2 Department of Dermatology, Kawasaki Hospital, Kawasaki Medical School, Okayama, Japan, 3 Department of Dermatology, Keio University School of Medicine, Tokyo, Japan, 4 Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan and 5 Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are closely related, but clinically distinct, autoimmune blistering diseases caused by autoantibodies against desmoglein (Dsg) 1 and Dsg3, respectively. By using ethylenediaminetetraacetic acid (EDTA)-treated Dsg3 enzyme-linked immunosorbent assay (ELISA), we have confirmed in PV patients that the pathogenicity of anti-Dsg3 antibodies is mainly determined by the amount of anti-Dsg3 antibodies against the calcium dependent epitopes of Dsg3. In this study, we have established EDTA-treated Dsg1 ELISA and exfoliative toxin (ET)-treated Dsg1 ELISA based on the structural characteristics of Dsg1. By using EDTA-treated and ET-treated ELISAs, we evaluated five PF patients who retained high serum levels of anti-Dsg1 antibodies in the inactive phase. Sera were obtained in both active and inactive phases. To map the epitopes of anti-Dsg1 antibodies, immunoprecipitation-immunoblotting was performed by using a set of Dsg1/Dsg2 domain swapped molecules. Anti-Dsg1 antibodies against the calcium dependent epitopes of Dsg1 were the predominant antibodies in both active and inactive phases. The proportion of anti-Dsg1 antibodies against the calcium dependent epitopes was not changed upon shifting to the inactive phase. The results of immunoprecipitation-immunoblotting showed that most of the anti-Dsg1 antibodies bound to the extracellular domains (EC) 1-2 of Dsg1. In PF, both pathogenic and nonpathogenic epitopes were located in the EC1-2 of Dsg1. The disease activity might be differentially controlled by the antibodies between PF and PV depending on the presence or absence of the nonpathogenic epitope.
Tissue antigen expression levels control the fundamental decision between tolerance and autoimmunity DF Pinheiro1, MM Maurano1, MM Klicznik1, R Holly1, G Achatz-Straussberger1, J Thalhamer1, A Abbas2, MD Rosenblum2 and IK Gratz1 1 Molecular Biology, University of Salzburg, Salzburg, Austria and 2 University of California, San Francisco, San Francisco, CA Immune homeostasis in peripheral tissues is achieved by maintaining a delicate balance between pathogenic effector T cells (Teff) and protective regulatory T cells (Treg). The factors responsible for maintaining this balance e especially in the target tissues e are largely unknown. We aimed to study immune regulatory mechanisms in T cell mediated skin inflammation. To this end we established two mouse models that feature tetracycline-inducible expression of chicken ovalbumin (Ova) in the epidermis. In these we can transfer naı¨ve Ovaspecific CD4+ DO11 T cells and follow their activation and differentiation into cytokineproducing Teff cells and Foxp3+ Treg cells in vivo. Expression of antigen (Ag) in the skin elicits a T cell dependent inflammatory dermatitis that can be controlled by in vivo generated peripheral Treg cells. We found that high doses of tissue-Ag lead to T cell proliferation, production of effector cytokines, increased T cell receptor (TCR) signaling, and a blockade in peripheral Treg cell differentiation, which was lifted at lower Ag doses. Reducing TCR signaling with small molecule inhibitors at high-dose Ag conditions can equally lift the blockade Treg generation in vivo, leading to the regulation of tissue inflammation. These findings suggest that initial TCR signal strength determined by the dose of tissue-Ag can be crucial in the decision of Teff versus pTreg differentiation. We conclude that tissue antigen helps to maintain the appropriate ratios of Ag-specific Teff to Treg cells in peripheral tissues with remarkable consequences on clinical outcome.
S206 Journal of Investigative Dermatology (2016), Volume 136
WITHDRAWN
Influence of mouse beta-defensin 14 on skin barrier repair and bacterial growth E Proksch, C Neumann, J Harder and N Graumann Dermatology, University of Kiel, Kiel, Germany The mouse orthologue of human beta-defensin 3, mouse beta-defensin 14 (mBD-14), has antimicrobial and immunomodulatory activities. Epidermal proliferation and inflammatory signals are important for skin barrier repair. We examined whether a deficiency in mBD-14 expression in a mouse model or vice versa excess topical supply of the protein influences permeability barrier repair and bacterial growth. We induced skin permeability barrier disruption by tape stripping in mBD-14 deficient mice and determined barrier repair as recovery in TEWL. Also, recombinant mBD-14 was applied topically on back skin of mBD-14 deficient and normal mice after skin barrier disruption. In addition, mBD-14 deficient mouse skin was infected with Staphylococcus aureus. The infected skin was treated with several concentrations of mBD14. After 24 hours skin bacterial load was determined and biopsy samples for histology and immuno-histology were obtained. In mBD-14-deficient compared to wild type mice barrier repair was delayed. Topical application of recombinant mBD-14 partially reversed the delay in permeability barrier repair. Barrier disruption resulted in an inflammatory cell infiltrate and IL-1 expression in wild type but less in mBD-14 deficient mice. The infection rate, bacterial count and number of skin neutrophils were unchanged in mBD-14 deficient compared to control mice. Application of various concentrations of recombinant mBD-14 didn’t influence the infection rate in mBD-14 deficient or in control mice. We suggest that the delay in permeability barrier repair in mBD-14 deficient mice may be related to the known chemoattractant and proinflammatory activity of this defensin. Defensins also interact with TLR4 and TLR9, regulatory T-cells and ion channels. Surprisingly, mBD-14 didn’t influence bacterial growth. In general, mice get seldom infected with Staphylococcus aureus and may have a redundant system to fight this bacterial species.
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Corticotropin-releasing hormone receptor blocker suppresses cytokine expression induced by cathelicidine peptides H Lee and S Lee CHA Bundang Medical Center, CHA University, Seongnam-si, Korea (the Republic of) Rosacea is a chronic inflammatory skin condition that manifests unique inflammatory responses to normal environmental stimuli such as ultraviolet light radiation, heat and stress. The pathophysiology of rosacea appears to be defined by consistently abnormal innate immune activity, abnormal barrier function, and neurogenic dysregulation. The corticotropin releasing hormone (CRH), a neuropeptide originally isolated from the hypothalamus, is also produced by the skin, where it can act as a regulatory element of local neuroendocrine interactions. CRH has been reported to play a role in the inflammation by stimulating release of proinflammatory cytokines, such as IL-1b, IL-6, IL-8 and vascular endothelial growth factor. However, there have been no reports regarding the functional role of CRH in rosacea. We investigated if CRH receptor blocker could influence on inflammation induced by cathelicidin, which had a major role in rosacea. We cultured normal human keratinocytes in low Ca2+ condition (0.06mM) and high Ca2+ condition (1.6mM) and treated them with CRH and cathelicidine peptides (3.2mM) or CRH alone. Then we added CRHR-1 inhibitor to both groups. Supernatants were collected and IL-8 and TNF-a production were analyzed by ELISA. IL-8 and TNF-a release were increased in keratinocytes co-stimulated with CRH and cathelicidine compared to the cells treated with cathelicidine alone or CRH alone, especially in cells cultured in high Ca2+ condition. IL-8 and TNF-a release were suppressed by CRHR-1 inhibitors. This observation demonstrated possible immunomodulatory role of CRH in rosacea.
Hyaluronic acid oligosaccharides suppress cytokine expression induced by cathelicidine peptides H Lee and S Lee Department of Dermatology, CHA Bundang Medical Center, CHA University, Seongnam-si, Korea (the Republic of) Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. The pathophysiology of rosacea appears to be defined by consistently abnormal innate immune activity such as increased expression of cathelicidin, kallikrein 5 and Toll-like receptor (TLR) 2, abnormal barrier function, and neurogenic dysregulation. Hyaluronic acid (HA) is a high molecular weight linear glycosaminoglycan composed of repeating disaccharide subunits of D-glucuronic acid and N-acetyl glucosamine. The function of HA was initially thought to be mainly structural, but this paradigm changed when receptors to HA and its ability to regulate multiple functions were found. HA binds several different proteins that are able to influence immune function such as CD44, TLR2 and TLR4. Recently, it has been reported that HA oligosaccharides (oligo-HA) could suppress TLRdependent cytokine expression. In this study we investigated if oligo-HA could influence on inflammation induced by cathelicidin, which had a major role in rosacea. We cultured normal human keratinocytes in low Ca2+ condition (0.06mM) and high Ca2+ condition (1.6mM) and treated them with the cathelicidine peptides (3.2mM) and oligo-HA (10mg/ml) or the cathelicidine peptides alone. Supernatants were collected and IL-8 and TNF-a production were analyzed by ELISA. IL-8 and TNF-a release were suppressed in keratinocytes co-stimulated with oligo-HA and cathelicidine peptides compared to the cells treated with cathelicidine peptides alone, especially in cells cultured in high Ca2+ condition. This observation demonstrated possible immunomodulatory role of oligo-HA in rosacea.
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Patients with pemphigus foliaceus may retain antibody reactivity against calcium dependent epitopes of desmoglein 1 in remission K Kamiya1, Y Aoyama2, J Yamagami3, O Yamasaki4, Y Tokura5 and K Iwatsuki4 1 Department of Dermatology, Jichi Medical University, Shimotsuke, Japan, 2 Department of Dermatology, Kawasaki Hospital, Kawasaki Medical School, Okayama, Japan, 3 Department of Dermatology, Keio University School of Medicine, Tokyo, Japan, 4 Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan and 5 Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are closely related, but clinically distinct, autoimmune blistering diseases caused by autoantibodies against desmoglein (Dsg) 1 and Dsg3, respectively. By using ethylenediaminetetraacetic acid (EDTA)-treated Dsg3 enzyme-linked immunosorbent assay (ELISA), we have confirmed in PV patients that the pathogenicity of anti-Dsg3 antibodies is mainly determined by the amount of anti-Dsg3 antibodies against the calcium dependent epitopes of Dsg3. In this study, we have established EDTA-treated Dsg1 ELISA and exfoliative toxin (ET)-treated Dsg1 ELISA based on the structural characteristics of Dsg1. By using EDTA-treated and ET-treated ELISAs, we evaluated five PF patients who retained high serum levels of anti-Dsg1 antibodies in the inactive phase. Sera were obtained in both active and inactive phases. To map the epitopes of anti-Dsg1 antibodies, immunoprecipitation-immunoblotting was performed by using a set of Dsg1/Dsg2 domain swapped molecules. Anti-Dsg1 antibodies against the calcium dependent epitopes of Dsg1 were the predominant antibodies in both active and inactive phases. The proportion of anti-Dsg1 antibodies against the calcium dependent epitopes was not changed upon shifting to the inactive phase. The results of immunoprecipitation-immunoblotting showed that most of the anti-Dsg1 antibodies bound to the extracellular domains (EC) 1-2 of Dsg1. In PF, both pathogenic and nonpathogenic epitopes were located in the EC1-2 of Dsg1. The disease activity might be differentially controlled by the antibodies between PF and PV depending on the presence or absence of the nonpathogenic epitope.
Tissue antigen expression levels control the fundamental decision between tolerance and autoimmunity DF Pinheiro1, MM Maurano1, MM Klicznik1, R Holly1, G Achatz-Straussberger1, J Thalhamer1, A Abbas2, MD Rosenblum2 and IK Gratz1 1 Molecular Biology, University of Salzburg, Salzburg, Austria and 2 University of California, San Francisco, San Francisco, CA Immune homeostasis in peripheral tissues is achieved by maintaining a delicate balance between pathogenic effector T cells (Teff) and protective regulatory T cells (Treg). The factors responsible for maintaining this balance e especially in the target tissues e are largely unknown. We aimed to study immune regulatory mechanisms in T cell mediated skin inflammation. To this end we established two mouse models that feature tetracycline-inducible expression of chicken ovalbumin (Ova) in the epidermis. In these we can transfer naı¨ve Ovaspecific CD4+ DO11 T cells and follow their activation and differentiation into cytokineproducing Teff cells and Foxp3+ Treg cells in vivo. Expression of antigen (Ag) in the skin elicits a T cell dependent inflammatory dermatitis that can be controlled by in vivo generated peripheral Treg cells. We found that high doses of tissue-Ag lead to T cell proliferation, production of effector cytokines, increased T cell receptor (TCR) signaling, and a blockade in peripheral Treg cell differentiation, which was lifted at lower Ag doses. Reducing TCR signaling with small molecule inhibitors at high-dose Ag conditions can equally lift the blockade Treg generation in vivo, leading to the regulation of tissue inflammation. These findings suggest that initial TCR signal strength determined by the dose of tissue-Ag can be crucial in the decision of Teff versus pTreg differentiation. We conclude that tissue antigen helps to maintain the appropriate ratios of Ag-specific Teff to Treg cells in peripheral tissues with remarkable consequences on clinical outcome.
S206 Journal of Investigative Dermatology (2016), Volume 136
WITHDRAWN
Influence of mouse beta-defensin 14 on skin barrier repair and bacterial growth E Proksch, C Neumann, J Harder and N Graumann Dermatology, University of Kiel, Kiel, Germany The mouse orthologue of human beta-defensin 3, mouse beta-defensin 14 (mBD-14), has antimicrobial and immunomodulatory activities. Epidermal proliferation and inflammatory signals are important for skin barrier repair. We examined whether a deficiency in mBD-14 expression in a mouse model or vice versa excess topical supply of the protein influences permeability barrier repair and bacterial growth. We induced skin permeability barrier disruption by tape stripping in mBD-14 deficient mice and determined barrier repair as recovery in TEWL. Also, recombinant mBD-14 was applied topically on back skin of mBD-14 deficient and normal mice after skin barrier disruption. In addition, mBD-14 deficient mouse skin was infected with Staphylococcus aureus. The infected skin was treated with several concentrations of mBD14. After 24 hours skin bacterial load was determined and biopsy samples for histology and immuno-histology were obtained. In mBD-14-deficient compared to wild type mice barrier repair was delayed. Topical application of recombinant mBD-14 partially reversed the delay in permeability barrier repair. Barrier disruption resulted in an inflammatory cell infiltrate and IL-1 expression in wild type but less in mBD-14 deficient mice. The infection rate, bacterial count and number of skin neutrophils were unchanged in mBD-14 deficient compared to control mice. Application of various concentrations of recombinant mBD-14 didn’t influence the infection rate in mBD-14 deficient or in control mice. We suggest that the delay in permeability barrier repair in mBD-14 deficient mice may be related to the known chemoattractant and proinflammatory activity of this defensin. Defensins also interact with TLR4 and TLR9, regulatory T-cells and ion channels. Surprisingly, mBD-14 didn’t influence bacterial growth. In general, mice get seldom infected with Staphylococcus aureus and may have a redundant system to fight this bacterial species.