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247 Disruption of the epidermal barrier induces regulatory T cells in an IL-33 dependent fashion
Inflammation, Immunity and Infection | ABSTRACTS 244
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Efficacy of kappa-opioid receptor agonist and mu-opioid receptor antagonist to treat itch in imiquimod-induced psoriasis-like dermatitis model N Takahashi1, M Tominaga1, Y Kamata1, Y Umehara1, H Matsuda1, H Ogawa2 and K Takamori2 1 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan and 2 Department of Dermatology, Juntendo University Graduate School of Medicine, Tokyo, Japan The majority of psoriasis patients suffer from antihistamine-resistant itch. However, therapeutic options to reduce such itch are few, and the pathogenesis of pruritus remains unclear. We previously reported that opioid systems may be involved in the pathogenesis of psoriatic itch. Here, we constructed a psoriasis-like model of scratching behavior and investigated the possible mechanism of itch in this model. Psoriasis-like lesions were induced in the skin of C57BL/6J mice by repeated topical application of imiquimod cream (IMQ) for five days (IMQmice) inducing scratching bouts. Oral administration of bepotastine besilate, a histamine H1 receptor antagonist, did not affect the number of scratching bouts in IMQ-mice. We next examined mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) protein expression in epidermis, dorsal root ganglion (DRG) and spinal cord by Western blotting. MOR was increased in the epidermis, DRG and spinal cord of IMQ-mice. In contrast, KOR was reduced in DRG and spinal cord of IMQ-mice. Based on these results, we examined the effects of topical application of naloxone (a MOR antagonist), oral administration of asimadoline hydrochloride (a peripheral KOR agonist) and ICI-199,441 (a central KOR agonist) on scratching behavior in IMQ-mice. The numbers of scratching bouts were significantly decreased in both the topical naloxone and oral ICI-199,441-treated mice without affecting locomotor activity. Meanwhile, asimadoline hydrochloride did not affect the number of scratching bouts in IMQmice. In conclusion, scratching behavior is induced in an imiquimod-induced psoriasis-like dermatitis model. In this model, histamine H1 receptor was not involved in scratching behavior. In addition, these results suggest that peripheral MOR and central KOR may be promising as therapeutic targets for psoriatic itch.
Mechanisms of ER stress sensor calreticulin (CRT) exposure controls the destruction of melanocytes by enhanced CD8+ T cell killing P Song, L Liu, G Wang, C Li and T Gao Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Reactive oxygen species and autoimmunity interact synergistically, leading to the final destruction of melanocytes in vitiligo. The cytotoxic CD8+ T cells were considered to be both necessary and sufficient to mediate depigmentation in vitiligo. We previously reported that total CRT levels increased in melanocytes exposed to H2O2-induced oxidative stress, and CRT-treated PBMCs or stressed melanocytes expressed higher levels of IL-6 and TNF-a than untreated cells. However, it is not known the mechanisms that mediate CRT translocation in melanocyte apoptosis. This study aimed to investigate the signaling pathway that mediates ecto-CRT expression and the interrelationships between ecto-CRT and immune reactions during melanocyte destruction. We first performed immunofluorescence and observed that CRT expressed in the melanocytes membrane of the leading edge skin from vitiligo patients. Moreover, the endoplasmic reticulum stress response via PERK and eIF2a was found to be involved in the translocation of CRT to the melanocyte surface during H2O2-induced oxidative stress in vitro. Meanwhile, the phosphor-PERK expressed in the leading edge skin from vitiligo patients only. Furthermore, the H2O2-treated melanocytes which were coincubated with PBMCs of vitiligo patients expressed higher levels of IFN-g and CD8+ T cell proliferation (P<0.01) than untreated cells by using flow cytometry and ELISA; this effect was inhibited with PERK knockdown (P<0.05). In the same incubation model, the levels of dentritic cells mature markers CD80, CD86 and HLA-DR also expressed higher than untreated cells model. And when we use PERK siRNA to downregulate ecto-CRT expression, the levels of CD86 and HLA-DR were lower (P<0.05). In summary, we provide evidence that CRT exposure via the activation of PERK pathway under oxidative stress plays a significant role in immune reactions during melanocyte destruction. Understanding how the cytotoxic CD8+ T cells find the malanocytes specifically in vitiltigo, targeted treatment strategies.
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Prebiotic stimulation of innate immune system, skin barrier and staphylococci homeostasis S Gauthier2, L Costes2, F Legendre2, S Leoty-Okombi1, D Rival1 and V Andre´-Frei1 1 BASF Beauty Care Solutions, LYON, France and 2 INVIVOGEN, Toulouse, France Skin is our first line of defense against external assault. First, it represents a physical barrier made of cornified keratinocytes with intercellular lipids and tight junctions preventing skin from dehydration and environmental stresses. Second, it is a physical and immunological barrier against infection caused by imbalanced population of commensal germs. Particularly, keratinocytes are involved in skin innate immunity as they express Pattern Recognition Receptors (PRRs) like TLRs (Toll-Like receptors) and dectins crucial for protection against pathogens. Among skin disorders, dry skin or atopic dermatitis are affected by both skin inflammation / barrier defect, and staphylococci disequilibrium. Here we present the first holobiontic repair approach by addressing both skin immunity/barrier and Staphylococci homeostasis. At first we selected a biofermented yeast extract (BYE) for its ability to stimulate skin immunity through PRRs activation and skin barrier. The level of Dectin-1a or TLR-2 were quantified after 24h by measuring the levels of NF-kappa B-induced SEAP (secreted embryonic alkaline phosphatase) reporter gene using spectrophotometry. 7 fold and 3 fold respective increase of TLR-2 and Dectin-1 were observed on HEK-BlueÔ cell lines kappa. Skin barrier strengthening was evaluated on reconstructed epidermis treated by BYE during 10 days. Among total lipids increase, ceramides were particularly induced (+226%). In addition, keratinocytes tight junctions evaluation was performed by immunostaining. Then, the impact of BYE was evaluated on different staphylococci strains and we evidenced an increase of Staphylococcus hominis known to produce efficient antimicrobial peptides against Staphylococcus aureus. Using a biofermented yeast extract, we succeed to demonstrate convergent benefits for dry or atopic skin training the skin to defend itself against pathogens to a direct effect on skin flora equilibrium.
Disruption of the epidermal barrier induces regulatory T cells in an IL-33 dependent fashion A Bruhs, T Schwarz and A Schwarz Department of Dermatology, University of Kiel, Kiel, Germany Disruption of the epidermal barrier is associated with a variety of defense feedback mechanisms, e.g. the release of antimicrobial peptides which combat the invasion of microbes. Since allergens also penetrate more easily into disturbed skin we asked whether similar defense mechanisms avoiding the induction of contact allergies exist. To address this issue, C57BL/6J mice were tape stripped on their shaved backs before sensitization with 2,4,6-trinitro-1-chlorobenzene (TNCB). After 5 days ear challenge was performed and the contact hypersensitivity (CHS) reaction was measured. This was significantly reduced in the tape stripped group. The suppression appears to be due to the induction of regulatory T cells (Treg) as demonstrated by adoptive transfer experiments. Tape strip-induced Treg express Foxp3, as demonstrated in DEREG mice in which Foxp3 expressing Treg can be selectively depleted. Recently, IL-33 was discovered to induce Treg in the intestine. IL-33 is a member of the IL-1 family and is constitutively expressed in epithelial cells of barrier organs where it functions as endogenous signal (alarmin) in response to tissue damage. Immunofluorescence staining of ears revealed a strong induction of IL-33 upon tape stripping. To address whether IL-33 is involved in the induction of Treg in tape stripped animals, mice were left untreated or received a neutralizing IL-33 antibody i.p. before both groups were tape stripped.. Upon challenge 5 days after sensitization, mice which were tape stripped only revealed a significant reduction of ear swelling in comparison to positive controls, whereas the CHS response in the tape stripped recipients of the anti-IL-33 antibody was not impaired. Together, these data indicate that the disturbance of the epidermal barrier suppresses the adaptive immune response via induction of Foxp3+ Treg and that this process in major parts may be mediated by IL-33. These findings indicate another compensatory defense mechanism which might lower the risk of the development of contact allergies in disrupted skin.
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Cell populations interacting with thermoresponsive nanocarriers: targeting of antiinflammatory drugs to skin F Rancan1, M Giulbudagian2, J Jurisch1, J Stanko1, H Volkmann1, U Blume-Peytavi1, M Calderon2 and A Vogt1 1 Department of Dermatology and Allergy, Charite´ e Universita¨tsmedizin Berlin, Berlin, Germany and 2 Institute for Chemistry and Biochemistry, Freie Universita¨t Berlin, Berlin, Germany Inflammatory dendritic cells plays a central role in skin conditions like psoriasis and atopic dermatitis. Since these cells are prone to take-up particulate material, there is a rational for the use of nanocarriers to target drugs to these cells, especially in inflamed skin, where the skin barrier is compromised and nanocarriers have easier access to the viable skin layers. Thermo-responsive nanogels (tNGs), loaded with fluorochromes or tacrolimus as model antiinflammatory drug, were applied topically on human skin explants after mechanical disruption of the stratum corneum. Skin penetration of both drug and tNGs as well as the expression of key cytokines were investigated. In order to identify which cell populations are involved in the uptake of tNGs, cells were isolated and identified by means of flow cytometry analysis. Drug penetration to the epidermis and dermis was significantly higher in skin with disrupted barrier as compared to skin with intact barrier. Different drug delivery outcomes were detected when comparing standard with nanogel formulations. We found that after barrier disruption, penetrated tNGs were internalized by different cell populations of both epidermis and dermis cells. Whereas antigen presenting cells of the dermis (HLA-DR+, CD206+) were found to take-up penetrated tNGs already after short incubation time (2 h), Langerhans cells (CD1a+) were found to be associated with tNG after longer incubation time (16 h) and only when considerable amounts of tNGs had crossed skin barrier. We conclude that improved local delivery of anti-inflammatory drugs as well as the targeting of skin immune active cells by means of nanocarriers such as tNGs represent promising strategies for the topical treatment of skin inflammatory conditions.
Contribution of IgE autoantibodies to the pathogenesis of bullous pemphigoid PC Freire, P Heil and G Stingl Department of Dermatology, Medical University of Vienna, Vienna, Austria Bullous pemphigoid (BP) is an auto-immune blistering disease that has consistently been associated with IgG autoantibodies and complement activation. Additionally, the frequent presence of urticarial plaques in these patients points to an involvement of IgE, corroborated by both ex- and in-vivo animal and human studies. The mechanisms by which IgE contributes to the pathogenesis of BP are however still not understood. Thus, the goal of this on-going study is to investigate the presence and pathogenicity of IgE autoantibodies in BP patients. In line with previous literature, we have detected, via ELISA, significantly higher levels of NC16a- (p < 0,0001) and BP230-specific (p < 0,005) IgE in BP sera comparing with healthy controls. Consistent with a class-switch phenomenon, IgG and IgE share the same dominant epitopes, as demonstrated using overlapping peptide sequences of BP180. Furthermore, we have found IgE in perilesional skin of 21 out of 32 (66 %) BP patients. This IgE was not found at the DEJ, but instead on the surface of mast cells and eosinophils, most likely bound as an immune complex. We have evidence that the high-affinity receptor for IgE is the primary molecule involved in this interaction and that eosinophils are expressing FcεRI in BP patients. Finally, using direct immunofluorescence we have detected dermal BP180 fragments that appear to colocalize with IgE+ eosinophils, a finding that offers a new hypothesis for IgE’s mechanism of action. We have therefore confirmed the association between IgE and BP and propose a pathway of disease pathogenesis alternative to IgG and complement.
www.jidonline.org S203
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Efficacy of kappa-opioid receptor agonist and mu-opioid receptor antagonist to treat itch in imiquimod-induced psoriasis-like dermatitis model N Takahashi1, M Tominaga1, Y Kamata1, Y Umehara1, H Matsuda1, H Ogawa2 and K Takamori2 1 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan and 2 Department of Dermatology, Juntendo University Graduate School of Medicine, Tokyo, Japan The majority of psoriasis patients suffer from antihistamine-resistant itch. However, therapeutic options to reduce such itch are few, and the pathogenesis of pruritus remains unclear. We previously reported that opioid systems may be involved in the pathogenesis of psoriatic itch. Here, we constructed a psoriasis-like model of scratching behavior and investigated the possible mechanism of itch in this model. Psoriasis-like lesions were induced in the skin of C57BL/6J mice by repeated topical application of imiquimod cream (IMQ) for five days (IMQmice) inducing scratching bouts. Oral administration of bepotastine besilate, a histamine H1 receptor antagonist, did not affect the number of scratching bouts in IMQ-mice. We next examined mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) protein expression in epidermis, dorsal root ganglion (DRG) and spinal cord by Western blotting. MOR was increased in the epidermis, DRG and spinal cord of IMQ-mice. In contrast, KOR was reduced in DRG and spinal cord of IMQ-mice. Based on these results, we examined the effects of topical application of naloxone (a MOR antagonist), oral administration of asimadoline hydrochloride (a peripheral KOR agonist) and ICI-199,441 (a central KOR agonist) on scratching behavior in IMQ-mice. The numbers of scratching bouts were significantly decreased in both the topical naloxone and oral ICI-199,441-treated mice without affecting locomotor activity. Meanwhile, asimadoline hydrochloride did not affect the number of scratching bouts in IMQmice. In conclusion, scratching behavior is induced in an imiquimod-induced psoriasis-like dermatitis model. In this model, histamine H1 receptor was not involved in scratching behavior. In addition, these results suggest that peripheral MOR and central KOR may be promising as therapeutic targets for psoriatic itch.
Mechanisms of ER stress sensor calreticulin (CRT) exposure controls the destruction of melanocytes by enhanced CD8+ T cell killing P Song, L Liu, G Wang, C Li and T Gao Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Reactive oxygen species and autoimmunity interact synergistically, leading to the final destruction of melanocytes in vitiligo. The cytotoxic CD8+ T cells were considered to be both necessary and sufficient to mediate depigmentation in vitiligo. We previously reported that total CRT levels increased in melanocytes exposed to H2O2-induced oxidative stress, and CRT-treated PBMCs or stressed melanocytes expressed higher levels of IL-6 and TNF-a than untreated cells. However, it is not known the mechanisms that mediate CRT translocation in melanocyte apoptosis. This study aimed to investigate the signaling pathway that mediates ecto-CRT expression and the interrelationships between ecto-CRT and immune reactions during melanocyte destruction. We first performed immunofluorescence and observed that CRT expressed in the melanocytes membrane of the leading edge skin from vitiligo patients. Moreover, the endoplasmic reticulum stress response via PERK and eIF2a was found to be involved in the translocation of CRT to the melanocyte surface during H2O2-induced oxidative stress in vitro. Meanwhile, the phosphor-PERK expressed in the leading edge skin from vitiligo patients only. Furthermore, the H2O2-treated melanocytes which were coincubated with PBMCs of vitiligo patients expressed higher levels of IFN-g and CD8+ T cell proliferation (P<0.01) than untreated cells by using flow cytometry and ELISA; this effect was inhibited with PERK knockdown (P<0.05). In the same incubation model, the levels of dentritic cells mature markers CD80, CD86 and HLA-DR also expressed higher than untreated cells model. And when we use PERK siRNA to downregulate ecto-CRT expression, the levels of CD86 and HLA-DR were lower (P<0.05). In summary, we provide evidence that CRT exposure via the activation of PERK pathway under oxidative stress plays a significant role in immune reactions during melanocyte destruction. Understanding how the cytotoxic CD8+ T cells find the malanocytes specifically in vitiltigo, targeted treatment strategies.
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Prebiotic stimulation of innate immune system, skin barrier and staphylococci homeostasis S Gauthier2, L Costes2, F Legendre2, S Leoty-Okombi1, D Rival1 and V Andre´-Frei1 1 BASF Beauty Care Solutions, LYON, France and 2 INVIVOGEN, Toulouse, France Skin is our first line of defense against external assault. First, it represents a physical barrier made of cornified keratinocytes with intercellular lipids and tight junctions preventing skin from dehydration and environmental stresses. Second, it is a physical and immunological barrier against infection caused by imbalanced population of commensal germs. Particularly, keratinocytes are involved in skin innate immunity as they express Pattern Recognition Receptors (PRRs) like TLRs (Toll-Like receptors) and dectins crucial for protection against pathogens. Among skin disorders, dry skin or atopic dermatitis are affected by both skin inflammation / barrier defect, and staphylococci disequilibrium. Here we present the first holobiontic repair approach by addressing both skin immunity/barrier and Staphylococci homeostasis. At first we selected a biofermented yeast extract (BYE) for its ability to stimulate skin immunity through PRRs activation and skin barrier. The level of Dectin-1a or TLR-2 were quantified after 24h by measuring the levels of NF-kappa B-induced SEAP (secreted embryonic alkaline phosphatase) reporter gene using spectrophotometry. 7 fold and 3 fold respective increase of TLR-2 and Dectin-1 were observed on HEK-BlueÔ cell lines kappa. Skin barrier strengthening was evaluated on reconstructed epidermis treated by BYE during 10 days. Among total lipids increase, ceramides were particularly induced (+226%). In addition, keratinocytes tight junctions evaluation was performed by immunostaining. Then, the impact of BYE was evaluated on different staphylococci strains and we evidenced an increase of Staphylococcus hominis known to produce efficient antimicrobial peptides against Staphylococcus aureus. Using a biofermented yeast extract, we succeed to demonstrate convergent benefits for dry or atopic skin training the skin to defend itself against pathogens to a direct effect on skin flora equilibrium.
Disruption of the epidermal barrier induces regulatory T cells in an IL-33 dependent fashion A Bruhs, T Schwarz and A Schwarz Department of Dermatology, University of Kiel, Kiel, Germany Disruption of the epidermal barrier is associated with a variety of defense feedback mechanisms, e.g. the release of antimicrobial peptides which combat the invasion of microbes. Since allergens also penetrate more easily into disturbed skin we asked whether similar defense mechanisms avoiding the induction of contact allergies exist. To address this issue, C57BL/6J mice were tape stripped on their shaved backs before sensitization with 2,4,6-trinitro-1-chlorobenzene (TNCB). After 5 days ear challenge was performed and the contact hypersensitivity (CHS) reaction was measured. This was significantly reduced in the tape stripped group. The suppression appears to be due to the induction of regulatory T cells (Treg) as demonstrated by adoptive transfer experiments. Tape strip-induced Treg express Foxp3, as demonstrated in DEREG mice in which Foxp3 expressing Treg can be selectively depleted. Recently, IL-33 was discovered to induce Treg in the intestine. IL-33 is a member of the IL-1 family and is constitutively expressed in epithelial cells of barrier organs where it functions as endogenous signal (alarmin) in response to tissue damage. Immunofluorescence staining of ears revealed a strong induction of IL-33 upon tape stripping. To address whether IL-33 is involved in the induction of Treg in tape stripped animals, mice were left untreated or received a neutralizing IL-33 antibody i.p. before both groups were tape stripped.. Upon challenge 5 days after sensitization, mice which were tape stripped only revealed a significant reduction of ear swelling in comparison to positive controls, whereas the CHS response in the tape stripped recipients of the anti-IL-33 antibody was not impaired. Together, these data indicate that the disturbance of the epidermal barrier suppresses the adaptive immune response via induction of Foxp3+ Treg and that this process in major parts may be mediated by IL-33. These findings indicate another compensatory defense mechanism which might lower the risk of the development of contact allergies in disrupted skin.
248
249
Cell populations interacting with thermoresponsive nanocarriers: targeting of antiinflammatory drugs to skin F Rancan1, M Giulbudagian2, J Jurisch1, J Stanko1, H Volkmann1, U Blume-Peytavi1, M Calderon2 and A Vogt1 1 Department of Dermatology and Allergy, Charite´ e Universita¨tsmedizin Berlin, Berlin, Germany and 2 Institute for Chemistry and Biochemistry, Freie Universita¨t Berlin, Berlin, Germany Inflammatory dendritic cells plays a central role in skin conditions like psoriasis and atopic dermatitis. Since these cells are prone to take-up particulate material, there is a rational for the use of nanocarriers to target drugs to these cells, especially in inflamed skin, where the skin barrier is compromised and nanocarriers have easier access to the viable skin layers. Thermo-responsive nanogels (tNGs), loaded with fluorochromes or tacrolimus as model antiinflammatory drug, were applied topically on human skin explants after mechanical disruption of the stratum corneum. Skin penetration of both drug and tNGs as well as the expression of key cytokines were investigated. In order to identify which cell populations are involved in the uptake of tNGs, cells were isolated and identified by means of flow cytometry analysis. Drug penetration to the epidermis and dermis was significantly higher in skin with disrupted barrier as compared to skin with intact barrier. Different drug delivery outcomes were detected when comparing standard with nanogel formulations. We found that after barrier disruption, penetrated tNGs were internalized by different cell populations of both epidermis and dermis cells. Whereas antigen presenting cells of the dermis (HLA-DR+, CD206+) were found to take-up penetrated tNGs already after short incubation time (2 h), Langerhans cells (CD1a+) were found to be associated with tNG after longer incubation time (16 h) and only when considerable amounts of tNGs had crossed skin barrier. We conclude that improved local delivery of anti-inflammatory drugs as well as the targeting of skin immune active cells by means of nanocarriers such as tNGs represent promising strategies for the topical treatment of skin inflammatory conditions.
Contribution of IgE autoantibodies to the pathogenesis of bullous pemphigoid PC Freire, P Heil and G Stingl Department of Dermatology, Medical University of Vienna, Vienna, Austria Bullous pemphigoid (BP) is an auto-immune blistering disease that has consistently been associated with IgG autoantibodies and complement activation. Additionally, the frequent presence of urticarial plaques in these patients points to an involvement of IgE, corroborated by both ex- and in-vivo animal and human studies. The mechanisms by which IgE contributes to the pathogenesis of BP are however still not understood. Thus, the goal of this on-going study is to investigate the presence and pathogenicity of IgE autoantibodies in BP patients. In line with previous literature, we have detected, via ELISA, significantly higher levels of NC16a- (p < 0,0001) and BP230-specific (p < 0,005) IgE in BP sera comparing with healthy controls. Consistent with a class-switch phenomenon, IgG and IgE share the same dominant epitopes, as demonstrated using overlapping peptide sequences of BP180. Furthermore, we have found IgE in perilesional skin of 21 out of 32 (66 %) BP patients. This IgE was not found at the DEJ, but instead on the surface of mast cells and eosinophils, most likely bound as an immune complex. We have evidence that the high-affinity receptor for IgE is the primary molecule involved in this interaction and that eosinophils are expressing FcεRI in BP patients. Finally, using direct immunofluorescence we have detected dermal BP180 fragments that appear to colocalize with IgE+ eosinophils, a finding that offers a new hypothesis for IgE’s mechanism of action. We have therefore confirmed the association between IgE and BP and propose a pathway of disease pathogenesis alternative to IgG and complement.
www.jidonline.org S203