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8 agonists stimulate plasmacytoid dendritic cells to initiate a Th17-deviated acute contact dermatitis in humans
Inflammation, Immunity and Infection | ABSTRACTS 484
485
Lichen planus pemphigoides is a chimera of lichen planus and bullous pemphigoid F Solimani1, A Schmidt2, R Eming1, M Hertl1 and T Schmidt1 1 Department of Dermatology and Allergology, Philipps-University Marburg, Marburg, Germany and 2 Department of Pathology, Philipps-University Marburg, Marburg, Germany Occasionally, lichen planus (LP), a common chronic inflammatory skin disorder of unknown etiology transforms into a bullous phenotype reminiscent of bullous pemphigoid (BP) and is associated with IgG autoantibodies against BP180. We here studied three female patients with LPP with regard to peripheral blood T cell recognition of BP180. LPP patient 1 (P1) showed lichenoid striae of the buccal mucosa, polygonal lichenoid papules on the dorsa of the extremities, C3 deposits at the dermal-epidermal BMZ by DIF and IgG against BP180 (128 RE/ml) by ELISA. Patient P2 showed tense bullae, partly on lichenoid plaques on the trunk, C3 deposits at the dermal-epidermal BMZ by DIF and IgG against BP180 (65 RE/ml) by ELISA. Patient P3 had lichenoid erosions of the buccal mucosa and extensive polygonal papules on the trunk and extremities, C3 deposits at the dermal-epidermal BMZ (DIF), and IgG against BP180 (577 RE/ml) by ELISA. All the three LPP patients showed a mixed Th1/Th2 cell infiltrate of lesional skin (CD3+/T-bet+ 45.3% 7.44, CD4+/GATA3+ 68.8% 6.22) which was accompanied by a mixed peripheral blood Th1/Th2 cellular response to the immunodominant NH2 of BP180 (Elispot analysis: ratio number of IFNg+/IL-5+ spots 2.87 2.03). In comparison, the Th1/Th2 ratio was significantly higher in LP (Elispot analysis: ratio number of IFNg+/IL-5+ spots 13.1 10.03) and similar in BP (Elispot analysis: ratio number of IFNg+/IL5+ spots 5.6 4.38). Thus, LPP shares features of LP (Th1-driven) and BP (Th2-dominated).
Optimization of cytokine milieu to reproduce atopic dermatitis-related gene expression in HaCaT keratinocytes J Roh1, H Kim1, J Baek1, H Ryu1, S Lee1 and Y Jung2 1 Dermaology, Gachon University Gil Medical Center, Incheon, Korea (the Republic of) and 2 Microbiology, Gachon University School of Medicine, Incheon, Korea (the Republic of) Although atopic dermatitis (AD) has been characterized by predominant Th2 cytokine production, Th1 cells are also involved in AD pathogenesis. This study was to identify in vitro cytokine milieu that can accurately reproduce the expression profile of genes important in AD pathogenesis. mRNA levels of CCL22 (MDC), CCL17 (TARC), IL5, IL13, FLG, and LOR were evaluated by qRT-PCR in skin samples from six AD patients, 12 healthy controls, and in HaCaT cells cultured in the presence of various combinations of Th1 (TNF-a, 10 ng/ml; and/ or IFN-g, 10 ng/ml) and Th2 (IL-4, 50 ng/ml) cytokines. Cytokine stimulation effects on HaCaT cell growth were examined by phase-contrast microscopy and water-soluble tetrazolium salt assay. HaCaT cell viability was not altered by treatment with TNF-a, IFN-g, or IL-4 alone, or by treatment with TNF-a and IL-4 in combination, while TNF-a and IFN-g in combination significantly reduced the viability. We compared the gene expression profiles of human AD skin samples and in vitro HaCaT cells in various cytokine treatment settings. Expression of Th2 genes (CCL22, CCL17, IL5 and IL13) was increased in human AD skin lesions, while cornified cell envelop-related genes, FLG and LOR, was reduced. Interestingly, similar HaCaT cell gene expression profiles were observed when stimulated with Th1 cytokines, TNF-a and/ or IFN-g, but not with Th2 cytokine, IL-4, implying that Th1 stimulation is required to reproduce the AD-like features in HaCaT cells. Current data suggest that Th1 and Th2 cytokines do not function dichotomously, and a complicated inflammatory network drives ADlike changes. Therefore, in further in vitro experiments using HaCaT cells to study AD-related genes, stimulation with various cytokine combinations not limited to Th2 polarization would be necessary for optimal gene expression levels.
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Short course model of oxazolone-induced dermatitis in the nape of mice: Characterisation and modulation with topical drugs M Pont, C Carcasona, A Blanco, G Tarraso´n, N Godessart and A Gavalda` R&D Centre, Almirall S.A., Barcelona, Spain Oxazolone (Oxa)-induced chronic skin inflammation in mice is commonly used as a model of atopic dermatitis. The disease can be evoked in the ears, the back or the nape in different strains of mice. The duration of the assay depends on the number of Oxa challenges, which ranges from 3 to 12. We aimed to develop a short course model to be used for the screening of anti-inflammatory drugs using pruritus as an early readout of efficacy. Following sensitisation, C57BL/6 mice received Oxa challenges in the nape every other day starting on day 8 post-sensitisation. Histological evaluation showed increased inflammation up to challenge 3, and subepidermal fibrosis from challenge 5. Thus, we selected the 3-challenge model for further studies. After challenge 3, lesional skin showed moderate epidermal hyperplasia with hyperkeratosis, scabs and a mixed cellular dermatitis. Lesions displayed skin barrier impairment and increased expression of the Th2 cytokines, IL-4 and IL-13. Enhanced pruritus was also evident after the 3rd challenge. For a pharmacological characterisation of the model, we used commercial formulations of corticosteroids (clobetasol, betamethasone and hydrocortisone) and tacrolimus. Treatments were applied once daily from day 8 to 12. Skin fold thickness and TEWL were measured daily. Pruritus was assessed on day 12, 2 h after challenge. On day 13 skin biopsies were collected for analysis. All treatments significantly reversed the induced histopathological changes. Cytokine levels decreased and TEWL improved. Except for hydrocortisone, the rest of treatments inhibited scratching over 65%. For some parameters, the effect of corticosteroids correlated with their potency. Skin atrophy was evident with clobetasol and betamethasone. Overall, the model described here replicates the main features of atopic dermatitis, including pruritus, and responds to topical drugs used in the clinical practice. The duration of the model makes it suitable for the screening of novel anti-inflammatory drugs.
Phosphorylated MLKL and cleaved caspase8 are useful for early diagnosis of Stevens-Johnson syndrome and toxic epidermal necrolysis A Hasegawa, M Maehara, N Yamazaki, Y Shigehara and R Abe Niigata University, Niigata, Japan Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening cutaneous adverse drug reactions. It is difficult to distinguish erythema multiforme (EM) and SJS/TEN in early stage. Although keratinocytes have been suggested to die as apoptosis, our recent study revealed that necroptosis, programmed necrosis, also contribute to keratinocyte death in SJS/TEN. We hypothesized that analyzing the form of keratinocyte death is useful for early diagnosis of SJS/TEN. The skin samples were obtained from non-bullous erythematus lesion of the patient who suspected drug eruption in the acute phase. We included 4 patients diagnosed as SJS/TEN and 10 patients diagnosed as EM. Anti-phosphorylated MLKL (pMLKL) antibody was used to detect necroptosis, and anti-cleaved caspase8 (cCas8) antibody was used to detect apoptosis. Both of pMLKL(+) cell (3.782.70%) and cCas8(+) cell (17.84.94%) were shown in the 4 SJS/TEN patients. In EM patients, pMLKL(+) cell was shown in 4 patients (1.472.56%) and cCas8(+) cell was shown in 2 patients (1.092.15%). In 4 all cases diagnosed as SJS/TEN, both of pMLKL and cCas8 cell were positive. In contrast, it was only 1 case that both pMLKL and cCas8 were positive and either pMLKL or cCas8 was positive in 4 cases in EM. The number of pMLKL(+) plus cCas8(+) cell were statistically higher than the number of death keratinocyte detected by HE staining (SJS/TEN: 13.425.39%)(EM: 1.021.38%). The results revealed that both of necroptosis and apoptosis are occurred in keratinocyte death in SJS/TEN. Although, in EM, both of necroptosis and apoptosis are shown, the amount of cell death is lower than SJS/TEN. It is suggested that the frequency of cell death is important for differentiation between SJS and EM. pMLKL and cCas8 may be able to detect earlier stages of cell death than HE staining, and it is expected that pMLKL and cCas8 can be the marker of SJS/TEN.
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Continuous sampling of immune cells in the skin by dermal open flow microperfusion T Birngruber1, B Prietl2, M Bodenlenz1, P Florian4, A Subramaniam3, S Kainz1, G Rauter1, TR Pieber1,2 and F Sinner1,2 1 HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft mbH, Graz, Austria, 2 Division of Endocrinology and Diabetology, Medical University of Graz, Graz, Austria, 3 Sanofi-Genzyme, Framingham, MA and 4 Sanofi, Frankfurt, Germany Immune cells and biologic response modulators like cytokines play a major role in immune responses in the skin. Currently, skin inflammation is studied by highly invasive biopsy methods in clinical studies. We investigated whether the minimally invasive dermal Open Flow Microperfusion (dOFM) enables continuous sampling of immune cells from inflamed and healthy skin in a psoriasis rat model. Psoriasis-like skin inflammation was induced on a skin test site of Sprague-Dawley rats by applying a daily dose of topical imiquimod. A daily dose of oral Dexamethasone was additionally administered to a subgroup of rats to prevent the imiquimod-induced skin inflammation. After 8 days, dermal interstitial fluid was sampled from imiquimod-treated skin and from untreated skin with dOFM sampling probes. The samples were stained with antibodies which were specific for the cell subtypes and analyzed by flow cytometry (FACS). A well-quantifiable amount of CD45-positive cells (granulocytes, lymphocytes, monocytes) was found in the dOFM samples. Staining also identified CD3 positive T-cells, CD8 positive cytotoxic T-cells, CD4 positive T-helper cells and IL17 positive cells. The amount and type of cells in dOFM samples derived from inflamed skin differed from that of untreated skin and of the skin of Dexamethasone-treated animals. This study illustrates the feasibility of the minimally-invasive sampling method dOFM to continuously sample immune cells from inflamed and healthy living tissue. dOFM has the potential to enable a combined investigation of locally released cytokines and the involved immune cells in clinical and preclinical in vivo studies of inflammatory diseases.
TLR7/8 agonists stimulate plasmacytoid dendritic cells to initiate a Th17-deviated acute contact dermatitis in humans N Garzorz-Stark1, F Lauffer1, L Krause2, O Groß3, C Traidl-Hoffmann4, F Theis2, C SchmidtWeber5, T Biedermann1, S Eyerich5 and K Eyerich1 1 Department of Dermatology and Allergy, Technical University of Munich, Munich, Germany, 2 Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany, 3 Technical University of Munich, Department of Clinical Chemistry, Munich, Germany, 4 UNIKAT, Technical University of Augsburg, Munich, Germany and 5 ZAUM e Center of Allergy and Environment, Technical University of Munich and Hemholtz Center, Munich, Germany A standardized human model to study early pathogenic events in psoriasis is missing. Activation of Toll-like receptor 7/8 by topical application of imiquimod is the most commonly used mouse model of psoriasis. Here, we investigated the potential of a human imiquimod patch test model to resemble human psoriasis. We demonstrate imiquimod induces a monomorphic and self-limited inflammatory response independent of the disease background. The clinical and histologic phenotype as well as transcriptome of imiquimodinduced inflammation resembles an acute contact dermatitis rather than psoriasis. Nevertheless, the imiquimod model mimics hallmarks of psoriasis. Plasmacytoid dendritic cells (pDC) are primary sensors of imiquimod, responding with stress signals and pro-inflammatory cytokine production. This cascade results in a Th17 immune response with IL-23 as a key driver. In a proof-of-concept setting, systemic treatment with ustekinumab dramatically diminished the imiquimod-induced inflammation. Taken together, in humans imiquimod induces contact dermatitis with the unicity that pDC are the primary sensors, leading to an IL23/Th17 deviation. Despite these shortcomings, the human imiquimod model might be useful to investigate early pathogenic events and prove molecular concepts in psoriasis.
www.jidonline.org S275
485
Lichen planus pemphigoides is a chimera of lichen planus and bullous pemphigoid F Solimani1, A Schmidt2, R Eming1, M Hertl1 and T Schmidt1 1 Department of Dermatology and Allergology, Philipps-University Marburg, Marburg, Germany and 2 Department of Pathology, Philipps-University Marburg, Marburg, Germany Occasionally, lichen planus (LP), a common chronic inflammatory skin disorder of unknown etiology transforms into a bullous phenotype reminiscent of bullous pemphigoid (BP) and is associated with IgG autoantibodies against BP180. We here studied three female patients with LPP with regard to peripheral blood T cell recognition of BP180. LPP patient 1 (P1) showed lichenoid striae of the buccal mucosa, polygonal lichenoid papules on the dorsa of the extremities, C3 deposits at the dermal-epidermal BMZ by DIF and IgG against BP180 (128 RE/ml) by ELISA. Patient P2 showed tense bullae, partly on lichenoid plaques on the trunk, C3 deposits at the dermal-epidermal BMZ by DIF and IgG against BP180 (65 RE/ml) by ELISA. Patient P3 had lichenoid erosions of the buccal mucosa and extensive polygonal papules on the trunk and extremities, C3 deposits at the dermal-epidermal BMZ (DIF), and IgG against BP180 (577 RE/ml) by ELISA. All the three LPP patients showed a mixed Th1/Th2 cell infiltrate of lesional skin (CD3+/T-bet+ 45.3% 7.44, CD4+/GATA3+ 68.8% 6.22) which was accompanied by a mixed peripheral blood Th1/Th2 cellular response to the immunodominant NH2 of BP180 (Elispot analysis: ratio number of IFNg+/IL-5+ spots 2.87 2.03). In comparison, the Th1/Th2 ratio was significantly higher in LP (Elispot analysis: ratio number of IFNg+/IL-5+ spots 13.1 10.03) and similar in BP (Elispot analysis: ratio number of IFNg+/IL5+ spots 5.6 4.38). Thus, LPP shares features of LP (Th1-driven) and BP (Th2-dominated).
Optimization of cytokine milieu to reproduce atopic dermatitis-related gene expression in HaCaT keratinocytes J Roh1, H Kim1, J Baek1, H Ryu1, S Lee1 and Y Jung2 1 Dermaology, Gachon University Gil Medical Center, Incheon, Korea (the Republic of) and 2 Microbiology, Gachon University School of Medicine, Incheon, Korea (the Republic of) Although atopic dermatitis (AD) has been characterized by predominant Th2 cytokine production, Th1 cells are also involved in AD pathogenesis. This study was to identify in vitro cytokine milieu that can accurately reproduce the expression profile of genes important in AD pathogenesis. mRNA levels of CCL22 (MDC), CCL17 (TARC), IL5, IL13, FLG, and LOR were evaluated by qRT-PCR in skin samples from six AD patients, 12 healthy controls, and in HaCaT cells cultured in the presence of various combinations of Th1 (TNF-a, 10 ng/ml; and/ or IFN-g, 10 ng/ml) and Th2 (IL-4, 50 ng/ml) cytokines. Cytokine stimulation effects on HaCaT cell growth were examined by phase-contrast microscopy and water-soluble tetrazolium salt assay. HaCaT cell viability was not altered by treatment with TNF-a, IFN-g, or IL-4 alone, or by treatment with TNF-a and IL-4 in combination, while TNF-a and IFN-g in combination significantly reduced the viability. We compared the gene expression profiles of human AD skin samples and in vitro HaCaT cells in various cytokine treatment settings. Expression of Th2 genes (CCL22, CCL17, IL5 and IL13) was increased in human AD skin lesions, while cornified cell envelop-related genes, FLG and LOR, was reduced. Interestingly, similar HaCaT cell gene expression profiles were observed when stimulated with Th1 cytokines, TNF-a and/ or IFN-g, but not with Th2 cytokine, IL-4, implying that Th1 stimulation is required to reproduce the AD-like features in HaCaT cells. Current data suggest that Th1 and Th2 cytokines do not function dichotomously, and a complicated inflammatory network drives ADlike changes. Therefore, in further in vitro experiments using HaCaT cells to study AD-related genes, stimulation with various cytokine combinations not limited to Th2 polarization would be necessary for optimal gene expression levels.
486
487
Short course model of oxazolone-induced dermatitis in the nape of mice: Characterisation and modulation with topical drugs M Pont, C Carcasona, A Blanco, G Tarraso´n, N Godessart and A Gavalda` R&D Centre, Almirall S.A., Barcelona, Spain Oxazolone (Oxa)-induced chronic skin inflammation in mice is commonly used as a model of atopic dermatitis. The disease can be evoked in the ears, the back or the nape in different strains of mice. The duration of the assay depends on the number of Oxa challenges, which ranges from 3 to 12. We aimed to develop a short course model to be used for the screening of anti-inflammatory drugs using pruritus as an early readout of efficacy. Following sensitisation, C57BL/6 mice received Oxa challenges in the nape every other day starting on day 8 post-sensitisation. Histological evaluation showed increased inflammation up to challenge 3, and subepidermal fibrosis from challenge 5. Thus, we selected the 3-challenge model for further studies. After challenge 3, lesional skin showed moderate epidermal hyperplasia with hyperkeratosis, scabs and a mixed cellular dermatitis. Lesions displayed skin barrier impairment and increased expression of the Th2 cytokines, IL-4 and IL-13. Enhanced pruritus was also evident after the 3rd challenge. For a pharmacological characterisation of the model, we used commercial formulations of corticosteroids (clobetasol, betamethasone and hydrocortisone) and tacrolimus. Treatments were applied once daily from day 8 to 12. Skin fold thickness and TEWL were measured daily. Pruritus was assessed on day 12, 2 h after challenge. On day 13 skin biopsies were collected for analysis. All treatments significantly reversed the induced histopathological changes. Cytokine levels decreased and TEWL improved. Except for hydrocortisone, the rest of treatments inhibited scratching over 65%. For some parameters, the effect of corticosteroids correlated with their potency. Skin atrophy was evident with clobetasol and betamethasone. Overall, the model described here replicates the main features of atopic dermatitis, including pruritus, and responds to topical drugs used in the clinical practice. The duration of the model makes it suitable for the screening of novel anti-inflammatory drugs.
Phosphorylated MLKL and cleaved caspase8 are useful for early diagnosis of Stevens-Johnson syndrome and toxic epidermal necrolysis A Hasegawa, M Maehara, N Yamazaki, Y Shigehara and R Abe Niigata University, Niigata, Japan Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening cutaneous adverse drug reactions. It is difficult to distinguish erythema multiforme (EM) and SJS/TEN in early stage. Although keratinocytes have been suggested to die as apoptosis, our recent study revealed that necroptosis, programmed necrosis, also contribute to keratinocyte death in SJS/TEN. We hypothesized that analyzing the form of keratinocyte death is useful for early diagnosis of SJS/TEN. The skin samples were obtained from non-bullous erythematus lesion of the patient who suspected drug eruption in the acute phase. We included 4 patients diagnosed as SJS/TEN and 10 patients diagnosed as EM. Anti-phosphorylated MLKL (pMLKL) antibody was used to detect necroptosis, and anti-cleaved caspase8 (cCas8) antibody was used to detect apoptosis. Both of pMLKL(+) cell (3.782.70%) and cCas8(+) cell (17.84.94%) were shown in the 4 SJS/TEN patients. In EM patients, pMLKL(+) cell was shown in 4 patients (1.472.56%) and cCas8(+) cell was shown in 2 patients (1.092.15%). In 4 all cases diagnosed as SJS/TEN, both of pMLKL and cCas8 cell were positive. In contrast, it was only 1 case that both pMLKL and cCas8 were positive and either pMLKL or cCas8 was positive in 4 cases in EM. The number of pMLKL(+) plus cCas8(+) cell were statistically higher than the number of death keratinocyte detected by HE staining (SJS/TEN: 13.425.39%)(EM: 1.021.38%). The results revealed that both of necroptosis and apoptosis are occurred in keratinocyte death in SJS/TEN. Although, in EM, both of necroptosis and apoptosis are shown, the amount of cell death is lower than SJS/TEN. It is suggested that the frequency of cell death is important for differentiation between SJS and EM. pMLKL and cCas8 may be able to detect earlier stages of cell death than HE staining, and it is expected that pMLKL and cCas8 can be the marker of SJS/TEN.
488
489
Continuous sampling of immune cells in the skin by dermal open flow microperfusion T Birngruber1, B Prietl2, M Bodenlenz1, P Florian4, A Subramaniam3, S Kainz1, G Rauter1, TR Pieber1,2 and F Sinner1,2 1 HEALTH - Institute for Biomedicine and Health Sciences, Joanneum Research Forschungsgesellschaft mbH, Graz, Austria, 2 Division of Endocrinology and Diabetology, Medical University of Graz, Graz, Austria, 3 Sanofi-Genzyme, Framingham, MA and 4 Sanofi, Frankfurt, Germany Immune cells and biologic response modulators like cytokines play a major role in immune responses in the skin. Currently, skin inflammation is studied by highly invasive biopsy methods in clinical studies. We investigated whether the minimally invasive dermal Open Flow Microperfusion (dOFM) enables continuous sampling of immune cells from inflamed and healthy skin in a psoriasis rat model. Psoriasis-like skin inflammation was induced on a skin test site of Sprague-Dawley rats by applying a daily dose of topical imiquimod. A daily dose of oral Dexamethasone was additionally administered to a subgroup of rats to prevent the imiquimod-induced skin inflammation. After 8 days, dermal interstitial fluid was sampled from imiquimod-treated skin and from untreated skin with dOFM sampling probes. The samples were stained with antibodies which were specific for the cell subtypes and analyzed by flow cytometry (FACS). A well-quantifiable amount of CD45-positive cells (granulocytes, lymphocytes, monocytes) was found in the dOFM samples. Staining also identified CD3 positive T-cells, CD8 positive cytotoxic T-cells, CD4 positive T-helper cells and IL17 positive cells. The amount and type of cells in dOFM samples derived from inflamed skin differed from that of untreated skin and of the skin of Dexamethasone-treated animals. This study illustrates the feasibility of the minimally-invasive sampling method dOFM to continuously sample immune cells from inflamed and healthy living tissue. dOFM has the potential to enable a combined investigation of locally released cytokines and the involved immune cells in clinical and preclinical in vivo studies of inflammatory diseases.
TLR7/8 agonists stimulate plasmacytoid dendritic cells to initiate a Th17-deviated acute contact dermatitis in humans N Garzorz-Stark1, F Lauffer1, L Krause2, O Groß3, C Traidl-Hoffmann4, F Theis2, C SchmidtWeber5, T Biedermann1, S Eyerich5 and K Eyerich1 1 Department of Dermatology and Allergy, Technical University of Munich, Munich, Germany, 2 Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany, 3 Technical University of Munich, Department of Clinical Chemistry, Munich, Germany, 4 UNIKAT, Technical University of Augsburg, Munich, Germany and 5 ZAUM e Center of Allergy and Environment, Technical University of Munich and Hemholtz Center, Munich, Germany A standardized human model to study early pathogenic events in psoriasis is missing. Activation of Toll-like receptor 7/8 by topical application of imiquimod is the most commonly used mouse model of psoriasis. Here, we investigated the potential of a human imiquimod patch test model to resemble human psoriasis. We demonstrate imiquimod induces a monomorphic and self-limited inflammatory response independent of the disease background. The clinical and histologic phenotype as well as transcriptome of imiquimodinduced inflammation resembles an acute contact dermatitis rather than psoriasis. Nevertheless, the imiquimod model mimics hallmarks of psoriasis. Plasmacytoid dendritic cells (pDC) are primary sensors of imiquimod, responding with stress signals and pro-inflammatory cytokine production. This cascade results in a Th17 immune response with IL-23 as a key driver. In a proof-of-concept setting, systemic treatment with ustekinumab dramatically diminished the imiquimod-induced inflammation. Taken together, in humans imiquimod induces contact dermatitis with the unicity that pDC are the primary sensors, leading to an IL23/Th17 deviation. Despite these shortcomings, the human imiquimod model might be useful to investigate early pathogenic events and prove molecular concepts in psoriasis.
www.jidonline.org S275