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336 Massive expansion of clonotypic and polycytotoxic CD8+ T cells in toxic epidermal necrolysis
ABSTRACTS | Inflammation, Immunity and Infection 334
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Role of RIP kinase signalling in the development of skin inflammation in mice with keratinocyte-specific IKK deficiency S Kumari, T Van and M Pasparakis Institute for Genetics, CECAD Research Center, Cologne, Germany The skin forms a life-sustaining structural and immunological barrier at the interface between the body and the environment. Skin immune homeostasis depends on tightly regulated interactions between epithelial, stromal and immune cells and commensal and environmental microorganisms. IKK/NF-kappaB signaling controls inflammation and cell death and plays an important role in the regulation of tissue homeostasis. Mice with epidermis-specific deletion of IKK2 (IKK2E-KO) or NEMO (NEMOE-KO) developed a progressive severe TNF-dependent inflammatory skin disease resulting in early postnatal death, suggesting that IKK/NF-kB signalling in keratinocytes critically controls skin immune homeostasis. We investigated the role of apoptotic and necroptotic cell death in the pathogenesis of skin inflammation in mice with keratinocyte-specific IKK deficiency. We found that IKK2E-KO mice crossed into the RIPK1D138N genetic background were fully protected from skin lesion developments, demonstrating that RIPK1 kinase activity is essential for the development of skin inflammation in this model. In addition, RIPK3 or MLKL deficiency strongly delayed and ameliorated the skin lesions in IKK2E-KO mice, suggesting that keratinocyte necroptosis plays an important role in triggering the inflammatory skin lesions. Importantly, deficiency of both FADD and RIPK3 fully protected IKK2E-KO mice from skin inflammation, showing that combined inhibition of both apoptosis and necroptosis fully prevents the disease. These results show that epidermisspecific IKK deficiency triggers skin inflammation by sensitizing keratinocytes to TNF-mediated cell death by both apoptosis and necroptosis.
Use of the hurley staging system for the assessment of hidradenitis suppurativa disease severity in 2 phase 3 clinical trials A Martorell1, AB Gottlieb2, J Maa3 and GD Mulder3 1 Hospital of Manises, Valencia, Spain, 2 Tufts University School of Medicine, Boston, MA and 3 AbbVie, Inc., North Chicago, IL The Hurley staging system is commonly used to classify the severity of hidradenitis suppurativa (HS). Hurley staging (I to III) is non-quantitative, does not assess the extent of inflammation, and may not be suitable for evaluating treatment effect. This analysis pooled baseline data from 2 phase 3 clinical trials to assess the relationship between Hurley staging and objective disease severity measures. Both studies (PIONEER I and II) enrolled adults with at least a 1-year history of moderate-to-severe HS. Patients were randomized to receive treatment with adalimumab or placebo for 12 weeks (Period A). Baseline data from the 2 trials were integrated and stratified by baseline Hurley stage. Mean lesion counts, Patient’s Global Assessment of Skin Pain Numeric Rating Scale (NRS) scores (0 to 10, least to worst), and Dermatology Life Quality index (DLQI) scores were calculated. The integrated analysis included 633 randomized patients, of which 339 had Hurley stage II HS and 294 had Hurley stage III HS. At baseline, patients with Hurley stage II HS had an average of 1.72.5 abscesses, 9.99.5 inflammatory nodules, and 1.82.9 draining fistulas, and an AN count of 11.610.3. Their mean skin pain NRS scores and DLQI scores were 4.32.6 and 14.17.2, respectively. Patients classified as having more severe, Hurley stage III disease had similar mean baseline counts of abscesses (3.43.9), inflammatory nodules (10.711.7), draining fistulas (6.05.5), and AN count (14.113.1). The mean skin pain NRS (5.32.6) and DLQI (16.77.0) scores for patients with Hurley stage III HS were also similar to those patients with Hurley stage II. Baseline Hurley staging did not correspond with differences in lesion counts, skin pain scores, or quality of life scores among 633 patients in 2 clinical trials. Newer, more quantitative methods of HS assessment may be preferred.
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Massive expansion of clonotypic and polycytotoxic CD8+ T cells in toxic epidermal necrolysis AP Villani2, A Rozie`res1, B Bensaı¨d1, F Albert1, V Mutez1, M Pallardy3, J Maryanski4, J Nicolas1, O Kanagawa1 and M Vocanson1 1 INSERM U1111 - CIRI, Lyon, France, 2 Dermatology department - University hospital E. Herriot, Lyon, France, 3 INSERM UMR 996, Chaˆtenay-Mallabry, Paris, France and 4 University, Nice Sophia Antipolis, Nice, France Toxic epidermal necrolysis (TEN) is a life-threatening and blistering adverse drug reaction, characterized by massive epidermal necrosis. Diverse studies have reported that the onset of TEN correlates with skin infiltration by cytotoxic lymphocytes (T, NK cells) and inflammatory monocytes. To further characterize the phenotype of skin-infiltrating lymphocytes at the acute phase of TEN, we conducted a prospective study on the blood and blister fluids from 14 TEN patients, using flow and mass cytometry, as well as next generation TCR sequencing. Our results confirm that conventional CD8+ T cells (CD45+TCRab+CD8b+) and at a less extend of CD4+ T cells, were the main leucocyte subsets found in recent TEN blisters. Consequently, the CD4/CD8 ratio was inversed in blisters (mean: 0.8) compared to blood (mean: 2). However, we failed to repeatedly detect NK (CD45+TCRab+NKP46+) or NKT cells (CD45+TCRab+TCRVa24+) in TEN blisters. Strikingly, deep sequencing of the T cell receptor CDR3 repertoire revealed massive expansion of rare CDR3 clonotypes in blister cells of 6 TEN patients, which were confirmed at TCR-Vb usage level by flow cytometry. Over-represented TCR-Vb+ blister cells were mainly effector memory CD8+CD45RA-CD27+ T cells and displayed a poly-cytotoxic phenotype since they co-expressed Granulysin, Granzyme B, Granzyme A, Perforin and TWEAK, as demonstrated by mass cytometry. By comparison, the TCR repertoire bias and poly-cytotoxic phenotype were far less marked in the cells extracted from the inflammed skin of DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms) and maculopapular exanthema patients. Our results higlight a massive expansion of clonotypic and poly-cytotoxic CD8+ T cells in the blisters of TEN patients, which could explain the severity of this life-threatening disease.
The double-stranded RNA induces IL-33 expression through activation of EGFR, ERK, and p38 in normal human epidermal keratinocytes M Jin1, M Komine1, H Tsuda1, S Tominaga2 and M Ohtsuki1 1 Dermatology, Jichi Medical University, Shimotsuke-shi, Japan and 2 Biochemistry, Jichi Medical University, Shimotsukeshi, Japan The skin provides the first line of defense against external stimulus. Various environmental stimuli influence epidermis, including ultraviolet (UV) irradiation, virus, bacteria, and mechanical stimuli. IL-33 is a member of IL-1 cytokine family that constitutively expressed in endothelial cells and epithelial cells of tissues. IL-33 can enhance Th2 immune reaction and work as a nuclear factor. Viral infection is known to induce Toll-like receptor 3 (TLR3) activation through double-stranded RNA (dsRNA), and Poly I:C is a synthetic dsRNA frequently used as a TLR3 ligand. We aimed to investigate whether TLR3 activation induces IL-33 expression in normal human epidermal keratinocytes (NHEKs) and examine the mechanism of its induction. NHEKs were stimulated with Poly I:C, the ligand for TLR3, at different concentrations and for different time periods. The expression levels of mRNA for IL33, IFN-a, and IFN-b were assayed using quantitative real-time PCR (TaqMan Gene Expression Assay). The expression of IL-33 was induced in NHEKs with the stimulation of Poly I:C and suppressed by the addition of TLR3 inhibitor. The induction of IL-33 by Poly I:C was doseand time- dependent. Induction of IL-33 was stronger when cells were confluent than when they were subconfluent. The expression of IL-33 stimulated by Poly I:C peaked at 8 hours, was suppressed at 24 through 48 hours, and was induced again at 96 hours. IFNa and IFNb were strongly induced by Poly I:C, which peaked at 48 and 4 hours, respectively. We used antiIFNb antibody and anti-IFNa antibody aiming at blocking each signal, but the induction of IL33 in 8, 96 hours were not suppressed. Inhibitors for phosphorylation of extracellular signal regulated kinase (ERK), epidermal growth factor receptor (EGFR), and p38 inhibited the induction of IL-33 efficiently. These results suggested that IL-33 was induced through EGFR, ERK and p38 activation, but not through the production of IFNs by Poly I:C.
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Basophils and mast cells are crucial for reactions due to epicutaneous sensitization to ovalbumin R Yu1, K Igawa1, Y Handa1, T Munetsugu1, T Satoh2 and H Yokozeki1 1 Dermatology, Tokyo Medical and Dental University, Bunkyo-ku, Japan and 2 Dermatology, National Defense Medical College, Tokorozawa, Japan The prevalence of food allergies worldwide has increased recently. Epicutaneous sensitization to antigen could be a method to study food allergy. To clarify the mechanisms of food allergy, we established a mouse model of epicutaneous sensitization using ovalbumin. BALB/c mice were sensitized by three-time application of ovalbumin to tape-stripped skin (1-week sensitization at 2-week intervals) and oral challenge of ovalbumin undertaken. Rectal temperature was monitored. Blood and tissue (skin and jejunum) of challenged mice were taken. Numbers of mast cells (MCs) and basophils were counted. Serum and/or tissue levels of ovalbuminspecific IgE and IgG antibodies and several cytokines were measured using enzyme-linked immunoassay kits. MC- and basophil-depletion experiments were undertaken. In ovalbuminepicutaneous sensitized and orally challenged mice, systemic anaphylaxis (as evidenced by reduced rectal temperature) was observed. Levels of ovalbumin-specific IgE and IgG antibodies were increased in these mice, as were increased number of MCs and basophils. Serum levels of mast cell protease 1 were increased significantly. Basophil- and MC-depletion experiments revealed that they both participate in reactions. Increased production of thymic stromal lymphopoietin (TSLP) at skin sites of OVA-sensitization was noted. We speculate that TSLP produced from epidermal cells during antigen sensitization can enable basophils to promote a T-helper (Th)2 immune reaction, leading to and systemic anaphylaxis by antigenspecific IgE-bearing MCs. This TSLPebasophilseMC axis could be a novel therapeutic target against food allergy.
S218 Journal of Investigative Dermatology (2016), Volume 136
Swertiamarin: a promising new treatment for Rosacea C Bezivin1, J Attia3, M De Torsiac1 and E Loing2 1 Lucas Meyer Cosmetics- IFF, Champlan, France, 2 Lucas Meyer Cosmetics-IFF, Quebec, QC, Canada and 3 Lucas Meyer CosmeticsIFF, Toulouse, France Rosacea is a common chronic inflammatory skin disease that occurs at any age and characterized by skin inflammation, change to the skin’s connective tissue and dilatation of the blood vessels. Some of these different symptoms are linked to the TLR/NF-kB pathway: interleukins and MMPs productions. Swertiamarin, a secoiridoid glycoside, has been previously studied for its capacity to inhibit NF-kB pathway in arthritis and regulated proinflammatory cytokines and enzymes. Due to this activity, an evaluation of the molecule on Rosacea has been carried out on the different key parameters (inflammation, extracellular matrix modulation) implied in the disease. In vitro evaluation of the activity of swertiamarin has been done on the inflammation parameter, IL-6 release, using immortalized HaCaT human cell line and dosed by Elisa assay. Then, effects on fibronectin and versican synthesis were investigated on normal human dermal fibroblasts. Clinical evaluation of the molecule was performed on 25 women aged between 46 and 65 years old for 28 days by using the skin analysis imaging system able to quantify red areas. In vitro, swertiamarin decreased the release of IL-6, a proinflammatory cytokine, by 46% at the concentration of 0.0025%. Moreover, the treatment increased fibronectin synthesis by 171% at the concentration of 0.005% and versican by 67% at the concentration of 0.0025% compared to the control. These data were confirmed by clinical study: 0.025% of pure swertiamarin improved the main sign of Rosacea after 28 days of treatment, with a significant decrease of 12% of the skin redness. Swertiamarin is able to increase dermal matrix proteins synthesis and decrease the release of cytokines involved in the inflammatory TLR/NF-kB pathway. Swertiamarin has proven to be a good candidate to fight against the symptoms of Rosacea.
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Role of RIP kinase signalling in the development of skin inflammation in mice with keratinocyte-specific IKK deficiency S Kumari, T Van and M Pasparakis Institute for Genetics, CECAD Research Center, Cologne, Germany The skin forms a life-sustaining structural and immunological barrier at the interface between the body and the environment. Skin immune homeostasis depends on tightly regulated interactions between epithelial, stromal and immune cells and commensal and environmental microorganisms. IKK/NF-kappaB signaling controls inflammation and cell death and plays an important role in the regulation of tissue homeostasis. Mice with epidermis-specific deletion of IKK2 (IKK2E-KO) or NEMO (NEMOE-KO) developed a progressive severe TNF-dependent inflammatory skin disease resulting in early postnatal death, suggesting that IKK/NF-kB signalling in keratinocytes critically controls skin immune homeostasis. We investigated the role of apoptotic and necroptotic cell death in the pathogenesis of skin inflammation in mice with keratinocyte-specific IKK deficiency. We found that IKK2E-KO mice crossed into the RIPK1D138N genetic background were fully protected from skin lesion developments, demonstrating that RIPK1 kinase activity is essential for the development of skin inflammation in this model. In addition, RIPK3 or MLKL deficiency strongly delayed and ameliorated the skin lesions in IKK2E-KO mice, suggesting that keratinocyte necroptosis plays an important role in triggering the inflammatory skin lesions. Importantly, deficiency of both FADD and RIPK3 fully protected IKK2E-KO mice from skin inflammation, showing that combined inhibition of both apoptosis and necroptosis fully prevents the disease. These results show that epidermisspecific IKK deficiency triggers skin inflammation by sensitizing keratinocytes to TNF-mediated cell death by both apoptosis and necroptosis.
Use of the hurley staging system for the assessment of hidradenitis suppurativa disease severity in 2 phase 3 clinical trials A Martorell1, AB Gottlieb2, J Maa3 and GD Mulder3 1 Hospital of Manises, Valencia, Spain, 2 Tufts University School of Medicine, Boston, MA and 3 AbbVie, Inc., North Chicago, IL The Hurley staging system is commonly used to classify the severity of hidradenitis suppurativa (HS). Hurley staging (I to III) is non-quantitative, does not assess the extent of inflammation, and may not be suitable for evaluating treatment effect. This analysis pooled baseline data from 2 phase 3 clinical trials to assess the relationship between Hurley staging and objective disease severity measures. Both studies (PIONEER I and II) enrolled adults with at least a 1-year history of moderate-to-severe HS. Patients were randomized to receive treatment with adalimumab or placebo for 12 weeks (Period A). Baseline data from the 2 trials were integrated and stratified by baseline Hurley stage. Mean lesion counts, Patient’s Global Assessment of Skin Pain Numeric Rating Scale (NRS) scores (0 to 10, least to worst), and Dermatology Life Quality index (DLQI) scores were calculated. The integrated analysis included 633 randomized patients, of which 339 had Hurley stage II HS and 294 had Hurley stage III HS. At baseline, patients with Hurley stage II HS had an average of 1.72.5 abscesses, 9.99.5 inflammatory nodules, and 1.82.9 draining fistulas, and an AN count of 11.610.3. Their mean skin pain NRS scores and DLQI scores were 4.32.6 and 14.17.2, respectively. Patients classified as having more severe, Hurley stage III disease had similar mean baseline counts of abscesses (3.43.9), inflammatory nodules (10.711.7), draining fistulas (6.05.5), and AN count (14.113.1). The mean skin pain NRS (5.32.6) and DLQI (16.77.0) scores for patients with Hurley stage III HS were also similar to those patients with Hurley stage II. Baseline Hurley staging did not correspond with differences in lesion counts, skin pain scores, or quality of life scores among 633 patients in 2 clinical trials. Newer, more quantitative methods of HS assessment may be preferred.
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Massive expansion of clonotypic and polycytotoxic CD8+ T cells in toxic epidermal necrolysis AP Villani2, A Rozie`res1, B Bensaı¨d1, F Albert1, V Mutez1, M Pallardy3, J Maryanski4, J Nicolas1, O Kanagawa1 and M Vocanson1 1 INSERM U1111 - CIRI, Lyon, France, 2 Dermatology department - University hospital E. Herriot, Lyon, France, 3 INSERM UMR 996, Chaˆtenay-Mallabry, Paris, France and 4 University, Nice Sophia Antipolis, Nice, France Toxic epidermal necrolysis (TEN) is a life-threatening and blistering adverse drug reaction, characterized by massive epidermal necrosis. Diverse studies have reported that the onset of TEN correlates with skin infiltration by cytotoxic lymphocytes (T, NK cells) and inflammatory monocytes. To further characterize the phenotype of skin-infiltrating lymphocytes at the acute phase of TEN, we conducted a prospective study on the blood and blister fluids from 14 TEN patients, using flow and mass cytometry, as well as next generation TCR sequencing. Our results confirm that conventional CD8+ T cells (CD45+TCRab+CD8b+) and at a less extend of CD4+ T cells, were the main leucocyte subsets found in recent TEN blisters. Consequently, the CD4/CD8 ratio was inversed in blisters (mean: 0.8) compared to blood (mean: 2). However, we failed to repeatedly detect NK (CD45+TCRab+NKP46+) or NKT cells (CD45+TCRab+TCRVa24+) in TEN blisters. Strikingly, deep sequencing of the T cell receptor CDR3 repertoire revealed massive expansion of rare CDR3 clonotypes in blister cells of 6 TEN patients, which were confirmed at TCR-Vb usage level by flow cytometry. Over-represented TCR-Vb+ blister cells were mainly effector memory CD8+CD45RA-CD27+ T cells and displayed a poly-cytotoxic phenotype since they co-expressed Granulysin, Granzyme B, Granzyme A, Perforin and TWEAK, as demonstrated by mass cytometry. By comparison, the TCR repertoire bias and poly-cytotoxic phenotype were far less marked in the cells extracted from the inflammed skin of DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms) and maculopapular exanthema patients. Our results higlight a massive expansion of clonotypic and poly-cytotoxic CD8+ T cells in the blisters of TEN patients, which could explain the severity of this life-threatening disease.
The double-stranded RNA induces IL-33 expression through activation of EGFR, ERK, and p38 in normal human epidermal keratinocytes M Jin1, M Komine1, H Tsuda1, S Tominaga2 and M Ohtsuki1 1 Dermatology, Jichi Medical University, Shimotsuke-shi, Japan and 2 Biochemistry, Jichi Medical University, Shimotsukeshi, Japan The skin provides the first line of defense against external stimulus. Various environmental stimuli influence epidermis, including ultraviolet (UV) irradiation, virus, bacteria, and mechanical stimuli. IL-33 is a member of IL-1 cytokine family that constitutively expressed in endothelial cells and epithelial cells of tissues. IL-33 can enhance Th2 immune reaction and work as a nuclear factor. Viral infection is known to induce Toll-like receptor 3 (TLR3) activation through double-stranded RNA (dsRNA), and Poly I:C is a synthetic dsRNA frequently used as a TLR3 ligand. We aimed to investigate whether TLR3 activation induces IL-33 expression in normal human epidermal keratinocytes (NHEKs) and examine the mechanism of its induction. NHEKs were stimulated with Poly I:C, the ligand for TLR3, at different concentrations and for different time periods. The expression levels of mRNA for IL33, IFN-a, and IFN-b were assayed using quantitative real-time PCR (TaqMan Gene Expression Assay). The expression of IL-33 was induced in NHEKs with the stimulation of Poly I:C and suppressed by the addition of TLR3 inhibitor. The induction of IL-33 by Poly I:C was doseand time- dependent. Induction of IL-33 was stronger when cells were confluent than when they were subconfluent. The expression of IL-33 stimulated by Poly I:C peaked at 8 hours, was suppressed at 24 through 48 hours, and was induced again at 96 hours. IFNa and IFNb were strongly induced by Poly I:C, which peaked at 48 and 4 hours, respectively. We used antiIFNb antibody and anti-IFNa antibody aiming at blocking each signal, but the induction of IL33 in 8, 96 hours were not suppressed. Inhibitors for phosphorylation of extracellular signal regulated kinase (ERK), epidermal growth factor receptor (EGFR), and p38 inhibited the induction of IL-33 efficiently. These results suggested that IL-33 was induced through EGFR, ERK and p38 activation, but not through the production of IFNs by Poly I:C.
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Basophils and mast cells are crucial for reactions due to epicutaneous sensitization to ovalbumin R Yu1, K Igawa1, Y Handa1, T Munetsugu1, T Satoh2 and H Yokozeki1 1 Dermatology, Tokyo Medical and Dental University, Bunkyo-ku, Japan and 2 Dermatology, National Defense Medical College, Tokorozawa, Japan The prevalence of food allergies worldwide has increased recently. Epicutaneous sensitization to antigen could be a method to study food allergy. To clarify the mechanisms of food allergy, we established a mouse model of epicutaneous sensitization using ovalbumin. BALB/c mice were sensitized by three-time application of ovalbumin to tape-stripped skin (1-week sensitization at 2-week intervals) and oral challenge of ovalbumin undertaken. Rectal temperature was monitored. Blood and tissue (skin and jejunum) of challenged mice were taken. Numbers of mast cells (MCs) and basophils were counted. Serum and/or tissue levels of ovalbuminspecific IgE and IgG antibodies and several cytokines were measured using enzyme-linked immunoassay kits. MC- and basophil-depletion experiments were undertaken. In ovalbuminepicutaneous sensitized and orally challenged mice, systemic anaphylaxis (as evidenced by reduced rectal temperature) was observed. Levels of ovalbumin-specific IgE and IgG antibodies were increased in these mice, as were increased number of MCs and basophils. Serum levels of mast cell protease 1 were increased significantly. Basophil- and MC-depletion experiments revealed that they both participate in reactions. Increased production of thymic stromal lymphopoietin (TSLP) at skin sites of OVA-sensitization was noted. We speculate that TSLP produced from epidermal cells during antigen sensitization can enable basophils to promote a T-helper (Th)2 immune reaction, leading to and systemic anaphylaxis by antigenspecific IgE-bearing MCs. This TSLPebasophilseMC axis could be a novel therapeutic target against food allergy.
S218 Journal of Investigative Dermatology (2016), Volume 136
Swertiamarin: a promising new treatment for Rosacea C Bezivin1, J Attia3, M De Torsiac1 and E Loing2 1 Lucas Meyer Cosmetics- IFF, Champlan, France, 2 Lucas Meyer Cosmetics-IFF, Quebec, QC, Canada and 3 Lucas Meyer CosmeticsIFF, Toulouse, France Rosacea is a common chronic inflammatory skin disease that occurs at any age and characterized by skin inflammation, change to the skin’s connective tissue and dilatation of the blood vessels. Some of these different symptoms are linked to the TLR/NF-kB pathway: interleukins and MMPs productions. Swertiamarin, a secoiridoid glycoside, has been previously studied for its capacity to inhibit NF-kB pathway in arthritis and regulated proinflammatory cytokines and enzymes. Due to this activity, an evaluation of the molecule on Rosacea has been carried out on the different key parameters (inflammation, extracellular matrix modulation) implied in the disease. In vitro evaluation of the activity of swertiamarin has been done on the inflammation parameter, IL-6 release, using immortalized HaCaT human cell line and dosed by Elisa assay. Then, effects on fibronectin and versican synthesis were investigated on normal human dermal fibroblasts. Clinical evaluation of the molecule was performed on 25 women aged between 46 and 65 years old for 28 days by using the skin analysis imaging system able to quantify red areas. In vitro, swertiamarin decreased the release of IL-6, a proinflammatory cytokine, by 46% at the concentration of 0.0025%. Moreover, the treatment increased fibronectin synthesis by 171% at the concentration of 0.005% and versican by 67% at the concentration of 0.0025% compared to the control. These data were confirmed by clinical study: 0.025% of pure swertiamarin improved the main sign of Rosacea after 28 days of treatment, with a significant decrease of 12% of the skin redness. Swertiamarin is able to increase dermal matrix proteins synthesis and decrease the release of cytokines involved in the inflammatory TLR/NF-kB pathway. Swertiamarin has proven to be a good candidate to fight against the symptoms of Rosacea.