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314 PepFect6-miRNA-146a nanocomplexes inhibit inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis
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T cell epitopes of BPAG2 antigen and the epitope-based pathogenesis of bullous pemphigoid J Zhang, H Fang, E Dang, Q Li and G Wang Dermatology, Xijing Hospital, Xi’an, China Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease characterized by autoantibodies directed against BPAG2, a transmembrane protein of epidermal basal cells. The pathogenesis of BP has not been elucidated completely, further researches are needed to understand the pathogenesis of BP. The purpose of this study is to identify the T cell epitope of BPAG2 and to understand the course of humoral immune response in BP. As IL-4 secretion is a marker of Th2 antigen-specific activation, PBMCs from 43 BP patients were collected and tested for IL-4 cytokine responses to a complete panel of 22 overlapping peptides spanning the BP180-NC16A domain by ELISPOT assay. Results revealed that IL-4 responses were significantly stronger for three peptides, P14, P18 and P21, in 27 BP patients, which representative aa492 to aa513 in NC16A domain. We next studied the function of three epitopes. Flow cytometry analysis showed that those three epitopes were able to stimulated BP patient’s T proliferation. ELISA analysis the levels of sCD23 in PBMCs of BP patients culture supernatant showed that after stimulating by those three epitopes, patients ‘s B cells were activated. Comparing the epitopes of BP patients in progression stage and recovery stage of disease respectively, we found that epitopes were changed in different stage, and the number of epitopes were elevated in the progression stage compared to the recovery stage. Thus, there exists “epitope spreading”, and the appeared new epitopes may be associated with disease recurrence. In conclusion, our research identified three Th2 cell epitopes, those epitopes have the ability to stimulated BP patients’ T cells proliferation and B cells activation pathogenic autoantibodies production. There also existed “epitope spreading” phenomenon, and the appeared new epitopes may be associated with disease recurrence. This results may contribute to the understanding the pathogenesis of BP, providing new ideas and the scientific evidences about the treatment of BP.
A role of spinal glial cells in pruritus of atopic dermatitis model mouse M Tominaga1, K Torigoe2, H Ogawa3 and K Takamori3 1 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan, 2 Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Tokyo, Japan and 3 Department of Dermatology, Juntendo University Graduate School of Medicine, Tokyo, Japan The role of spinal glia in the intractable itch of skin diseases such as atopic dermatitis (AD) remains unclear. Here, we examined antipruritic effects of intrathecal or oral administration of minocycline, an inhibitor of microglial activation, and immunohistochemically examined the distribution of spinal microglia and astrocytes during development of AD-like symptoms in AD-like model NC/Nga mice. A dermatophagoides farinae body (Dfb) ointment-induced AD-like mouse model (Dfb-NC/Nga) was used. Dermatitis was induced by repeated application of Dfb ointment. Dfb-NC/Nga mice were treated by intrathecal administration of saline or minocycline three times a week for 2 weeks. Saline or minocycline was administered orally by syringe twice per day for 3 weeks. Scratching behavior was monitored for 12 h using SCLABA-Real system. Distributions of spinal microglia and astrocytes in the Dfb-NC/Nga mice were examined immunohistochemically with anti-Iba1 and anti-GFAP antibodies, respectively. Dermatitis was induced by application of the Dfb, and numbers of scratching bouts and spinal Iba1+ microglia were increased in Dfb-NC/Nga mice. Intrathecal minocycline dose-dependently reduced scratching bouts, concomitant with decreased number of Iba1+ microglia in Dfb-NC/Nga mice. Intrathecal minocycline improved dermatitis, and oral administration of high-dose minocycline improved dermatitis and showed a tendency to reduce the numbers of scratching bouts and spinal Iba1+ microglia in these mice. In the development of AD, the numbers of microglia and astrocytes were increased in the spinal cord of Dfb-NC/Nga mice. Spinal microglia may be a therapeutic target for the pruritus associated with AD. Spinal astrocytes may be also involved in the pathological process of intractable itch in this model.
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Roles of brain-natriuretic peptide- and gastrin-releasing peptide-responsive neurons in spinal transmission of itchRoles of brain-natriuretic peptide- and gastrin-releasing peptideresponsive neurons in spinal transmission of itch F Kusube1, M Tominaga2, H Kawasaki2, F Yamakura3, H Naito4, H Ogawa5, Y Tomooka1 and K Takamori5 1 Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Katsusika-ku, Japan, 2 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan, 3 Juntendo University Faculty of International Liberal Arts, Tokyo, Japan, 4 Institute of Health and Sports Science & Medicine, Juntendo University, Inzai, Japan and 5 Department of Dermatology, Juntendo University Graduate School of Medicine, Urayasu, Japan Brain-natriuretic peptide (BNP) receptor (Npra)-expressing spinal neurons and gastrin releasing peptide receptor (GRPR)-expressing spinal neurons are critically involved in signaling itch. However, the pathway of spinal itch transmission involving BNP and GRP is still under debate. Using in vivo electrophysiology in anesthetized mice, spinal superfusion of BNP and GRP was performed in chloroquine (histamine-independent pruritogen)-responsive and histamine-responsive dorsal horn neurons. Furthermore, we compared itch-related scratching bouts in response to intrathecal injection of BNP or GRP. Electrophysiological analyses revealed 4.8% of chloroquine-responsive neurons responded to BNP and GRP, 9.5% to BNP, and 33.3% to GRP, indicating almost half of chloroquine-responsive neurons were unresponsive to both BNP and GRP. In contrast, 0% of histamine-responsive neurons responded to BNP, whereas 30% responded to spinal GRP. In addition, 70% of histamineresponsive neurons were unresponsive to both BNP and GRP. Differences in the time-course and frequency of scratching responses evoked by intrathecal BNP and GRP were observed. These results suggest that 1) BNP-Npra and GRP-GRPR signaling pathways of spinal itch transmission differ, 2) spinal BNP may contribute little to histaminergic itch, and 3) multiple neurotransmitters are involved in spinal itch transmission.
PepFect6-miRNA-146a nanocomplexes inhibit inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis ¨ Langel3, V Jaks2, M Pooga2 E Urgard1, A Lorents2, M Klaas2, K Padarik2, J Viil2, T Runnel1, U and A Rebane1 1 Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia, 2 Insititute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia and 3 Department of Neurochemistry, Stockholm University, Stockholm, Sweden The skin is a difficult to access tissue for efficient delivery of therapeutic RNA oligonucleotides. miR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the NF-kB pathway in various cell types, including skin keratinocytes. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. In this study, we tested the capacity of PF6 to form noncovalent nanocomplexes with miR-146a mimics and their effect on inflammatory responses in human primary keratinocytes and mouse model of irritant contact dermatitis. We demonstrate that PF6 forms unimodal nanocomplexes with miR-146a mimics. The PF6-miR-146a nanocomplexes efficiently entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-kB pathway and the genes known to be activated by NF-kB, such as CCL5 and IL-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines, such as IL-6, CCL11 and mouse homolog for IL8, CXCL1, in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.
S214 Journal of Investigative Dermatology (2016), Volume 136
Modulation of neutrophil extracellular trap formation by Dimethylfumarat J Hoffmann, K Schaekel, A Enk and E Hadaschik Hautklinik Heidelberg, Heidelberg, Germany Fumaric acid esters (FAE) are successfully used in the treatment of psoriasis and multiple sclerosis. Neutrophil (polymorphonuclear) granulocytes (PMN) are considered a histologic hallmark of psoriasis and were shown to be among the first cells infiltrating nascent psoriatic lesions. In the present study, we investigated the predisposition of PMN from psoriasis patients, including patients treated with FAE, and healthy donors to form NETs in response to Phorbol myristate acetate (PMA) stimulation. While we found no significant difference between NET formation of PMN from psoriatic patients and healthy donors, PMN from the subgroup of FAE treated psoriasis patients had a lower NET rate than the other donors combined. Using healthy donor PMN, we could reproduce a consistent and dose dependent inhibitory effect of the FAE dimethylfumarate (DMF) on NET formation to PMA. The inhibitory effect of DMF could be effectively blocked by L-Glutathion substitution and involved the reduction of reactive oxygen species production. DMF effects were stimulus specific, as NET formation to other stimuli was not reduced. In conclusion, we report DMF as a potent modulator of PMN function, in particular NET formation. These mechanisms may contribute to the beneficiary effects of FAE.
Air pollution activates Aryl hydrocarbon receptor of undifferentiated keratinocytes, leading to atopic dermatitis-like pathologies T Hidaka1, EH Kobayashi2, T Suzuki2 and M Yamamoto2 1 Dermatology, Tohoku Univ., Sendai, Japan and 2 Medical Chemistry, Tohoku Univ., Sendai, Japan The increase in the prevalence of atopic dermatitis (AD) is reported to be correlated with air pollution levels. Polycyclic aromatic hydrocarbons (PAHs) were identified as the component responsible for the air pollutant-induced inflammation. PAHs exert their biological effects via binding to a transcription factor, aryl hydrocarbon receptor (AhR). We have previously established the transgenic mice whose keratinocytes express the constitutive active form of AhR (AhR-CA mice) and reported AhR-CA mice show barrier dysfunction, Th2-type skin inflammation and alloknesis with epidermal hyperinnervation. However, it remained unknown whether air pollution-induced AhR activation induce AD-like pathologies observed in AhR-CA mice, since AhR has diverse functions depending on the type of ligands. To explorer the effect of chronic AhR activation by AhR ligands, we treated the skin of AhRflox/flox or AhRflox/flox::K5-Cre mice with 7,12-Dimethylbenz[a]anthracene (DMBA), a not-degradable PAHs included in air pollutants, and 6-Formylindolo[3,2-b]carbazole (FICZ), a rapidly metabolized-natural AhR ligand in the skin. The skin of DMBA-applied AhRflox/flox mice exhibited scaling, T cell and mast cell infiltration, upregulated Tslp, Il33 and Il4ra gene expression and elevated serum IgE level. Furthermore, scratching behavior, alloknesis and epidermal hyperinnervation were also observed in DMBA-applied AhRflox/flox mice. In contrast, almost all of these pathologies were not observed in DMBA-applied AhRflox/flox::K5Cre mice nor FICZ-applied mice. To investigate the mechanism of different effects between DMBA and FICZ, we exposed both ligands to primary human keratinocytes either in differentiated or un-differentiated status induced by different Ca2+ load. AD-associated genes, including TSLP and IL33, were upregulated only when DMBA was exposed to un-differeniated keratinocytes. These results suggest air pollutants penetrate through the skin and activate AhR in keratinocytes at the basal layer, leading to AD-like pathologies.
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T cell epitopes of BPAG2 antigen and the epitope-based pathogenesis of bullous pemphigoid J Zhang, H Fang, E Dang, Q Li and G Wang Dermatology, Xijing Hospital, Xi’an, China Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease characterized by autoantibodies directed against BPAG2, a transmembrane protein of epidermal basal cells. The pathogenesis of BP has not been elucidated completely, further researches are needed to understand the pathogenesis of BP. The purpose of this study is to identify the T cell epitope of BPAG2 and to understand the course of humoral immune response in BP. As IL-4 secretion is a marker of Th2 antigen-specific activation, PBMCs from 43 BP patients were collected and tested for IL-4 cytokine responses to a complete panel of 22 overlapping peptides spanning the BP180-NC16A domain by ELISPOT assay. Results revealed that IL-4 responses were significantly stronger for three peptides, P14, P18 and P21, in 27 BP patients, which representative aa492 to aa513 in NC16A domain. We next studied the function of three epitopes. Flow cytometry analysis showed that those three epitopes were able to stimulated BP patient’s T proliferation. ELISA analysis the levels of sCD23 in PBMCs of BP patients culture supernatant showed that after stimulating by those three epitopes, patients ‘s B cells were activated. Comparing the epitopes of BP patients in progression stage and recovery stage of disease respectively, we found that epitopes were changed in different stage, and the number of epitopes were elevated in the progression stage compared to the recovery stage. Thus, there exists “epitope spreading”, and the appeared new epitopes may be associated with disease recurrence. In conclusion, our research identified three Th2 cell epitopes, those epitopes have the ability to stimulated BP patients’ T cells proliferation and B cells activation pathogenic autoantibodies production. There also existed “epitope spreading” phenomenon, and the appeared new epitopes may be associated with disease recurrence. This results may contribute to the understanding the pathogenesis of BP, providing new ideas and the scientific evidences about the treatment of BP.
A role of spinal glial cells in pruritus of atopic dermatitis model mouse M Tominaga1, K Torigoe2, H Ogawa3 and K Takamori3 1 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan, 2 Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Tokyo, Japan and 3 Department of Dermatology, Juntendo University Graduate School of Medicine, Tokyo, Japan The role of spinal glia in the intractable itch of skin diseases such as atopic dermatitis (AD) remains unclear. Here, we examined antipruritic effects of intrathecal or oral administration of minocycline, an inhibitor of microglial activation, and immunohistochemically examined the distribution of spinal microglia and astrocytes during development of AD-like symptoms in AD-like model NC/Nga mice. A dermatophagoides farinae body (Dfb) ointment-induced AD-like mouse model (Dfb-NC/Nga) was used. Dermatitis was induced by repeated application of Dfb ointment. Dfb-NC/Nga mice were treated by intrathecal administration of saline or minocycline three times a week for 2 weeks. Saline or minocycline was administered orally by syringe twice per day for 3 weeks. Scratching behavior was monitored for 12 h using SCLABA-Real system. Distributions of spinal microglia and astrocytes in the Dfb-NC/Nga mice were examined immunohistochemically with anti-Iba1 and anti-GFAP antibodies, respectively. Dermatitis was induced by application of the Dfb, and numbers of scratching bouts and spinal Iba1+ microglia were increased in Dfb-NC/Nga mice. Intrathecal minocycline dose-dependently reduced scratching bouts, concomitant with decreased number of Iba1+ microglia in Dfb-NC/Nga mice. Intrathecal minocycline improved dermatitis, and oral administration of high-dose minocycline improved dermatitis and showed a tendency to reduce the numbers of scratching bouts and spinal Iba1+ microglia in these mice. In the development of AD, the numbers of microglia and astrocytes were increased in the spinal cord of Dfb-NC/Nga mice. Spinal microglia may be a therapeutic target for the pruritus associated with AD. Spinal astrocytes may be also involved in the pathological process of intractable itch in this model.
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Roles of brain-natriuretic peptide- and gastrin-releasing peptide-responsive neurons in spinal transmission of itchRoles of brain-natriuretic peptide- and gastrin-releasing peptideresponsive neurons in spinal transmission of itch F Kusube1, M Tominaga2, H Kawasaki2, F Yamakura3, H Naito4, H Ogawa5, Y Tomooka1 and K Takamori5 1 Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Katsusika-ku, Japan, 2 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan, 3 Juntendo University Faculty of International Liberal Arts, Tokyo, Japan, 4 Institute of Health and Sports Science & Medicine, Juntendo University, Inzai, Japan and 5 Department of Dermatology, Juntendo University Graduate School of Medicine, Urayasu, Japan Brain-natriuretic peptide (BNP) receptor (Npra)-expressing spinal neurons and gastrin releasing peptide receptor (GRPR)-expressing spinal neurons are critically involved in signaling itch. However, the pathway of spinal itch transmission involving BNP and GRP is still under debate. Using in vivo electrophysiology in anesthetized mice, spinal superfusion of BNP and GRP was performed in chloroquine (histamine-independent pruritogen)-responsive and histamine-responsive dorsal horn neurons. Furthermore, we compared itch-related scratching bouts in response to intrathecal injection of BNP or GRP. Electrophysiological analyses revealed 4.8% of chloroquine-responsive neurons responded to BNP and GRP, 9.5% to BNP, and 33.3% to GRP, indicating almost half of chloroquine-responsive neurons were unresponsive to both BNP and GRP. In contrast, 0% of histamine-responsive neurons responded to BNP, whereas 30% responded to spinal GRP. In addition, 70% of histamineresponsive neurons were unresponsive to both BNP and GRP. Differences in the time-course and frequency of scratching responses evoked by intrathecal BNP and GRP were observed. These results suggest that 1) BNP-Npra and GRP-GRPR signaling pathways of spinal itch transmission differ, 2) spinal BNP may contribute little to histaminergic itch, and 3) multiple neurotransmitters are involved in spinal itch transmission.
PepFect6-miRNA-146a nanocomplexes inhibit inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis ¨ Langel3, V Jaks2, M Pooga2 E Urgard1, A Lorents2, M Klaas2, K Padarik2, J Viil2, T Runnel1, U and A Rebane1 1 Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia, 2 Insititute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia and 3 Department of Neurochemistry, Stockholm University, Stockholm, Sweden The skin is a difficult to access tissue for efficient delivery of therapeutic RNA oligonucleotides. miR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the NF-kB pathway in various cell types, including skin keratinocytes. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. In this study, we tested the capacity of PF6 to form noncovalent nanocomplexes with miR-146a mimics and their effect on inflammatory responses in human primary keratinocytes and mouse model of irritant contact dermatitis. We demonstrate that PF6 forms unimodal nanocomplexes with miR-146a mimics. The PF6-miR-146a nanocomplexes efficiently entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-kB pathway and the genes known to be activated by NF-kB, such as CCL5 and IL-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines, such as IL-6, CCL11 and mouse homolog for IL8, CXCL1, in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.
S214 Journal of Investigative Dermatology (2016), Volume 136
Modulation of neutrophil extracellular trap formation by Dimethylfumarat J Hoffmann, K Schaekel, A Enk and E Hadaschik Hautklinik Heidelberg, Heidelberg, Germany Fumaric acid esters (FAE) are successfully used in the treatment of psoriasis and multiple sclerosis. Neutrophil (polymorphonuclear) granulocytes (PMN) are considered a histologic hallmark of psoriasis and were shown to be among the first cells infiltrating nascent psoriatic lesions. In the present study, we investigated the predisposition of PMN from psoriasis patients, including patients treated with FAE, and healthy donors to form NETs in response to Phorbol myristate acetate (PMA) stimulation. While we found no significant difference between NET formation of PMN from psoriatic patients and healthy donors, PMN from the subgroup of FAE treated psoriasis patients had a lower NET rate than the other donors combined. Using healthy donor PMN, we could reproduce a consistent and dose dependent inhibitory effect of the FAE dimethylfumarate (DMF) on NET formation to PMA. The inhibitory effect of DMF could be effectively blocked by L-Glutathion substitution and involved the reduction of reactive oxygen species production. DMF effects were stimulus specific, as NET formation to other stimuli was not reduced. In conclusion, we report DMF as a potent modulator of PMN function, in particular NET formation. These mechanisms may contribute to the beneficiary effects of FAE.
Air pollution activates Aryl hydrocarbon receptor of undifferentiated keratinocytes, leading to atopic dermatitis-like pathologies T Hidaka1, EH Kobayashi2, T Suzuki2 and M Yamamoto2 1 Dermatology, Tohoku Univ., Sendai, Japan and 2 Medical Chemistry, Tohoku Univ., Sendai, Japan The increase in the prevalence of atopic dermatitis (AD) is reported to be correlated with air pollution levels. Polycyclic aromatic hydrocarbons (PAHs) were identified as the component responsible for the air pollutant-induced inflammation. PAHs exert their biological effects via binding to a transcription factor, aryl hydrocarbon receptor (AhR). We have previously established the transgenic mice whose keratinocytes express the constitutive active form of AhR (AhR-CA mice) and reported AhR-CA mice show barrier dysfunction, Th2-type skin inflammation and alloknesis with epidermal hyperinnervation. However, it remained unknown whether air pollution-induced AhR activation induce AD-like pathologies observed in AhR-CA mice, since AhR has diverse functions depending on the type of ligands. To explorer the effect of chronic AhR activation by AhR ligands, we treated the skin of AhRflox/flox or AhRflox/flox::K5-Cre mice with 7,12-Dimethylbenz[a]anthracene (DMBA), a not-degradable PAHs included in air pollutants, and 6-Formylindolo[3,2-b]carbazole (FICZ), a rapidly metabolized-natural AhR ligand in the skin. The skin of DMBA-applied AhRflox/flox mice exhibited scaling, T cell and mast cell infiltration, upregulated Tslp, Il33 and Il4ra gene expression and elevated serum IgE level. Furthermore, scratching behavior, alloknesis and epidermal hyperinnervation were also observed in DMBA-applied AhRflox/flox mice. In contrast, almost all of these pathologies were not observed in DMBA-applied AhRflox/flox::K5Cre mice nor FICZ-applied mice. To investigate the mechanism of different effects between DMBA and FICZ, we exposed both ligands to primary human keratinocytes either in differentiated or un-differentiated status induced by different Ca2+ load. AD-associated genes, including TSLP and IL33, were upregulated only when DMBA was exposed to un-differeniated keratinocytes. These results suggest air pollutants penetrate through the skin and activate AhR in keratinocytes at the basal layer, leading to AD-like pathologies.