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304 Tonicity signals drive and anti-inflammatory Th17 cell properties
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Tonicity signals drive and anti-inflammatory Th17 cell properties J Matthias2, R Noster3, M Poltorak1, D Soll1 and C Zielinski1 1 Technical University of Munich, Munich, Germany, 2 Technical University of Munich, Munich, Germany and 3 Charite, Berlin, Germany Sodium chloride has recently been demonstrated to strongly enhance the generation of Th17 cells in both mouse and man, which provided mechanistic evidence for the epidemiological correlation of enhanced dietary salt intake and the increasing incidence of autoimmune diseases. Our data support the Th17 cell promoting effect of sodium chloride on human Th17 cell priming. However, Th17 cells acquired anti-inflammatory properties upon stimulation in sodium chloride enriched micromilieus. In addition, sodium chloride also strongly suppressed cytotoxic functions of human T cells. Together, our data suggest that despite the reported association of salt with induction of pathogenic Th17 cells, sodium chloride instead is a potent inducer of the anti-inflammatory Th17 cell subset and also paralyzes cytotoxic T cell properties. Therefore, our data suggest that reduction of sodium chloride levels by either dietary restriction or specific targeting of downstream signaling events could counteract T cell immunoparalysis and exhaustion in settings of chronic inflammatory skin diseases or malignancies.
Heat shock protein 60 & 70 expression in psoriasis vulgaris before and after NB-UVB therapy A Kotb Dermatology Research, National Research Center, Cairo, Egypt Psoriasis is a common chronic, relapsing, non-infectious inflammatory skin disease. Heat shock proteins (Hsps) play an important role in essential functions in cells under physiological conditions and play a major role in protecting cells against damage in stressful conditions & they appeared in some studies to play an important role in pathogenesis of many inflammatory skin conditions including psoriasis, atopic dermatitis and systemic lupus erythrematoses. The aim was to detect the expression of HSP60 & HSP70 in psoriasis and its correlation to the disease severity and to review potential role of HSP60 & HSP70 in pathogenesis and therapy of psoriasis. Patients with Psoriasis Vulgaris were treated with NB-UVB & the skin biopsies were taken before & after therapy. The immunoreactivity of HSP60 & HSP70 was detected using IRIDI score. It was confirmed that HSP60 & HSP70 was expressed in psoriatic lesions and their expression is increased with the disease severity but not related to either age or sex & it decreased markedly after exposure to NB-UVB twice weekly for 12 weeks. In conclusion, HSP60 & HSP70 play a role in the pathogenesis of psoriasis & both can be used as a target for therapy.
Efficacy of Dermatophagoides farinae-specific subcutaneous immunotherapy for mouse atopic dermatitis model S Kim2, J Shin1, J Noh1, C Park1, J Kim2, H Kim1, J Lee1, H Chu1 and K Lee1 1 Dermatology, Cutaneous Biology Research Institute, Yonsei Univerisity College of Medicine, Seoul, Korea (the Republic of) and 2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea (the Republic of) Subcutaneous immunotherapy (SCIT) is a clinically effective treatment in atopic dermatitis (AD). However, there was no mouse model to understand the mechanism of house dust mite immunotherapy in atopic dermatitis. The aim of this study was to establish a mouse model of SCIT mouse model of house dust mite induced atopic dermatitis. Female NC/Nga mice were treated with Dermatophagoides farinae (D.farinae) body extract ointment for 4 weeks to induce atopic dermatitis like skin lesion. Then we separated the mice into 2 groups, control group and immunotherapy (IT) group. Both groups were continuously treated with D.farinae body extract ointment for 4 weeks, however, only IT group was treated with 8 injections of D.farinae (100 ug subcutaneous) twice a week along with D.farinae body extract ointment treatment. Subsequently, we measured total IgE and IgG4, D.farinaeespecific IgE, level of cytokines using Flow Cytometry and qRT-PCR. In mice sera, the level of total IgE and D.farinae-specific IgE was decreased in IT group than control group at 3 weeks from the beginning of immunotherapy, while the level of IgG4 was remarkably increased in IT group. In mice splenocyte, expression of IL-4 in CD4+ T cells was more decreased in IT group than control group. Population of Foxp3 in CD4+CD25+ T cells was also increased in IT group than control group, and expression of IL-10 in regulatory T cells and NK cells was increased in IT group. Lastly, IT group made a strong contrast with control group at the relative mRNA levels of IL-10, IL-17 and IFN-gamma. In the present study, we have established a house dust mite SCIT mouse model and demonstrated that house dust mite SCIT might improve atopic dermatitis-like skin lesion by decreasing total IgE and increasing the IgG4 and Il-10. Using this model, it will be possible to find novel way to potentiate the effects of allergen immunotherapy.
Establishment of D. farinae-loaded microneedle patches for allergen specific immunotherapy in allergic diseases J Kim1, J Shin2, C Park2, J Noh2, S Kim1, H Kim2, J Lee2, H Chu2 and K Lee2 1 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea (the Republic of) and 2 Dermatology & Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea (the Republic of) Allergen-specific immunotherapy which induces allergen-specific tolerance is an effective treatment modality in atopic patients. However, frequent visit over 3 years results to the low compliance. To overcome this limitation, sublingual immunotherapy has been developed, however, the effects in atopic dermatitis is controversial. Therefore more convenient and effective allergen delivery is necessary. The aim of this study is to develop allergen loaded microneedle patches and to confirm the stability and allergenicity of allergen in the hyaluronic acid (HA) microneedle. Also, safety of the allergen loaded microneedle patches was investigated in mouse model. Dermatophagoides farinae (D. farinae) extract was loaded in microneedle patches in the concentration of 10 mg, 2 mg, and 0.2 mg/patch. D. farinae-loaded HA microneedle patches were dissolved and then stability of the allergen was validated. Female BALB/c mice were shaved and D. farinae-loaded HA microneedle patches were applied twice a week for 4 weeks. Protein concentrations were maintained in D. farinaeloaded HA microneedle patches. Inhibition ELISA analysis revealed that there was no decrease of allergenicity after loading D. farinae into the HA microneedle patches. Also, 4week application of D. farinae-loaded HA microneedle patches does not showed any adverse effects. However, 4-week application of high dose allergen-loaded patches (10 mg/patch) increased the total IgE level. We found that the allergenicity of D. farinae is maintained after loaded into the HA microneedle patches. And high dose allergen-loaded HA microneedle patches were able to sensitize BALB/c mice. Therefore, we suggest that D. farinae-loaded HA microneedle patches successfully delivered allergen into the skin and this patches might be useful in the treatment for allergic diseases.
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Triptolide, triptonide and celastrol alleviates the proliferation and IFN-g-induced immune dysfunction of keratinocytes by inhibiting miR-17-92 Y An, W Zhang, S Guo, T Gao and C Li Department of Dermatology, Xijing Hospital, Xi’an, China Psoriasis is an autoimmune skin disease, in which the activation of keratinocytes induced by cytokines plays a crucial role in the pathogenesis. Tripterygium wilfordii Hook. f. is a traditional Chinese medicine that can be used for psoriasis therapy for its immunosuppressive effects. However, whether Tripterygium wilfordii Hook. f. can directly repress the cytokinesinduced activation of keratinocytes in psoriasis is not known. In the present study, we initially treated normal human keratinocytes with seven active constituents of Tripterygium wilfordii Hook. f. for 24 hours and then performed CCK-8 assay. It turned out that three compounds, including triptolide, triptonide and celastrol, could successfully inhibit the proliferation of keratinocytes with the effective concentration at 5 nM. Furthermore, the mRNA and protein level of STAT1, an important inflammatory transcriptional factor in psoriasis keratinocytes, as well as the expression of phosphorylated STAT1 could be down-regulated in IFN-g-stimulated keratinocytes by the three compounds, respectively. Moreover, the level of multiple chemokines, including CXCL1, CXCL9, CXCL10 and CXCL16, were all reduced by the pretreatment of the three compounds in keratinocytes stimulated by IFN-g for 24 hours. Finally, triptolide, triptonide and celastrol could inhibit the expression of miR-17-92, a miRNA that had been proved to promote the proliferation of keratinocytes and the production of chemokines by maintaining the activation of STAT1 signaling pathway in our previous research. Taken together, our results suggested that triptolide, triptonide and celastrol could inhibit the proliferation and the cytokines-induced immune dysfunction of keratinocytes possibly through the suppression of miR-17-92. Based on our findings, topical application of triptolide, triptonide and celastrol could inhibit the activation of epidermal keratinocytes in psoriasis and may be applied in the therapy of psoriasis in the future.
Collagen XVII is the major autoantigen in mucous membrane pemphigoid K Izumi, W Nishie, M Nishimura, H Ujiie, H Iwata and H Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Mucous membrane pemphigoid (MMP) is a heterogeneous autoimmune blistering disorder which mainly affects mucosal regions. Various molecules of the basement membrane zone, including collagen XVII (COL17), BP230, a6b4 integrin, laminin-332 and collagen VII, have been reported as autoantigens in MMP. Based on the reactivity of indirect immunofluorescence using 1M NaCl salt-split skin (ss-IIF), MMP autoantibodies (autoAbs) can be classified into two groups. AutoAbs targeting COL17, BP230 and a6b4 integrin react with the epidermal side, whereas those targeting laminin-332 or collagen VII react with the dermal side. Previous studies using immunoblotting or ELISA with bacterial recombinant proteins have shown that around 50-70% of MMP autoAbs targeting the epidermal side in ss-IIF react with the C-terminus of COL17, which is thought to be the main epitope for this type of MMP. However, other regions of COL17 can be targeted by MMP autoAbs. Based on these findings, we hypothesize that an ELISA using full-length recombinant COL17 (a full-length COL17 ELISA) will be useful for detecting MMP autoAbs, because the system can detect autoAbs targeting entire regions of COL17. Recombinant full-length COL17 was purified from HEK293 cells, and a full-length COL17 ELISA was performed using 17 MMP sera. Of these 17 sera, autoAbs from 10 cases and 2 cases, respectively, reacted with the epidermal or the dermal side in ss-IIF. One case reacted with both the epidermal and the dermal sides, and 4 cases showed no reactivity in ss-IIF. The full-length COL17 ELISA detected IgG autoAbs in 12 of 17 MMP cases (70.6%), indicating that COL17 is the major autoantigen in MMP. Notably, all 11 MMP cases in which autoAbs reacted with epidermal side in ss-IIF showed positive reactivity by full-length COL17 ELISA, suggesting that MMP autoAbs reacting with the epidermal side in ss-IIF target COL17. In conclusion, the present study shows that COL17 is the main autoantigen in MMP, especially in cases with autoAbs targeting the epidermal side in ss-IIF.
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Tonicity signals drive and anti-inflammatory Th17 cell properties J Matthias2, R Noster3, M Poltorak1, D Soll1 and C Zielinski1 1 Technical University of Munich, Munich, Germany, 2 Technical University of Munich, Munich, Germany and 3 Charite, Berlin, Germany Sodium chloride has recently been demonstrated to strongly enhance the generation of Th17 cells in both mouse and man, which provided mechanistic evidence for the epidemiological correlation of enhanced dietary salt intake and the increasing incidence of autoimmune diseases. Our data support the Th17 cell promoting effect of sodium chloride on human Th17 cell priming. However, Th17 cells acquired anti-inflammatory properties upon stimulation in sodium chloride enriched micromilieus. In addition, sodium chloride also strongly suppressed cytotoxic functions of human T cells. Together, our data suggest that despite the reported association of salt with induction of pathogenic Th17 cells, sodium chloride instead is a potent inducer of the anti-inflammatory Th17 cell subset and also paralyzes cytotoxic T cell properties. Therefore, our data suggest that reduction of sodium chloride levels by either dietary restriction or specific targeting of downstream signaling events could counteract T cell immunoparalysis and exhaustion in settings of chronic inflammatory skin diseases or malignancies.
Heat shock protein 60 & 70 expression in psoriasis vulgaris before and after NB-UVB therapy A Kotb Dermatology Research, National Research Center, Cairo, Egypt Psoriasis is a common chronic, relapsing, non-infectious inflammatory skin disease. Heat shock proteins (Hsps) play an important role in essential functions in cells under physiological conditions and play a major role in protecting cells against damage in stressful conditions & they appeared in some studies to play an important role in pathogenesis of many inflammatory skin conditions including psoriasis, atopic dermatitis and systemic lupus erythrematoses. The aim was to detect the expression of HSP60 & HSP70 in psoriasis and its correlation to the disease severity and to review potential role of HSP60 & HSP70 in pathogenesis and therapy of psoriasis. Patients with Psoriasis Vulgaris were treated with NB-UVB & the skin biopsies were taken before & after therapy. The immunoreactivity of HSP60 & HSP70 was detected using IRIDI score. It was confirmed that HSP60 & HSP70 was expressed in psoriatic lesions and their expression is increased with the disease severity but not related to either age or sex & it decreased markedly after exposure to NB-UVB twice weekly for 12 weeks. In conclusion, HSP60 & HSP70 play a role in the pathogenesis of psoriasis & both can be used as a target for therapy.
Efficacy of Dermatophagoides farinae-specific subcutaneous immunotherapy for mouse atopic dermatitis model S Kim2, J Shin1, J Noh1, C Park1, J Kim2, H Kim1, J Lee1, H Chu1 and K Lee1 1 Dermatology, Cutaneous Biology Research Institute, Yonsei Univerisity College of Medicine, Seoul, Korea (the Republic of) and 2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea (the Republic of) Subcutaneous immunotherapy (SCIT) is a clinically effective treatment in atopic dermatitis (AD). However, there was no mouse model to understand the mechanism of house dust mite immunotherapy in atopic dermatitis. The aim of this study was to establish a mouse model of SCIT mouse model of house dust mite induced atopic dermatitis. Female NC/Nga mice were treated with Dermatophagoides farinae (D.farinae) body extract ointment for 4 weeks to induce atopic dermatitis like skin lesion. Then we separated the mice into 2 groups, control group and immunotherapy (IT) group. Both groups were continuously treated with D.farinae body extract ointment for 4 weeks, however, only IT group was treated with 8 injections of D.farinae (100 ug subcutaneous) twice a week along with D.farinae body extract ointment treatment. Subsequently, we measured total IgE and IgG4, D.farinaeespecific IgE, level of cytokines using Flow Cytometry and qRT-PCR. In mice sera, the level of total IgE and D.farinae-specific IgE was decreased in IT group than control group at 3 weeks from the beginning of immunotherapy, while the level of IgG4 was remarkably increased in IT group. In mice splenocyte, expression of IL-4 in CD4+ T cells was more decreased in IT group than control group. Population of Foxp3 in CD4+CD25+ T cells was also increased in IT group than control group, and expression of IL-10 in regulatory T cells and NK cells was increased in IT group. Lastly, IT group made a strong contrast with control group at the relative mRNA levels of IL-10, IL-17 and IFN-gamma. In the present study, we have established a house dust mite SCIT mouse model and demonstrated that house dust mite SCIT might improve atopic dermatitis-like skin lesion by decreasing total IgE and increasing the IgG4 and Il-10. Using this model, it will be possible to find novel way to potentiate the effects of allergen immunotherapy.
Establishment of D. farinae-loaded microneedle patches for allergen specific immunotherapy in allergic diseases J Kim1, J Shin2, C Park2, J Noh2, S Kim1, H Kim2, J Lee2, H Chu2 and K Lee2 1 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea (the Republic of) and 2 Dermatology & Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea (the Republic of) Allergen-specific immunotherapy which induces allergen-specific tolerance is an effective treatment modality in atopic patients. However, frequent visit over 3 years results to the low compliance. To overcome this limitation, sublingual immunotherapy has been developed, however, the effects in atopic dermatitis is controversial. Therefore more convenient and effective allergen delivery is necessary. The aim of this study is to develop allergen loaded microneedle patches and to confirm the stability and allergenicity of allergen in the hyaluronic acid (HA) microneedle. Also, safety of the allergen loaded microneedle patches was investigated in mouse model. Dermatophagoides farinae (D. farinae) extract was loaded in microneedle patches in the concentration of 10 mg, 2 mg, and 0.2 mg/patch. D. farinae-loaded HA microneedle patches were dissolved and then stability of the allergen was validated. Female BALB/c mice were shaved and D. farinae-loaded HA microneedle patches were applied twice a week for 4 weeks. Protein concentrations were maintained in D. farinaeloaded HA microneedle patches. Inhibition ELISA analysis revealed that there was no decrease of allergenicity after loading D. farinae into the HA microneedle patches. Also, 4week application of D. farinae-loaded HA microneedle patches does not showed any adverse effects. However, 4-week application of high dose allergen-loaded patches (10 mg/patch) increased the total IgE level. We found that the allergenicity of D. farinae is maintained after loaded into the HA microneedle patches. And high dose allergen-loaded HA microneedle patches were able to sensitize BALB/c mice. Therefore, we suggest that D. farinae-loaded HA microneedle patches successfully delivered allergen into the skin and this patches might be useful in the treatment for allergic diseases.
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309
Triptolide, triptonide and celastrol alleviates the proliferation and IFN-g-induced immune dysfunction of keratinocytes by inhibiting miR-17-92 Y An, W Zhang, S Guo, T Gao and C Li Department of Dermatology, Xijing Hospital, Xi’an, China Psoriasis is an autoimmune skin disease, in which the activation of keratinocytes induced by cytokines plays a crucial role in the pathogenesis. Tripterygium wilfordii Hook. f. is a traditional Chinese medicine that can be used for psoriasis therapy for its immunosuppressive effects. However, whether Tripterygium wilfordii Hook. f. can directly repress the cytokinesinduced activation of keratinocytes in psoriasis is not known. In the present study, we initially treated normal human keratinocytes with seven active constituents of Tripterygium wilfordii Hook. f. for 24 hours and then performed CCK-8 assay. It turned out that three compounds, including triptolide, triptonide and celastrol, could successfully inhibit the proliferation of keratinocytes with the effective concentration at 5 nM. Furthermore, the mRNA and protein level of STAT1, an important inflammatory transcriptional factor in psoriasis keratinocytes, as well as the expression of phosphorylated STAT1 could be down-regulated in IFN-g-stimulated keratinocytes by the three compounds, respectively. Moreover, the level of multiple chemokines, including CXCL1, CXCL9, CXCL10 and CXCL16, were all reduced by the pretreatment of the three compounds in keratinocytes stimulated by IFN-g for 24 hours. Finally, triptolide, triptonide and celastrol could inhibit the expression of miR-17-92, a miRNA that had been proved to promote the proliferation of keratinocytes and the production of chemokines by maintaining the activation of STAT1 signaling pathway in our previous research. Taken together, our results suggested that triptolide, triptonide and celastrol could inhibit the proliferation and the cytokines-induced immune dysfunction of keratinocytes possibly through the suppression of miR-17-92. Based on our findings, topical application of triptolide, triptonide and celastrol could inhibit the activation of epidermal keratinocytes in psoriasis and may be applied in the therapy of psoriasis in the future.
Collagen XVII is the major autoantigen in mucous membrane pemphigoid K Izumi, W Nishie, M Nishimura, H Ujiie, H Iwata and H Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Mucous membrane pemphigoid (MMP) is a heterogeneous autoimmune blistering disorder which mainly affects mucosal regions. Various molecules of the basement membrane zone, including collagen XVII (COL17), BP230, a6b4 integrin, laminin-332 and collagen VII, have been reported as autoantigens in MMP. Based on the reactivity of indirect immunofluorescence using 1M NaCl salt-split skin (ss-IIF), MMP autoantibodies (autoAbs) can be classified into two groups. AutoAbs targeting COL17, BP230 and a6b4 integrin react with the epidermal side, whereas those targeting laminin-332 or collagen VII react with the dermal side. Previous studies using immunoblotting or ELISA with bacterial recombinant proteins have shown that around 50-70% of MMP autoAbs targeting the epidermal side in ss-IIF react with the C-terminus of COL17, which is thought to be the main epitope for this type of MMP. However, other regions of COL17 can be targeted by MMP autoAbs. Based on these findings, we hypothesize that an ELISA using full-length recombinant COL17 (a full-length COL17 ELISA) will be useful for detecting MMP autoAbs, because the system can detect autoAbs targeting entire regions of COL17. Recombinant full-length COL17 was purified from HEK293 cells, and a full-length COL17 ELISA was performed using 17 MMP sera. Of these 17 sera, autoAbs from 10 cases and 2 cases, respectively, reacted with the epidermal or the dermal side in ss-IIF. One case reacted with both the epidermal and the dermal sides, and 4 cases showed no reactivity in ss-IIF. The full-length COL17 ELISA detected IgG autoAbs in 12 of 17 MMP cases (70.6%), indicating that COL17 is the major autoantigen in MMP. Notably, all 11 MMP cases in which autoAbs reacted with epidermal side in ss-IIF showed positive reactivity by full-length COL17 ELISA, suggesting that MMP autoAbs reacting with the epidermal side in ss-IIF target COL17. In conclusion, the present study shows that COL17 is the main autoantigen in MMP, especially in cases with autoAbs targeting the epidermal side in ss-IIF.
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