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038 Hyaluronan oligosaccharides induce suppressive effect to chronic allergic dermatitis
Adaptive and Auto-Immunity | ABSTRACTS 037
038
Possible role of autophagy in scleroderma T Mori1 and T Yamamoto2 1 Fukushima Medical University / Japan, Fukushima, Japan and 2 Fukushima Med Univ, Fukushima, Japan Autophagy is an essential intracellular self-degradation system to maintain the homeostatic balance between the synthesis, degradation and recycling of cellular proteins and organelles. Recently, it is reported that autophagy is associated with systemic lupus erythematosus, rheumatoid arthritis and idiopathic pulmonary fibrosis. The association with autophagy and scleroderma (SSc) is also suspected, however, the role of autophagy in the pathogenesis of tissue fibrosis is still unclarified, and the association of which phase of scleroderma is largely unknown. Therefore, we investigated the role of autophagy in the pathogenesis of SSc by using skin and lung samples of bleomycin (BLM)-induced SSc murine model and human SSc skin. BLM or phosphate-buffered saline (PBS) was injected into shaved back of C3H/HeJ mice for 2 or 4 weeks. Also, we used skin samples of edematous phase SSc and those of sclerotic phase SSc. We carried out haematoxylin and eosin (HE) stain for evaluation of skin sclerosis and immunofluorescent stain by anti microtubule-associated protein 1 light chain 3 (LC3) antibody, which is the most common autophagy marker. We evaluated the number of LC3positive dots in fibroblasts in the mice samples (skin and lung) and the human skin samples (both edematous and sclerotic phase). There was no clear difference between the number of LC3-positive dots of skin samples from PBS mice and those from BLM mice qualitatively. We are currently investigating the number of LC3-positive dots of mice lung tissues and human skin samples in more numbers.
Hyaluronan oligosaccharides induce suppressive effect to chronic allergic dermatitis J Muto1, D Watanabe1 and RL Gallo2 1 Department of Dermatology, Aichi Medical University School of Medicine, Nagakute, Japan and 2 UCSD, San Diego, CA Hyaluronan (HA) has been implicated in wound healing and inflammation, and its biological properties are thought to be dependent on its molecular size. Previously it has been shown that HA oligomer administration in vivo modulates contact hypersensitivity responses. In this study we investigated if HA oligomer could influence inflammation of chronic allergic dermatitits. The function of a tetramer form of HA (oligo-HA) was examined in a chronic dermatitis model induced by repeated DNFB application. This dermatitis model produces Atopic Dermatitis (AD)-like skin inflammation with high serum IgE levels in an IL-4 dependent manner. Wild-type (WT) mice were painted with either DNFB or vehicle on both ears once a week over 29 days. Oligo-HA cream (1 %) or vehicle alone was applied topically daily from day 15 for 2 weeks. To quantify the inflammatory responses in this model, ear thickness was measured 24 hours after each DNFB painting and cytokine responses quantified in local tissues. Topical application of oligo-HA significantly suppressed the increase in ear thickness after the fifth DNFB painting (p < 0.001). Histological evaluation of skin tissues by hematoxylin and eosin staining confirmed that AD-like skin lesions were significantly alleviated and the number of polymorphonuclear leukocytes (PMNs) in the ears of DNFB treated mice was significantly decreased by oligo-HA application (p < 0.01). Futhermore, we found the mRNA expression of IL-4 (p < 0.05) and MIP-2 (p < 0.001) in ears of DNFB treated mice were significantly suppressed by oligo-HA application when measured by quantitative real-time PCR. The elevation of serum IgE levels in DNFB treated mice were also attenuated (p < 0.01). These data show that topical application of oligo-HA induces the suppressive effect to ADlike dermatitis by affecting the inflammatory status in skin and can influence clinical outcome.
039
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Recapitulation of ectopic lymphocytes aggregation and B cell phenotype in the skin lesions of patients with pemphigus S Zhou1, Z Liu1, J Zheng1 and M Pan2 1 Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China and 2 Department of Dermatology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ectopic lymphoid aggregates could be observed in several autoimmune diseases (e.g. rheumatoid arthritis). Pemphigus is an organ specific autoimmune disease and caused by circulating antibodies. Little is known about the features and function of lesional lymphocytes. The aim of this study is to illustrate the lymphoid neogenesis and B cell phenotype in pemphigus skin lesions. Methods: 197 HE-stained sections of pemphigus vulgaris (PV) and pemphigus foliaceus (PF) skin lesions in our department were collected. The histological grading of lymphoid aggregates was assessed according to a published grading system with grade 1 aggregates displaying a radial cell number between 2 and 5 cells, grade 2 between 6 and 10 and grade 3 greater than 10. Immunohistochemistry staining for CD3, CD20 and CD138 was proceeded and T, B and plasma cells were checked in selected 29 cases. Meanwhile, the B cells and plasma cells were compared between lesional skin and peripheral blood from the same patient by flow cytometry. Results: Grade 1 lymphoid neogenesis was observed in 195 of 197 cases (99.0 %), grade 2 in 133 of 197 (67.5 %) and grade 3 in 23 of 197 (11.7 %). There was no significant difference between PV and PF. The increasing B cell and plasma cells could be detected by immunohistochemistry in parallel to the grade of lymphoid aggregates. Compared to the peripheral blood, the lesional CD27+ B cell and plasma cell were significantly increased. Conclusions: Ectopic lymphoid aggregates could be detected in the skin lesion of pemphigus and the major B cell subset are memory B cells which might play a pathogenic role in the local immune response.
Metabolomic profiling of psoriasis skin reveals localized cortisol deficiency resulting in maintenance of inflammatory state and disruption of epidermal differentiation X Xing1, MK Sarkar1, A Tsoi2, N Kaplan3, Y Liang1, P Hoover3, JJ Voorhees1, RP Nair1, JT Elder1, I Budunova4, S Getsios3 and JE Gudjonsson1 1 University of Michigan, Ann Arbor, MI, 2 Dept. of Dermatology, U. of Michigan, Ann Arbor, MI, 3 Northwestern University, Chicago, IL and 4 Northwestern University Feinberg School of Medicine, Chicago, IL The factors involved in maintaining inflammatory state in psoriatic skin remain poorly understood. To address this knowledge gap, we performed metabolomic and transcriptomic profiling of skin from 6 lesional (PP) and non-lesional (PN) psoriatic skin, and 6 healthy (NN) individuals. We identified 377 metabolites altered between NN, PN and PP skin(p < 0.05). Amongst the most decreased compounds in PP skin were cortisone and cortisol (357 and 1057-fold decreased, p < 0.0001). In contrast serum cortisol levels were similar in psoriatic (n¼37) and healthy controls (n¼43). Genes involved in cortisol biosynthesis were expressed in NN skin but suppressed in PP skin (HSD11B1, p < 0.0001, confirmed by IHC) coincident with decreased nuclear localization of the glucocorticoid receptor. Using a 3-D human epidermis culture model, we uncovered a differentiation-dependent increase in HSD11B1 expression (p < 0.05) and cortisol consumption (2.9-fold decrease compared to baseline, n¼3, p < 0.01). When starved of exogenous steroids, epidermal differentiation was markedly altered (LOR, FLG, p < 0.05) and associated with increased mRNA expression and secretion of pro-inflammatory mediators including CXCL9 and CXCL10 (n¼3, p < 0.05). Notably, in vivo application of topical glucocorticoids led to rapid restoration of glucocorticoid biosynthesis gene expression coincident with normalization of epidermal differentiation (n¼3).These data demonstrate that steroids have a dual role in the epidermis acting as a critical factor in facilitating epidermal differentiation and as a suppressor of inflammatory responses. The localized steroid deficiency in PP skin is likely to maintain inflammatory state and impede restoration of normal epidermal differentiation.
041
042
Regulatory T cells drive stem cell differentiation during skin barrier repair A Mathur, B Zirak, M Lowe, A Abbas and M Rosenblum University of San Francisco California, San Francisco, CA Epithelial regeneration requires the coordinated responses of stem cells and immune cells. Mechanisms that link immune and stem cell function during epidermal repair are largely unknown. CD4+FoxP3+ regulatory T-cells (Tregs) play a major role in tissue repair in nonlymphoid organs, suggesting that Tregs may influence tissue-specific stem cell functions. Here, we demonstrate that selective ablation of Tregs leads to sustained barrier defects indicated by elevated transepidermal water loss, defective epidermal stratification and keratinocyte differentiation following injury. Hair follicle (HF) stem cell fate-mapping studies revealed that cells derived from Lgr5+ stem cells substantially contribute to the regenerating epidermis in barrier injured mice. Ablation of Tregs resulted in retention of Lgr5-derived cells in the HF bulge, indicating that the egress of Lgr5 derived cells to repair injured skin requires Tregs. Whole transcriptome RNA-sequencing of Lgr5-derived cells isolated from mice in the absence or presence of Tregs revealed a dominant role for Tregs in promoting HF stem cell differentiation toward a keratinized epidermis. In contrast, Lgr5-derived cells from mice lacking Tregs retain a quiescent stem cell phenotype. Mechanistically, Tregs facilitate stem cell differentiation by controlling CXCL5 dependent influx of neutrophils and inflammatory monocytes. CXCL5 neutralization in Treg-depleted animals lead to improved barrier function and partial restoration of Lgr5-derived cells in the epidermis after injury. These data indicate that Tregs facilitate an ‘alternate’ fate decision for HF stem cells toward an epidermal phenotype following skin injury by regulating the CXCL5 pathway. Our results elucidate novel mechanisms by which skin immune cells influence stem cell function to facilitate barrier repair.
Aggregation of Dsg-specific lymphocytes and antibody production in the lesions of pemphigus vulgaris patients H Yuan1 and M Pan2 1 Department of Dermatology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Philadelphia, PA and 2 Department of Dermatology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Pemphigus is a skin and mucosal membranes-targeting autoimmune bullous disorder, in which it is often believed that circulating anti-Desmoglein (Dsg) antibodies lead to the pathogenesis. However, the role of local infiltration of immune cells in the lesional skin has never been investigated. In this study, we examined the local immune response from the lesions of pemphigus vulgaris (PV) patients. Histologically, we found there existed a large number of B lymphocytes, T lymphocytes, and plasma cells in the skin lesions by immunohistochemistry and immunofluorescence staining. By flow cytometry and ELISPOT assay, a significantly increased number of Dsg1 and Dsg3-specific B cells could be identified. Additionally, anti-Dsg1 and Dsg3 antibodies could be detected from the supernatant of in vitro cultures with isolated lymphocytes from the lesional skin, and the titers of antibodies showed positive correlation with the Pemphigus Disease Area Index (PDAI). Furthermore, CD3+ T cells were also found accumulated in PV lesion in a large amount, and the majority of them were CD4+ T helper cells, capable of producing IL-21 and IL-17a, but not typical T follicular helper cells (Tfh) that express Bcl-6 and CXCR5. To determine the gene profile from the local lesions, we extracted RNA from infiltrating lymphocytes of PV for the microarray analysis. The results revealed that a few genes were up-regulated, such as CCL19, CCL21 and CXCL13, which were also confirmed by Real-time PCR. These cytokines were recognized as cytokines related to lymphoid tissue formation. Collectively, our results have suggested a critical role of local lymphocytic aggregation and formation of tertiary lymphoid organ (TLO)like structure in the pathogenesis of pemphigus.
www.jidonline.org S7
038
Possible role of autophagy in scleroderma T Mori1 and T Yamamoto2 1 Fukushima Medical University / Japan, Fukushima, Japan and 2 Fukushima Med Univ, Fukushima, Japan Autophagy is an essential intracellular self-degradation system to maintain the homeostatic balance between the synthesis, degradation and recycling of cellular proteins and organelles. Recently, it is reported that autophagy is associated with systemic lupus erythematosus, rheumatoid arthritis and idiopathic pulmonary fibrosis. The association with autophagy and scleroderma (SSc) is also suspected, however, the role of autophagy in the pathogenesis of tissue fibrosis is still unclarified, and the association of which phase of scleroderma is largely unknown. Therefore, we investigated the role of autophagy in the pathogenesis of SSc by using skin and lung samples of bleomycin (BLM)-induced SSc murine model and human SSc skin. BLM or phosphate-buffered saline (PBS) was injected into shaved back of C3H/HeJ mice for 2 or 4 weeks. Also, we used skin samples of edematous phase SSc and those of sclerotic phase SSc. We carried out haematoxylin and eosin (HE) stain for evaluation of skin sclerosis and immunofluorescent stain by anti microtubule-associated protein 1 light chain 3 (LC3) antibody, which is the most common autophagy marker. We evaluated the number of LC3positive dots in fibroblasts in the mice samples (skin and lung) and the human skin samples (both edematous and sclerotic phase). There was no clear difference between the number of LC3-positive dots of skin samples from PBS mice and those from BLM mice qualitatively. We are currently investigating the number of LC3-positive dots of mice lung tissues and human skin samples in more numbers.
Hyaluronan oligosaccharides induce suppressive effect to chronic allergic dermatitis J Muto1, D Watanabe1 and RL Gallo2 1 Department of Dermatology, Aichi Medical University School of Medicine, Nagakute, Japan and 2 UCSD, San Diego, CA Hyaluronan (HA) has been implicated in wound healing and inflammation, and its biological properties are thought to be dependent on its molecular size. Previously it has been shown that HA oligomer administration in vivo modulates contact hypersensitivity responses. In this study we investigated if HA oligomer could influence inflammation of chronic allergic dermatitits. The function of a tetramer form of HA (oligo-HA) was examined in a chronic dermatitis model induced by repeated DNFB application. This dermatitis model produces Atopic Dermatitis (AD)-like skin inflammation with high serum IgE levels in an IL-4 dependent manner. Wild-type (WT) mice were painted with either DNFB or vehicle on both ears once a week over 29 days. Oligo-HA cream (1 %) or vehicle alone was applied topically daily from day 15 for 2 weeks. To quantify the inflammatory responses in this model, ear thickness was measured 24 hours after each DNFB painting and cytokine responses quantified in local tissues. Topical application of oligo-HA significantly suppressed the increase in ear thickness after the fifth DNFB painting (p < 0.001). Histological evaluation of skin tissues by hematoxylin and eosin staining confirmed that AD-like skin lesions were significantly alleviated and the number of polymorphonuclear leukocytes (PMNs) in the ears of DNFB treated mice was significantly decreased by oligo-HA application (p < 0.01). Futhermore, we found the mRNA expression of IL-4 (p < 0.05) and MIP-2 (p < 0.001) in ears of DNFB treated mice were significantly suppressed by oligo-HA application when measured by quantitative real-time PCR. The elevation of serum IgE levels in DNFB treated mice were also attenuated (p < 0.01). These data show that topical application of oligo-HA induces the suppressive effect to ADlike dermatitis by affecting the inflammatory status in skin and can influence clinical outcome.
039
040
Recapitulation of ectopic lymphocytes aggregation and B cell phenotype in the skin lesions of patients with pemphigus S Zhou1, Z Liu1, J Zheng1 and M Pan2 1 Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China and 2 Department of Dermatology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Ectopic lymphoid aggregates could be observed in several autoimmune diseases (e.g. rheumatoid arthritis). Pemphigus is an organ specific autoimmune disease and caused by circulating antibodies. Little is known about the features and function of lesional lymphocytes. The aim of this study is to illustrate the lymphoid neogenesis and B cell phenotype in pemphigus skin lesions. Methods: 197 HE-stained sections of pemphigus vulgaris (PV) and pemphigus foliaceus (PF) skin lesions in our department were collected. The histological grading of lymphoid aggregates was assessed according to a published grading system with grade 1 aggregates displaying a radial cell number between 2 and 5 cells, grade 2 between 6 and 10 and grade 3 greater than 10. Immunohistochemistry staining for CD3, CD20 and CD138 was proceeded and T, B and plasma cells were checked in selected 29 cases. Meanwhile, the B cells and plasma cells were compared between lesional skin and peripheral blood from the same patient by flow cytometry. Results: Grade 1 lymphoid neogenesis was observed in 195 of 197 cases (99.0 %), grade 2 in 133 of 197 (67.5 %) and grade 3 in 23 of 197 (11.7 %). There was no significant difference between PV and PF. The increasing B cell and plasma cells could be detected by immunohistochemistry in parallel to the grade of lymphoid aggregates. Compared to the peripheral blood, the lesional CD27+ B cell and plasma cell were significantly increased. Conclusions: Ectopic lymphoid aggregates could be detected in the skin lesion of pemphigus and the major B cell subset are memory B cells which might play a pathogenic role in the local immune response.
Metabolomic profiling of psoriasis skin reveals localized cortisol deficiency resulting in maintenance of inflammatory state and disruption of epidermal differentiation X Xing1, MK Sarkar1, A Tsoi2, N Kaplan3, Y Liang1, P Hoover3, JJ Voorhees1, RP Nair1, JT Elder1, I Budunova4, S Getsios3 and JE Gudjonsson1 1 University of Michigan, Ann Arbor, MI, 2 Dept. of Dermatology, U. of Michigan, Ann Arbor, MI, 3 Northwestern University, Chicago, IL and 4 Northwestern University Feinberg School of Medicine, Chicago, IL The factors involved in maintaining inflammatory state in psoriatic skin remain poorly understood. To address this knowledge gap, we performed metabolomic and transcriptomic profiling of skin from 6 lesional (PP) and non-lesional (PN) psoriatic skin, and 6 healthy (NN) individuals. We identified 377 metabolites altered between NN, PN and PP skin(p < 0.05). Amongst the most decreased compounds in PP skin were cortisone and cortisol (357 and 1057-fold decreased, p < 0.0001). In contrast serum cortisol levels were similar in psoriatic (n¼37) and healthy controls (n¼43). Genes involved in cortisol biosynthesis were expressed in NN skin but suppressed in PP skin (HSD11B1, p < 0.0001, confirmed by IHC) coincident with decreased nuclear localization of the glucocorticoid receptor. Using a 3-D human epidermis culture model, we uncovered a differentiation-dependent increase in HSD11B1 expression (p < 0.05) and cortisol consumption (2.9-fold decrease compared to baseline, n¼3, p < 0.01). When starved of exogenous steroids, epidermal differentiation was markedly altered (LOR, FLG, p < 0.05) and associated with increased mRNA expression and secretion of pro-inflammatory mediators including CXCL9 and CXCL10 (n¼3, p < 0.05). Notably, in vivo application of topical glucocorticoids led to rapid restoration of glucocorticoid biosynthesis gene expression coincident with normalization of epidermal differentiation (n¼3).These data demonstrate that steroids have a dual role in the epidermis acting as a critical factor in facilitating epidermal differentiation and as a suppressor of inflammatory responses. The localized steroid deficiency in PP skin is likely to maintain inflammatory state and impede restoration of normal epidermal differentiation.
041
042
Regulatory T cells drive stem cell differentiation during skin barrier repair A Mathur, B Zirak, M Lowe, A Abbas and M Rosenblum University of San Francisco California, San Francisco, CA Epithelial regeneration requires the coordinated responses of stem cells and immune cells. Mechanisms that link immune and stem cell function during epidermal repair are largely unknown. CD4+FoxP3+ regulatory T-cells (Tregs) play a major role in tissue repair in nonlymphoid organs, suggesting that Tregs may influence tissue-specific stem cell functions. Here, we demonstrate that selective ablation of Tregs leads to sustained barrier defects indicated by elevated transepidermal water loss, defective epidermal stratification and keratinocyte differentiation following injury. Hair follicle (HF) stem cell fate-mapping studies revealed that cells derived from Lgr5+ stem cells substantially contribute to the regenerating epidermis in barrier injured mice. Ablation of Tregs resulted in retention of Lgr5-derived cells in the HF bulge, indicating that the egress of Lgr5 derived cells to repair injured skin requires Tregs. Whole transcriptome RNA-sequencing of Lgr5-derived cells isolated from mice in the absence or presence of Tregs revealed a dominant role for Tregs in promoting HF stem cell differentiation toward a keratinized epidermis. In contrast, Lgr5-derived cells from mice lacking Tregs retain a quiescent stem cell phenotype. Mechanistically, Tregs facilitate stem cell differentiation by controlling CXCL5 dependent influx of neutrophils and inflammatory monocytes. CXCL5 neutralization in Treg-depleted animals lead to improved barrier function and partial restoration of Lgr5-derived cells in the epidermis after injury. These data indicate that Tregs facilitate an ‘alternate’ fate decision for HF stem cells toward an epidermal phenotype following skin injury by regulating the CXCL5 pathway. Our results elucidate novel mechanisms by which skin immune cells influence stem cell function to facilitate barrier repair.
Aggregation of Dsg-specific lymphocytes and antibody production in the lesions of pemphigus vulgaris patients H Yuan1 and M Pan2 1 Department of Dermatology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Philadelphia, PA and 2 Department of Dermatology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Pemphigus is a skin and mucosal membranes-targeting autoimmune bullous disorder, in which it is often believed that circulating anti-Desmoglein (Dsg) antibodies lead to the pathogenesis. However, the role of local infiltration of immune cells in the lesional skin has never been investigated. In this study, we examined the local immune response from the lesions of pemphigus vulgaris (PV) patients. Histologically, we found there existed a large number of B lymphocytes, T lymphocytes, and plasma cells in the skin lesions by immunohistochemistry and immunofluorescence staining. By flow cytometry and ELISPOT assay, a significantly increased number of Dsg1 and Dsg3-specific B cells could be identified. Additionally, anti-Dsg1 and Dsg3 antibodies could be detected from the supernatant of in vitro cultures with isolated lymphocytes from the lesional skin, and the titers of antibodies showed positive correlation with the Pemphigus Disease Area Index (PDAI). Furthermore, CD3+ T cells were also found accumulated in PV lesion in a large amount, and the majority of them were CD4+ T helper cells, capable of producing IL-21 and IL-17a, but not typical T follicular helper cells (Tfh) that express Bcl-6 and CXCR5. To determine the gene profile from the local lesions, we extracted RNA from infiltrating lymphocytes of PV for the microarray analysis. The results revealed that a few genes were up-regulated, such as CCL19, CCL21 and CXCL13, which were also confirmed by Real-time PCR. These cytokines were recognized as cytokines related to lymphoid tissue formation. Collectively, our results have suggested a critical role of local lymphocytic aggregation and formation of tertiary lymphoid organ (TLO)like structure in the pathogenesis of pemphigus.
www.jidonline.org S7