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probiotics and active microbial structures on human primary keratinocytes
ABSTRACTS | Inflammation, Immunity and Infection 274
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AP1S3 mutations cause cutaneous autoinflammation by disrupting autophagy and upregulating IL-36 production in keratinocytes SK Mahil1, N Setta-Kaffetzi1, R Trembath1, CH Smith1, P Di Meglio2, J Barker1 and F Capon1 1 Division of Genetics and Molecular Medicine, King’s College London, London, United Kingdom and 2 Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom Although skin involvement is a prominent and defining characteristic of autoinflammatory disorders (AID), the pathways and cell types that drive the cutaneous features of AID are poorly defined. Our study sought to address this issue by investigating the molecular mechanisms underlying the onset of pustular psoriasis, an autoinflammatory condition with predominant dermatological manifestations. The focus of the study was on AP1S3, a disease gene which encodes a protein involved in membrane trafficking and autophagosome formation. AP1S3 expression was measured in disease relevant cell types and the consequences of AP1S3 deficiency were determined in a variety of experimental systems and in patient cells. AP1S3 expression was found to be distinctively elevated in keratinocytes. In these cells, AP1S3 knockout disrupted autophagy, causing abnormal accumulation of p62, an adaptor protein that mediates NF-kB activation. As a consequence, AP1S3 deficient cells up-regulated IL-1 signalling and over-expressed IL-36a, an important mediator of skin inflammation. These abnormal expression profiles were recapitulated by inhibition of autophagy with 3-MA and verified in the keratinocytes of patients harbouring AP1S3 mutations. These findings demonstrate that keratinocytes play a key role in the pathogenesis of skin autoinflammation. They also identify impaired keratinocyte autophagy and IL-36 over-production as important pathogenic pathways, which could be targeted by novel therapeutics.
Kallikrein 5 and eosinophil infiltration participate in itching of mycosis fungoides K Shimizu1, T Andoh2, Y Yoshihisa1, M Mizawa1, T Makino1 and T Shimizu1 1 Dermatology, University of Toyama, Toyama, Japan and 2 Applied Pharmaceutical Sciences, University of Toyama, Toyama, Japan Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas (CTCL). Itching can be a destressing or even debilitating symptom for the patients with CTCL. However, the itching of MF is unrelieved by conventional therapy using anti-histamines, suggesting that histamine is not the main pruritogen for the itching. Therefore, the underlying mechanisms of itching in patients with MF are still unclear. To determine the underlying mechanism of itching in MF, in this study, we investigated the clinical and histopathological features in patients with MF. Thirty patients with MF visited our hospital were investigated. The grade of itching was classified by three categories as follows; none, moderate itching, and strong itching. As pathological analysis, the skin sections were stained with HematoxylinEosin. In addition, the immunostaining was performed using specific antibodies for itchrelated factors, such as IL-31 and kallikrein 5 (KLK5), a serine protease. The activity of serine proteases in the skin sections was determined using enzyme histochemical analysis. The patients without and with itching were 40% and 60% (moderate itching: 40%, strong itching: 20%), respectively. The number of eosinophils infiltrated into the skin was increased in the group with strong itching. Although IL-31 released from Th2 cells is suggested to play a role in itching of MF, the level of IL-31-immunoreactivity in the skin of our patients with and without itching was not different. On the other hand, in the skin of the patients, both KLK5-immunoreactivity and the serine protease activity were increased depending on the degree of itching. Taken together, these results suggest that at least KLK5 and eosinophil infiltration may be involved in itching in patients with MF.
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Immuno-modulatory effects of pre-/probiotics and active microbial structures on human primary keratinocytes C Schlumprecht1, DC Dittlein1, J van Bergenhenegouwen2, J Garssen2, D Haller3 and C Traidl-Hoffmann1 1 Institute of Environmental Medicine, UNIKA-T, TUM and Helmholtz Zentrum Mu¨nchen, Augsburg, Germany, 2 Department of Immunology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands and 3 Chair for Biofunctionality, ZIEL, CDD, TUM, Freising, Weihenstephan, Germany As interface between the body and the environment the skin has to ensure defense against environmental insults. This is achieved by a crosstalk between epithelial and immune cells as well as the skin’s microbiota via cell-cell contact or soluble mediators. The release of mediators is assumed to be influenced by pre-, probiotics or active microbial structures. However, the underlying mode of action or a direct contribution to skin health is not clear to date. Therefore, we investigated whether pre-/probiotics or active microbial structures have a direct effect on keratinocytes and on inflammatory skin reactions. Primary human keratinocytes (KCs) from healthy donors and atopic dermatitis patients were stimulated with a mixture of non-digestible short-chain galactooligosaccharides (GOS) and long-chain fructo-oligosaccharides (FOS) alone or in combination with either lactic acid bacteria (LAB) or lactocepin, a cell envelope protease of LAB. To simulate inflammatory conditions, KCs were costimulated with IFN-g/TNF-a. GOS/FOS decreased the IFN-g/TNF-a-induced secretion of IP-10 and CCL-5 in KCs. This inhibitory effect was more pronounced when GOS/FOS was combined with LAB while the strongest effect was observed in the presence of lactocepin. Moreover, the release of Galectin-9, a known supporter of regulatory T cell functions, was enhanced in response to GOS/FOS. The results imply that pre-/probiotics as well as active microbial structures can influence inflammatory processes in KCs and can act as regulatory compounds in inflammatory cutaneous responses. This supports our hypothesis that these compounds might have therapeutic potential in allergic-inflammatory skin diseases such as atopic eczema.
Prolonged incubation period after initial HIV infection is mediated by CTL activation and Treg cell suppression induced by Langerhans cells T Matsuzawa, T Kawamura, Y Ogawa, R Aoki and S Shimada Dermatology, University of Yamanashi, Yamanashi, Japan Langerhans cells (LCs) are one of the initial target cells for HIV. However, little is known on the modulation of HIV-specific immune responses by HIV-infected LCs, such as the induction of HIV-specific CD8+ T cells and CD4+ regulatory T (Treg) cells, both of which play important roles in controlling HIV replication in infected individuals. To investigate the inducibility of HIV-specific CD8+ T cells by HIV-infected LCs, naive CD8+ T cells from HLA-A0201 healthy donors (n ¼ 3) were co-cultured with HIVBaL-infected autologous monocyte-derived LCs (mLCs) or monocyte-derived DCs (mDCs) as a control. Seven days later, primed CD8+ T cells were restimulated by HIVBaL-infected autologous mLCs/mDCs for seven days. The number of HIV gag tetramer+CD8+ T cells induced by HIVBaL-infected mLCs was significantly higher than that induced by mDCs. Moreover, IFN-g production of HIV gag tetramer+CD8+ T cells induced by HIVBaL-infected mLCs was significantly higher than that of tetramer-CD8+ T cells. Likewise, ex vivo HIVBaL-infected human epidermal LCs induced IFN-g-producing HIVspecific CD8+ T cells (n ¼ 2). Next, to investigate the inducibility of Treg cells by HIV-infected LCs, naive CD4+ T cells were co-cultured with HIVBaL-infected autologous mLCs or epidermal LCs for six days (n ¼ 3 each). Treg cells have recently been classified into three subpopulations: FoxP3hiCD45RA, FoxP3loCD45RA+, and FoxP3loCD45RA cells. The number of FoxP3hiCD45RA Treg cells, but not the other two subpopulations, induced by HIV-infected mLCs or epidermal LCs was significantly lower than that induced by uninfected mLCs or epidermal LCs. The ability to suppress T-cell proliferation was comparable between Treg cells induced by HIV-infected mLCs and HIV-uninfected mLCs. Taking these results together, HIV-infected LCs trigger beneficial anti-HIV immune responses in the initial HIV infection phase, thereby contributing to the prolonged incubation period from initial HIV infection to AIDS onset.
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High burden of Merkel cell polyomavirus DNA in the nonlesional, sun-exposed skin of patients with Merkel cell carcinoma K Nakajima1, Y Hashida2, T Shiga1, H Nakajima1, M Daibata2 and S Sano1 1 Department of Dermatology, Kochi Medical School, Kochi University, Nankoku, Japan and 2 Department of Microbiology and Infection, Kochi Medical School, Kochi University, Nankoku, Japan Merkel cell carcinoma (MCC), one of the most aggressive skin cancers, develops in sunexposed skin of the elderly and is linked to infection with the Merkel cell polyomavirus (MCPyV). However, the pathogenic association of ultraviolet light irradiation with the MCPyV infection in patients with MCC remains unclear. Recently, we elucidated that the Japanese healthy donors older than 40 years harbored the MCPyV DNA particularly in the sun-exposed skin (Hashida, et al. J Infect Dis, 2016, 213:1708). Here, we investigated patients with MCC for the MCPyV loads in the uninvolved skin by comparing between sun-exposed and non sunexposed areas and also by comparing with controls including age-matched healthy donors and patients with other skin cancers other than MCC. (Methods) Enrolled were; six patients with MCC, in which the MCPyV was integrated, age-matched 30 healthy donors, and 19 patients with non-MCC skin cancers, including BCCs, SCCs, malignant melanomas. Skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time PCR, and the PCR products were subjected to DNA sequencing. (Results) MCPyV DNA levels were significantly higher in swabs obtained from the nonlesional skins of patients with MCC compared with those from age-matched healthy individuals and patients with other cutaneous cancers. Furthermore, the viral DNA burdens were generally higher in sun-exposed than in non sun-exposed skins in the three cohorts. Whereas MCPyV strains obtained from the nonlesional skin of patients with MCC had gene sequences without structural alterations, those of the MCC-derived strains showed truncating mutations or deletions. (Conclusion) Findings of this study suggested that MCC might develop in individuals harboring higher MCPyV load in the skin, where the MCPyV virome might be affected by ultraviolet exposure.
S208 Journal of Investigative Dermatology (2016), Volume 136
Skin dendritic cells show increased migration to lymph nodes in CD73 deficient mice leading to exacerbated contact hypersensitivity reactions A Pushkakevskaya, C Vilches, A Enk and K Mahnke Dpt. of Dermatology, University Hospital Heidelberg, Heidelberg, Germany The ecto-50 -nucleotidase CD73 is expressed by different cell types in skin and plays a critical role in conversion of extracellular ATP to adenosine (ADO). Therefore it modulates the balance of pro-inflammatory (ATP) or anti-inflammatory (ADO) signals in the skin. To assess the role of CD73 during contact hypersensitivity reactions (CHS), we sensitized groups of wildtype (wt) and CD73 deficient (CD73-/-) mice with 1% TNCB at the abdomen. After challenge the ear swelling response (ESR) was measured. We recorded 3-times higher ESR in CD73-/mice as compared to wt controls and immunohistology of ears from CD73-/- mice revealed increased infiltration by T-lymphocytes. In comparison in irritative dermatitis induced by croton oil, no differences between wt and CD73-/- mice were apparent. When sensitizing mice by injecting either hapten-pulsed CD11c+ lymph node-derived dendritic cells (DCs) or in vitro generated bone marrow-derived DCs, no difference in ESR was recorded between wt and CD73-/- mice. Thus, these data indicate that in particular the epicutaneous sensitization is augmented by CD73 deficiency. As for epicutaneous sensitization migration of skin DCs to draining lymph nodes (dLN) is mandatory, we next analyzed the numbers of different skin DC subsets in dLN after sensitization. We found significantly increased frequencies of skin derived MHCII++CD11c+CD207+ DCs in dLN of CD73-/- mice as compared to controls. These cells were potent inducers of T cell proliferation in vitro. Thus, these data indicate that absence of CD73 and its catalytic product ADO stimulates DC migration into dLN during CHS reactions, leading to increased sensitization. Thereby modulation of ATP-ADO turnover may provide a tool to ameliorate CHS reactions.
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AP1S3 mutations cause cutaneous autoinflammation by disrupting autophagy and upregulating IL-36 production in keratinocytes SK Mahil1, N Setta-Kaffetzi1, R Trembath1, CH Smith1, P Di Meglio2, J Barker1 and F Capon1 1 Division of Genetics and Molecular Medicine, King’s College London, London, United Kingdom and 2 Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom Although skin involvement is a prominent and defining characteristic of autoinflammatory disorders (AID), the pathways and cell types that drive the cutaneous features of AID are poorly defined. Our study sought to address this issue by investigating the molecular mechanisms underlying the onset of pustular psoriasis, an autoinflammatory condition with predominant dermatological manifestations. The focus of the study was on AP1S3, a disease gene which encodes a protein involved in membrane trafficking and autophagosome formation. AP1S3 expression was measured in disease relevant cell types and the consequences of AP1S3 deficiency were determined in a variety of experimental systems and in patient cells. AP1S3 expression was found to be distinctively elevated in keratinocytes. In these cells, AP1S3 knockout disrupted autophagy, causing abnormal accumulation of p62, an adaptor protein that mediates NF-kB activation. As a consequence, AP1S3 deficient cells up-regulated IL-1 signalling and over-expressed IL-36a, an important mediator of skin inflammation. These abnormal expression profiles were recapitulated by inhibition of autophagy with 3-MA and verified in the keratinocytes of patients harbouring AP1S3 mutations. These findings demonstrate that keratinocytes play a key role in the pathogenesis of skin autoinflammation. They also identify impaired keratinocyte autophagy and IL-36 over-production as important pathogenic pathways, which could be targeted by novel therapeutics.
Kallikrein 5 and eosinophil infiltration participate in itching of mycosis fungoides K Shimizu1, T Andoh2, Y Yoshihisa1, M Mizawa1, T Makino1 and T Shimizu1 1 Dermatology, University of Toyama, Toyama, Japan and 2 Applied Pharmaceutical Sciences, University of Toyama, Toyama, Japan Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas (CTCL). Itching can be a destressing or even debilitating symptom for the patients with CTCL. However, the itching of MF is unrelieved by conventional therapy using anti-histamines, suggesting that histamine is not the main pruritogen for the itching. Therefore, the underlying mechanisms of itching in patients with MF are still unclear. To determine the underlying mechanism of itching in MF, in this study, we investigated the clinical and histopathological features in patients with MF. Thirty patients with MF visited our hospital were investigated. The grade of itching was classified by three categories as follows; none, moderate itching, and strong itching. As pathological analysis, the skin sections were stained with HematoxylinEosin. In addition, the immunostaining was performed using specific antibodies for itchrelated factors, such as IL-31 and kallikrein 5 (KLK5), a serine protease. The activity of serine proteases in the skin sections was determined using enzyme histochemical analysis. The patients without and with itching were 40% and 60% (moderate itching: 40%, strong itching: 20%), respectively. The number of eosinophils infiltrated into the skin was increased in the group with strong itching. Although IL-31 released from Th2 cells is suggested to play a role in itching of MF, the level of IL-31-immunoreactivity in the skin of our patients with and without itching was not different. On the other hand, in the skin of the patients, both KLK5-immunoreactivity and the serine protease activity were increased depending on the degree of itching. Taken together, these results suggest that at least KLK5 and eosinophil infiltration may be involved in itching in patients with MF.
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Immuno-modulatory effects of pre-/probiotics and active microbial structures on human primary keratinocytes C Schlumprecht1, DC Dittlein1, J van Bergenhenegouwen2, J Garssen2, D Haller3 and C Traidl-Hoffmann1 1 Institute of Environmental Medicine, UNIKA-T, TUM and Helmholtz Zentrum Mu¨nchen, Augsburg, Germany, 2 Department of Immunology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands and 3 Chair for Biofunctionality, ZIEL, CDD, TUM, Freising, Weihenstephan, Germany As interface between the body and the environment the skin has to ensure defense against environmental insults. This is achieved by a crosstalk between epithelial and immune cells as well as the skin’s microbiota via cell-cell contact or soluble mediators. The release of mediators is assumed to be influenced by pre-, probiotics or active microbial structures. However, the underlying mode of action or a direct contribution to skin health is not clear to date. Therefore, we investigated whether pre-/probiotics or active microbial structures have a direct effect on keratinocytes and on inflammatory skin reactions. Primary human keratinocytes (KCs) from healthy donors and atopic dermatitis patients were stimulated with a mixture of non-digestible short-chain galactooligosaccharides (GOS) and long-chain fructo-oligosaccharides (FOS) alone or in combination with either lactic acid bacteria (LAB) or lactocepin, a cell envelope protease of LAB. To simulate inflammatory conditions, KCs were costimulated with IFN-g/TNF-a. GOS/FOS decreased the IFN-g/TNF-a-induced secretion of IP-10 and CCL-5 in KCs. This inhibitory effect was more pronounced when GOS/FOS was combined with LAB while the strongest effect was observed in the presence of lactocepin. Moreover, the release of Galectin-9, a known supporter of regulatory T cell functions, was enhanced in response to GOS/FOS. The results imply that pre-/probiotics as well as active microbial structures can influence inflammatory processes in KCs and can act as regulatory compounds in inflammatory cutaneous responses. This supports our hypothesis that these compounds might have therapeutic potential in allergic-inflammatory skin diseases such as atopic eczema.
Prolonged incubation period after initial HIV infection is mediated by CTL activation and Treg cell suppression induced by Langerhans cells T Matsuzawa, T Kawamura, Y Ogawa, R Aoki and S Shimada Dermatology, University of Yamanashi, Yamanashi, Japan Langerhans cells (LCs) are one of the initial target cells for HIV. However, little is known on the modulation of HIV-specific immune responses by HIV-infected LCs, such as the induction of HIV-specific CD8+ T cells and CD4+ regulatory T (Treg) cells, both of which play important roles in controlling HIV replication in infected individuals. To investigate the inducibility of HIV-specific CD8+ T cells by HIV-infected LCs, naive CD8+ T cells from HLA-A0201 healthy donors (n ¼ 3) were co-cultured with HIVBaL-infected autologous monocyte-derived LCs (mLCs) or monocyte-derived DCs (mDCs) as a control. Seven days later, primed CD8+ T cells were restimulated by HIVBaL-infected autologous mLCs/mDCs for seven days. The number of HIV gag tetramer+CD8+ T cells induced by HIVBaL-infected mLCs was significantly higher than that induced by mDCs. Moreover, IFN-g production of HIV gag tetramer+CD8+ T cells induced by HIVBaL-infected mLCs was significantly higher than that of tetramer-CD8+ T cells. Likewise, ex vivo HIVBaL-infected human epidermal LCs induced IFN-g-producing HIVspecific CD8+ T cells (n ¼ 2). Next, to investigate the inducibility of Treg cells by HIV-infected LCs, naive CD4+ T cells were co-cultured with HIVBaL-infected autologous mLCs or epidermal LCs for six days (n ¼ 3 each). Treg cells have recently been classified into three subpopulations: FoxP3hiCD45RA, FoxP3loCD45RA+, and FoxP3loCD45RA cells. The number of FoxP3hiCD45RA Treg cells, but not the other two subpopulations, induced by HIV-infected mLCs or epidermal LCs was significantly lower than that induced by uninfected mLCs or epidermal LCs. The ability to suppress T-cell proliferation was comparable between Treg cells induced by HIV-infected mLCs and HIV-uninfected mLCs. Taking these results together, HIV-infected LCs trigger beneficial anti-HIV immune responses in the initial HIV infection phase, thereby contributing to the prolonged incubation period from initial HIV infection to AIDS onset.
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High burden of Merkel cell polyomavirus DNA in the nonlesional, sun-exposed skin of patients with Merkel cell carcinoma K Nakajima1, Y Hashida2, T Shiga1, H Nakajima1, M Daibata2 and S Sano1 1 Department of Dermatology, Kochi Medical School, Kochi University, Nankoku, Japan and 2 Department of Microbiology and Infection, Kochi Medical School, Kochi University, Nankoku, Japan Merkel cell carcinoma (MCC), one of the most aggressive skin cancers, develops in sunexposed skin of the elderly and is linked to infection with the Merkel cell polyomavirus (MCPyV). However, the pathogenic association of ultraviolet light irradiation with the MCPyV infection in patients with MCC remains unclear. Recently, we elucidated that the Japanese healthy donors older than 40 years harbored the MCPyV DNA particularly in the sun-exposed skin (Hashida, et al. J Infect Dis, 2016, 213:1708). Here, we investigated patients with MCC for the MCPyV loads in the uninvolved skin by comparing between sun-exposed and non sunexposed areas and also by comparing with controls including age-matched healthy donors and patients with other skin cancers other than MCC. (Methods) Enrolled were; six patients with MCC, in which the MCPyV was integrated, age-matched 30 healthy donors, and 19 patients with non-MCC skin cancers, including BCCs, SCCs, malignant melanomas. Skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time PCR, and the PCR products were subjected to DNA sequencing. (Results) MCPyV DNA levels were significantly higher in swabs obtained from the nonlesional skins of patients with MCC compared with those from age-matched healthy individuals and patients with other cutaneous cancers. Furthermore, the viral DNA burdens were generally higher in sun-exposed than in non sun-exposed skins in the three cohorts. Whereas MCPyV strains obtained from the nonlesional skin of patients with MCC had gene sequences without structural alterations, those of the MCC-derived strains showed truncating mutations or deletions. (Conclusion) Findings of this study suggested that MCC might develop in individuals harboring higher MCPyV load in the skin, where the MCPyV virome might be affected by ultraviolet exposure.
S208 Journal of Investigative Dermatology (2016), Volume 136
Skin dendritic cells show increased migration to lymph nodes in CD73 deficient mice leading to exacerbated contact hypersensitivity reactions A Pushkakevskaya, C Vilches, A Enk and K Mahnke Dpt. of Dermatology, University Hospital Heidelberg, Heidelberg, Germany The ecto-50 -nucleotidase CD73 is expressed by different cell types in skin and plays a critical role in conversion of extracellular ATP to adenosine (ADO). Therefore it modulates the balance of pro-inflammatory (ATP) or anti-inflammatory (ADO) signals in the skin. To assess the role of CD73 during contact hypersensitivity reactions (CHS), we sensitized groups of wildtype (wt) and CD73 deficient (CD73-/-) mice with 1% TNCB at the abdomen. After challenge the ear swelling response (ESR) was measured. We recorded 3-times higher ESR in CD73-/mice as compared to wt controls and immunohistology of ears from CD73-/- mice revealed increased infiltration by T-lymphocytes. In comparison in irritative dermatitis induced by croton oil, no differences between wt and CD73-/- mice were apparent. When sensitizing mice by injecting either hapten-pulsed CD11c+ lymph node-derived dendritic cells (DCs) or in vitro generated bone marrow-derived DCs, no difference in ESR was recorded between wt and CD73-/- mice. Thus, these data indicate that in particular the epicutaneous sensitization is augmented by CD73 deficiency. As for epicutaneous sensitization migration of skin DCs to draining lymph nodes (dLN) is mandatory, we next analyzed the numbers of different skin DC subsets in dLN after sensitization. We found significantly increased frequencies of skin derived MHCII++CD11c+CD207+ DCs in dLN of CD73-/- mice as compared to controls. These cells were potent inducers of T cell proliferation in vitro. Thus, these data indicate that absence of CD73 and its catalytic product ADO stimulates DC migration into dLN during CHS reactions, leading to increased sensitization. Thereby modulation of ATP-ADO turnover may provide a tool to ameliorate CHS reactions.