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603 Autophagy and skin aging
ABSTRACTS | Innate Immunity, Microbiology, Inflammation 600
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Microbial-sensing liver Vg6+ gd T-cells modulate cutaneous inflammation E Merrill, M Constantinides, S Nakajima and Y Belkaid NIAID, Bethesda, MD Each body barrier surfaces is colonized by site specific microbiota. Whether the microbiota at one barrier surface can influence immunity and inflammation at other barrier surfaces is only beginning to be understood. Here we explored the possibility that gut bacteria can affect the cutaneous inflammatory response to imiquimod. To address this question, we colonized germ-free animals with gut-specific commensals and found that mice raised in absence of live microbes develop less inflammation following imiquimod treatment compared to gut bacteria colonized mice. Notably, the increased inflammatory response in gut bacteria colonized mice was associated with IL17+ Vg6+ gd T-cells, but not Vg4+ gd T-cells, ab T-cells, or ILCs. To determine the source of Vg6+ gd T-cells, various gut-associated tissues were assessed and Vg6+ gd T-cells were identified as major producers of IL17a in liver and spleen compared to other gut-associated organs, and this IL17a production is dependent on the microbiota. To assess the physiological relevance of this skin/liver axis we explored the possibility that Vg6+ gd T-cells may play a role in clearance of extracellular microbes during bacteremia. Indeed, in E. coli sepsis, liver Vg6+ gd T-cells, but not Vg6+ gd T-cells in other organs, responded within 24 hours. Taken together, IL17+ Vg6+ gd T-cells may be part of an evolutionarily conserved system where barrier breach leads to bacteremia, which leads to accumulation of IL17+ Vg6+ gd T-cells in the liver. In a feedback mechanism, the expanded IL17+ Vg6+ gd T-cells can traffic to barrier sites where they are capable of protecting against subsequent microbial invasion. Conversely, this mechanism can become pathogenic when intestinal leakiness leads to excess stimulation, followed by over-responsiveness in peripheral tissues and exacerbated inflammation.
Phenotype and functions of ribotype-specific Th17 cells targeting propionibacterium acnes GW Agak, S Kao, D Moon, K Ouyang and J Kim University of California, Los Angeles, Los Angeles, CA Propionibacterium acnes strains may contribute differently to skin health and disease. However, the immune phenotype and functions of Th17 cells induced by healthy (PH) vs. acne (PA) skin-associated P. acnes strains is currently unknown. Herein, we evaluated the cytokine profiles and functional activity of PH and PA-specific Th17 clones targeting P. acnes. We generated CD4+ T cell clones that express Th17 associated genes, and upon activation secrete IL-17 and other Th17-associated cytokines. We further classified these clones into protective (prTh17) phenotype, secreting IL-17 and IL-10 vs. the pathogenic (paTh17)phenotype, secreting IL-17 and IFN-g. PH-associated strains induced mainly the PrTh17 phenotype in comparison to PA, and supernatants derived from activated prTh17 cells but not paTh17, demonstrate antimicrobial activity against P. acnes in vitro. In addition, results from electron microscopic studies revealed that supernatants derived from activated prTh17 cells have robust membrane activity against P. acnes, and complete breaches in the bacterial cell envelope are observed, with externalized cytoplasmic contents characteristic of lysis. Taken together, our data suggest that P. acnes strains may modulate the CD4+ T cell responses to generate a heterogeneous population of Th17 cells that may contribute to the pathogenesis of acne vulgaris.
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The consequence of the loss of major cornified envelope protein in a mouse model of psoriasis Y Ishitsuka1, DR Roop2, T Ogawa1, N Okiyama1, Y Fujisawa1 and M Fujimoto1 1 Department of Dermatology, University of Tsukuba, Tsukuba, Ibaraki, Japan and 2 University of Colorado Anschutz Medical Campus, Aurora, CO Epidermal keratinocytes contribute to the innate defense system by making a physical/ mechanical barrier, the stratum corneum. In psoriasis, the expression levels of epidermal differentiation complex (EDC) genes including loricrin (LOR) are often reduced. One of the well-characterized psoriasis susceptibility loci is on chromosome 1q (PSORS4), in which EDC genes are clustered, and a linkage between EDC gene deletions with the HLACw6 was found, highlighting an importance of epidermal barrier defects in psoriasis pathogenesis. Although LOR is a major cornified cell envelope (CE) protein, LOR knockout (LKO) mice did not exhibit a postnatal epidermal barrier defect due to the induction of a compensatory repair process in utero. Interestingly, we recently found that LKO mice had impaired crosslinking of filaggrin and keratin 1/10 in their CEs, resulting in reduced mechanical stability of the epidermis. Since mechanical stress/trauma can contribute to psoriasis induction and exacerbation, we wanted to evaluate the impact of the loss LOR in a mouse model of psoriasis. Topical application of imiquimod on LKO mice resulted in a severe inflammatory phenotype; ear skin had augmented swelling, and back skin had enhanced erythema and hyperkeratosis, which was accompanied by increased transcriptional expression levels of keratin 6 and involucrin. Moreover, applying mechanical stress on ear skin resulted in increased transcriptional expression levels of inflammatory cytokines including interleukin23 and tumor necrosis factor-a. Taken together, these results indicate that epidermal barrier defects combined with mechanical stress/trauma can exacerbate psoriasis.
Spatial heterogeneity and functional diversity of innate lymphoid cells in the skin T Kobayashi1, A Truong1, H Shih1, T Doebel1, B Voisin1, T Woodring1, S Sohn1, E Kennedy1, J Jo1, K Moro2, W Leonard1, H Kong1 and K Nagao1 1 NIH, Bethesda, MD and 2 RIKEN-IMS, Yokohama, Kanagawa, Japan Innate lymphoid cells (ILCs) orchestrate host defense and tissue homeostasis by initiating and modulating immunological cascades. In contrast to extensive research on ILCs in the gut and lung, the characteristics and functions of ILCs in skin remain incompletely understood. Here, we found that anatomically different compartments of skin harbor IL-2Rg-dependent ILCs with distinct identities and functions. IL-5- and IL-13-producing group 2 ILCs (ILC2), which express transcription factor GATA3, were enriched in the subcutaneous layer. ILCs in dermis contained ILC2 and IL-17A-producing group 3 ILCs (ILC3). On the other hand, RORgt+ nontypical ILCs with minimal capacity to produce cytokines were identified in epidermis. The distinctiveness of ILC signatures in different niches of skin was further delineated by differential transcriptomic profiles (RNA-seq) and chromatin landscape (ATAC-seq). Whereas all ILCs in skin expressed IL-7Ra and were absent in Il7ra-/- mice, IL-7 was dispensable for epidermal ILCs. Interestingly, the absence of TSLP significantly reduced ILCs in epidermis, and the lack of both IL-7 and TSLP resulted in complete absence of epidermal ILCs, demonstrating cooperative effect of IL-7 and TSLP on the lineage commitment specific for epidermal ILCs. Furthermore, among the ILC subsets, specific absence of epidermal ILCs in Ccr6-/- mice indicated a requirement of CCL20-CCR6 axis on epidermotropism. Finally, Ccr6-/- mice displayed impaired clearance of topically applied S. aureus, suggesting a role for epidermal ILCs, and perhaps other lymphoid cells, in the regulation of commensal microbes. These results establishes a detailed framework of skin ILCs, which highlights skin layerspecific identities of ILCs, including their distinct cytokine and chemokine dependencies and functions.
S104 Journal of Investigative Dermatology (2017), Volume 137
Autophagy and skin aging H Kim, H Kim, S Cho and J Lee Incheon St. Mary’s Hospital, The Catholic University of Korea, Incheon, Kyonggi-do, Republic of Korea Autophagy is an intracellular degradative system that is believed to be involved in the aging process. However, the contribution of autophagy to age-related changes in the human skin is yet unclear. In this talk, I will be presenting my study data on the relationship between autophagy and skin aging. For monitoring autophagosomes, transmission electron microscopy and biochemical detection of LC3-II was performed on skin fibroblasts and keratinocytes derived from the young and old population. As to monitor autophagic activity, LC3 turnover assay was done and the amount of total selective substrate (p62) was evaluated.
Inflammatory responses of cutaneous cells and inflammatory cells against S. aureus C Mainzer1, S Bordes2, B Closs2, P Elias3, W Holleran3 and Y Uchida4 1 SILAB Inc., Hazlet, NJ, 2 R&D Department SILAB, Brive, France, Brive, Limousin, France, 3 Department of Dermatology, School of Medicine, University of California, San Francisco, USA, San Francisco, CA and 4 VA Med Ctr/UCSF, San Francisco, CA Staphylococcus (S) aureus is a transient microbe of normal skin, but when the epidermal permeability barrier is compromised, it can invade with virulent and inflammatory consequences. Prior studies have demonstrated a direct effect of S. aureus on epidermal cytokine production, leading to widespread responses in other cutaneous cells types. We assessed here how S. aureus provokes downstream inflammation. Specifically, we investigated the effects of conditioned medium from normal cultured human keratinocytes (KC) or fibroblasts, after treatment with heat-killed S. aureus (HKSA) on cytokine/chemokine production by dendritic cells (DC), prepared from normal human monocytes by GM-CSF and IL-4-induced differentiation. Following incubation with KC conditioned medium, mRNA production of IL-1b and IL-8 by DC increased markedly (by 9.13.5-fold and 5.02.7-fold, respectively). IL-12 expression increased in DC following incubation with fibroblast conditioned medium (14.47.6-fold). It is well known that IL-1b and IL-8 recruit innate immune cells (e.g., neutrophils and monocytes), while IL-12 activates Th1 cells. Hence, these results indicate that KC and fibroblasts play divergent, potentially complementary roles in provoking inflammatory responses following exposure to S. aureus.
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Microbial-sensing liver Vg6+ gd T-cells modulate cutaneous inflammation E Merrill, M Constantinides, S Nakajima and Y Belkaid NIAID, Bethesda, MD Each body barrier surfaces is colonized by site specific microbiota. Whether the microbiota at one barrier surface can influence immunity and inflammation at other barrier surfaces is only beginning to be understood. Here we explored the possibility that gut bacteria can affect the cutaneous inflammatory response to imiquimod. To address this question, we colonized germ-free animals with gut-specific commensals and found that mice raised in absence of live microbes develop less inflammation following imiquimod treatment compared to gut bacteria colonized mice. Notably, the increased inflammatory response in gut bacteria colonized mice was associated with IL17+ Vg6+ gd T-cells, but not Vg4+ gd T-cells, ab T-cells, or ILCs. To determine the source of Vg6+ gd T-cells, various gut-associated tissues were assessed and Vg6+ gd T-cells were identified as major producers of IL17a in liver and spleen compared to other gut-associated organs, and this IL17a production is dependent on the microbiota. To assess the physiological relevance of this skin/liver axis we explored the possibility that Vg6+ gd T-cells may play a role in clearance of extracellular microbes during bacteremia. Indeed, in E. coli sepsis, liver Vg6+ gd T-cells, but not Vg6+ gd T-cells in other organs, responded within 24 hours. Taken together, IL17+ Vg6+ gd T-cells may be part of an evolutionarily conserved system where barrier breach leads to bacteremia, which leads to accumulation of IL17+ Vg6+ gd T-cells in the liver. In a feedback mechanism, the expanded IL17+ Vg6+ gd T-cells can traffic to barrier sites where they are capable of protecting against subsequent microbial invasion. Conversely, this mechanism can become pathogenic when intestinal leakiness leads to excess stimulation, followed by over-responsiveness in peripheral tissues and exacerbated inflammation.
Phenotype and functions of ribotype-specific Th17 cells targeting propionibacterium acnes GW Agak, S Kao, D Moon, K Ouyang and J Kim University of California, Los Angeles, Los Angeles, CA Propionibacterium acnes strains may contribute differently to skin health and disease. However, the immune phenotype and functions of Th17 cells induced by healthy (PH) vs. acne (PA) skin-associated P. acnes strains is currently unknown. Herein, we evaluated the cytokine profiles and functional activity of PH and PA-specific Th17 clones targeting P. acnes. We generated CD4+ T cell clones that express Th17 associated genes, and upon activation secrete IL-17 and other Th17-associated cytokines. We further classified these clones into protective (prTh17) phenotype, secreting IL-17 and IL-10 vs. the pathogenic (paTh17)phenotype, secreting IL-17 and IFN-g. PH-associated strains induced mainly the PrTh17 phenotype in comparison to PA, and supernatants derived from activated prTh17 cells but not paTh17, demonstrate antimicrobial activity against P. acnes in vitro. In addition, results from electron microscopic studies revealed that supernatants derived from activated prTh17 cells have robust membrane activity against P. acnes, and complete breaches in the bacterial cell envelope are observed, with externalized cytoplasmic contents characteristic of lysis. Taken together, our data suggest that P. acnes strains may modulate the CD4+ T cell responses to generate a heterogeneous population of Th17 cells that may contribute to the pathogenesis of acne vulgaris.
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The consequence of the loss of major cornified envelope protein in a mouse model of psoriasis Y Ishitsuka1, DR Roop2, T Ogawa1, N Okiyama1, Y Fujisawa1 and M Fujimoto1 1 Department of Dermatology, University of Tsukuba, Tsukuba, Ibaraki, Japan and 2 University of Colorado Anschutz Medical Campus, Aurora, CO Epidermal keratinocytes contribute to the innate defense system by making a physical/ mechanical barrier, the stratum corneum. In psoriasis, the expression levels of epidermal differentiation complex (EDC) genes including loricrin (LOR) are often reduced. One of the well-characterized psoriasis susceptibility loci is on chromosome 1q (PSORS4), in which EDC genes are clustered, and a linkage between EDC gene deletions with the HLACw6 was found, highlighting an importance of epidermal barrier defects in psoriasis pathogenesis. Although LOR is a major cornified cell envelope (CE) protein, LOR knockout (LKO) mice did not exhibit a postnatal epidermal barrier defect due to the induction of a compensatory repair process in utero. Interestingly, we recently found that LKO mice had impaired crosslinking of filaggrin and keratin 1/10 in their CEs, resulting in reduced mechanical stability of the epidermis. Since mechanical stress/trauma can contribute to psoriasis induction and exacerbation, we wanted to evaluate the impact of the loss LOR in a mouse model of psoriasis. Topical application of imiquimod on LKO mice resulted in a severe inflammatory phenotype; ear skin had augmented swelling, and back skin had enhanced erythema and hyperkeratosis, which was accompanied by increased transcriptional expression levels of keratin 6 and involucrin. Moreover, applying mechanical stress on ear skin resulted in increased transcriptional expression levels of inflammatory cytokines including interleukin23 and tumor necrosis factor-a. Taken together, these results indicate that epidermal barrier defects combined with mechanical stress/trauma can exacerbate psoriasis.
Spatial heterogeneity and functional diversity of innate lymphoid cells in the skin T Kobayashi1, A Truong1, H Shih1, T Doebel1, B Voisin1, T Woodring1, S Sohn1, E Kennedy1, J Jo1, K Moro2, W Leonard1, H Kong1 and K Nagao1 1 NIH, Bethesda, MD and 2 RIKEN-IMS, Yokohama, Kanagawa, Japan Innate lymphoid cells (ILCs) orchestrate host defense and tissue homeostasis by initiating and modulating immunological cascades. In contrast to extensive research on ILCs in the gut and lung, the characteristics and functions of ILCs in skin remain incompletely understood. Here, we found that anatomically different compartments of skin harbor IL-2Rg-dependent ILCs with distinct identities and functions. IL-5- and IL-13-producing group 2 ILCs (ILC2), which express transcription factor GATA3, were enriched in the subcutaneous layer. ILCs in dermis contained ILC2 and IL-17A-producing group 3 ILCs (ILC3). On the other hand, RORgt+ nontypical ILCs with minimal capacity to produce cytokines were identified in epidermis. The distinctiveness of ILC signatures in different niches of skin was further delineated by differential transcriptomic profiles (RNA-seq) and chromatin landscape (ATAC-seq). Whereas all ILCs in skin expressed IL-7Ra and were absent in Il7ra-/- mice, IL-7 was dispensable for epidermal ILCs. Interestingly, the absence of TSLP significantly reduced ILCs in epidermis, and the lack of both IL-7 and TSLP resulted in complete absence of epidermal ILCs, demonstrating cooperative effect of IL-7 and TSLP on the lineage commitment specific for epidermal ILCs. Furthermore, among the ILC subsets, specific absence of epidermal ILCs in Ccr6-/- mice indicated a requirement of CCL20-CCR6 axis on epidermotropism. Finally, Ccr6-/- mice displayed impaired clearance of topically applied S. aureus, suggesting a role for epidermal ILCs, and perhaps other lymphoid cells, in the regulation of commensal microbes. These results establishes a detailed framework of skin ILCs, which highlights skin layerspecific identities of ILCs, including their distinct cytokine and chemokine dependencies and functions.
S104 Journal of Investigative Dermatology (2017), Volume 137
Autophagy and skin aging H Kim, H Kim, S Cho and J Lee Incheon St. Mary’s Hospital, The Catholic University of Korea, Incheon, Kyonggi-do, Republic of Korea Autophagy is an intracellular degradative system that is believed to be involved in the aging process. However, the contribution of autophagy to age-related changes in the human skin is yet unclear. In this talk, I will be presenting my study data on the relationship between autophagy and skin aging. For monitoring autophagosomes, transmission electron microscopy and biochemical detection of LC3-II was performed on skin fibroblasts and keratinocytes derived from the young and old population. As to monitor autophagic activity, LC3 turnover assay was done and the amount of total selective substrate (p62) was evaluated.
Inflammatory responses of cutaneous cells and inflammatory cells against S. aureus C Mainzer1, S Bordes2, B Closs2, P Elias3, W Holleran3 and Y Uchida4 1 SILAB Inc., Hazlet, NJ, 2 R&D Department SILAB, Brive, France, Brive, Limousin, France, 3 Department of Dermatology, School of Medicine, University of California, San Francisco, USA, San Francisco, CA and 4 VA Med Ctr/UCSF, San Francisco, CA Staphylococcus (S) aureus is a transient microbe of normal skin, but when the epidermal permeability barrier is compromised, it can invade with virulent and inflammatory consequences. Prior studies have demonstrated a direct effect of S. aureus on epidermal cytokine production, leading to widespread responses in other cutaneous cells types. We assessed here how S. aureus provokes downstream inflammation. Specifically, we investigated the effects of conditioned medium from normal cultured human keratinocytes (KC) or fibroblasts, after treatment with heat-killed S. aureus (HKSA) on cytokine/chemokine production by dendritic cells (DC), prepared from normal human monocytes by GM-CSF and IL-4-induced differentiation. Following incubation with KC conditioned medium, mRNA production of IL-1b and IL-8 by DC increased markedly (by 9.13.5-fold and 5.02.7-fold, respectively). IL-12 expression increased in DC following incubation with fibroblast conditioned medium (14.47.6-fold). It is well known that IL-1b and IL-8 recruit innate immune cells (e.g., neutrophils and monocytes), while IL-12 activates Th1 cells. Hence, these results indicate that KC and fibroblasts play divergent, potentially complementary roles in provoking inflammatory responses following exposure to S. aureus.