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502 Antiseptics elicit personalized alterations to skin microbial communities
ABSTRACTS | Innate Immunity, Inflammation & Microbiology 497
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The microbiome modulates cytokine production in the skin through epigenetic control of histone acetylation J Sanford, CE Huang and RL Gallo Dermatology, University of California, San Diego, La Jolla, CA The skin microbiome influences host cell behavior, but the molecular mechanisms responsible for this are incompletely understood. Many bacteria produce short-chain fatty acids (SCFAs) that in turn inhibit histone deacetylase (HDAC) activity in host tissues, an important mechanism for epigenetic control of cell function. We hypothesized that Propionibacterium acnes (P. acnes), a prominent member of the skin microbiome, can generate these metabolites and may influence cutaneous inflammation through their effects on histone acetylation. We found by GC-MS that P. acnes generates high levels of propionic acid and valeric acid only when cultured under conditions favoring fermentation, an environment similar to the occluded follicle. We confirmed that these molecules increased levels of histone acetylation by Western blot of H3K9ac and H3K27ac and ChIP-qPCR of H3K9ac in the IL-6 promoter. Using qPCR, RNA-sequencing, ELISA and multiplex analysis we observed that while stimulation of human keratinocytes (NHEK) with HDAC inhibitors alone had no effect on cytokine production, in the presence of TLR2 and TLR3 ligands, the addition of HDAC inhibitors greatly increased the production of many cytokines (i.e. IL-1b, IL-6, IL-8, TNFa, and CCL5) compared to the TLR ligands alone. In direct contrast, these HDAC inhibitors suppressed TLRstimulated inductions of these cytokines from monocytes and dendritic cells. Inhibition of HDAC activity in mouse skin confirmed these responses in vivo. The mechanism of inhibition in PBMCs and DCs was consistent with previous findings demonstrating increased expression of Mi-2b and activation of the NuRD co-repressor complex, events that were absent in NHEK. siRNA screening of NHEK identified the target of HDAC inhibition to be HDAC8 and HDAC9. These results indicate that the local microenvironment and microbe-derived SCFAs influence inflammatory gene expression in a cell type-specific manner, thus potentially explaining selective inflammation in diseases such as acne.
Gene expression signature in the bone marrow and skin of Ptpn6-insufficient mice with neutrophilic dermatosis-like disease (NDLD) A Nesterovitch1, Z Arbieva3, D Toth2, M Tharp1 and T Glant2 1 Dermatology, Rush University Medical Center, Chicago, IL, 2 Department of Orthopedic Surgery (Section of Molecular Medicine), Biochemistry and Internal Medicine, Rush University Medical Center, Chicago, IL and 3 Core Genomics Facility, University of Illinois at Chicago, Chicago, IL We recently described a new spontaneous mutation in the protein tyrosine phosphatase, nonreceptor type-6 (Ptpn6) gene. Irradiated syngeneic wild-type mice developed the same neutrophilic dermatosis-like disease (NDLD) after adoptive transfer of bone marrow cells from Ptpn6meb2/meb2 mutant mice. As Ptpn6 is involved in a myriad of signaling pathways, we used a global approach with microarray technology for the first time to characterize changes in the bone marrow and skin of motheaten-type mice. A total number of 1,511 probe sets in the bone marrow showed at least two-fold changes with FDR < 0.05, of which 256 probe sets had over four-fold changes. A group of 63 genes in the bone marrow of NDLD mice had more than a 4-fold change with FDR < 0.0001. From 503 genes encoding proteins with ITIM motif that binds to Ptpn6, 109 were up-regulated and 83 were down-regulated. We found that genes encoding hematopoietic receptors, neutrophil chemoattractants, Toll-like receptors (Tlr1, Tlr2 and Tlr4) and C-type lectin innate immunity receptors (Clec4a2, Clec4a3, Clec4n and Clec4e) were significantly up-regulated in both NDLD bone marrow and skin. The IL1b gene but not IL1awas also significantly overexpressed in skin samples, confirming the importance of the IL-1/TLR pathway in the development of early skin inflammation in NDLD mice.
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Interferon-inducible protein 16 contributes to psoriasis by modulating cytokine production in keratinocytes T Cao, S Shao, B Li, J Lei and G Wang Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Psoriasis is a common immune-mediated chronic inflammatory disease in which keratinocytes play a critical role. Recent studies showed that interferon-inducible protein 16 (IFI16), an innate immune system sensor, is overexpressed in the keratinocytes of psoriasis patients. However, the mechanism of its function in psoriasis was still largely unknown. We detected the expression level of IFI16 in separated epidermis and dermis of both psoriasis lesions and imiquimod (IMQ)-induced psoriasis-like mice. Then, we treated primary human keratinocytes with psoriasis-related cytokines to explore how IFI16 was activated in psoriasis. We also transfected primary human keratinocytes with a plasmid overexpressing IFI16 or a siRNA construct targeting IFI16, and examined the mechanism of IFI16-mediated keratinocyte activation. Further, we blocked p204—reported as the mouse ortholog of human IFI16—in a mouse model of IMQ-induced psoriasis to identify the function of IFI16 in the pathogenesis of psoriasis. We found that IFI16 was overexpressed in epidermal keratinocytes but not in the dermis of psoriasis patients and IMQ-induced mice. In addition, psoriasis-related cytokines— including interferon g (IFN-g), tumor necrosis factor a (TNF-a), and interleukin 17 (IL-17) and IL-22—induced IFI16 expression in primary human keratinocytes via activation of signal transducer and activator of transcription 3 signaling. We also observed that IFI16 activated the nuclear factor kB pathway, leading to the production of chemokine ligand 10 (CXCL10) and chemokine ligand 20 (CCL20) both in vitro and in vivo. Importantly, knocking down p204 in mice with IMQ-induced psoriasis improved epidermal hyperplasia. Our findings reveal that IFI16 promotes the development of psoriasis by inducing keratinocyte activation, and can therefore serve as a potential therapeutic target in its treatment.
Staphylococcal LTA-induced miR-143 inhibits Propionibacterium acnes-mediated inflammatory response in skin Y Lai East China Normal University, Shanghai, China Staphylococcus epidermidis (S. epidermidis) plays a critical role in modulating cutaneous inflammatory responses in skin. Although S. epidermidis has been shown to co-colonize with Propionibacterium acnes (P. acnes) in acne lesions, it is unclear whether S. epidermidis is involved in the regulation of P. acnes-induced inflammatory responses. In this study we demonstrated that S. epidermidis inhibited P. acnes-induced inflammation in skin. P. acnes induced the expression of IL-6 and TNF-a via the activation of TLR2 in both keratinocytes and mouse ears. Staphylococcal LTA activated TLR2 to induce miR-143 in keratinocytes, and miR-143, in turn, directly targeted 3’ UTR of TLR2 to decrease the stability of TLR2 mRNA and then decreased TLR2 protein, thus inhibiting P. acnes-induced pro-inflammatory cytokines. The inhibitory effect of miR-143 was further confirmed in vivo as the administration of miR-143 antagomir into mouse ears abrogated the inhibitory effect of LTA on P. acnes-induced inflammation in skin. Taken together, these observations demonstrate that staphylococcal LTA inhibits P. acnes-induced inflammation via the induction of miR-143, and suggest that local modulation of inflammatory responses by S. epidermidis at the site of acne vulgaris might be a beneficial therapeutic strategy for management of P. acnes-induced inflammation.
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Increased lipocalin-2 contributes to the pathogenesis of psoriasis by modulating neutrophil chemotaxis and cytokine secretion S Shao, T Cao, L Jin, B Li, H Fang and G Wang Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China, Xi’an, China The pathogenesis of psoriasis, a chronic inflammatory skin disease, is not fully understood yet. Antibacterial proteins not only protect skin from bacterial infections, but also mediate inflammation; however, the precise mechanism is not well defined. Our study aims to elucidate the role of lipocalin-2, an antimicrobial protein, in the pathogenesis of psoriasis. Our findings show that lipocalin-2 is highly expressed in the lesional skin of psoriasis patients. Neutralizing lipocalin-2 alleviates epidermal hyperplasia and inflammation, and especially neutrophil infiltration in an imiquimod-treated murine model. In vitro lipocalin-2 stimulates human neutrophils to produce vital proinflammatory mediators, such as interleukin (IL)-6, IL-8, tumor necrosis factor(TNF)-a, and IL-1a via a specific receptor, 24p3R, on neutrophils, activating the downstream extracellular signal-regulated kinase (Erk)-1/2 and p38-mitogenactivated protein kinase (MAPK) signaling pathways. Furthermore, our findings show that lipocalin-2 induces neutrophil chemotaxis, which is both gradient dependent and associated with Erk1/2 and p38 MAPK signaling pathways in vitro. We also identify that the sources of lipocalin-2 are keratinocytes and neutrophils in the lesional skin of psoriasis patients. Taken together, our findings reveal that lipocalin-2 participates in the pathogenesis of psoriasis by modulating neutrophils, and can be a potential target for psoriasis treatment.
S88 Journal of Investigative Dermatology (2016), Volume 136
Antiseptics elicit personalized alterations to skin microbial communities A SanMiguel, J Meisel, J Horwinski, Q Zheng and EA Grice Dermatology, University of Pennsylvania, Philadelphia, PA Antimicrobial drugs are commonly employed to inhibit the growth of pathogenic microorganisms on the skin. These treatments are rarely narrow in spectrum and act upon a range of members of our commensal microbiota. Only recently have these effects been fully appreciated, as previous research has relied on isolated, culture-dependent systems that cannot account for the complex growth requirements of whole bacterial communities, and often exclude numerous affected species. This is especially true at the skin surface in which antisepsis is used with the expectation of complete bacterial sterilization. Indeed, many antiseptics boast efficacy rates >99% when used against pathogenic microorganisms in artificial colonization and culture experiments, though other studies have reported modest effects when measuring the response of cutaneous microorganisms in their natural environments. Herein, we describe the first steps toward determining the true effect of antiseptics on a community-wide scale through the use of 16S rRNA gene sequencing in a longitudinal human treatment study. We report a personalized effect of treatment that is highly dependent upon starting community composition. Substantial shifts in resident structure and bacterial load were observed in a subset of the population, whereas another subset exhibited a strong degree of resilience. The persistence of these changes over time was also evaluated, illustrating dynamic succession with distinct levels of periodicity. Few antiseptic-specific effects were observed among study participants; rather most variations were the result of generalized perturbation. In all, this suggests that antiseptic treatments, while potent in vitro, may have a more modest effect on cutaneous bacteria in their natural environment. Moreover, it appears that certain communities may respond more readily than others, an important consideration when evaluating the context and use of these antibacterial treatments.
498
The microbiome modulates cytokine production in the skin through epigenetic control of histone acetylation J Sanford, CE Huang and RL Gallo Dermatology, University of California, San Diego, La Jolla, CA The skin microbiome influences host cell behavior, but the molecular mechanisms responsible for this are incompletely understood. Many bacteria produce short-chain fatty acids (SCFAs) that in turn inhibit histone deacetylase (HDAC) activity in host tissues, an important mechanism for epigenetic control of cell function. We hypothesized that Propionibacterium acnes (P. acnes), a prominent member of the skin microbiome, can generate these metabolites and may influence cutaneous inflammation through their effects on histone acetylation. We found by GC-MS that P. acnes generates high levels of propionic acid and valeric acid only when cultured under conditions favoring fermentation, an environment similar to the occluded follicle. We confirmed that these molecules increased levels of histone acetylation by Western blot of H3K9ac and H3K27ac and ChIP-qPCR of H3K9ac in the IL-6 promoter. Using qPCR, RNA-sequencing, ELISA and multiplex analysis we observed that while stimulation of human keratinocytes (NHEK) with HDAC inhibitors alone had no effect on cytokine production, in the presence of TLR2 and TLR3 ligands, the addition of HDAC inhibitors greatly increased the production of many cytokines (i.e. IL-1b, IL-6, IL-8, TNFa, and CCL5) compared to the TLR ligands alone. In direct contrast, these HDAC inhibitors suppressed TLRstimulated inductions of these cytokines from monocytes and dendritic cells. Inhibition of HDAC activity in mouse skin confirmed these responses in vivo. The mechanism of inhibition in PBMCs and DCs was consistent with previous findings demonstrating increased expression of Mi-2b and activation of the NuRD co-repressor complex, events that were absent in NHEK. siRNA screening of NHEK identified the target of HDAC inhibition to be HDAC8 and HDAC9. These results indicate that the local microenvironment and microbe-derived SCFAs influence inflammatory gene expression in a cell type-specific manner, thus potentially explaining selective inflammation in diseases such as acne.
Gene expression signature in the bone marrow and skin of Ptpn6-insufficient mice with neutrophilic dermatosis-like disease (NDLD) A Nesterovitch1, Z Arbieva3, D Toth2, M Tharp1 and T Glant2 1 Dermatology, Rush University Medical Center, Chicago, IL, 2 Department of Orthopedic Surgery (Section of Molecular Medicine), Biochemistry and Internal Medicine, Rush University Medical Center, Chicago, IL and 3 Core Genomics Facility, University of Illinois at Chicago, Chicago, IL We recently described a new spontaneous mutation in the protein tyrosine phosphatase, nonreceptor type-6 (Ptpn6) gene. Irradiated syngeneic wild-type mice developed the same neutrophilic dermatosis-like disease (NDLD) after adoptive transfer of bone marrow cells from Ptpn6meb2/meb2 mutant mice. As Ptpn6 is involved in a myriad of signaling pathways, we used a global approach with microarray technology for the first time to characterize changes in the bone marrow and skin of motheaten-type mice. A total number of 1,511 probe sets in the bone marrow showed at least two-fold changes with FDR < 0.05, of which 256 probe sets had over four-fold changes. A group of 63 genes in the bone marrow of NDLD mice had more than a 4-fold change with FDR < 0.0001. From 503 genes encoding proteins with ITIM motif that binds to Ptpn6, 109 were up-regulated and 83 were down-regulated. We found that genes encoding hematopoietic receptors, neutrophil chemoattractants, Toll-like receptors (Tlr1, Tlr2 and Tlr4) and C-type lectin innate immunity receptors (Clec4a2, Clec4a3, Clec4n and Clec4e) were significantly up-regulated in both NDLD bone marrow and skin. The IL1b gene but not IL1awas also significantly overexpressed in skin samples, confirming the importance of the IL-1/TLR pathway in the development of early skin inflammation in NDLD mice.
499
500
Interferon-inducible protein 16 contributes to psoriasis by modulating cytokine production in keratinocytes T Cao, S Shao, B Li, J Lei and G Wang Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Psoriasis is a common immune-mediated chronic inflammatory disease in which keratinocytes play a critical role. Recent studies showed that interferon-inducible protein 16 (IFI16), an innate immune system sensor, is overexpressed in the keratinocytes of psoriasis patients. However, the mechanism of its function in psoriasis was still largely unknown. We detected the expression level of IFI16 in separated epidermis and dermis of both psoriasis lesions and imiquimod (IMQ)-induced psoriasis-like mice. Then, we treated primary human keratinocytes with psoriasis-related cytokines to explore how IFI16 was activated in psoriasis. We also transfected primary human keratinocytes with a plasmid overexpressing IFI16 or a siRNA construct targeting IFI16, and examined the mechanism of IFI16-mediated keratinocyte activation. Further, we blocked p204—reported as the mouse ortholog of human IFI16—in a mouse model of IMQ-induced psoriasis to identify the function of IFI16 in the pathogenesis of psoriasis. We found that IFI16 was overexpressed in epidermal keratinocytes but not in the dermis of psoriasis patients and IMQ-induced mice. In addition, psoriasis-related cytokines— including interferon g (IFN-g), tumor necrosis factor a (TNF-a), and interleukin 17 (IL-17) and IL-22—induced IFI16 expression in primary human keratinocytes via activation of signal transducer and activator of transcription 3 signaling. We also observed that IFI16 activated the nuclear factor kB pathway, leading to the production of chemokine ligand 10 (CXCL10) and chemokine ligand 20 (CCL20) both in vitro and in vivo. Importantly, knocking down p204 in mice with IMQ-induced psoriasis improved epidermal hyperplasia. Our findings reveal that IFI16 promotes the development of psoriasis by inducing keratinocyte activation, and can therefore serve as a potential therapeutic target in its treatment.
Staphylococcal LTA-induced miR-143 inhibits Propionibacterium acnes-mediated inflammatory response in skin Y Lai East China Normal University, Shanghai, China Staphylococcus epidermidis (S. epidermidis) plays a critical role in modulating cutaneous inflammatory responses in skin. Although S. epidermidis has been shown to co-colonize with Propionibacterium acnes (P. acnes) in acne lesions, it is unclear whether S. epidermidis is involved in the regulation of P. acnes-induced inflammatory responses. In this study we demonstrated that S. epidermidis inhibited P. acnes-induced inflammation in skin. P. acnes induced the expression of IL-6 and TNF-a via the activation of TLR2 in both keratinocytes and mouse ears. Staphylococcal LTA activated TLR2 to induce miR-143 in keratinocytes, and miR-143, in turn, directly targeted 3’ UTR of TLR2 to decrease the stability of TLR2 mRNA and then decreased TLR2 protein, thus inhibiting P. acnes-induced pro-inflammatory cytokines. The inhibitory effect of miR-143 was further confirmed in vivo as the administration of miR-143 antagomir into mouse ears abrogated the inhibitory effect of LTA on P. acnes-induced inflammation in skin. Taken together, these observations demonstrate that staphylococcal LTA inhibits P. acnes-induced inflammation via the induction of miR-143, and suggest that local modulation of inflammatory responses by S. epidermidis at the site of acne vulgaris might be a beneficial therapeutic strategy for management of P. acnes-induced inflammation.
501
502
Increased lipocalin-2 contributes to the pathogenesis of psoriasis by modulating neutrophil chemotaxis and cytokine secretion S Shao, T Cao, L Jin, B Li, H Fang and G Wang Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China, Xi’an, China The pathogenesis of psoriasis, a chronic inflammatory skin disease, is not fully understood yet. Antibacterial proteins not only protect skin from bacterial infections, but also mediate inflammation; however, the precise mechanism is not well defined. Our study aims to elucidate the role of lipocalin-2, an antimicrobial protein, in the pathogenesis of psoriasis. Our findings show that lipocalin-2 is highly expressed in the lesional skin of psoriasis patients. Neutralizing lipocalin-2 alleviates epidermal hyperplasia and inflammation, and especially neutrophil infiltration in an imiquimod-treated murine model. In vitro lipocalin-2 stimulates human neutrophils to produce vital proinflammatory mediators, such as interleukin (IL)-6, IL-8, tumor necrosis factor(TNF)-a, and IL-1a via a specific receptor, 24p3R, on neutrophils, activating the downstream extracellular signal-regulated kinase (Erk)-1/2 and p38-mitogenactivated protein kinase (MAPK) signaling pathways. Furthermore, our findings show that lipocalin-2 induces neutrophil chemotaxis, which is both gradient dependent and associated with Erk1/2 and p38 MAPK signaling pathways in vitro. We also identify that the sources of lipocalin-2 are keratinocytes and neutrophils in the lesional skin of psoriasis patients. Taken together, our findings reveal that lipocalin-2 participates in the pathogenesis of psoriasis by modulating neutrophils, and can be a potential target for psoriasis treatment.
S88 Journal of Investigative Dermatology (2016), Volume 136
Antiseptics elicit personalized alterations to skin microbial communities A SanMiguel, J Meisel, J Horwinski, Q Zheng and EA Grice Dermatology, University of Pennsylvania, Philadelphia, PA Antimicrobial drugs are commonly employed to inhibit the growth of pathogenic microorganisms on the skin. These treatments are rarely narrow in spectrum and act upon a range of members of our commensal microbiota. Only recently have these effects been fully appreciated, as previous research has relied on isolated, culture-dependent systems that cannot account for the complex growth requirements of whole bacterial communities, and often exclude numerous affected species. This is especially true at the skin surface in which antisepsis is used with the expectation of complete bacterial sterilization. Indeed, many antiseptics boast efficacy rates >99% when used against pathogenic microorganisms in artificial colonization and culture experiments, though other studies have reported modest effects when measuring the response of cutaneous microorganisms in their natural environments. Herein, we describe the first steps toward determining the true effect of antiseptics on a community-wide scale through the use of 16S rRNA gene sequencing in a longitudinal human treatment study. We report a personalized effect of treatment that is highly dependent upon starting community composition. Substantial shifts in resident structure and bacterial load were observed in a subset of the population, whereas another subset exhibited a strong degree of resilience. The persistence of these changes over time was also evaluated, illustrating dynamic succession with distinct levels of periodicity. Few antiseptic-specific effects were observed among study participants; rather most variations were the result of generalized perturbation. In all, this suggests that antiseptic treatments, while potent in vitro, may have a more modest effect on cutaneous bacteria in their natural environment. Moreover, it appears that certain communities may respond more readily than others, an important consideration when evaluating the context and use of these antibacterial treatments.