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303 The adaptive response of mesenchymal stem cells to danger signals regulate neutrophil activation in cutaneous wounds
ABSTRACTS | Inflammation, Immunity and Infection 298
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Fli1-deficient adipocytes promote the spontaneous development of dermal fibrosis and vasculopathy mimicking systemic sclerosis through adipocytes-to-myofibroblast transdifferentiation and defective vasculogenesis T Miyagawa1, Y Asano1, R Saigusa1, K Nakamura1, M Hirabayashi1, T Yamashita1, S Miura1, T Taniguchi1, A Yoshizaki1, M Trojanowska2 and S Sato1 1 Dermatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan and 2 Boston University School of Medicine, Boston, MA Systemic sclerosis (SSc) is a multisystem chronic disease manifesting autoimmunity, vasculopathy, and extensive organ fibrosis. Although its pathogenesis still remains unknown, adipokines have recently caught much attention as a family of cytokines regulating the three cardinal pathological features of this disease. On the other hand, our studies have demonstrated that the deficiency of transcription factor Fli1 induces SSc-like phenotypes in various cells, such as dermal fibroblasts, endothelial cells, macrophages, and epithelial cells. Therefore, we generated adipocyte-specific Fli1 knockout (Fli1 AdipoKO) mice (Fli1flox/flox;Adipo-Cre) and investigated whether these mice recapitulate the three cardinal features of SSc. Of note, Fli1 AdipoKO mice spontaneously developed dermal fibrosis at the age of 3 months. In double immunofluorescence for a-smooth muscle actin and perilipin, double positive fusiform cells were evident throughout the deep dermis in Fli1 AdipoKO mice, suggesting the promotion of adipocyte-to-myofibroblast transdifferentiation. In addition, vascular structural and functional abnormalities, such as arteriolar stenosis and increased permeability, were remarkable in Fli1 AdipoKO mice. Importantly, bone marrowderived mesenchymal stem cells, a precursor of pro-angiogenic hematopoietic cells, exhibited de-differentiated phenotype characterized by a-smooth muscle actin downregulation and Rgs5 up-regulation, suggesting the contribution of defective vasculogenesis to the development of vascular abnormalities. Taken together, these results indicate that Fli1deficient adipocytes may be involved in the development of dermal fibrosis and vasculopathy recapitulating SSc, suggesting a potential contribution of phenotypically altered adipocytes to the development of SSc.
Development of a novel, perceived stress-sensitive humanized mouse model of atopic dermatitis A Gilhar1, A Keren1, Y Ullmann1 and R Paus2 1 Technion, Israel and 2 Centre for Dermatology Research Centre, University of Manchester, UK Optimal animal models for human atopic dermatitis (AD), which fully recapitulate key characteristics of AD such as, reduced filaggrin and increased TSLP expression, increased number of epidermal Langerhans, eosinophils, mast cells and a dermal Th2 cytokine profile, ideally in human skin, urgently to be developed, not the least for preclinical R&D. Here we report the development of a novel, humanized mouse model of AD, in which xenotransplanted healthy human skin on SCID mice are i.d injected one month after transplantation with autologous PBMCs that were pre-stimulated in vitro by a combination of the Th2-type cytokine, IL-4, and LPS. Features of AD were observed within 14 days after the injection. Given that psychoemotional stress triggers flare-ups in many AD patients, we next tested the effect of perceived (sonic) stress, in the humanized AD mouse model during a dexamethasone-induced remission phase. This showed the reappearance of typical AD lesions in human skin xenotransplants 14 days after exposure to sound stress for 24 hours, thus further documenting that perfectly recapitulates human AD. Finally, we tested the therapeutic response of established human xenotransplant AD lesions to classical and novel AD treatment regimens. A complete normalization of AD lesions in human skin grafts was observed in mice treated i.p with the NK1 antagonist, aprepitant, as well as with topical dexamethasone or tacrolimus compared to vehicle-treated mice, while AD lesions were completely unresponsiveness to oral treatment with the PDE4 inhibitor, apremilast. This contradicts the claimed ineffectiveness of aprepitant in a (likely misleading) murine “AD” model, but is perfectly in line with therapeutic effects of aprepitant, glucocorticosteroids and tacrolimus in AD patients. Thus, the novel humanized AD mouse model constitutes a major advance in preclinical AD research, and promises to be more predictive for the clinical response seen with candidate AD treatment strategies than any other preclinical AD model.
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Tonicity signals drive immunoparalysis and anti-inflammatory Th17 cell properties J Matthias and C Zielinski TU Munich, Munich, Germany Sodium chloride has recently been demonstrated to strongly enhance the generation of Th17 cells in both mouse and man. This finding has helped consolidate the epidemiological correlation of enhanced dietary salt intake with the increase in the incidence of autoimmune diseases such as multiple sclerosis. Our data support the Th17 cell promoting effect of sodium chloride on human Th17 cell priming. However, Th17 cells acquired anti-inflammatory properties upon stimulation in sodium chloride enriched micromilieus as evidenced by strong induction of FOXP3 expression and TGF-b production within IL-17 and ROR-gt positive T cells. In addition, sodium chloride also strongly suppressed cytotoxic functions of human T cells and induced T cell exhaustion. This functional modulation occurred at sodium chloride concentrations that are in the range of physiological skin and lymph levels but highly elevated compared to the blood. Tissue salt concentrations are also prone to a wide range of fluctuations depending on dietary habits, which suggests that sodium chloride can have an in vivo impact on functionalities of tissue residing T cells. Together, our data suggest that despite the reported association of salt with induction of pathogenic Th17 cells, sodium chloride instead is a potent inducer of the anti-inflammatory Th17 cell subset and also paralyzes cytotoxic T cell properties in humans. Therefore, our data suggest that reduction of sodium chloride levels by either dietary restriction or specific targeting of downstream signaling events could counteract T cell immunoparalysis and exhaustion in settings of malignancies or chronic infections.
The psoriasis-associated IL-17A induces and cooperates with IL-36 cytokines to control keratinocyte differentiation and function CM Pfaff, D Kluwig, Y Marquardt, K Fietkau, B Lu¨scher and JM Baron Uniklinik RWTH Aachen, Aachen, Germany The proinflammatory IL-17A has been identified as a key cytokine in the pathogenesis of psoriasis. The molecular consequences of IL-17A signaling in the skin are only partially understood. Therefore, we evaluated the IL-17A effects on organotypic 3D skin models. We used psoriasis models developed with normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts from psoriatic lesions of patients and control models containing cells from healthy donors. Stimulation with IL-17A led to a defective differentiation of keratinocytes in both models. Microarray analysis revealed down-regulation of genes important for epidermal differentiation and skin barrier formation and an increased expression of antimicrobial peptides (AMPs) upon IL-17A stimulation. Furthermore we found an up-regulation of the IL36 cytokine family in IL-17A treated models. Thus we speculated that the IL-17A effects were at least in part mediated by the induction of IL-36 cytokines. Indeed their application was sufficient to induce the expression of genes encoding different AMPs although to a lower extent compared to IL-17A. We could show that IL-36 cytokines are produced and secreted upon IL-17A stimulation in NHEKs and 3D models and that these proteins are unprocessed and therefore almost inactive. We were able to activate the released IL-36g by addition of recombinant neutrophil elastase and detected an increased expression of genes encoding AMPs, IL-36 cytokines and IL-17C. Furthermore the activated IL-36g in combination with IL17A showed synergistic effects on gene expression in NHEKs. In conclusion we found that IL17A disturbed differentiation in control and psoriasis 3D skin models. The analysis of the downstream effects revealed that inactive IL-36 cytokines are produced but require proteolytic activation to enhance the IL-17A effects in a synergistic manner. This suggests an amplification cycle that involves IL-17A, IL-36 and activating proteases providing a molecular explanation for a persistent psoriatic phenotype.
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A novel population of skin-tropic T cells with a potential role in wound healing MM Klicznik1, T Duhen2, S Motley2, B Hoellbacher1,2, A Sir3, R Reitsamer3, EM Murauer4, DJ Campbell2 and IK Gratz1,4 1 Molecular Biology, University of Salzburg, Salzburg, Austria, 2 Benaroya Research Institute, Seattle, WA, 3 Breast Center, University Hosptial Salzburg, Salzburg, Austria and 4 EB House Austria, Paracelsus Medical University, Salzburg, Austria The skin functions as the primary barrier between an organism and the outside world. Although it is home to a robust and diverse community of commensal microorganisms, it must also prevent entry of environmental toxins, irritants, and numerous viral, bacterial and parasitic pathogens. To effectively maintain its barrier function, the skin must undergo rapid and highly efficient tissue repair when damaged or compromised, and this is associated with local antimicrobial and inflammatory responses that help prevent or combat concurrent infection. Consistent with its functions in maintaining barrier integrity and preventing infection, the skin is home to a number of specialized T cell populations. Using 33-parameter CyTOF (Cytometry by time-of-flight) analysis and standard flow cytometry we have identified a novel population of CLA+CD4+ T cells in the peripheral blood of healthy subjects that expresses the CD103 integrin and produces the novel cytokine combination of IL-22, GMCSF and IL-13 upon activation (CD103+CLAhi cells). Interestingly, this cytokine combination is also produced by epidermal CD103+CLAhiCD69+ TRM in the skin itself. Based on the ability of IL-22 to promote keratinocyte proliferation, migration and production of antimicrobial peptides, and GM-CSF and IL-13 to act on fibroblasts, and to mediate the differentiation of monocytes and macrophages into ‘alternatively activated’ cells that promote tissue repair, we are currently testing the impact of CD103+CLAhi cells on skin repair functions. In summary, we have identified a circulating population that resembles skin TRM and might be useful in studying (recirculating) skin TRM.
S244 Journal of Investigative Dermatology (2017), Volume 137
The adaptive response of mesenchymal stem cells to danger signals regulate neutrophil activation in cutaneous wounds S Munir, K Singh, A BASU and K Scharffetter-Kochanek Dermatology and Allergic Diseases, Ulm University, Ulm, Germany Mesenchymal stem cells (MSCs) are most likely endowed with the capacity to sense environmental cues like infection and generate an integrated adaptive response in the interest of tissue protection. While neutrophils are important in initial phases of wound healing to effectively cleanse tissue debris and avoid microbial infections after wounding, their unrestrained activation may lead to tissue break down and delayed wound healing. In the present study, we wished to investigate how MSCs adaptively regulate neutrophils function under conditions of wound infection, where MSC suppression of neutrophil functions would rather be detrimental. Here, we evaluated the adaptive response of adipose derived MSCs (ADMSCs) on activated neutrophil functions in the presence and absence of pathogen-associated molecular patterns (PAMP); lipopolysaccharide (LPS) mimicking an infectious wound environment. Of note, LPS-treated AD-MSCs substantially augment neutrophil activation resulting in an increased neutrophil extracellular trap (NET) formation and increased ROS production as opposed to MSCs suppression of activated neutrophils under “non-infectious” conditions. To further explore whether toll like receptor-4 (TLR-4) present on MSCs surface, is involved in the adaptive response of AD-MSC, we specifically silenced the TLR-4 receptor gene. Our results show that TLR-4 silenced MSCs upon LPS treatment failed to activate neutrophils, subsequent NET formation and ROS production, indicating a causal role for TLR-4-dependent sensing LPS, subsequent signaling and shaping the adaptive response. RNAseq analysis, RT PCR, antibody arrays, and factor specific ELISA of MSCs cultured in the presence or absence of LPS uncovered GCP-2 and IL-8, which are known to recruit and activate neutrophils. Collectively, we identified the mechanism underlying the master role of MSCs in the control of infectious cues and tissue integrity. Our data may even hold promise to be therapeutically exploited for the benefit of patients with difficult-to-treat and/or infected wounds.
299
Fli1-deficient adipocytes promote the spontaneous development of dermal fibrosis and vasculopathy mimicking systemic sclerosis through adipocytes-to-myofibroblast transdifferentiation and defective vasculogenesis T Miyagawa1, Y Asano1, R Saigusa1, K Nakamura1, M Hirabayashi1, T Yamashita1, S Miura1, T Taniguchi1, A Yoshizaki1, M Trojanowska2 and S Sato1 1 Dermatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan and 2 Boston University School of Medicine, Boston, MA Systemic sclerosis (SSc) is a multisystem chronic disease manifesting autoimmunity, vasculopathy, and extensive organ fibrosis. Although its pathogenesis still remains unknown, adipokines have recently caught much attention as a family of cytokines regulating the three cardinal pathological features of this disease. On the other hand, our studies have demonstrated that the deficiency of transcription factor Fli1 induces SSc-like phenotypes in various cells, such as dermal fibroblasts, endothelial cells, macrophages, and epithelial cells. Therefore, we generated adipocyte-specific Fli1 knockout (Fli1 AdipoKO) mice (Fli1flox/flox;Adipo-Cre) and investigated whether these mice recapitulate the three cardinal features of SSc. Of note, Fli1 AdipoKO mice spontaneously developed dermal fibrosis at the age of 3 months. In double immunofluorescence for a-smooth muscle actin and perilipin, double positive fusiform cells were evident throughout the deep dermis in Fli1 AdipoKO mice, suggesting the promotion of adipocyte-to-myofibroblast transdifferentiation. In addition, vascular structural and functional abnormalities, such as arteriolar stenosis and increased permeability, were remarkable in Fli1 AdipoKO mice. Importantly, bone marrowderived mesenchymal stem cells, a precursor of pro-angiogenic hematopoietic cells, exhibited de-differentiated phenotype characterized by a-smooth muscle actin downregulation and Rgs5 up-regulation, suggesting the contribution of defective vasculogenesis to the development of vascular abnormalities. Taken together, these results indicate that Fli1deficient adipocytes may be involved in the development of dermal fibrosis and vasculopathy recapitulating SSc, suggesting a potential contribution of phenotypically altered adipocytes to the development of SSc.
Development of a novel, perceived stress-sensitive humanized mouse model of atopic dermatitis A Gilhar1, A Keren1, Y Ullmann1 and R Paus2 1 Technion, Israel and 2 Centre for Dermatology Research Centre, University of Manchester, UK Optimal animal models for human atopic dermatitis (AD), which fully recapitulate key characteristics of AD such as, reduced filaggrin and increased TSLP expression, increased number of epidermal Langerhans, eosinophils, mast cells and a dermal Th2 cytokine profile, ideally in human skin, urgently to be developed, not the least for preclinical R&D. Here we report the development of a novel, humanized mouse model of AD, in which xenotransplanted healthy human skin on SCID mice are i.d injected one month after transplantation with autologous PBMCs that were pre-stimulated in vitro by a combination of the Th2-type cytokine, IL-4, and LPS. Features of AD were observed within 14 days after the injection. Given that psychoemotional stress triggers flare-ups in many AD patients, we next tested the effect of perceived (sonic) stress, in the humanized AD mouse model during a dexamethasone-induced remission phase. This showed the reappearance of typical AD lesions in human skin xenotransplants 14 days after exposure to sound stress for 24 hours, thus further documenting that perfectly recapitulates human AD. Finally, we tested the therapeutic response of established human xenotransplant AD lesions to classical and novel AD treatment regimens. A complete normalization of AD lesions in human skin grafts was observed in mice treated i.p with the NK1 antagonist, aprepitant, as well as with topical dexamethasone or tacrolimus compared to vehicle-treated mice, while AD lesions were completely unresponsiveness to oral treatment with the PDE4 inhibitor, apremilast. This contradicts the claimed ineffectiveness of aprepitant in a (likely misleading) murine “AD” model, but is perfectly in line with therapeutic effects of aprepitant, glucocorticosteroids and tacrolimus in AD patients. Thus, the novel humanized AD mouse model constitutes a major advance in preclinical AD research, and promises to be more predictive for the clinical response seen with candidate AD treatment strategies than any other preclinical AD model.
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Tonicity signals drive immunoparalysis and anti-inflammatory Th17 cell properties J Matthias and C Zielinski TU Munich, Munich, Germany Sodium chloride has recently been demonstrated to strongly enhance the generation of Th17 cells in both mouse and man. This finding has helped consolidate the epidemiological correlation of enhanced dietary salt intake with the increase in the incidence of autoimmune diseases such as multiple sclerosis. Our data support the Th17 cell promoting effect of sodium chloride on human Th17 cell priming. However, Th17 cells acquired anti-inflammatory properties upon stimulation in sodium chloride enriched micromilieus as evidenced by strong induction of FOXP3 expression and TGF-b production within IL-17 and ROR-gt positive T cells. In addition, sodium chloride also strongly suppressed cytotoxic functions of human T cells and induced T cell exhaustion. This functional modulation occurred at sodium chloride concentrations that are in the range of physiological skin and lymph levels but highly elevated compared to the blood. Tissue salt concentrations are also prone to a wide range of fluctuations depending on dietary habits, which suggests that sodium chloride can have an in vivo impact on functionalities of tissue residing T cells. Together, our data suggest that despite the reported association of salt with induction of pathogenic Th17 cells, sodium chloride instead is a potent inducer of the anti-inflammatory Th17 cell subset and also paralyzes cytotoxic T cell properties in humans. Therefore, our data suggest that reduction of sodium chloride levels by either dietary restriction or specific targeting of downstream signaling events could counteract T cell immunoparalysis and exhaustion in settings of malignancies or chronic infections.
The psoriasis-associated IL-17A induces and cooperates with IL-36 cytokines to control keratinocyte differentiation and function CM Pfaff, D Kluwig, Y Marquardt, K Fietkau, B Lu¨scher and JM Baron Uniklinik RWTH Aachen, Aachen, Germany The proinflammatory IL-17A has been identified as a key cytokine in the pathogenesis of psoriasis. The molecular consequences of IL-17A signaling in the skin are only partially understood. Therefore, we evaluated the IL-17A effects on organotypic 3D skin models. We used psoriasis models developed with normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts from psoriatic lesions of patients and control models containing cells from healthy donors. Stimulation with IL-17A led to a defective differentiation of keratinocytes in both models. Microarray analysis revealed down-regulation of genes important for epidermal differentiation and skin barrier formation and an increased expression of antimicrobial peptides (AMPs) upon IL-17A stimulation. Furthermore we found an up-regulation of the IL36 cytokine family in IL-17A treated models. Thus we speculated that the IL-17A effects were at least in part mediated by the induction of IL-36 cytokines. Indeed their application was sufficient to induce the expression of genes encoding different AMPs although to a lower extent compared to IL-17A. We could show that IL-36 cytokines are produced and secreted upon IL-17A stimulation in NHEKs and 3D models and that these proteins are unprocessed and therefore almost inactive. We were able to activate the released IL-36g by addition of recombinant neutrophil elastase and detected an increased expression of genes encoding AMPs, IL-36 cytokines and IL-17C. Furthermore the activated IL-36g in combination with IL17A showed synergistic effects on gene expression in NHEKs. In conclusion we found that IL17A disturbed differentiation in control and psoriasis 3D skin models. The analysis of the downstream effects revealed that inactive IL-36 cytokines are produced but require proteolytic activation to enhance the IL-17A effects in a synergistic manner. This suggests an amplification cycle that involves IL-17A, IL-36 and activating proteases providing a molecular explanation for a persistent psoriatic phenotype.
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A novel population of skin-tropic T cells with a potential role in wound healing MM Klicznik1, T Duhen2, S Motley2, B Hoellbacher1,2, A Sir3, R Reitsamer3, EM Murauer4, DJ Campbell2 and IK Gratz1,4 1 Molecular Biology, University of Salzburg, Salzburg, Austria, 2 Benaroya Research Institute, Seattle, WA, 3 Breast Center, University Hosptial Salzburg, Salzburg, Austria and 4 EB House Austria, Paracelsus Medical University, Salzburg, Austria The skin functions as the primary barrier between an organism and the outside world. Although it is home to a robust and diverse community of commensal microorganisms, it must also prevent entry of environmental toxins, irritants, and numerous viral, bacterial and parasitic pathogens. To effectively maintain its barrier function, the skin must undergo rapid and highly efficient tissue repair when damaged or compromised, and this is associated with local antimicrobial and inflammatory responses that help prevent or combat concurrent infection. Consistent with its functions in maintaining barrier integrity and preventing infection, the skin is home to a number of specialized T cell populations. Using 33-parameter CyTOF (Cytometry by time-of-flight) analysis and standard flow cytometry we have identified a novel population of CLA+CD4+ T cells in the peripheral blood of healthy subjects that expresses the CD103 integrin and produces the novel cytokine combination of IL-22, GMCSF and IL-13 upon activation (CD103+CLAhi cells). Interestingly, this cytokine combination is also produced by epidermal CD103+CLAhiCD69+ TRM in the skin itself. Based on the ability of IL-22 to promote keratinocyte proliferation, migration and production of antimicrobial peptides, and GM-CSF and IL-13 to act on fibroblasts, and to mediate the differentiation of monocytes and macrophages into ‘alternatively activated’ cells that promote tissue repair, we are currently testing the impact of CD103+CLAhi cells on skin repair functions. In summary, we have identified a circulating population that resembles skin TRM and might be useful in studying (recirculating) skin TRM.
S244 Journal of Investigative Dermatology (2017), Volume 137
The adaptive response of mesenchymal stem cells to danger signals regulate neutrophil activation in cutaneous wounds S Munir, K Singh, A BASU and K Scharffetter-Kochanek Dermatology and Allergic Diseases, Ulm University, Ulm, Germany Mesenchymal stem cells (MSCs) are most likely endowed with the capacity to sense environmental cues like infection and generate an integrated adaptive response in the interest of tissue protection. While neutrophils are important in initial phases of wound healing to effectively cleanse tissue debris and avoid microbial infections after wounding, their unrestrained activation may lead to tissue break down and delayed wound healing. In the present study, we wished to investigate how MSCs adaptively regulate neutrophils function under conditions of wound infection, where MSC suppression of neutrophil functions would rather be detrimental. Here, we evaluated the adaptive response of adipose derived MSCs (ADMSCs) on activated neutrophil functions in the presence and absence of pathogen-associated molecular patterns (PAMP); lipopolysaccharide (LPS) mimicking an infectious wound environment. Of note, LPS-treated AD-MSCs substantially augment neutrophil activation resulting in an increased neutrophil extracellular trap (NET) formation and increased ROS production as opposed to MSCs suppression of activated neutrophils under “non-infectious” conditions. To further explore whether toll like receptor-4 (TLR-4) present on MSCs surface, is involved in the adaptive response of AD-MSC, we specifically silenced the TLR-4 receptor gene. Our results show that TLR-4 silenced MSCs upon LPS treatment failed to activate neutrophils, subsequent NET formation and ROS production, indicating a causal role for TLR-4-dependent sensing LPS, subsequent signaling and shaping the adaptive response. RNAseq analysis, RT PCR, antibody arrays, and factor specific ELISA of MSCs cultured in the presence or absence of LPS uncovered GCP-2 and IL-8, which are known to recruit and activate neutrophils. Collectively, we identified the mechanism underlying the master role of MSCs in the control of infectious cues and tissue integrity. Our data may even hold promise to be therapeutically exploited for the benefit of patients with difficult-to-treat and/or infected wounds.