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431 Live demodex mites modulate the inflammatory responses of sebocytes and PBMCs
ABSTRACTS | Inflammation, Immunity and Infection 430
431
Preclinical evaluation of a novel non-steroidal glucocorticoid receptor agonist with low systemic exposure and reduced potential for skin atrophy H Norsgaard, M Stahlhut, S Eirefelt, N Svitacheva, PS Johnson, M Krogh-Madsen, TT Marstrand, DA Ewald, KN Dack and AK Ollerstam LEO Pharma, Ballerup, Denmark Topical corticosteroids are widely used efficacious drugs but are associated with both local and systemic adverse effects. With the aim to provide an effective but safer topical treatment, we here describe a novel non-steroidal glucocorticoid receptor (GR) agonist with low systemic exposure and reduced potential for skin atrophy induction. LEO 134310 was shown to be a full GR agonist with high affinity for GR and low nanomolar potency in an inflammatory functional assay in human PBMCs. GR was the only nuclear hormone receptor affected by LEO 134310 in a panel of nuclear receptors, including the mineralocorticoid receptor, a receptor which has been associated with induction of skin atrophy. In blood and liver LEO 134310 is rapidly inactivated at a labile site, leading to high systemic clearance. In accordance with this profile, topical treatment with LEO 134310 in a TPA-induced dermatitis mouse model resulted in comparable efficacy to betamethasone valerate (BMV), with no systemic adverse effects at maximally effective doses. In contrast, BMV and clobetasol propionate (CP) markedly reduced body weight at maximally effective doses after topical treatment. Furthermore, skin atrophy after 4-week application to minipigs was minimal after treatment with LEO 134310 compared with significant skin atrophy induced by treatment with CP and BMV. LEO 134310 showed similar levels of target engagement as BMV, measured by FKBP5 mRNA, but lower than CP. Finally, topical treatment with LEO 134310 in a human skin explant model resulted in full agonism, measured by GR target engagement, albeit at higher dosage strength than required for BMV and CP. In conclusion, LEO 134310A shows an improved therapeutic index in preclinical studies compared to traditional corticosteroids and may potentially offer an improved topical treatment solution for patients with atopic dermatitis and psoriasis.
Live demodex mites modulate the inflammatory responses of sebocytes and PBMCs S Gatault1, R Foley1, C Chaussade2, L Shiels1, D Piwnica2 and F Powell1 1 UCD Charles Institute of Dermatology, Dublin, Ireland and 2 Nestle Skin Health R&D, Sophia Antipolis, France Demodex folliculorum mites are the largest, most complex organisms of the cutaneous microbiome and reside in the pilosebaceous units of normal adult facial skin. Mites are recognized pathogens in animal cutaneous disease but their pathogenic potential in humans and their mode of interaction with host resident/immune cells is unknown. A role for mites in the pathogenesis of rosacea has been suggested. We investigated the secretion of inflammatory mediators by sebocytes and peripheral blood mononuclear cells co-cultured with human-derived live demodex mites. Demodex mites, extracted from human skin using the Modified Standardized Skin Surface Biopsy (MSSSB), were incubated with rat preputial sebocytes (RPSs) and human peripheral blood mononuclear cells (PBMCs) for 24 hours in three independent experiments. The release of inflammatory mediators by these cells was assessed by Luminex array, proteome profiler array and ELISAs. RPSs released significant levels of CXCL3 (P < 0.001), ICAM-1 (P < 0.01), IFNg (P < 0.001), TNFa (P < 0.01) and IL-6 (P < 0.01) after incubation for 24 hours with live Demodex mites. In the presence of live demodex mites, PBMCs do not release inflammatory cytokines during their initial (6 hours) exposure suggesting that mites possess downregulatory properties. However, after 24 hours, we observed a release of CCL2 and CXCL10 by PBMCs when they are incubated with live Demodex. After 24 hours exposure to mites both PBMCs and RPSs secrete cytokines. This is probably related to mite degeneration over time (up to 50% mortality at 24 hours) and consequent loss of downregulatory properties. The release of CXCL10 and MIF by PBMCs when exposed to ruptured dead mites (separate experiment) supports this concept. Understanding the interactions between live and degenerating Demodex mites and host cells are important to explore their potential role in the pathogenesis of human skin disease.
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The epidermal growth factor receptor is critical for the Staphylococcus aureus-mediated induction of human beta-defensin-3 in keratinocytes M Klee, F Salgado, F Rademacher, M Simanski and J Harder Dept. of Dermatology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Antimicrobial peptides (AMP) contribute to cutaneous defense due to their potent antimicrobial activity. A feature of many AMP expressed by keratinocytes is their inducibility by cytokines and microbial factors. There is increasing evidence that several skin infections may be associated with a dysregulation of AMP. Patients receiving anti-EGFR (epidermal growth factor receptor) therapy often suffer from cutaneous infection caused by Staphylococcus (S.) aureus. This is in line with recent studies indicating a pivotal role of the EGFR to mediate the expression of various AMP in keratinocytes. Human beta-defensin-3 (hBD-3) is an important AMP expressed by keratinocytes to control the growth of S. aureus. Since S. aureus is able to induce the expression of hBD-3 in keratinocytes we analyzed if the EGFR is involved in this induction. To this end we stimulated human primary keratinocytes with S. aureus in the presence of the anti-EGFR antibody cetuximab. Inactivation of the EGFR by cetuximab already resulted in a decreased constitutive hBD-3 gene expression as measured by real-time PCR. Simulation of the keratinocytes with living S. aureus or their culture supernatants resulted in an increased hBD-3 gene expression which was abrogated in the presence of cetuximab. Accordingly, the EGFR inhibitor AG1478 also inhibited the S. aureus-mediated hBD-3 induction. Induction of hBD-3 in keratinocytes treated with the EGFR ligand TGFalpha confirmed the EGFR-dependent induction of hBD-3. Treatment of a 3D skin equivalent with living S. aureus also resulted in an increased expression of hBD-3. This S. aureusmediated hBD-3 induction was decreased in skin equivalents treated with cetuximab. Together, these data further highlight the importance of the EGFR-pathway in cutaneous defense against S. aureus. Our data suggest that a failure of S. aureus-mediated hBD-3 induction in patients receiving anti-EGFR therapy may contribute to the increased S. aureus infection rate seen in these patients.
Combined loss of Trex2 and DNase1L2 nucleases leads to disrupted DNA degradation during lingual cornification and absence of inflammatory responses J Manils1, H Fischer4, J Climent2, E Casas3, J Bas2, T Vavouri3, J de Anta1, E Tschachler4, L Eckhart4 and C Soler1 1 Pathology and Experimental Therapeutics, Universitat de Barcelona, L’Hospitalet de Llobregat, Spain, 2 Immunology, Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, Spain, 3 Institute Germans Trias i Pujol, L’Hospitalet de Llobregat, Spain and 4 Research Division of Biology and Pathobiology of the Skin, Medical University of Vienna, Vienna, Austria Nuclear destruction is one of the main changes occurring throughout keratinocyte cornification, a differentiating-associated mode of programmed cell death. However, the mechanisms of DNA degradation during cornification remain unclear. Here, we investigated the effects in mice of simultaneous loss of the two keratinocyte-specific nucleases, the DNase1L2 endonuclease and the Trex2 exonuclease. Studies in single and double deficient mice have revealed that Trex2 and DNase1L2 contributed and cooperated in the DNA degradation process taking place during keratinocyte cornification in the tongue epithelium, whereas were dispensable for epidermal skin cornification. Notably, simultaneous loss of Trex2 and DNase1L2 led to synergistic accumulation of DNA fragments into the uppermost lingual epithelium layers, rather than a simple sum of single DNase1l2 and Trex2 loss phenotypes, pointing out these two nucleases as essential members of the DNA degrading machinery in lingual cornification process. Intriguingly, the abnormal accumulation of cytosolic DNA in lingual corneocytes was not associated to DNA-driven immune responses and inflammation, establishing that lingual epithelium displays physiological tolerance to aberrantly retained endogenous DNA. Keratinocyte immunological tolerance to self-DNA may be explained in part by the low expression levels of key DNA sensing and signalling genes. In sum, our data indicate a keratinocyte tissue-dependent essential interplay of Trex2 and DNase1L2 in DNA degradation during homeostatic cornification.
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Treatment with ixekizumab does not interfere with the efficacy of tetanus and pneumococcal vaccines in healthy subjects E Gomez, J Bishop, K Jackson, T Muram, D Phillips and S Wilhelm (non-author presenter) Eli Lilly and Company, Indianapolis, IN The aim of this study was to determine if the immune response to tetanus and pneumococcal vaccines in healthy subjects administered ixekizumab (IXE) was noninferior to control (Study ID: NCT02543918). In a Phase 1, randomised, open-label, parallel-group study, adult subjects received vaccinations alone (N¼42, control) or in combination with 160 mg IXE subcutaneously 2 weeks (wks) prior to and 80 mg IXE on the day of vaccination (N¼41, IXE). Anti-tetanus antibody levels (ATAb) and anti-pneumococcal antibody levels (APAb) were measured using validated assays at baseline, 2 and 4 wks post-vaccination. Response to tetanus vaccination was defined as ATAb 1.0 IU and a 1.5-fold increase if baseline was 1.0 IU or a 2.5-fold increase if baseline was >1.0 IU, 4 wks after vaccination. Response to pneumococcal vaccination was defined as a 2-fold increase from baseline in APAb against >50% of the 23 serotypes. The primary analysis was noninferiority of IXE to control on the difference of proportion of responders between groups using a 40% noninferiority margin. Multiple additional analyses of varying definitions of immune response were also performed. Safety and PK were evaluated. IXE (38 completers) was noninferior to control (41 completers) based on the difference in the proportion of responders to tetanus (1.4%; 90% CI: -16.6%, 19.2%) and pneumococcus (-0.8%; 90% CI: -12.9%, 11.0%). Twenty subjects (14 IXE, 6 control) reported 43 treatment-emergent adverse events; all were mild in severity. Headache, injection-site, erythema and fatigue were most common; reported in up to 5% of subjects. Median Tmax was 4 days, and mean Cmax was 17.2 mg/mL after the 160-mg dose. Treatment with IXE did not affect the humoral immune response to non-live vaccines. The IXE group showed noninferiority to control on the difference of proportion of responders between groups for both T-cell dependent (tetanus) and T-cell independent (pneumococcus) immune responses to non-live vaccines.
S266 Journal of Investigative Dermatology (2017), Volume 137
IRF7 and IRF8 have distinct effects on the pathogenesis of SLE F Miyagawa and H Asada Dermatology, Nara Medical Univerity, Kashihara, Japan Recent genome-wide association studies of systemic lupus erythematosus (SLE) patients have revealed that polymorphisms of interferon regulatory factor (IRF) 7 and IRF8 are associated with an increased risk of SLE but the precise role of these IRFs in SLE development is not fully understood. IRF7 and IRF8 belong to the IRF family of transcription factors that regulate transcription of type I Interferon (IFN) and the expression of IFN-stimulated genes. Using a murine model of SLE induced by 2,6,10,14-tetramethylpentadecane (TMPD), we previously reported that IRF7 knockout (KO) mice failed to produce autoantibodies such as anti-dsDNA, ssDNA, nRNP and Sm antibodies but developed glomerulonephritis. In contrast, we found that IRF8KO mice showed no production of autoantibodies along with reduced glomerulonephritis after TMPD injection. These results suggest that type I IFN pathway is critical for autoantibody production but not development of glomerulonephritis. In order to explore the mechanism responsible for tissue damage including glomerulonephritis in this experimental model of SLE, we analyzed the peritoneal cell infiltrate in these mice. We found that inflammatory monocytes were recruited into the peritoneum in wild-type (WT) and IRF7KO mice but not in IRF8KO mice 2 weeks after TMPD injection. Inflammatory monocytes, characterized by expression of Ly6C and CCR2, are known to be recruited to inflamed tissue and contribute to tissue damage. These results suggest that inflammatory monocytes trafficking to the peritoneum might be important event leading to tissue damage in this model.
431
Preclinical evaluation of a novel non-steroidal glucocorticoid receptor agonist with low systemic exposure and reduced potential for skin atrophy H Norsgaard, M Stahlhut, S Eirefelt, N Svitacheva, PS Johnson, M Krogh-Madsen, TT Marstrand, DA Ewald, KN Dack and AK Ollerstam LEO Pharma, Ballerup, Denmark Topical corticosteroids are widely used efficacious drugs but are associated with both local and systemic adverse effects. With the aim to provide an effective but safer topical treatment, we here describe a novel non-steroidal glucocorticoid receptor (GR) agonist with low systemic exposure and reduced potential for skin atrophy induction. LEO 134310 was shown to be a full GR agonist with high affinity for GR and low nanomolar potency in an inflammatory functional assay in human PBMCs. GR was the only nuclear hormone receptor affected by LEO 134310 in a panel of nuclear receptors, including the mineralocorticoid receptor, a receptor which has been associated with induction of skin atrophy. In blood and liver LEO 134310 is rapidly inactivated at a labile site, leading to high systemic clearance. In accordance with this profile, topical treatment with LEO 134310 in a TPA-induced dermatitis mouse model resulted in comparable efficacy to betamethasone valerate (BMV), with no systemic adverse effects at maximally effective doses. In contrast, BMV and clobetasol propionate (CP) markedly reduced body weight at maximally effective doses after topical treatment. Furthermore, skin atrophy after 4-week application to minipigs was minimal after treatment with LEO 134310 compared with significant skin atrophy induced by treatment with CP and BMV. LEO 134310 showed similar levels of target engagement as BMV, measured by FKBP5 mRNA, but lower than CP. Finally, topical treatment with LEO 134310 in a human skin explant model resulted in full agonism, measured by GR target engagement, albeit at higher dosage strength than required for BMV and CP. In conclusion, LEO 134310A shows an improved therapeutic index in preclinical studies compared to traditional corticosteroids and may potentially offer an improved topical treatment solution for patients with atopic dermatitis and psoriasis.
Live demodex mites modulate the inflammatory responses of sebocytes and PBMCs S Gatault1, R Foley1, C Chaussade2, L Shiels1, D Piwnica2 and F Powell1 1 UCD Charles Institute of Dermatology, Dublin, Ireland and 2 Nestle Skin Health R&D, Sophia Antipolis, France Demodex folliculorum mites are the largest, most complex organisms of the cutaneous microbiome and reside in the pilosebaceous units of normal adult facial skin. Mites are recognized pathogens in animal cutaneous disease but their pathogenic potential in humans and their mode of interaction with host resident/immune cells is unknown. A role for mites in the pathogenesis of rosacea has been suggested. We investigated the secretion of inflammatory mediators by sebocytes and peripheral blood mononuclear cells co-cultured with human-derived live demodex mites. Demodex mites, extracted from human skin using the Modified Standardized Skin Surface Biopsy (MSSSB), were incubated with rat preputial sebocytes (RPSs) and human peripheral blood mononuclear cells (PBMCs) for 24 hours in three independent experiments. The release of inflammatory mediators by these cells was assessed by Luminex array, proteome profiler array and ELISAs. RPSs released significant levels of CXCL3 (P < 0.001), ICAM-1 (P < 0.01), IFNg (P < 0.001), TNFa (P < 0.01) and IL-6 (P < 0.01) after incubation for 24 hours with live Demodex mites. In the presence of live demodex mites, PBMCs do not release inflammatory cytokines during their initial (6 hours) exposure suggesting that mites possess downregulatory properties. However, after 24 hours, we observed a release of CCL2 and CXCL10 by PBMCs when they are incubated with live Demodex. After 24 hours exposure to mites both PBMCs and RPSs secrete cytokines. This is probably related to mite degeneration over time (up to 50% mortality at 24 hours) and consequent loss of downregulatory properties. The release of CXCL10 and MIF by PBMCs when exposed to ruptured dead mites (separate experiment) supports this concept. Understanding the interactions between live and degenerating Demodex mites and host cells are important to explore their potential role in the pathogenesis of human skin disease.
432
433
The epidermal growth factor receptor is critical for the Staphylococcus aureus-mediated induction of human beta-defensin-3 in keratinocytes M Klee, F Salgado, F Rademacher, M Simanski and J Harder Dept. of Dermatology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Antimicrobial peptides (AMP) contribute to cutaneous defense due to their potent antimicrobial activity. A feature of many AMP expressed by keratinocytes is their inducibility by cytokines and microbial factors. There is increasing evidence that several skin infections may be associated with a dysregulation of AMP. Patients receiving anti-EGFR (epidermal growth factor receptor) therapy often suffer from cutaneous infection caused by Staphylococcus (S.) aureus. This is in line with recent studies indicating a pivotal role of the EGFR to mediate the expression of various AMP in keratinocytes. Human beta-defensin-3 (hBD-3) is an important AMP expressed by keratinocytes to control the growth of S. aureus. Since S. aureus is able to induce the expression of hBD-3 in keratinocytes we analyzed if the EGFR is involved in this induction. To this end we stimulated human primary keratinocytes with S. aureus in the presence of the anti-EGFR antibody cetuximab. Inactivation of the EGFR by cetuximab already resulted in a decreased constitutive hBD-3 gene expression as measured by real-time PCR. Simulation of the keratinocytes with living S. aureus or their culture supernatants resulted in an increased hBD-3 gene expression which was abrogated in the presence of cetuximab. Accordingly, the EGFR inhibitor AG1478 also inhibited the S. aureus-mediated hBD-3 induction. Induction of hBD-3 in keratinocytes treated with the EGFR ligand TGFalpha confirmed the EGFR-dependent induction of hBD-3. Treatment of a 3D skin equivalent with living S. aureus also resulted in an increased expression of hBD-3. This S. aureusmediated hBD-3 induction was decreased in skin equivalents treated with cetuximab. Together, these data further highlight the importance of the EGFR-pathway in cutaneous defense against S. aureus. Our data suggest that a failure of S. aureus-mediated hBD-3 induction in patients receiving anti-EGFR therapy may contribute to the increased S. aureus infection rate seen in these patients.
Combined loss of Trex2 and DNase1L2 nucleases leads to disrupted DNA degradation during lingual cornification and absence of inflammatory responses J Manils1, H Fischer4, J Climent2, E Casas3, J Bas2, T Vavouri3, J de Anta1, E Tschachler4, L Eckhart4 and C Soler1 1 Pathology and Experimental Therapeutics, Universitat de Barcelona, L’Hospitalet de Llobregat, Spain, 2 Immunology, Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, Spain, 3 Institute Germans Trias i Pujol, L’Hospitalet de Llobregat, Spain and 4 Research Division of Biology and Pathobiology of the Skin, Medical University of Vienna, Vienna, Austria Nuclear destruction is one of the main changes occurring throughout keratinocyte cornification, a differentiating-associated mode of programmed cell death. However, the mechanisms of DNA degradation during cornification remain unclear. Here, we investigated the effects in mice of simultaneous loss of the two keratinocyte-specific nucleases, the DNase1L2 endonuclease and the Trex2 exonuclease. Studies in single and double deficient mice have revealed that Trex2 and DNase1L2 contributed and cooperated in the DNA degradation process taking place during keratinocyte cornification in the tongue epithelium, whereas were dispensable for epidermal skin cornification. Notably, simultaneous loss of Trex2 and DNase1L2 led to synergistic accumulation of DNA fragments into the uppermost lingual epithelium layers, rather than a simple sum of single DNase1l2 and Trex2 loss phenotypes, pointing out these two nucleases as essential members of the DNA degrading machinery in lingual cornification process. Intriguingly, the abnormal accumulation of cytosolic DNA in lingual corneocytes was not associated to DNA-driven immune responses and inflammation, establishing that lingual epithelium displays physiological tolerance to aberrantly retained endogenous DNA. Keratinocyte immunological tolerance to self-DNA may be explained in part by the low expression levels of key DNA sensing and signalling genes. In sum, our data indicate a keratinocyte tissue-dependent essential interplay of Trex2 and DNase1L2 in DNA degradation during homeostatic cornification.
434
435
Treatment with ixekizumab does not interfere with the efficacy of tetanus and pneumococcal vaccines in healthy subjects E Gomez, J Bishop, K Jackson, T Muram, D Phillips and S Wilhelm (non-author presenter) Eli Lilly and Company, Indianapolis, IN The aim of this study was to determine if the immune response to tetanus and pneumococcal vaccines in healthy subjects administered ixekizumab (IXE) was noninferior to control (Study ID: NCT02543918). In a Phase 1, randomised, open-label, parallel-group study, adult subjects received vaccinations alone (N¼42, control) or in combination with 160 mg IXE subcutaneously 2 weeks (wks) prior to and 80 mg IXE on the day of vaccination (N¼41, IXE). Anti-tetanus antibody levels (ATAb) and anti-pneumococcal antibody levels (APAb) were measured using validated assays at baseline, 2 and 4 wks post-vaccination. Response to tetanus vaccination was defined as ATAb 1.0 IU and a 1.5-fold increase if baseline was 1.0 IU or a 2.5-fold increase if baseline was >1.0 IU, 4 wks after vaccination. Response to pneumococcal vaccination was defined as a 2-fold increase from baseline in APAb against >50% of the 23 serotypes. The primary analysis was noninferiority of IXE to control on the difference of proportion of responders between groups using a 40% noninferiority margin. Multiple additional analyses of varying definitions of immune response were also performed. Safety and PK were evaluated. IXE (38 completers) was noninferior to control (41 completers) based on the difference in the proportion of responders to tetanus (1.4%; 90% CI: -16.6%, 19.2%) and pneumococcus (-0.8%; 90% CI: -12.9%, 11.0%). Twenty subjects (14 IXE, 6 control) reported 43 treatment-emergent adverse events; all were mild in severity. Headache, injection-site, erythema and fatigue were most common; reported in up to 5% of subjects. Median Tmax was 4 days, and mean Cmax was 17.2 mg/mL after the 160-mg dose. Treatment with IXE did not affect the humoral immune response to non-live vaccines. The IXE group showed noninferiority to control on the difference of proportion of responders between groups for both T-cell dependent (tetanus) and T-cell independent (pneumococcus) immune responses to non-live vaccines.
S266 Journal of Investigative Dermatology (2017), Volume 137
IRF7 and IRF8 have distinct effects on the pathogenesis of SLE F Miyagawa and H Asada Dermatology, Nara Medical Univerity, Kashihara, Japan Recent genome-wide association studies of systemic lupus erythematosus (SLE) patients have revealed that polymorphisms of interferon regulatory factor (IRF) 7 and IRF8 are associated with an increased risk of SLE but the precise role of these IRFs in SLE development is not fully understood. IRF7 and IRF8 belong to the IRF family of transcription factors that regulate transcription of type I Interferon (IFN) and the expression of IFN-stimulated genes. Using a murine model of SLE induced by 2,6,10,14-tetramethylpentadecane (TMPD), we previously reported that IRF7 knockout (KO) mice failed to produce autoantibodies such as anti-dsDNA, ssDNA, nRNP and Sm antibodies but developed glomerulonephritis. In contrast, we found that IRF8KO mice showed no production of autoantibodies along with reduced glomerulonephritis after TMPD injection. These results suggest that type I IFN pathway is critical for autoantibody production but not development of glomerulonephritis. In order to explore the mechanism responsible for tissue damage including glomerulonephritis in this experimental model of SLE, we analyzed the peritoneal cell infiltrate in these mice. We found that inflammatory monocytes were recruited into the peritoneum in wild-type (WT) and IRF7KO mice but not in IRF8KO mice 2 weeks after TMPD injection. Inflammatory monocytes, characterized by expression of Ly6C and CCR2, are known to be recruited to inflamed tissue and contribute to tissue damage. These results suggest that inflammatory monocytes trafficking to the peritoneum might be important event leading to tissue damage in this model.