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379 IL-26: A new actor in allergic contact dermatitis
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Grass pollen-specific immunotherapy entails systemic increase of regulatory B cells and shift in Th17 cell compartments CA Jakwerth1, UM Zissler1, FM Gu¨rth1, Z Hajdu2, C Schmidt-Weber1 and AM Chaker2 1 ZAUM - Center of Allergy and Environment, Technische Universita¨t Mu¨nchen (TUM) and Helmholtz Center Munich, Member of the German Center for Lung Research (DZL), Munich, Germany and 2 ENT-Department, Klinikum rechts der Isar der Technischen Universita¨t Mu¨nchen (TUM), Munich, Germany IL-10-producing regulatory B cells (Bregs) are known to maintain regulatory capacities of T cells while restricting Th1 and Th17 differentiation. Recently, it has been shown that regulatory T cells (Tregs) can not only differentiate into effector Th17 cells via an intermediate subset expressing FoxP3 and IL-17 simultaneously, but also, that Th17 cells carry the ability to transdifferentiate “back” into Tregs. In this study, we extracted peripheral blood monocytes (PBMCs) from 20 allergic patients at several time points during an up-dosing period of grasspollen specific immunotherapy (SIT) and analyzed immune cell populations using flow cytometry. We prospectively monitored these patients over several years and collected further samples in and out of grass pollen season. Systemic T- and B-cell subsets were compared at baseline and six hours after the last maintenance top dose of SIT. We found a significant increase in IL-10-producing Bregs shortly after the last maintenance injection. On top, the IL10+/TNF-a+ ratio in B cells was significantly increased, which has been postulated to indicate their regulatory function even stronger than IL-10 production alone. The increased Breg population in the blood coincided with a significant decrease of effector Th17 cells and, notably, with a decrease in the IL-17-expressing CD4+FoxP3+ Treg population. We postulate that the SIT-triggered induction of Bregs leads to a shift of Th17 cells towards a rather regulatory phenotype, as not only the Th17 population, but also the intermediate IL-17+FoxP3+ Treg subset was significantly decreased shortly after the last maintenance injection. In conclusion, the frequency of Th17 cells and IL-17-producing regulatory T cells in the peripheral blood may represent early biomarkers of immunotherapy efficacy.
IL-17E favors the recruitment of neutrophils in psoriasis via macrophage activation L Senra, R Stalder, W Boehncke and N Brembilla Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland Psoriasis vulgaris is a chronic recurrent inflammatory skin disease, affecting approximately 2% of the population. We have recently found that IL-17E (also known as IL-25), a member of the IL-17 cytokine family, is over-expressed in lesional psoriatic skin when compared to non lesional and healthy donors. Within the psoriatic plaque, macrophages represent an important proportion of IL-17E+ cells infiltrating the dermis. In this study we investigated the biological effects of IL-17E on macrophages in psoriasis. By confocal analysis of the lesional skin form 6 psoriatic patients, we found that IL-17E co-localized in macrophages with its receptor IL-17RB and clathrin, suggesting its in vivo internalization via a receptor-induced clathrin-mediated mechanism. Phenotypically, IL-17E+ macrophages exhibit a mixed M1/M2 phenotype, being positive for both CLEC5A and CD163L1 markers. In vitro, monocytederived macrophages were unable to produce IL-17E mRNA and internalized the cytokine in a time-dependent manner. The internalization process was blocked in the presence of a clathrin specific inhibitor. M2-polarized macrophages, but not M1, expressed high level of IL17RB and responded to IL-17E dose-dependently by increasing the mRNA levels of inflammatory cytokines and chemokines preferentially favoring the recruitment of neutrophils. Of note, the number of IL-17E+ cells in lesional skin correlated with the PASI score and the number of neutrophils, while being inversely proportional to the number of infiltrating T cells. In vivo, intra-dermal injection of rmIL-17E in C57BL/6 mice induced severe dermal inflammation with neutrophil and eosinophil infiltration, in addition to a hyperplastic epidermis, compared to the PBS control group. Our data provide strong evidence for a novel pro-inflammatory role of IL-17E in psoriasis, possibly via activation of macrophages leading to a preferential recruitment of neutrophils.
Allergen-loaded strontium-doped hydroxyapatite spheres improve allergen-specific immunotherapy in mice M Garbani1, W Xia4, H Engkvist4, A Scheynius5, B Malissen3, M Maurer2, R Crameri1 and D Terhorst2 1 Swiss Institute of Allergy Foundation, Davos, Switzerland, 2 Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 3 Centre d’Immunologie Marseille-Luminy, Marseille, France, 4 A˚ngstro¨m Laboratory, Uppsala University, Uppsala, Sweden and 5 Karolinska Institutet, Stockholm, Sweden Immunomodulatory interventions play a key role in the treatment of infections and cancer as well as allergic diseases. Adjuvants such as micro- and nanoparticles are often added to immunomodulatory therapies to enhance the triggered immune response. Here, we report the immunological assessment of novel and economically manufactured microparticle adjuvants, namely strontium-doped hydroxyapatite porous spheres (SHAS), which we suggest for the use as adjuvant and carrier in allergen-specific immunotherapy (ASIT). Scanning electron microscopy revealed that the synthesis procedure developed for the production of SHAS results in a highly homogeneous population of spheres. SHAS bound and released proteins such as ovalbumin (OVA) or the major cat allergen Fel d 1. SHAS- OVA bound to human monocytederived dendritic cells (mdDCs) and did not have any necrotic or apoptotic effects even at high densities. In a murine model of ASIT for allergic asthmatic inflammation we found that OVA released from subcutaneously injected SHAS-OVA led to a sustained stimulation of both CD4+ and CD8+ T-cells. ASIT with SHAS-OVA as compared to soluble OVA resulted in similar humoral responses but in a higher efficacy as assessed by symptom scoring. We conclude that SHAS may constitute a suitable carrier and adjuvant for ASIT with great potential due to its unique protein-binding properties.
IL-26: A new actor in allergic contact dermatitis A Balato1, G Caiazzo2, F Ayala2, N Balato2, R Di Caprio2, G Monfrecola2, C Patruno2, A Raimondo2 and S Lembo3 1 Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy, 2 Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy and 3 Department of Medicine and Surgery, University of Salerno, Salerno, Italy The purpose of this study was to assess the possible interleukin (IL)-26 involvement in allergic contact dermatitis (ACD). The study population comprised 20 patients affected by ACD and 10 healthy controls. RNA and protein analysis was executed on skin biopsies and on peripheral blood mononuclear cells (PBMC). In addition, in vitro PBMC stimulation assays with toxic shock syndrome toxin (TSST-1) or nickel sulfate (NiSO4) were performed. Ex vivo assay was executed with ACD uninvolved skin exposed to the allergen (NiSO4) only or contemporarily to NiSO4 and PBMC isolated from the same subject. IL-26 gene (qRT-PCR) and protein (IHC) expression was enhanced in ACD skin compared to healthy skin (p<0.01). IL-26 was increased in PBMC from nickel-ACD patients respect to PBMC from healthy controls. In vitro PBMC stimulation assays showed that only TSST-1 treatment was able to induce a significant increase of IL-26 levels in nickel-ACD PBMC respect to healthy PBMC (p<0.05). Ex vivo model showed that there was no significant increase when non lesional ACD skin was exposed only to NiSO4, but a significant increase was obtained when non lesional ACD skin was exposed contemporarily to NiSO4 and PBMC (p<0.05), indicating that PBMC contribute to IL-26 production. In conclusion, our data show a possible role of IL-26 in the complex inflammatory scenario of ACD.
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Age dictates a steroid resistant cascade of wnt5a, transglutaminase-2 and leukotrienes in inflamed airways K Dietz1, M de los Reyes Jime´nez1, E Gollwitzer2, C Schmidt-Weber1, AM Chaker3, B Marsland2 and J Esser-von Bieren1 1 Center of Allergy and Environment (ZAUM), Technical University of Munich and HelmholtzCentre Munich, Munich, Germany, 2 Faculty of Biology and Medicine, Universite´ de Lausanne, Epalinges, Switzerland and 3 Department of Otolaryngology, Allergy Section, Klinikum rechts der Isar, TUM, Munich, Germany Airway remodeling is a detrimental and refractory event in asthma, which is driven by cytokines, growth factors and lipid mediators. Remodeling in asthma shows age-dependent manifestations such as increased basal membrane thickening in adults as compared to children, but explanatory mechanisms are lacking. The leukotriene (LT) and wingless/ integrase (wnt) pathways have been implicated in airway remodeling, but age-dependent expression profiles and common regulators have remained elusive. We studied age differences in LTs and wnt5a during airway inflammation in vivo and investigated regulatory pathways in human macrophages and primary bronchial epithelial cells (HBEC). LT levels in the airways of HDM-sensitized mice were increased in neonatal and adult, but reduced in juvenile mice. Compared to young mice, adult mice expressed higher levels of the remodeling factors sPLA2-X and wnt5a and the M2 macrophage marker TGM2. In young mice, wnt5a was exclusively localized to airway smooth muscle cells, whilst older mice showed epithelial expression of wnt5a. In human cells, IL-4 promoted epithelial wnt5a secretion, which upregulated macrophage TGM2 expression. In turn, TGM2 inhibition reduced LT release by HBEC and macrophages. Our findings reveal age differences in LT and wnt pathways during airway inflammation and identify a steroid-resistant mechanism of myeloidepithelial cell crosstalk, which may represent a therapeutic target for airway remodeling.
Minimal requirements for diagnosis of cutaneous pemphigoid. Comparative study of direct immunofluorescence, serology and clinical features in 316 patients J Meijer, E de Lang, G Diercks, H Pas and M Jonkman Department of Dermatology - Center for Blistering Diseases, University Medical Center Groningen, University of Groningen, Groningen, Netherlands In order to determine the minimal laboratory requirements for diagnosis of cutaneous pemphigoid, this single-center retrospective study assessed the diagnostic value of direct immunofluorescence (DIF) microscopy and of several (routine) immune serological tests, including indirect immunofluorescence microscopy on 1M NaCl-split skin (IIF SSS), IIF on monkey oesophagus (IIF MO), immunoblot and anti-BP180 NC16A and anti-BP230 ELISA’s. Paired data of 316 newly diagnosed patients with cutaneous pemphigoid with a mean age of 71.9 years were compared to 811 controls. In addition, the study assessed DIF serration pattern analysis and differences in biopsy sites for DIF microscopy in cutaneous pemphigoid, including the nonbullous subtype. DIF showed a sensitivity of 88.3% and specificity of 96.1%. Sensitivities of serological assays were 80.1% (IIF SSS), 60.2% (IIF MO), 74.6% (immunoblot), 68.6% (MBL ELISA anti-BP180 NC16A) and 52.4% (MBL ELISA anti-BP230), with high specificities in all tests. IIF SSS showed a very high positive predictive value (97.3%). Sensitivity of perilesional skin biopsies for DIF was significantly higher compared to lesional skin and unaffected skin. 20% of patients presented with the nonbullous subtype of cutaneous pemphigoid: with pruritus and absence of blisters. No preference in biopsy site for DIF was found in nonbullous cutaneous pemphigoid. BP230 was significantly more often targeted as autoantigen in patients with nonbullous cutaneous pemphigoid compared to pemphigoid patients with blisters. The current gold standard DIF misses approximately 10% of diagnoses of cutaneous pemphigoid, which additionally can be found with IIF SSS. Both ELISA’s showed low sensitivities and should not be recommended for screening or as standalone test for diagnosing cutaneous pemphigoid. The minimal required diagnostic tests for the diagnosis of cutaneous pemphigoid are DIF plus IIF SSS.
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Grass pollen-specific immunotherapy entails systemic increase of regulatory B cells and shift in Th17 cell compartments CA Jakwerth1, UM Zissler1, FM Gu¨rth1, Z Hajdu2, C Schmidt-Weber1 and AM Chaker2 1 ZAUM - Center of Allergy and Environment, Technische Universita¨t Mu¨nchen (TUM) and Helmholtz Center Munich, Member of the German Center for Lung Research (DZL), Munich, Germany and 2 ENT-Department, Klinikum rechts der Isar der Technischen Universita¨t Mu¨nchen (TUM), Munich, Germany IL-10-producing regulatory B cells (Bregs) are known to maintain regulatory capacities of T cells while restricting Th1 and Th17 differentiation. Recently, it has been shown that regulatory T cells (Tregs) can not only differentiate into effector Th17 cells via an intermediate subset expressing FoxP3 and IL-17 simultaneously, but also, that Th17 cells carry the ability to transdifferentiate “back” into Tregs. In this study, we extracted peripheral blood monocytes (PBMCs) from 20 allergic patients at several time points during an up-dosing period of grasspollen specific immunotherapy (SIT) and analyzed immune cell populations using flow cytometry. We prospectively monitored these patients over several years and collected further samples in and out of grass pollen season. Systemic T- and B-cell subsets were compared at baseline and six hours after the last maintenance top dose of SIT. We found a significant increase in IL-10-producing Bregs shortly after the last maintenance injection. On top, the IL10+/TNF-a+ ratio in B cells was significantly increased, which has been postulated to indicate their regulatory function even stronger than IL-10 production alone. The increased Breg population in the blood coincided with a significant decrease of effector Th17 cells and, notably, with a decrease in the IL-17-expressing CD4+FoxP3+ Treg population. We postulate that the SIT-triggered induction of Bregs leads to a shift of Th17 cells towards a rather regulatory phenotype, as not only the Th17 population, but also the intermediate IL-17+FoxP3+ Treg subset was significantly decreased shortly after the last maintenance injection. In conclusion, the frequency of Th17 cells and IL-17-producing regulatory T cells in the peripheral blood may represent early biomarkers of immunotherapy efficacy.
IL-17E favors the recruitment of neutrophils in psoriasis via macrophage activation L Senra, R Stalder, W Boehncke and N Brembilla Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland Psoriasis vulgaris is a chronic recurrent inflammatory skin disease, affecting approximately 2% of the population. We have recently found that IL-17E (also known as IL-25), a member of the IL-17 cytokine family, is over-expressed in lesional psoriatic skin when compared to non lesional and healthy donors. Within the psoriatic plaque, macrophages represent an important proportion of IL-17E+ cells infiltrating the dermis. In this study we investigated the biological effects of IL-17E on macrophages in psoriasis. By confocal analysis of the lesional skin form 6 psoriatic patients, we found that IL-17E co-localized in macrophages with its receptor IL-17RB and clathrin, suggesting its in vivo internalization via a receptor-induced clathrin-mediated mechanism. Phenotypically, IL-17E+ macrophages exhibit a mixed M1/M2 phenotype, being positive for both CLEC5A and CD163L1 markers. In vitro, monocytederived macrophages were unable to produce IL-17E mRNA and internalized the cytokine in a time-dependent manner. The internalization process was blocked in the presence of a clathrin specific inhibitor. M2-polarized macrophages, but not M1, expressed high level of IL17RB and responded to IL-17E dose-dependently by increasing the mRNA levels of inflammatory cytokines and chemokines preferentially favoring the recruitment of neutrophils. Of note, the number of IL-17E+ cells in lesional skin correlated with the PASI score and the number of neutrophils, while being inversely proportional to the number of infiltrating T cells. In vivo, intra-dermal injection of rmIL-17E in C57BL/6 mice induced severe dermal inflammation with neutrophil and eosinophil infiltration, in addition to a hyperplastic epidermis, compared to the PBS control group. Our data provide strong evidence for a novel pro-inflammatory role of IL-17E in psoriasis, possibly via activation of macrophages leading to a preferential recruitment of neutrophils.
Allergen-loaded strontium-doped hydroxyapatite spheres improve allergen-specific immunotherapy in mice M Garbani1, W Xia4, H Engkvist4, A Scheynius5, B Malissen3, M Maurer2, R Crameri1 and D Terhorst2 1 Swiss Institute of Allergy Foundation, Davos, Switzerland, 2 Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 3 Centre d’Immunologie Marseille-Luminy, Marseille, France, 4 A˚ngstro¨m Laboratory, Uppsala University, Uppsala, Sweden and 5 Karolinska Institutet, Stockholm, Sweden Immunomodulatory interventions play a key role in the treatment of infections and cancer as well as allergic diseases. Adjuvants such as micro- and nanoparticles are often added to immunomodulatory therapies to enhance the triggered immune response. Here, we report the immunological assessment of novel and economically manufactured microparticle adjuvants, namely strontium-doped hydroxyapatite porous spheres (SHAS), which we suggest for the use as adjuvant and carrier in allergen-specific immunotherapy (ASIT). Scanning electron microscopy revealed that the synthesis procedure developed for the production of SHAS results in a highly homogeneous population of spheres. SHAS bound and released proteins such as ovalbumin (OVA) or the major cat allergen Fel d 1. SHAS- OVA bound to human monocytederived dendritic cells (mdDCs) and did not have any necrotic or apoptotic effects even at high densities. In a murine model of ASIT for allergic asthmatic inflammation we found that OVA released from subcutaneously injected SHAS-OVA led to a sustained stimulation of both CD4+ and CD8+ T-cells. ASIT with SHAS-OVA as compared to soluble OVA resulted in similar humoral responses but in a higher efficacy as assessed by symptom scoring. We conclude that SHAS may constitute a suitable carrier and adjuvant for ASIT with great potential due to its unique protein-binding properties.
IL-26: A new actor in allergic contact dermatitis A Balato1, G Caiazzo2, F Ayala2, N Balato2, R Di Caprio2, G Monfrecola2, C Patruno2, A Raimondo2 and S Lembo3 1 Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy, 2 Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy and 3 Department of Medicine and Surgery, University of Salerno, Salerno, Italy The purpose of this study was to assess the possible interleukin (IL)-26 involvement in allergic contact dermatitis (ACD). The study population comprised 20 patients affected by ACD and 10 healthy controls. RNA and protein analysis was executed on skin biopsies and on peripheral blood mononuclear cells (PBMC). In addition, in vitro PBMC stimulation assays with toxic shock syndrome toxin (TSST-1) or nickel sulfate (NiSO4) were performed. Ex vivo assay was executed with ACD uninvolved skin exposed to the allergen (NiSO4) only or contemporarily to NiSO4 and PBMC isolated from the same subject. IL-26 gene (qRT-PCR) and protein (IHC) expression was enhanced in ACD skin compared to healthy skin (p<0.01). IL-26 was increased in PBMC from nickel-ACD patients respect to PBMC from healthy controls. In vitro PBMC stimulation assays showed that only TSST-1 treatment was able to induce a significant increase of IL-26 levels in nickel-ACD PBMC respect to healthy PBMC (p<0.05). Ex vivo model showed that there was no significant increase when non lesional ACD skin was exposed only to NiSO4, but a significant increase was obtained when non lesional ACD skin was exposed contemporarily to NiSO4 and PBMC (p<0.05), indicating that PBMC contribute to IL-26 production. In conclusion, our data show a possible role of IL-26 in the complex inflammatory scenario of ACD.
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Age dictates a steroid resistant cascade of wnt5a, transglutaminase-2 and leukotrienes in inflamed airways K Dietz1, M de los Reyes Jime´nez1, E Gollwitzer2, C Schmidt-Weber1, AM Chaker3, B Marsland2 and J Esser-von Bieren1 1 Center of Allergy and Environment (ZAUM), Technical University of Munich and HelmholtzCentre Munich, Munich, Germany, 2 Faculty of Biology and Medicine, Universite´ de Lausanne, Epalinges, Switzerland and 3 Department of Otolaryngology, Allergy Section, Klinikum rechts der Isar, TUM, Munich, Germany Airway remodeling is a detrimental and refractory event in asthma, which is driven by cytokines, growth factors and lipid mediators. Remodeling in asthma shows age-dependent manifestations such as increased basal membrane thickening in adults as compared to children, but explanatory mechanisms are lacking. The leukotriene (LT) and wingless/ integrase (wnt) pathways have been implicated in airway remodeling, but age-dependent expression profiles and common regulators have remained elusive. We studied age differences in LTs and wnt5a during airway inflammation in vivo and investigated regulatory pathways in human macrophages and primary bronchial epithelial cells (HBEC). LT levels in the airways of HDM-sensitized mice were increased in neonatal and adult, but reduced in juvenile mice. Compared to young mice, adult mice expressed higher levels of the remodeling factors sPLA2-X and wnt5a and the M2 macrophage marker TGM2. In young mice, wnt5a was exclusively localized to airway smooth muscle cells, whilst older mice showed epithelial expression of wnt5a. In human cells, IL-4 promoted epithelial wnt5a secretion, which upregulated macrophage TGM2 expression. In turn, TGM2 inhibition reduced LT release by HBEC and macrophages. Our findings reveal age differences in LT and wnt pathways during airway inflammation and identify a steroid-resistant mechanism of myeloidepithelial cell crosstalk, which may represent a therapeutic target for airway remodeling.
Minimal requirements for diagnosis of cutaneous pemphigoid. Comparative study of direct immunofluorescence, serology and clinical features in 316 patients J Meijer, E de Lang, G Diercks, H Pas and M Jonkman Department of Dermatology - Center for Blistering Diseases, University Medical Center Groningen, University of Groningen, Groningen, Netherlands In order to determine the minimal laboratory requirements for diagnosis of cutaneous pemphigoid, this single-center retrospective study assessed the diagnostic value of direct immunofluorescence (DIF) microscopy and of several (routine) immune serological tests, including indirect immunofluorescence microscopy on 1M NaCl-split skin (IIF SSS), IIF on monkey oesophagus (IIF MO), immunoblot and anti-BP180 NC16A and anti-BP230 ELISA’s. Paired data of 316 newly diagnosed patients with cutaneous pemphigoid with a mean age of 71.9 years were compared to 811 controls. In addition, the study assessed DIF serration pattern analysis and differences in biopsy sites for DIF microscopy in cutaneous pemphigoid, including the nonbullous subtype. DIF showed a sensitivity of 88.3% and specificity of 96.1%. Sensitivities of serological assays were 80.1% (IIF SSS), 60.2% (IIF MO), 74.6% (immunoblot), 68.6% (MBL ELISA anti-BP180 NC16A) and 52.4% (MBL ELISA anti-BP230), with high specificities in all tests. IIF SSS showed a very high positive predictive value (97.3%). Sensitivity of perilesional skin biopsies for DIF was significantly higher compared to lesional skin and unaffected skin. 20% of patients presented with the nonbullous subtype of cutaneous pemphigoid: with pruritus and absence of blisters. No preference in biopsy site for DIF was found in nonbullous cutaneous pemphigoid. BP230 was significantly more often targeted as autoantigen in patients with nonbullous cutaneous pemphigoid compared to pemphigoid patients with blisters. The current gold standard DIF misses approximately 10% of diagnoses of cutaneous pemphigoid, which additionally can be found with IIF SSS. Both ELISA’s showed low sensitivities and should not be recommended for screening or as standalone test for diagnosing cutaneous pemphigoid. The minimal required diagnostic tests for the diagnosis of cutaneous pemphigoid are DIF plus IIF SSS.
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