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6 gene expression in lesional skin of patients with chronic plaque type psoriasis
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UV-B treatment induces LRP5/6 gene expression in lesional skin of patients with chronic plaque type psoriasis R Slind Olsen2, A Duvetorp1, M Skarstedt3, J So¨derman3 and O Seifert1 1 Department of Clinical and Experimental Medicine, Linko¨ping University, Linko¨ping, Sweden, 2 Department of Medical and Health Sciences, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden and 3 Division of Medical Diagnostics, Ryhov Hospital, Jo¨nko¨ping, Sweden Low-density lipoprotein-related receptors 5 and 6 are transmembrane receptors with key functions in canonical Wnt signaling. Recent studies assign LRP5/6 anti-inflammatory properties and LRP5/6 gene alterations are linked to coronary artery disease, osteoporosis, cancer, and metabolic disease. The objective of this study was to investigate the expression of LRP5 and 6 in lesional and non-lesional skin, in serum and in peripheral blood mononuclear cells of patients with chronic plaque type psoriasis compared to control individuals. To investigate the effect of UV-B radiation, LRP5 and LRP6 skin gene expression was analyzed before and after narrow band UV-B treatment. Associations between single nucleotide polymorphisms for LRP5 and LRP6 and psoriasis were tested. Our results showed significantly decreased gene expression of LRP5 and 6 in lesional skin from patients with psoriasis compared to non-lesional skin and healthy control skin. Interestingly, nb UV-B treatment induced a significant increase in LRP5 and LRP6 gene expression in lesional skin. Immunohistochemistry did not reveal differences in protein expression of LRP5/6. LRP5 protein expression is significantly decreased in peripheral blood mononuclear cells of patients with psoriasis compared to control individuals. No significant differences of LRP5/6 serum levels were observed. LRP5 gene expression in peripheral blood cells is significantly decreased in patients with psoriasis. No significant differences in allele or genotype frequencies for LRP5 gene polymorphism rs3736228, rs634008 and rs4988300 and LRP6 gene polymorphism rs2302685 were found between patient and control group. Decreased LRP5/6 gene expression and upregulation after nb UV-B treatment suggest a possible role for LRP5/6 in psoriasis. LRP5 and 6 might be possible targets for new future treatment options.
Plasmacytoid dendritic cell-derived type I interferon drives flares of rosacea A Mylonas1, O Demaria1, S Meller3, H Friedrich3, B Homey3, AA Navarini2, J Di Domizio1, M Gilliet1 and C Conrad1 1 Dermatology, University Hospital Lausanne, Lausanne, Switzerland, 2 University Hospital Zurich, Zurich, Switzerland and 3 University Hospital Duesseldorf, Duesseldorf, Germany Rosacea is a common chronic inflammatory skin disease with a variety of clinical manifestations such as recurrent flares, centro-facial erythema, papules, and pustules. Antimicrobial peptides such as LL-37 have previously been described to be overexpressed in rosacea. Although there is increasing evidence of a role for antimicrobial peptides and the innate immune system in the pathogenesis of rosacea, the pathogenic mechanisms remain elusive. In a cohort of rosacea patients, we found a marked dermal accumulation of plasmacytoid dendritic cell (pDC) accompanied by significant overexpression of type-I interferon as compared to healthy controls. Using an established in-vivo model for rosacea, repetitive dermal injections of LL37 induced pDC infiltration of the skin and local type I interferon production, which was reduced upon pDC depletion. LL37 injections induced a predominant IL-23/Th17 expression profile, similar to what we found in the skin of our patient cohort. Intriguingly, induction of the IL-23/Th17 cytokines was largely dependent on pDCs and the induction of type I interferon, as both pDC-depletion and of type I interferon blockade inhibited IL-23 and Th17 cytokine expression. Furthermore, Bacillus oleronius, a bacterium that thrives in rosacea lesions, activated pDCs to produce type I interferon in-vitro. The presence of LL37 significantly increased this type I interferon production. Taken together, our data indicate that antimicrobial peptides along with microbial products in the skin of rosacea patients lead to rapid pDC-recruitment and -activation and that pDC-derived type I interferon plays a pathogenic role in inducing a predominant IL-23/Th17 cytokine expression thereby potentially driving flares of rosacea.
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Macrophage Migration Inhibitory Factor (MIF) promotes TH17 cell-driven psoriasiform dermatitis S Bezdek1, S Mousavi1, AM Hadenah1, D Zillikens1, R Bucala2 and CD Sadik1 1 Dermatology, Allergy, and Venereology, University of Lu¨beck, Lu¨beck, Germany and 2 Departments of Medicine and Pathology, Yale University School of Medicine, New Haven, CT Macrophage Migration Inhibitory Factor (MIF) is a unique protein that combines properties of cytokines, enzymes, hormones, and chaperones, and exerts pleotropic effects. In sum, its effects are mostly considered proinflammatory. Accordingly, MIF has been suggested to promote the pathogenesis of multiple inflammatory disease in diverse organs. Thus, it is elevated in the serum of psoriasis patients, and functional polymorphisms in the MIF gene are associated with enhanced susceptibility to psoriasis. The significance of these findings for the pathogenesis of psoriasis, however, has remained elusive. We therefore addressed the role of ˆ - (AIPD) and in the IL-23-induced MIF in psoriasis by examining Mif-/- mice in the AldaraO psoriasiform dermatitis model. Genetic deficiency in MIF reduced clinical severity of psoriasiform skin inflammation in the AIPD and in the IL-23-induced model by approximately 50%. On the histopathological level, psoriasiform skin lesions of Mif-/- mice showed less T cell and and macrophage infiltration in the skin. Likewise, epidermal proliferation as well as dermal neoangiogenesis were markedly attenuated. Our results suggest a significant role for MIF in the pathogenesis of plaque psoriasis and highlight it as a new therapeutic target in the treatment of the disease that may be selectively applied in those patients with high expression MIF risk alleles. With exogenous glucocorticoids known to upregulate MIF, the inhibition of MIF may have particular application when used adjunctly with topical corticosteroids.
Leucine-rich-a-2 glycoprotein, a biomarker and potential target of psoriasis therapy H Nakajima1, M Takaishi1, S Serada2, M Fujimoto2, T Naka3 and S Sano1 1 Dermatology, Kochi Medical School, Nankoku, Japan, 2 Laboratory of Immune signal, National Institute of Biomedical Innovation, Ibaraki, Japan and 3 Integrated Center for Advanced Medical Technologies, Kochi Medical School, Nankoku, Japan Recent study showed that serum levels of Leucine-rich-a-2 glycoprotein (LRG) were significantly higher in patients with autoimmune diseases such as rheumatoid arthritis, Crohn’s disease and Behc¸et disease. Here, we assessed whether LRG could be a biomarker and any pathogenic of psoriasis. Serum LRG levels were significantly increased in psoriasis patients even in those who were not positive for serum C-reactive protein (CRP), and well correlated with PASI score. Moreover, serum LRG levels showed superior sensitivity over CRP in patients with mild to moderate psoriasis based on receiver operating characteristic curve analysis. Furthermore, RT-PCR analysis revealed that psoriasis patients showed increased LRG in peripheral mononuclear cells and neutrophils, the latter of which expressed LRG marginally in normal controls, suggesting that neutrophils are relevant to be LRG source in psoriasis. We also showed that LRG was up-regulated in both human psoriatic skins and psoriasis-like lesions of K5.Stat3C transgenic mice, which were induced by tape stripping. Interestingly, LRG expression was enhanced after tape stripping on the back skin not only in the skin itself but also in the liver of K5.Stat3C mice. LRG mRNA and protein levels in the liver following tape stripping were more strongly increased in K5.Stat3C mice than in WT mice, suggesting the positive feedback loop between the skin and systemic LRG in psoriasis. To elucidate functional roles of LRG in psoriasis development, we crossed K5.Stat3C with LRG KO mice. LRG gene ablation attenuated the psoriatic features including epidermal hyperplasia, inflammatory cell infiltrates and psoriasis-related gene expression. In conclusion, LRG may be not only a useful biomarker but would be a therapeutic target for psoriasis.
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The character of TLN-58, newly discovered hCAP18 processing form, found in the lesion vesicle of palmoplantar pustulosis in the skin M Murakami1, K Kameda2, H Mori1, R Utsunomiya1, K Masuda1, K Shiraishi1, M Tohyama1 and K Sayama1 1 Dermatology, Ehime University, Toon, Japan and 2 Advanced Research support Center, Ehime University, Toon, Japan We have reported that Palmoplantar Pustulosis (PPP) vesicle (before pustule formation) was originated from eccrine sweat in the acrosyringium, and PPP vesicle contains antimicrobial peptides human cathelicidin (hCAP-18/LL-37) (JID 2010, PLOSONE 2014). In the latter report, we have shown that PPP vesicle contains additional small fragment of hCAP-18 sized about 7KDa but not been identified before. The principal aim of this study was to confirm the character of this extra fragment of hCAP-18 found in the PPP vesicle. Lesional PPP vesicle was collected from PPP patients under sterile condition, the endogenous hCAP-18/LL-37 was depleted with LL-37 antibody affinity column. The recombinant hCAP-18 peptide was prepared as a full-length human cathelicidin, and then the peptide was incubated with the depleted PPP vesicle. After incubation, the sample was processed for 15% SDS-PAGE, and the gel was visualized with Coomassie Blue staining. After cutting out the target band, the gel fragment was processed for the in-gel digestion and the protein sequencing. Normal keratinocyte was incubated with synthetic peptides of LL-37 and a newly discovered fragment to check the inflammatory activity, so that IL-17C, IL-8, IL-23, IL-1a, and IL-1b mRNAs expressions were evaluated with qRT-PCR. In addition, antibacterial activity against S. aureus, S. epidermidis, and Group A Streptococcus were also evaluated with solution killing assay. We could confirm that the small newly discovered form processed from recombinant hCAP-18 was TLN-58 (estimated MW 6858). The fragment showed the antimicrobial activity as LL-37 showed, and could induce higher mRNA expressions of IL-8 and IL-17 in normal human keratinocyte comparing with LL-37 induced. It is possible that this processing form has a role of antimicrobial peptide, and could be a candidate for the pathogenesis of continuous inflammation of PPP lesion in the skin.
Stimulation of the histamine 4 receptor increases the production of IL-5 in innate lymphoid cells K Schaper1, H Stark2, T Werfel1 and R Gutzmer1 1 Department for Dermatology and Allergy, Medical School Hannover, Hannover, Germany and 2 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Du¨sseldorf, Germany Allergic disorders, such as asthma or atopic dermatitis (AD) are associated with increased Th2 cytokines, while Th2 cells are known to be the major source. However, also other cell types, especially the novel discovered type 2 innate lymphoid cell subset (ILC2) produce high amounts of IL-5 and IL-13. While IL-5 serves mainly as stimulator of eosinophils, IL-13 also regulates cell-mediated immunity and thereby enhances airway hyper reactivity. ILC2 accumulate in tissue at the site of inflammation and it could be shown that the number of ILC2 is significantly elevated in skin of AD patients. Since histamine is also increased in allergic diseases and AD lesions, the present study aimed to identify the impact of histamine, especially with regard to the histamine 4 receptor (H4R) on the cytokine production of ILC2. We therefore isolated PBMCs from healthy subjects and depleted CD3+ cells. CD127+ cells were further enriched by means of magnetic cell separation. On stimulation with IL-2 and IL25 for five days CD3-CD127+ cells produced high amounts of IL-5 and IL-13, while the cytokine production of CD3+ cells was lower. CD3-CD127- did not produce detectable levels of IL-5 and IL-13 at all. Incubation of the cells with histamine during the five days of stimulation augmented the production of IL-5 and IL-13 in ILC2 significantly, but not in CD3+ cells. CD3-CD127+ cells incubated with ST1006, a selective H4R agonist, also increased the production of IL-5, while the IL-13 secretion was not altered. To determine histamine receptor expression, we sorted ILC2 (Lin-CD45highCD127+CRTH2+) and performed real-time PCR. Thereby we showed that pure ILC2 expressed histamine receptor 1 (H1R), H2R and H4R. Taken together, our study indicates a possible role for the H4R in the regulation of IL-5 in human ILC2. In allergic diseases blocking the H4R could therefore lead to a decrease of IL-5 and subsequently result in an anti-inflammatory effect.
www.jidonline.org S205
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UV-B treatment induces LRP5/6 gene expression in lesional skin of patients with chronic plaque type psoriasis R Slind Olsen2, A Duvetorp1, M Skarstedt3, J So¨derman3 and O Seifert1 1 Department of Clinical and Experimental Medicine, Linko¨ping University, Linko¨ping, Sweden, 2 Department of Medical and Health Sciences, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden and 3 Division of Medical Diagnostics, Ryhov Hospital, Jo¨nko¨ping, Sweden Low-density lipoprotein-related receptors 5 and 6 are transmembrane receptors with key functions in canonical Wnt signaling. Recent studies assign LRP5/6 anti-inflammatory properties and LRP5/6 gene alterations are linked to coronary artery disease, osteoporosis, cancer, and metabolic disease. The objective of this study was to investigate the expression of LRP5 and 6 in lesional and non-lesional skin, in serum and in peripheral blood mononuclear cells of patients with chronic plaque type psoriasis compared to control individuals. To investigate the effect of UV-B radiation, LRP5 and LRP6 skin gene expression was analyzed before and after narrow band UV-B treatment. Associations between single nucleotide polymorphisms for LRP5 and LRP6 and psoriasis were tested. Our results showed significantly decreased gene expression of LRP5 and 6 in lesional skin from patients with psoriasis compared to non-lesional skin and healthy control skin. Interestingly, nb UV-B treatment induced a significant increase in LRP5 and LRP6 gene expression in lesional skin. Immunohistochemistry did not reveal differences in protein expression of LRP5/6. LRP5 protein expression is significantly decreased in peripheral blood mononuclear cells of patients with psoriasis compared to control individuals. No significant differences of LRP5/6 serum levels were observed. LRP5 gene expression in peripheral blood cells is significantly decreased in patients with psoriasis. No significant differences in allele or genotype frequencies for LRP5 gene polymorphism rs3736228, rs634008 and rs4988300 and LRP6 gene polymorphism rs2302685 were found between patient and control group. Decreased LRP5/6 gene expression and upregulation after nb UV-B treatment suggest a possible role for LRP5/6 in psoriasis. LRP5 and 6 might be possible targets for new future treatment options.
Plasmacytoid dendritic cell-derived type I interferon drives flares of rosacea A Mylonas1, O Demaria1, S Meller3, H Friedrich3, B Homey3, AA Navarini2, J Di Domizio1, M Gilliet1 and C Conrad1 1 Dermatology, University Hospital Lausanne, Lausanne, Switzerland, 2 University Hospital Zurich, Zurich, Switzerland and 3 University Hospital Duesseldorf, Duesseldorf, Germany Rosacea is a common chronic inflammatory skin disease with a variety of clinical manifestations such as recurrent flares, centro-facial erythema, papules, and pustules. Antimicrobial peptides such as LL-37 have previously been described to be overexpressed in rosacea. Although there is increasing evidence of a role for antimicrobial peptides and the innate immune system in the pathogenesis of rosacea, the pathogenic mechanisms remain elusive. In a cohort of rosacea patients, we found a marked dermal accumulation of plasmacytoid dendritic cell (pDC) accompanied by significant overexpression of type-I interferon as compared to healthy controls. Using an established in-vivo model for rosacea, repetitive dermal injections of LL37 induced pDC infiltration of the skin and local type I interferon production, which was reduced upon pDC depletion. LL37 injections induced a predominant IL-23/Th17 expression profile, similar to what we found in the skin of our patient cohort. Intriguingly, induction of the IL-23/Th17 cytokines was largely dependent on pDCs and the induction of type I interferon, as both pDC-depletion and of type I interferon blockade inhibited IL-23 and Th17 cytokine expression. Furthermore, Bacillus oleronius, a bacterium that thrives in rosacea lesions, activated pDCs to produce type I interferon in-vitro. The presence of LL37 significantly increased this type I interferon production. Taken together, our data indicate that antimicrobial peptides along with microbial products in the skin of rosacea patients lead to rapid pDC-recruitment and -activation and that pDC-derived type I interferon plays a pathogenic role in inducing a predominant IL-23/Th17 cytokine expression thereby potentially driving flares of rosacea.
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Macrophage Migration Inhibitory Factor (MIF) promotes TH17 cell-driven psoriasiform dermatitis S Bezdek1, S Mousavi1, AM Hadenah1, D Zillikens1, R Bucala2 and CD Sadik1 1 Dermatology, Allergy, and Venereology, University of Lu¨beck, Lu¨beck, Germany and 2 Departments of Medicine and Pathology, Yale University School of Medicine, New Haven, CT Macrophage Migration Inhibitory Factor (MIF) is a unique protein that combines properties of cytokines, enzymes, hormones, and chaperones, and exerts pleotropic effects. In sum, its effects are mostly considered proinflammatory. Accordingly, MIF has been suggested to promote the pathogenesis of multiple inflammatory disease in diverse organs. Thus, it is elevated in the serum of psoriasis patients, and functional polymorphisms in the MIF gene are associated with enhanced susceptibility to psoriasis. The significance of these findings for the pathogenesis of psoriasis, however, has remained elusive. We therefore addressed the role of ˆ - (AIPD) and in the IL-23-induced MIF in psoriasis by examining Mif-/- mice in the AldaraO psoriasiform dermatitis model. Genetic deficiency in MIF reduced clinical severity of psoriasiform skin inflammation in the AIPD and in the IL-23-induced model by approximately 50%. On the histopathological level, psoriasiform skin lesions of Mif-/- mice showed less T cell and and macrophage infiltration in the skin. Likewise, epidermal proliferation as well as dermal neoangiogenesis were markedly attenuated. Our results suggest a significant role for MIF in the pathogenesis of plaque psoriasis and highlight it as a new therapeutic target in the treatment of the disease that may be selectively applied in those patients with high expression MIF risk alleles. With exogenous glucocorticoids known to upregulate MIF, the inhibition of MIF may have particular application when used adjunctly with topical corticosteroids.
Leucine-rich-a-2 glycoprotein, a biomarker and potential target of psoriasis therapy H Nakajima1, M Takaishi1, S Serada2, M Fujimoto2, T Naka3 and S Sano1 1 Dermatology, Kochi Medical School, Nankoku, Japan, 2 Laboratory of Immune signal, National Institute of Biomedical Innovation, Ibaraki, Japan and 3 Integrated Center for Advanced Medical Technologies, Kochi Medical School, Nankoku, Japan Recent study showed that serum levels of Leucine-rich-a-2 glycoprotein (LRG) were significantly higher in patients with autoimmune diseases such as rheumatoid arthritis, Crohn’s disease and Behc¸et disease. Here, we assessed whether LRG could be a biomarker and any pathogenic of psoriasis. Serum LRG levels were significantly increased in psoriasis patients even in those who were not positive for serum C-reactive protein (CRP), and well correlated with PASI score. Moreover, serum LRG levels showed superior sensitivity over CRP in patients with mild to moderate psoriasis based on receiver operating characteristic curve analysis. Furthermore, RT-PCR analysis revealed that psoriasis patients showed increased LRG in peripheral mononuclear cells and neutrophils, the latter of which expressed LRG marginally in normal controls, suggesting that neutrophils are relevant to be LRG source in psoriasis. We also showed that LRG was up-regulated in both human psoriatic skins and psoriasis-like lesions of K5.Stat3C transgenic mice, which were induced by tape stripping. Interestingly, LRG expression was enhanced after tape stripping on the back skin not only in the skin itself but also in the liver of K5.Stat3C mice. LRG mRNA and protein levels in the liver following tape stripping were more strongly increased in K5.Stat3C mice than in WT mice, suggesting the positive feedback loop between the skin and systemic LRG in psoriasis. To elucidate functional roles of LRG in psoriasis development, we crossed K5.Stat3C with LRG KO mice. LRG gene ablation attenuated the psoriatic features including epidermal hyperplasia, inflammatory cell infiltrates and psoriasis-related gene expression. In conclusion, LRG may be not only a useful biomarker but would be a therapeutic target for psoriasis.
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The character of TLN-58, newly discovered hCAP18 processing form, found in the lesion vesicle of palmoplantar pustulosis in the skin M Murakami1, K Kameda2, H Mori1, R Utsunomiya1, K Masuda1, K Shiraishi1, M Tohyama1 and K Sayama1 1 Dermatology, Ehime University, Toon, Japan and 2 Advanced Research support Center, Ehime University, Toon, Japan We have reported that Palmoplantar Pustulosis (PPP) vesicle (before pustule formation) was originated from eccrine sweat in the acrosyringium, and PPP vesicle contains antimicrobial peptides human cathelicidin (hCAP-18/LL-37) (JID 2010, PLOSONE 2014). In the latter report, we have shown that PPP vesicle contains additional small fragment of hCAP-18 sized about 7KDa but not been identified before. The principal aim of this study was to confirm the character of this extra fragment of hCAP-18 found in the PPP vesicle. Lesional PPP vesicle was collected from PPP patients under sterile condition, the endogenous hCAP-18/LL-37 was depleted with LL-37 antibody affinity column. The recombinant hCAP-18 peptide was prepared as a full-length human cathelicidin, and then the peptide was incubated with the depleted PPP vesicle. After incubation, the sample was processed for 15% SDS-PAGE, and the gel was visualized with Coomassie Blue staining. After cutting out the target band, the gel fragment was processed for the in-gel digestion and the protein sequencing. Normal keratinocyte was incubated with synthetic peptides of LL-37 and a newly discovered fragment to check the inflammatory activity, so that IL-17C, IL-8, IL-23, IL-1a, and IL-1b mRNAs expressions were evaluated with qRT-PCR. In addition, antibacterial activity against S. aureus, S. epidermidis, and Group A Streptococcus were also evaluated with solution killing assay. We could confirm that the small newly discovered form processed from recombinant hCAP-18 was TLN-58 (estimated MW 6858). The fragment showed the antimicrobial activity as LL-37 showed, and could induce higher mRNA expressions of IL-8 and IL-17 in normal human keratinocyte comparing with LL-37 induced. It is possible that this processing form has a role of antimicrobial peptide, and could be a candidate for the pathogenesis of continuous inflammation of PPP lesion in the skin.
Stimulation of the histamine 4 receptor increases the production of IL-5 in innate lymphoid cells K Schaper1, H Stark2, T Werfel1 and R Gutzmer1 1 Department for Dermatology and Allergy, Medical School Hannover, Hannover, Germany and 2 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Du¨sseldorf, Germany Allergic disorders, such as asthma or atopic dermatitis (AD) are associated with increased Th2 cytokines, while Th2 cells are known to be the major source. However, also other cell types, especially the novel discovered type 2 innate lymphoid cell subset (ILC2) produce high amounts of IL-5 and IL-13. While IL-5 serves mainly as stimulator of eosinophils, IL-13 also regulates cell-mediated immunity and thereby enhances airway hyper reactivity. ILC2 accumulate in tissue at the site of inflammation and it could be shown that the number of ILC2 is significantly elevated in skin of AD patients. Since histamine is also increased in allergic diseases and AD lesions, the present study aimed to identify the impact of histamine, especially with regard to the histamine 4 receptor (H4R) on the cytokine production of ILC2. We therefore isolated PBMCs from healthy subjects and depleted CD3+ cells. CD127+ cells were further enriched by means of magnetic cell separation. On stimulation with IL-2 and IL25 for five days CD3-CD127+ cells produced high amounts of IL-5 and IL-13, while the cytokine production of CD3+ cells was lower. CD3-CD127- did not produce detectable levels of IL-5 and IL-13 at all. Incubation of the cells with histamine during the five days of stimulation augmented the production of IL-5 and IL-13 in ILC2 significantly, but not in CD3+ cells. CD3-CD127+ cells incubated with ST1006, a selective H4R agonist, also increased the production of IL-5, while the IL-13 secretion was not altered. To determine histamine receptor expression, we sorted ILC2 (Lin-CD45highCD127+CRTH2+) and performed real-time PCR. Thereby we showed that pure ILC2 expressed histamine receptor 1 (H1R), H2R and H4R. Taken together, our study indicates a possible role for the H4R in the regulation of IL-5 in human ILC2. In allergic diseases blocking the H4R could therefore lead to a decrease of IL-5 and subsequently result in an anti-inflammatory effect.
www.jidonline.org S205