RESUMO
Segmented filamentous bacteria (SFB) are members of the commensal intestinal microbiome. They are known to contribute to the postnatal maturation of the gut immune system, but also to augment inflammatory conditions in chronic diseases such as Crohn's disease. Living primary tissue slices are ultrathin multicellular sections of the intestine and provide a unique opportunity to analyze tissue-specific immune responses ex vivo. This study aimed to investigate whether supplementation of the gut flora with SFB promotes T helper 17 (Th17) cell responses in primary intestinal tissue slices ex vivo. Primary tissue slices were prepared from the small intestine of healthy Taconic mice with SFB-positive and SFB-negative microbiomes and stimulated with anti-CD3/CD28 or Concanavalin A. SFB-positive and -negative mice exhibited distinct microbiome compositions and Th17 cell frequencies in the intestine and complex microbiota including SFB induced up to 15-fold increase in Th17 cell-associated mediators, serum amyloid A (SAA), and immunoglobulin A (IgA) responses ex vivo. This phenotype could be transmitted by co-housing of mice. Our findings highlight that changes in the gut microbiome can be observed in primary intestinal tissue slices ex vivo. This makes the system very attractive for disease modeling and assessment of new therapies.
Assuntos
Microbioma Gastrointestinal , Homeostase , Células Th17 , Animais , Células Th17/imunologia , Camundongos , Microbioma Gastrointestinal/imunologia , Homeostase/imunologia , Camundongos Endogâmicos C57BL , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologiaRESUMO
BACKGROUND: Current diagnostics for the detection of pancreato-biliary cancers (PBCs) need to be optimized. We therefore propose that methylated cell-free DNA (cfDNA) derived from non-invasive liquid biopsies serves as a novel biomarker with the ability to discriminate pancreato-biliary cancers from non-cancer pancreatitis patients. METHODS: Differentially methylated regions (DMRs) from plasma cfDNA between PBCs, pancreatitis and clinical control samples conditions were identified by next-generation sequencing after enrichment using methyl-binding domains and database searches to generate a discriminatory panel for a hybridization and capture assay with subsequent targeted high throughput sequencing. RESULTS: The hybridization and capture panel, covering around 74 kb in total, was applied to sequence a cohort of 25 PBCs, 25 pancreatitis patients, 25 clinical controls, and seven cases of Intraductal Papillary Mucinous Neoplasia (IPMN). An unbiased machine learning approach identified the 50 most discriminatory methylation markers for the discrimination of PBC from pancreatitis and controls resulting in an AUROC of 0.85 and 0.88 for a training (n = 45) and a validation (n = 37) data set, respectively. The panel was also able to distinguish high grade from low grade IPMN samples. CONCLUSIONS: We present a proof of concept for a methylation biomarker panel with better performance and improved discriminatory power than the current clinical marker CA19-9 for the discrimination of pancreato-biliary cancers from non-cancerous pancreatitis patients and clinical controls. This workflow might be used in future diagnostics for the detection of precancerous lesions, e.g. the identification of high grade IPMNs vs. low grade IPMNs.
Assuntos
Carcinoma Ductal Pancreático , Ácidos Nucleicos Livres , Neoplasias Intraductais Pancreáticas , Neoplasias Pancreáticas , Pancreatite , Humanos , Biomarcadores Tumorais/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pancreatite/diagnóstico , Pancreatite/genética , Biópsia Líquida , Carcinoma Ductal Pancreático/patologiaRESUMO
Via the histamine H4 -receptor (H4 R), histamine promotes the pathogenesis of experimental allergic asthma in mice. Application of H4 R antagonists during sensitization as well as during provocation reduces the severity of the disease. However, the specific cell types functionally expressing H4 R in experimental allergic asthma have not been well characterized in vivo. In this study, we identified the cell type(s) responsible for H4 R activity in experimental asthma and related physiological mechanisms. Using H4 R-deficient mice, we studied the role of H4 R in the sensitization and effector phase. DCs lacking H4 R expression during the in vitro sensitization reaction resulted in effector T cells unable to induce an entire eosinophilic inflammation in the lung upon adoptive transfer in vivo. Recipient mice lacking H4 R expression, which were adoptively transferred with H4 R(+/+) T cells polarized in the presence of H4 R(+/+) DCs, showed reduced signs of inflammation and ameliorated lung function. Here, we provide in vivo evidence that in experimental asthma in mice the H4 R specifically regulates activation of DCs during sensitization, while in the effector phase the H4 R is active in cells involved in the activation of eosinophils, and possibly other cells. A putative therapy targeting the H4 R may be an option for asthma patients developing IL-5-dependent eosinophilia.
Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Inflamação/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores Histamínicos/imunologia , Transferência Adotiva , Alérgenos/imunologia , Animais , Asma/induzido quimicamente , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Antígeno CD11c/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Histamina/metabolismo , Interleucina-5/imunologia , Pulmão/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/genética , Receptores Histamínicos H4 , Células Th2/imunologia , Células Th2/transplanteRESUMO
Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo.
Assuntos
CMP Cíclico/metabolismo , Nucleotídeos Cíclicos/metabolismo , Uridina Monofosfato/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em TandemRESUMO
In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.
Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/biossíntese , Glucosiltransferases/metabolismo , Nucleotídeos Cíclicos/biossíntese , Nucleotidiltransferases/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Uridina Monofosfato/biossíntese , Apoptose , Sobrevivência CelularRESUMO
Abdominal sepsis triggers the transition of microorganisms from the gut to the peritoneum and bloodstream. Unfortunately, there is a limitation of methods and biomarkers to reliably study the emergence of pathobiomes and to monitor their respective dynamics. Three-month-old CD-1 female mice underwent cecal ligation and puncture (CLP) to induce abdominal sepsis. Serial and terminal endpoint specimens were collected for fecal, peritoneal lavage, and blood samples within 72 h. Microbial species compositions were determined by NGS of (cell-free) DNA and confirmed by microbiological cultivation. As a result, CLP induced rapid and early changes of gut microbial communities, with a transition of pathogenic species into the peritoneum and blood detected at 24 h post-CLP. NGS was able to identify pathogenic species in a time course-dependent manner in individual mice using cfDNA from as few as 30 microliters of blood. Absolute levels of cfDNA from pathogens changed rapidly during acute sepsis, demonstrating its short half-life. Pathogenic species and genera in CLP mice significantly overlapped with pathobiomes from septic patients. The study demonstrated that pathobiomes serve as reservoirs following CLP for the transition of pathogens into the bloodstream. Due to its short half-life, cfDNA can serve as a precise biomarker for pathogen identification in blood.
RESUMO
During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen-specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcgamma receptor (FcgammaR)-mediated antigen uptake and enhance antigen presentation resulting in augmented T-cell proliferation. We examined the impact of antigen presentation and T-cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene-deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcgammaR-deficient mice. Lung DC of WT, but not FcgammaR-deficient mice, induced increased antigen-specific CD4+ T-cell proliferation when pulsed with anti-OVA IgG immune complexes. Intranasal application of anti-OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen-specific T-cell proliferation in vivo. Finally, antigen-specific IgG in the serum of sensitized mice led to a significant increase of antigen-specific CD4+ T-cell proliferation induced by WT, but not FcgammaR-deficient, lung DC. We conclude that FcgammaR-mediated enhanced antigen presentation and T-cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.
Assuntos
Apresentação de Antígeno , Hiper-Reatividade Brônquica/imunologia , Células Dendríticas/imunologia , Imunoglobulina G/imunologia , Pulmão/imunologia , Receptores de IgG/imunologia , Transferência Adotiva , Animais , Asma/imunologia , Feminino , Inflamação , Pulmão/patologia , Ativação Linfocitária , Linfocinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
CpG-containing oligonucleotides (CpG) have been shown to reduce key features of allergic airway inflammation in mouse models. Given the inhibitory effects of CpG treatment on Ag presentation of subsequently encountered Ags via MHC class I and II molecules by dendritic cells (DC), we hypothesized that intranasal CpG treatment would lead to reduced Ag-specific T cell stimulation in the lung-draining lymph nodes, thereby reducing the inflammatory response in sensitized mice. Intranasal CpG administration led to phenotypic maturation of lung and mediastinal lymph node DC as determined by expression of MHC class II, CD80, and CD86. This was accompanied by a significant reduction in the proliferation of adoptively transferred Ag-specific CD4(+) and CD8(+) T cells in mediastinal lymph nodes, when CpG was given before inhalative OVA challenges. DC obtained from mediastinal lymph nodes of CpG-treated mice before OVA inhalation led to reduced T cell stimulation via MHC class I and II molecules. In addition, CpG diminished airway eosinophilia and pulmonary infiltration after sensitization or following adoptive transfer of Ag-specific Th2 cells. These results were explained by reduced CCL21 expression and inhibition of lung DC migration following CpG administration, which could be restored by transfer of bone marrow-derived DC, because CpG had no major impact on the constitutive MHC class II Ag presentation of protein-derived Ag by lung tissue-derived DC. We conclude that CpG treatment can effectively impair the DC-mediated Ag transport from the lungs to the lymph nodes, resulting in reduced T cell activation and blunted airway inflammation.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Mediadores da Inflamação/administração & dosagem , Pulmão/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/terapia , Linfócitos T/imunologia , Alérgenos/efeitos adversos , Alérgenos/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Ilhas de CpG/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Feminino , Imunofenotipagem , Mediadores da Inflamação/fisiologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipersensibilidade Respiratória/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
Pollen starch granules (PSGs) are allergen particles that get into contact with pulmonary surfactant and phagocytes in the terminal airways. In this study, the effects of surfactant protein D (SP-D) on the interaction of PSGs with phagocytes and on the pulmonary clearance of PSGs were determined. Fluorescently labeled PSGs were incubated in vitro with murine lung macrophages or dendritic cells (DCs) ± recombinant rat SP-D (rrSP-D). In addition, the effect of SP-D on uptake of PSGs by lung macrophages and DCs was studied in vivo. Furthermore, PSGs were instilled in Balb/c mice and the effects of SP-D on total lung clearance were assessed by optical imaging. SP-D treatment increased the number of PSG-positive macrophages and DCs in vitro. Furthermore, SP-D accelerated uptake/binding by alveolar macrophages and reduced the number of PSG-positive tissue macrophages and DCs at 24 hours. However, SP-D did not affect total lung clearance of PSGs and it did not enhance the T-cell proliferation induced by PSG-positive DCs. In conclusion, SP-D increased PSG-positive cells in vitro and accelerated PSG binding/uptake in vivo. The observed effects were limited to cellular clearance mechanisms and did not affect the total clearance of PSGs from the lung.
Assuntos
Alérgenos/metabolismo , Pulmão/efeitos dos fármacos , Depuração Mucociliar/efeitos dos fármacos , Pólen/metabolismo , Proteína D Associada a Surfactante Pulmonar/farmacologia , Amido/metabolismo , Alérgenos/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Citometria de Fluxo , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Pólen/imunologia , Ratos , Proteínas Recombinantes , Amido/imunologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
T cells and T cell derived cytokines are involved in the complex pathogenesis of asthma. The role of the cytokine IL-18 however, is not clearly defined so far. On the one hand side IL-18 induces Th1-type cytokines and thereby might counter-regulate Th2-mediated allergic asthma. On the other hand IL-18 also bears pro-inflammatory effects possibly enhancing experimental asthma. In order to elucidate the role of IL-18 in allergic pulmonary inflammation typical symptoms were compared after induction of experimental asthma in IL-18(-/-) and in wild type mice. Asthma was induced using ovalbumin (OVA) as allergen for sensitization and challenge. Sham sensitized and OVA challenged mice served as controls. Bronchoalveolar lavage-fluid cytology, leukocyte infiltration in lung tissues, serum levels of OVA-specific IgE and cytokines, and lung function were analyzed. Clear differences could be observed between control and asthmatic mice, both in wild type and IL-18(-/-) animals. Surprisingly, no differences were found between asthmatic wild type and IL-18(-/-) mice. Thus, in contrast to conflicting data in the literature IL-18 did not suppress or enhance the pulmonary allergic immune response in a murine experimental model of asthma.
Assuntos
Asma/imunologia , Interleucina-18/sangue , Animais , Asma/patologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Imunização , Imunoglobulina E/sangue , Interleucina-17/sangue , Interleucina-5/sangue , Contagem de Leucócitos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/farmacologia , Pletismografia TotalRESUMO
BACKGROUND: Heroin-assisted treatment (HAT) is a new form of treatment for heroin-dependent patients not responding to conventional interventions such as methadone maintenance treatment. No pregnancies or births under HAT have been reported until now. CASE: The pregnancy course of a 31-year-old severely dependent multi-morbid woman receiving HAT and the birth of a healthy baby after premature delivery is described. HAT helped to reduce the use of illicit substances both before and during pregnancy. The neonatal abstinence syndrome was clinically well compensated. CONCLUSION: HAT seems to be feasible in pregnant women and normal birth is possible under HAT, which therefore may act as a harm reduction measure for polydrug-using pregnant women not responding to methadone maintenance treatment.
Assuntos
Heroína/uso terapêutico , Síndrome de Abstinência Neonatal/diagnóstico , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Adulto , Feminino , Heroína/efeitos adversos , Humanos , Recém-Nascido , Masculino , Síndrome de Abstinência Neonatal/etiologia , Síndrome de Abstinência Neonatal/terapia , Gravidez , Complicações na Gravidez/etiologia , Transtornos Relacionados ao Uso de Substâncias/complicaçõesRESUMO
BACKGROUND: Murine models assist in elucidating the pathogenesis of allergic asthma and evaluation of new therapeutic strategies. We aimed to assess the requirement of boostering needed in the BL/6 murine asthma model and its influence on DC populations in lungs and bronchial lymph nodes. METHODS AND RESULTS: Two injections of OVA+alum - one sensitization and one booster - followed by two aerosol challenges were sufficient to induce a distinct asthma-like inflammation in BL/6 mice, including significant increased immunoglobulin (IgE) level, influx of eosinophils in the airway lumen, and evident histopathology. Using this protocol, CD11chighMHC-II+ DC counts in lungs and lymph nodes doubled with no changes of CD8+ DC in the lungs but increase in lung-draining lymph nodes. CONCLUSIONS: Given the site-specific changes of dendritic cell (DC) subpopulations during allergic asthma we propose a distinct regulation of antigen transport and antigen presentation in the murine asthma model.
Assuntos
Asma/imunologia , Brônquios/imunologia , Células Dendríticas/imunologia , Alérgenos/imunologia , Animais , Asma/patologia , Asma/fisiopatologia , Brônquios/patologia , Brônquios/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Citocinas/sangue , Células Dendríticas/patologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Imunoglobulina E/sangue , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Testes de Função RespiratóriaRESUMO
BACKGROUND: A major goal of heroin-assisted treatment in Switzerland has been to reduce the drug-related mortality of heroin users. Therefore, a continuous monitoring of deaths under treatment is essential. AIMS: To assess mortality of participants in heroin-assisted treatment in Switzerland over a 7-year period from 1994 to 2000, and to compare this mortality to the general population and to other populations of opioid users, as reported in the literature. METHOD: Estimation of person years under heroin-assisted treatment from the complete case registry of heroin-assisted treatment in Switzerland. Estimation of standardized mortality ratios comparing the population in treatment to the Swiss population (standardized to the year 2000). RESULTS: Over the 7-year period, the crude death rate of patients in heroin-assisted treatment, and including one month after discharge from treatment, was 1% per year. The standardized mortality ratio for the entire observation period was 9.7 (95% C.I. 7.3-12.8), with females having higher standardized mortality ratios (SMR 17.2) than males (SMR 8.4). There was no clear time trend. CONCLUSION: Mortality in heroin-assisted treatment was low compared to the mortality rate of Swiss opioid users 1990s (estimated to be between 2.5 and 3%). It was also low compared to mortality rates of opioid users in other maintenance treatments in other countries as reported in the literature. The SMR was also lower than that reported in the only meta-analysis in the literature: 13.2 (95% C.I. 12.3-14.1). The low mortality rate is all the more noteworthy as heroin-assisted treatment in Switzerland included only refractory opioid addicts with existing severe somatic and/or mental problems. DECLARATION OF INTEREST: No conflicts of interest declared.
Assuntos
Dependência de Heroína/tratamento farmacológico , Dependência de Heroína/mortalidade , Heroína/uso terapêutico , Entorpecentes/uso terapêutico , Centros de Tratamento de Abuso de Substâncias , Causas de Morte , Feminino , Humanos , Masculino , Metadona/uso terapêutico , Fatores Sexuais , Suíça/epidemiologiaRESUMO
Asthma is the consequence of allergic inflammation in the lung compartments and lung-draining lymph nodes. Dendritic cells initiate and promote T cell response and drive it to immunity or allergy. However, their modes of action during asthma are poorly understood. In this study, an allergic inflammation with ovalbumin was induced in 38 mice versus 42 control animals. After ovalbumin aerosol challenge, conventional dendritic cells (CD11c/MHCII/CD8) were isolated from the lungs and the draining lymph nodes by means of magnetic cell sorting followed by fluorescence-activated cell sorting. A comparative transcriptional analysis was performed using gene arrays. In general, many transcripts are up- and downregulated in the CD8(-) dendritic cells of the allergic inflamed lung tissue, whereas few genes are regulated in CD8(+) dendritic cells. The dendritic cells of the lymph nodes also showed minor transcriptional changes. The data support the relevance of the CD8(-) conventional dendritic cells but do not exclude distinct functions of the small population of CD8(+) dendritic cells, such as cross presentation of external antigen. So far, this is the first approach performing gene arrays in dendritic cells obtained from lung tissue and lung-draining lymph nodes of asthmatic-like mice.
RESUMO
Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are well-established second messengers, whereas the physiological role of the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) is poorly understood. Certain mammalian nucleotidyl cyclases (NCs) and bacterial NC toxins can generate cCMP and cUMP. Human HEK293 cells and rat B103 neuroblastoma cells are of neuronal origin and possess high basal concentrations of cCMP and cUMP that can be attributed to soluble adenylyl cyclase activity. These data prompted us to conduct a systematic analysis of basal nucleoside 3',5'-cyclic monophosphate (cNMP) concentrations across the tree of life. cCMP and cUMP were identified in many mammalian cell lines and primary cells. cNMP patterns varied broadly among cells, and in several systems, cCMP and cUMP concentrations were quite high. Prokaryotes, fungi, amoeba and invertebrates lacked cCMP and cUMP, whereas cAMP was found across the tree of life. High cCMP and cUMP concentrations were found in astrocytes. The distinct cNMP patterns support specific second messenger roles of cCMP and cUMP, specifically in astrocytes.
Assuntos
Astrócitos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Animais , Células Cultivadas , Cricetinae , AMP Cíclico/metabolismo , CMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fungos/metabolismo , Haplorrinos , Humanos , Invertebrados/metabolismo , Plantas/metabolismo , Células Procarióticas/metabolismo , Ratos , Especificidade da Espécie , Uridina Monofosfato/metabolismoRESUMO
BACKGROUND: Histamine drives pruritus in allergic skin diseases which clinically constitutes a most disruptive symptom. Skin pathology in allergic skin diseases is crucially influenced by different T-helper subsets. However, the contribution of different histamine-receptors to T-helper cell dependent skin pathology has not been definitively answered. Models which can specifically address the functional role of T-helper subsets and the mediators involved are therefore valuable to gain further insights into molecular pathways which contribute to allergic skin disease. They might also be helpful to probe amendable therapeutic interventions such as histamine-receptor antagonism. OBJECTIVE: Establishing an adoptive transfer model for antigen-specific Th cells, we aimed to delineate the role of histamine H1- and H4-receptors in Th2-dependent skin inflammation. METHODS: In-vitro differentiated and OVA primed Th2 cells were adoptively transferred into congenic recipient mice. In vivo treatment with specific histamine H1- and H4-receptor antagonists was performed to analyze the contribution of these histamine-receptors to Th2-dependent skin pathology in our model. Analysis four days after epicutaneous challenge comprised skin histology, flow cytometric detection of transferred T-helper cells and analysis of antigen-cytokine profiles in skin-draining lymph nodes. RESULTS: Use of specific H1- and H4-receptor antagonists revealed a crucial role for H1- and H4-receptors for Th2 migration and cytokine secretion in a Th2-driven model of skin inflammation. While H1- and H4-receptor antagonists both reduced Th2 recruitment to the site of challenge, local cytokine responses in skin-draining lymph nodes were only reduced by the combined application of H1- and H4-receptor antagonists and mast cell counts remained altogether unchanged by either H1R-, H4R- or combined antagonism. CONCLUSION: Our model demonstrates a role for H1- and H4-receptors in Th2 cell infiltration and cytokine secretion in allergic skin diseases and suggests further studies to evaluate these findings for therapeutic approaches.
Assuntos
Epitopos/imunologia , Inflamação/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos/metabolismo , Pele/imunologia , Pele/patologia , Células Th2/imunologia , Transferência Adotiva , Animais , Contagem de Células , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Feminino , Antagonistas dos Receptores Histamínicos/farmacologia , Inflamação/patologia , Mastócitos/imunologia , Camundongos Endogâmicos BALB C , Receptores Histamínicos H4 , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, the histamine H4 receptor (H4R)-selective ligand JNJ 7777120 reduces asthma-like symptoms. A sole antagonistic function of JNJ 7777120 at the murine H4R has, however, been questioned in the literature. Therefore, in the present study, we aimed at analyzing the effects of JNJ 7777120 in comparison to that of the H3/4R-selective antagonist thioperamide. Experimental murine asthma was induced by sensitization and provocation of BALB/c mice with ovalbumine (OVA). JNJ 7777120, thioperamide, or JNJ 5207852, an H3R-selective antagonist which was used to dissect H3R- and H4R-mediated activities of thioperamide, were injected subcutaneously during sensitization and effects were analyzed after provocation. Pharmacokinetic analyses revealed shortest t1/2 values in both plasma and lung tissue and lowest maximal concentration in lung tissue for JNJ 7777120 in comparison to thioperamide and JNJ 5207852. Nevertheless, JNJ 7777120 reduced serum titers of allergen-specific (anti-OVA) IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar lavage fluid. In contrast, thioperamide reduced only eosinophilia in bronchoalveolar lavage fluid, while anti-OVA IgE concentrations and lung infiltrations remained unaffected. JNJ 5207852 had no effect on these parameters. JNJ 7777120 provides beneficial effects in experimental murine asthma, which, however, could only partially be mimicked by thioperamide, despite more favorable pharmacokinetics. Thus, whether these effects of JNJ 7777120 are entirely attributable to an antagonistic activity at the murine H4R or whether an agonistic activity is also involved has to be reconsidered.