RESUMO
Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol, and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases.
Assuntos
Antígenos CD1/imunologia , Dermatite de Contato/imunologia , Células T Matadoras Naturais/imunologia , Linfócitos T/imunologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Apresentação de Antígeno , Benzoquinonas/farmacologia , Linhagem Celular , Células Dendríticas/imunologia , Dinitroclorobenzeno/farmacologia , Eugenol/análogos & derivados , Eugenol/farmacologia , Humanos , Lipídeos/imunologia , Ativação Linfocitária , Monócitos/efeitos dos fármacos , Resorcinóis/farmacologia , Pele/imunologiaRESUMO
Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.
Assuntos
Antígenos Virais/imunologia , Fatores de Transcrição Forkhead/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/genética , Influenza Humana/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The effects of air pollutants, particularly polycyclic aromatic hydrocarbons (PAHs), on the skin microbiome remain poorly understood. Thus, to better understand the interplay between air pollutants, microbiomes, and skin conditions, we applied metagenomics and metabolomics to analyze the effects of PAHs in air pollution on the skin microbiomes of over 120 subjects residing in two cities in China with different levels of air pollution. RESULTS: The skin microbiomes differentiated into two cutotypes (termed 1 and 2) with distinct taxonomic, functional, resistome, and metabolite compositions as well as skin phenotypes that transcended geography and host factors. High PAH exposure was linked to dry skin and cutotype 2, which was enriched with species with potential biodegradation functions and had reduced correlation network structure integrity. The positive correlations identified between dominant taxa, key functional genes, and metabolites in the arginine biosynthesis pathway in cutotype 1 suggest that arginine from bacteria contributes to the synthesis of filaggrin-derived natural moisturizing factors (NMFs), which provide hydration for the skin, and could explain the normal skin phenotype observed. In contrast, no correlation with the arginine biosynthesis pathway was observed in cutotype 2, which indicates the limited hydration functions of NMFs and explains the observed dry skin phenotype. In addition to dryness, skin associated with cutotype 2 appeared prone to other adverse conditions such as inflammation. CONCLUSIONS: This study revealed the roles of PAHs in driving skin microbiome differentiation into cutotypes that vary extensively in taxonomy and metabolic functions and may subsequently lead to variations in skin-microbe interactions that affect host skin health. An improved understanding of the roles of microbiomes on skin exposed to air pollutants can aid the development of strategies that harness microbes to prevent undesirable skin conditions. Video Abstract.
Assuntos
Poluentes Atmosféricos , Microbiota , Hidrocarbonetos Policíclicos Aromáticos , Pele/química , Poluentes Atmosféricos/análise , Biodegradação Ambiental , Microbiota/genética , Monitoramento AmbientalRESUMO
Proteinase-activated receptor 2 (PAR(2)) is widely expressed in the respiratory tract and is an integral component of the host antimicrobial defense system. The principal aim of this study was to investigate the influence of a PAR(2)-activating peptide, SLIGRL, on influenza A virus (IAV)-induced pathogenesis in mice. Intranasal inoculation of BALB/c mice with influenza A/PR/8/34 virus caused time-dependent increases in the number of pulmonary leukocytes (recovered from bronchoalveolar lavage fluid), marked airway histopathology characterized by extensive epithelial cell damage, airway hyper-responsiveness to the bronchoconstrictor methacholine, and elevated levels of inflammatory chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2) and cytokines (interferon-γ). It is noteworthy that these IAV-induced effects were dose-dependently attenuated in mice treated with a PAR(2)-activating peptide, SLIGRL, at the time of IAV inoculation. However, SLIGRL also inhibited IAV-induced increases in pulmonary leukocytes in PAR(2)-deficient mice, indicating these antiviral actions were not mediated by PAR(2). The potency order obtained for a series of structural analogs of SLIGRL for anti-IAV activity (IGRL > SLIGRL > LSIGRL >2-furoyl-LIGRL) was also inconsistent with a PAR(2)-mediated effect. In further mechanistic studies, SLIGRL inhibited IAV-induced propagation in ex vivo perfused segments of trachea from wild-type or PAR(2)(-/-) mice, but did not inhibit viral attachment or replication in Madin-Darby canine kidney cells and chorioallantoic membrane cells, which are established hosts for IAV. In summary, SLIGRL protected mice from IAV infection independently of PAR(2) and independently of direct inhibition of IAV attachment or replication, potentially through the activation of endogenous antiviral pathways within the mouse respiratory tract.
Assuntos
Antivirais/uso terapêutico , Vírus da Influenza A/efeitos dos fármacos , Oligopeptídeos/uso terapêutico , Infecções por Orthomyxoviridae/tratamento farmacológico , Receptor PAR-2/metabolismo , Animais , Antivirais/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Citocinas/imunologia , Cães , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Contagem de Leucócitos , Leucócitos/citologia , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Eletrônica de Varredura , Oligopeptídeos/administração & dosagem , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Receptor PAR-2/genética , Testes de Função Respiratória , Traqueia/efeitos dos fármacos , Traqueia/ultraestrutura , Traqueia/virologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Ultraviolet exposure has profound effect on the dermal connective tissue of human skin. OBJECTIVE: We aimed to develop and validate an evaluation method/methodology using a full-thickness reconstructed skin model, to assess the anti-photoaging efficacy of cosmetic ingredients and sunscreen formulas by blending multi relevant biological endpoints including the newly developed dermal collagen quantification method with Multi-photon microscopy. METHODS: The response of ex vivo human skin to UVA exposure was first characterized with multiphoton microscopy. Reconstructed full-thickness skin models was then used to reproduce the data and to create a proof-of-concept study by treating the models with sunscreen prototypes A or B, which differ on their UVA absorption properties, and systemic Vitamin C (Vit C). After exposure to UVA, the collagen density was quantified via multiphoton microscopy with automatic imaging processing. Histology, fibroblasts number, metalloprotease 1 (MMP1) secretion were also assessed. RESULTS: UVA exposure induced pronounced reduction in collagen density and increased MMP1 secretion within both ex vivo human skin and reconstructed skin. Histological damage and fibroblast disappearance was observed with reconstructed skin. Within the proof-of-concept study prototype B, possessing higher UVA filtration, gave better protection than prototype A on the UV associated biological markers, and association with Vit C boosted sunscreen formula efficacy. CONCLUSIONS: The photoaging evaluation method, consists of multi biological markers as well as dermal collagen quantification, is a relevant mean to assess the pre-clinical efficacy of anti-photoaging ingredients and sunscreen products. This approach is also beneficial for evaluating the efficacy of sunscreens and photoprotective ingredients.
Assuntos
Colágeno , Avaliação Pré-Clínica de Medicamentos/métodos , Microscopia/métodos , Envelhecimento da Pele/efeitos dos fármacos , Protetores Solares , Humanos , Estudo de Prova de Conceito , Raios Ultravioleta/efeitos adversosRESUMO
Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN-γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN-γ-stimulated normal human bronchial epithelial cells (HBEC) using a 2-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS. IFN-γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN-γ, in lung tissues from an IFN-γ-predominant influenza A virus-infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN-γ-induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN-γ-induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub-G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN-γ-induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide-double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase-dependent manner. Our results reveal a novel function for ApoL2 in conferring anti-apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN-γ, in maintaining airway epithelial layer integrity.
Assuntos
Apolipoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Interferon gama/farmacologia , Lipoproteínas HDL/metabolismo , Mucosa Respiratória/citologia , Animais , Apolipoproteínas/genética , Apolipoproteínas L , Células Cultivadas , Fragmentação do DNA , Células Epiteliais/citologia , Humanos , Lipoproteínas HDL/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Glycosaminoglycans (GAGs) are long, linear polysaccharides comprised of repeating disaccharide units with pleiotropic biological functions, with the non-sulfated GAG hyaluronic acid (HA), and sulfated GAGs dermatan sulfate, chondroitin sulfate, heparan sulfate, keratan sulfate, and to a lesser extent heparin all being expressed in skin. Their ability to regulate keratinocyte proliferation and differentiation, inflammatory processes and extracellular matrix composition and quality demonstrates their critical role in regulating skin physiology. Similarly, the water-binding properties of GAGs and structural qualities, particularly for HA, are crucial for maintaining proper skin form and hydration. The biological importance of GAGs, as well as extensive evidence that their properties and functions are altered in both chronological and extrinsic skin aging, makes them highly promising targets to improve cosmetic skin quality. Within the present review, we examine the cutaneous biological activity of GAGs alongside the protein complexes they form called proteoglycans and summarize the age-related changes of these molecules in skin. We also examine current topical interventional approaches to modulate GAGs for improved skin quality such as direct exogenous administration of GAGs, with a particular interest in strategies targeted at potentiating GAG levels in skin through either attenuating GAG degradation or increasing GAG production.
RESUMO
BACKGROUND: Urban pollution is correlated with an increased prevalence of skin pigmentation disorders, however the physiological processes underlying this association are unclear. OBJECTIVES: To delineate the relationship between polycyclic aromatic hydrocarbons (PAHs), a key constituent of atmospheric pollution, and immunity/skin pigmentation pathways. METHODS: We exposed peripheral blood mononuclear cells (PBMC) to PAHs and performed cytokines/chemokine profiling. We then examined the effect of immune activation on pigmentation by co-culturing PBMC and Benzo(a)pyrene (BaP) with reconstructed human pigmented epidermis (RHPE). To study the mechanism, we treated keratinocytes with conditioned medium from BaP-exposed PBMC and studied DNA damage responses, aryl hydrocarbon receptor (AhR) activation and pro-pigmentation factor, proopiomelanocortin (POMC) secretion. RESULTS: PAHs induced up-regulation of inflammatory cytokines/chemokine in PBMC. Co-culturing of RHPE with PBMC+BaP resulted in increased melanin content and localization. BaP-conditioned medium significantly increased DNA damage, p53 stabilization, AhR activation and POMC secretion in keratinocytes. We found that IFNγ induced DNA damage, while TNFα and IL-8 potentiated POMC secretion in keratinocytes. Importantly, BaP-conditioned medium-induced DNA damage and POMC secretion is prevented by antioxidants vitamin E, vitamin C and sulforaphane, as well as the prototypical corticosteroid dexamethasone. Finally, vitamin C and sulforaphane enhanced the genome protective and depigmentation effects of dexamethasone, providing proof-of-concept for a combinatorial approach for the prevention and/or correction of PAH-induced pigment spots formation. CONCLUSION: Our study reveals the importance of systemic immunity in regulating PAH-induced skin pigmentation, and provide a new keratinocyte DNA damage response mechanistic target for the prevention or reversal of pollution-associated skin pigmentation.
Assuntos
Antioxidantes/farmacologia , Citocinas/metabolismo , Reparo do DNA , Hidrocarbonetos Policíclicos Aromáticos/imunologia , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/imunologia , Anti-Inflamatórios/farmacologia , Ácido Ascórbico/farmacologia , Benzo(a)pireno/farmacologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Dano ao DNA/efeitos dos fármacos , Dexametasona/farmacologia , Epiderme , Humanos , Fenômenos do Sistema Imunitário , Interferon gama/metabolismo , Interleucina-8/metabolismo , Isotiocianatos/farmacologia , Queratinócitos , Leucócitos Mononucleares , Melaninas/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Pró-Opiomelanocortina/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Sulfóxidos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Vitamina E/farmacologiaRESUMO
While it is well established that CD4(+) T lymphocytes play a crucial role in the initiation, progression and persistence of asthma, the role of CD8(+) T cells is less understood. CD8(+) T cells form functionally similar subsets which exhibit similar cytokine profiles as Th1 and Th2 cells, known as Tc1 and Tc2. Evidence from animal studies suggest that CD8(+) T cells are capable of regulating IgE production through the induction of IL-12 and IL-18 production in dendritic cells, and that CD8(+) T cells may act to moderate Th2 polarisation within the localised lymph nodes during allergic sensitisation. Such findings have led to the suggestion that Th1 polarising, CD8(+) T cell-inducing vaccines would inhibit the development of airway hyperresponsiveness (AHR) and Th2 cell infiltration. Despite these positive findings, the role of CD8(+) T cells within the lung remains poorly understood. While CD8(+) T cells, particularly those expressing the Tc1 phenotype, are capable of moderating inflammation and suppressing AHR, it has been postulated that Tc2 CD8(+) T cells predominate within established asthma and may act to amplify the inappropriate immune response which defines the condition. Within the clinic, the association between CD8(+) T cells and asthma is almost universally defined as injurious, further suggesting a prejudicial role for these cells within the established disease. CD8(+) T cells may be a valuable potential target for therapeutic intervention, either by potentiating their regulatory effects prior to the development of sensitisation, or through suppressing their pro-inflammatory properties within established atopy.
Assuntos
Antiasmáticos/farmacologia , Asma/imunologia , Linfócitos T CD8-Positivos/imunologia , Animais , Asma/tratamento farmacológico , Asma/fisiopatologia , Ensaios Clínicos como Assunto , Citocinas/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Pulmão/imunologia , Pulmão/fisiopatologia , FenótipoRESUMO
BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are of environmental and public health concerns and contribute to adverse skin attributes such as premature skin aging and pigmentary disorder. However, little information is available on the potential roles of chronic urban PAH pollutant exposure on the cutaneous microbiota. Given the roles of the skin microbiota have on healthy and undesirable skin phenotypes and the relationships between PAHs and skin properties, we hypothesize that exposure of PAHs may be associated with changes in the cutaneous microbiota. In this study, the skin microbiota of over two hundred Chinese individuals from two cities in China with varying exposure levels of PAHs were characterized by bacterial and fungal amplicon and shotgun metagenomics sequencing. RESULTS: Skin site and city were strong parameters in changing microbial communities and their assembly processes. Reductions of bacterial-fungal microbial network structural integrity and stability were associated with skin conditions (acne and dandruff). Multivariate analysis revealed associations between abundances of Propionibacterium and Malassezia with host properties and pollutant exposure levels. Shannon diversity increase was correlated to exposure levels of PAHs in a dose-dependent manner. Shotgun metagenomics analysis of samples (n = 32) from individuals of the lowest and highest exposure levels of PAHs further highlighted associations between the PAHs quantified and decrease in abundances of skin commensals and increase in oral bacteria. Functional analysis identified associations between levels of PAHs and abundance of microbial genes of metabolic and other pathways with potential importance in host-microbe interactions as well as degradation of aromatic compounds. CONCLUSIONS: The results in this study demonstrated the changes in composition and functional capacities of the cutaneous microbiota associated with chronic exposure levels of PAHs. Findings from this study will aid the development of strategies to harness the microbiota in protecting the skin against pollutants. Video Abstract.
Assuntos
Poluentes Ambientais/farmacologia , Microbiota/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Pele/efeitos dos fármacos , Pele/microbiologia , Adulto , China , Cidades , Monitoramento Ambiental , Poluentes Ambientais/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Hidrocarbonetos Policíclicos Aromáticos/administração & dosagemRESUMO
Stimulants of protease-activated receptor (PAR)(2) promote the generation of the bronchoprotective prostanoid prostaglandin (PG) E(2) by airway epithelial cells. In contrast, glucocorticoids reduce the levels of PGE(2) in airway epithelial cell cultures by concomitantly inhibiting pathways required for PGE(2) synthesis and facilitating pathways involved in PGE(2) inactivation. The aim of this study was to determine whether glucocorticoids inhibited PAR(2)-mediated, PGE(2)-dependent responses in epithelial cell cultures, in intact airway preparations, and in whole animals. In cultures of A549 cells, a PAR(2)-activating peptide SLI-GRL-NH(2) produced concentration and time-dependent increases in PGE(2) levels, which were significantly enhanced after exposure to lipopolysaccharide (LPS). However, SLIGRL-NH(2)-induced increases in PGE(2) levels were abolished by pretreatment of cells with the glucocorticoid, dexamethasone. In mouse isolated tracheal preparations, SLIGRL-NH(2) and PGE(2) induced concentration-dependent relaxation responses that were unaffected by dexamethasone, irrespective of whether dexamethasone exposure occurred in vitro or in vivo. Intranasal administration of LPS produced a pronounced increase in the numbers of neutrophils recovered from the bronchoalveolar lavage fluid of BALB/c mice. Numbers of recovered neutrophils were 40 to 60% lower in mice that received f-LIGRL-NH(2) (PAR(2)-activating peptide, 30 microg intranasally), PGE(2) (10 mugintranasally), or dexamethasone (1 mg/kg i.p.). In the combined presence of dexamethasone and f-LIGRL-NH(2) or dexamethasone and PGE(2), the number of neutrophils was suppressed further (80-83% lower). Thus, although dexamethasone abolished PAR(2)-mediated generation of PGE(2) in A549 cells, neither the smooth muscle relaxant nor the anti-inflammatory effects of PAR(2)-activating peptides (and PGE(2)) were diminished by in vitro or in vivo exposure to dexamethasone.
Assuntos
Dexametasona/farmacologia , Receptor PAR-2/fisiologia , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Animais , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Sistema Respiratório/efeitos dos fármacosRESUMO
Foxp3âºCD4⺠regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8⺠T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25âºFoxp3⺠regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4âºCD25⺠regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection.
Assuntos
Anticorpos Monoclonais/imunologia , Vírus da Influenza A/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Reguladores/imunologia , Doença Aguda , Animais , Anticorpos Monoclonais/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Embrião de Galinha , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Interferon gama/imunologia , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fatores de Tempo , Redução de Peso/efeitos dos fármacos , Redução de Peso/imunologiaRESUMO
We are interested in developing an airway explant culture system using sheep bronchi in which to establish respiratory viral infection and from which tissue can be used for functional, biochemical and immunohistochemical studies involving the endothelins (ETs). Freshly harvested sheep bronchial airway smooth muscle contains a homogeneous population of the ET(A) receptor. However, the potency of ET-1 and maximum contractile response of sheep bronchial explants to ET-1 increased with time in culture, despite these parameters remaining constant for carbachol in explants maintained for up to 48 h. The possibility that this was caused by changes in ET receptor density was assessed using light microscopic quantitative autoradiography. In view of the increased responsiveness to ET-1 in cultured explants, it was surprising to demonstrate a significant decrease in total ET receptor (59+/-6% compared with the initial value, n=4-5; P<0.01) and ET(A) receptor (51+/-2% compared with the initial value, n=4-5, P<0.01) density in sheep bronchial explants after 48 h. No ET(B) receptors were detected. Thus, the culture of sheep bronchial explants was associated with an increase in ET(A) receptor-mediated contractile function that was accompanied by a decrease in ET(A) receptor density. In addition, the structural integrity of the ciliated epithelium was preserved using this culture protocol, a feature that is critical to successful respiratory viral infection. The significant changes in ET receptor density and function in these bronchial explants must be carefully considered when assessing any effects of respiratory viral infection in this model.