RESUMO
Cerebral ischemia triggers a cascade of neuroinflammatory and peripheral immune responses that contribute to post-ischemic reperfusion injury. Prior work conducted in CNS ischemia models underscore the potential to harness non-antibiotic properties of tetracycline antibiotics for therapeutic benefit. In the present study, we explored the immunomodulatory effects of the tetracycline derivative 9-tert-butyl doxycycline (9-TB) in a mouse model of transient global ischemia that mimics immunologic aspects of the post-cardiac arrest syndrome. Pharmacokinetic studies performed in C57BL/6 mice demonstrate that within four hours after delivery, levels of 9-TB in the brain were 1.6 and 9.5-fold higher than those obtained using minocycline and doxycycline, respectively. Minocycline and 9-TB also dampened inflammation, measured by reduced TNFα-inducible, NF-κß-dependent luciferase activity in a microglial reporter line. Notably, daily 9-TB treatment following ischemia-reperfusion injury in vivo induced the retention of polymorphonuclear neutrophils (PMNs) within the spleen while simultaneously biasing CNS PMNs towards an anti-inflammatory (CD11bLowYm1+) phenotype. These studies indicate that aside from exhibiting enhanced CNS delivery, 9-TB alters both the trafficking and polarization of PMNs in the context of CNS ischemia-reperfusion injury.
Assuntos
Anti-Inflamatórios/farmacologia , Isquemia Encefálica/imunologia , Doxiciclina/farmacologia , Imunidade Inata/efeitos dos fármacos , Inflamação/prevenção & controle , Células Mieloides/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Antibacterianos/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologiaRESUMO
Lysophosphatidic acid (LPA) is a pleiotropic lipid signaling molecule associated with asthma pathobiology. LPA elicits its effects by binding to at least six known cell surface G protein-coupled receptors (LPA1-6) that are expressed in the lung in a cell type-specific manner. LPA2 in particular has emerged as an attractive therapeutic target in asthma because it appears to transduce inhibitory or cell-protective signals. We studied a novel and specific small molecule LPA2 agonist (2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl] benzoic acid [DBIBB]) in a mouse model of house dust mite-induced allergic airway inflammation. Mice injected with DBIBB developed significantly less airway and lung inflammation compared with vehicle-treated controls. Levels of lung Th2 cytokines were also significantly attenuated by DBIBB. We conclude that pharmacologic activation of LPA2 attenuates Th2-driven allergic airway inflammation in a mouse model of asthma. Targeting LPA receptor signaling holds therapeutic promise in allergic asthma.
Assuntos
Antiasmáticos/farmacologia , Anti-Inflamatórios/farmacologia , Asma/prevenção & controle , Pulmão/efeitos dos fármacos , Naftalimidas/farmacologia , Pneumonia/prevenção & controle , Receptores de Ácidos Lisofosfatídicos/agonistas , Sulfonamidas/farmacologia , Alérgenos , Animais , Antígenos de Dermatophagoides , Proteínas de Artrópodes , Asma/imunologia , Asma/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Diester Fosfórico Hidrolases/metabolismo , Pneumonia/imunologia , Pneumonia/metabolismo , Receptores de Ácidos Lisofosfatídicos/imunologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Fatores de TempoRESUMO
Lysophosphatidic acid (LPA) is a pleiotropic lipid molecule with potent effects on cell growth and motility. Major progress has been made in recent years in deciphering the mechanisms of LPA generation and how it acts on target cells. Most research has been conducted in other disciplines, but emerging data indicate that LPA has an important role to play in immunity. A key discovery was that autotaxin (ATX), an enzyme previously implicated in cancer cell motility, generates extracellular LPA from the precursor lysophosphatidylcholine. Steady-state ATX is expressed by only a few tissues, including high endothelial venules in lymph nodes, but inflammatory signals can upregulate ATX expression in different tissues. In this article, we review current thinking about the ATX/LPA axis in lymphocyte homing, as well as in models of allergic airway inflammation and asthma. New insights into the role of LPA in regulating immune responses should be forthcoming in the near future.
Assuntos
Quimiotaxia de Leucócito/imunologia , Lisofosfolipídeos/imunologia , Diester Fosfórico Hidrolases/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Remodelação das Vias Aéreas/imunologia , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Asma/imunologia , Movimento Celular/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Inflamação/imunologia , Isoxazóis/farmacologia , Tecido Linfoide/imunologia , Lisofosfolipídeos/análise , Camundongos , Diester Fosfórico Hidrolases/efeitos dos fármacos , Propionatos/farmacologia , Quimera por Radiação , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/imunologia , Transdução de Sinais/imunologia , Regulação para Cima , Vênulas/imunologiaRESUMO
Recent published studies have highlighted the complexity of the immune response to allergens, and the various asthma phenotypes that arise as a result. Although the interplay of regulatory and effector immune cells responding to allergen would seem to dictate the nature of the asthmatic response, little is known regarding how tolerance versus reactivity to allergen occurs in the lung. The vast majority of mouse models study allergen encounter in naive animals, and therefore exclude the possibility that previous encounters with allergen may influence future sensitization. To address this, we studied sensitization to the model allergen OVA in mice in the context of pre-existing tolerance to OVA. Allergen sensitization by either systemic administration of OVA with aluminum hydroxide or mucosal administration of OVA with low-dose LPS was suppressed in tolerized animals. However, higher doses of LPS induced a mixed Th2 and Th17 response to OVA in both naive and tolerized mice. Of interest, tolerized mice had more pronounced Th17-type inflammation than did naive mice receiving the same sensitization, suggesting pre-existing tolerance altered the inflammatory phenotype. These data show that a pre-existing tolerogenic immune response to allergen can affect subsequent sensitization in the lung. These findings have potential significance for understanding late-onset disease in individuals with severe asthma.
Assuntos
Asma/imunologia , Tolerância Imunológica , Pulmão/imunologia , Células Th17/imunologia , Células Th2/imunologia , Transferência Adotiva , Alérgenos/imunologia , Hidróxido de Alumínio/imunologia , Animais , Modelos Animais de Doenças , Imunidade nas Mucosas , Imunoglobulina G/imunologia , Inflamação/imunologia , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaRESUMO
Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/virologia , Proteína Quinase C/biossíntese , Vírus Sinciciais Respiratórios/patogenicidade , Técnicas de Cultura de Células , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Condutividade Elétrica , Humanos , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologiaRESUMO
Studies have demonstrated that bleach baths improve atopic dermatitis (AD) severity; however, the effects on itch, skin barrier, and cutaneous microbial composition are less clear. We examined whether bleach baths reduce itch, normalize skin barrier function, reduce S. aureus absolute abundance, and increase microbial diversity in adults with AD who were colonized with S. aureus on their non-lesional skin. This was an open label, non-randomized, controlled trial performed at a single academic center. Fifteen AD and five non-atopic healthy controls (NA) were instructed to take two bleach baths (0.005% NaClO; 5-10 min duration) per week for a total of 12 weeks as add-on therapy. Adults 18 to 65 years (inclusive) with mild to severe AD were recruited with EASI score > 6.0, S. aureus culture positivity, access to a bathtub, and ability and willingness to maintain current topical or systemic treatments. They were evaluated at baseline (before bleach baths), 6 weeks, and 12 weeks after the intervention of twice-weekly bleach baths. Efficacy measurements included EASI as well as 5-D Pruritus and ItchyQoL™. Transepidermal water loss (TEWL) and stratum corneum (SC) integrity assay were performed to assess the skin barrier. Skin dysbiosis was measured by S. aureus cultivation, S. aureus abundance (qPCR of thermonuclease gene), and V1-V3 16S rRNA gene sequencing on non-lesional and lesional AD skin. After 12 weeks of bleach baths, 8/15 (53.3%) AD subjects achieved an EASI50 and a significant reduction in itch as measured by 5-D pruritus and Itchy QoL. Eighty-seven percent reported improvements in sleep quality. At study entry, AD subjects had higher non-lesional TEWL values than NA subjects, and only AD subjects experienced a reduction with bleach baths (p = 0.006). Similarly, SC integrity improved as early as 6 weeks after bleach baths in AD subjects. Notably, bleach baths had no significant effect on S. aureus culture-positivity, qPCR absolute abundance, or microbial diversity. The addition of twice-weekly bleach baths improves investigator-assessed AD severity, patient-reported pruritus and sleep as well as physiological measures of skin barrier function in adult AD subjects while having no effect on qualitative and quantitative measures of cutaneous S. aureus. Trial Registration: ClinicalTrials.gov Identifier: NCT01996150, Date of registration: November 27th, 2013.
Assuntos
Dermatite Atópica , Adulto , Humanos , Banhos , Dermatite Atópica/terapia , Disbiose/terapia , Prurido/terapia , Qualidade de Vida , RNA Ribossômico 16S , Pele , Staphylococcus aureus , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , IdosoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out-derived primary microglia reveal reduced amyloid-ß phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-ß peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Região CA3 Hipocampal/patologia , Região CA3 Hipocampal/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Cruzamentos Genéticos , Feminino , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Microglia/patologia , Fagocitose , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Sinapses/metabolismo , Transgenes/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas tau/genéticaRESUMO
Systemic inflammation and multi-organ failure represent hallmarks of the post-cardiac arrest syndrome (PCAS) and predict severe neurological injury and often fatal outcomes. Current interventions for cardiac arrest focus on the reversal of precipitating cardiac pathologies and the implementation of supportive measures with the goal of limiting damage to at-risk tissue. Despite the widespread use of targeted temperature management, there remain no proven approaches to manage reperfusion injury in the period following the return of spontaneous circulation. Recent evidence has implicated the lung as a moderator of systemic inflammation following remote somatic injury in part through effects on innate immune priming. In this review, we explore concepts related to lung-dependent innate immune priming and its potential role in PCAS. Specifically, we propose and investigate the conceptual model of lung-brain coupling drawing from the broader literature connecting tissue damage and acute lung injury with cerebral reperfusion injury. Subsequently, we consider the role that interventions designed to short-circuit lung-dependent immune priming might play in improving patient outcomes following cardiac arrest and possibly other acute neurological injuries.
Assuntos
Pulmão/imunologia , Síndrome Pós-Parada Cardíaca/fisiopatologia , Traumatismo por Reperfusão/terapia , Encéfalo/patologia , Humanos , NeuroproteçãoRESUMO
Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, infective endocarditis, osteoarticular, pleuropulmonary, and device-related infections. Virulence factors secreted by S. aureus, including superantigens and cytotoxins, play significant roles in driving disease. The ability to identify virulence factors present at the site of infection will be an important tool in better identifying and understanding how specific virulence factors contribute to disease. Previously, virulence factor production has been determined by culturing S. aureus isolates and detecting the mRNA of specific virulence factors. We demonstrated for the first time that virulence factors can be directly detected at the protein level from human samples, removing the need to first culture isolated bacteria. Superantigens and cytotoxins were detected and quantified with a Western dot blot assay by using reconstituted skin swabs obtained from patients with atopic dermatitis. This methodology will significantly enhance our ability to investigate the complex host-microbe environment and the effects various therapies have on virulence factor production. Overall, the ability to directly quantify virulence factors present at the site of infection or colonization will enhance our understanding of S. aureus-related diseases and help identify optimal treatments.IMPORTANCE For the first time, we show that secreted staphylococcal virulence factors can be quantified at the protein level directly from skin swabs obtained from the skin of atopic dermatitis patients. This technique eliminates the need to culture Staphylococcus aureus and then test the strain's potential to produce secreted virulence factors. Our methodology shows that secreted virulence factors are present on the skin of atopic patients and provides a more accurate means of evaluating the physiological impact of S. aureus in inflammatory diseases such as atopic dermatitis.
Assuntos
Dermatite Atópica/complicações , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Fatores de Virulência/biossíntese , Dermatite Atópica/microbiologia , Humanos , Proteoma/análise , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Staphylococcus aureus/genética , Fatores de Virulência/genéticaRESUMO
Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1-6) in T cells. We studied the effects of 18â¶1 and 16â¶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2-/- CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.
Assuntos
Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Lisofosfolipídeos/farmacologia , Receptores de Ácidos Lisofosfatídicos/genética , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Expressão Gênica , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Knockout , Receptores de Ácidos Lisofosfatídicos/deficiência , Subpopulações de Linfócitos T/imunologiaRESUMO
Emerging evidence indicates that airway epithelial barrier function is compromised in asthma, a disease characterized by Th2-skewed immune response against inhaled allergens, but the mechanisms involved are not well understood. The purpose of this study was to investigate the effects of Th2-type cytokines on airway epithelial barrier function. 16HBE14o- human bronchial epithelial cells monolayers were grown on collagen coated Transwell inserts. The basolateral or apical surfaces of airway epithelia were exposed to human interleukin-4 (IL-4), IL-13, IL-25, IL-33, thymic stromal lymphopoietin (TSLP) alone or in combination at various concentrations and time points. We analyzed epithelial apical junctional complex (AJC) function by measuring transepithelial electrical resistance (TEER) and permeability to FITC-conjugated dextran over time. We analyzed AJC structure using immunofluorescence with antibodies directed against key junctional components including occludin, ZO-1, ß-catenin and E-cadherin. Transepithelial resistance was significantly decreased after both basolateral and apical exposure to IL-4. Permeability to 3 kDa dextran was also increased in IL-4-exposed cells. Similar results were obtained with IL-13, but none of the innate type 2 cytokines examined (TSLP, IL-25 or IL-33) significantly affected barrier function. IL-4 and IL-13-induced barrier dysfunction was accompanied by reduced expression of membrane AJC components but not by induction of claudin- 2. Enhanced permeability caused by IL-4 was not affected by wortmannin, an inhibitor of PI3 kinase signaling, but was attenuated by a broad spectrum inhibitor of janus associated kinases. Our study indicates that IL-4 and IL-13 have disruptive effect on airway epithelial barrier function. Th2-cytokine induced epithelial barrier dysfunction may contribute to airway inflammation in allergic asthma.