RESUMEN
CD4 T cells can differentiate into multiple effector subsets, including ThCTL that mediate MHC class II-restricted cytotoxicity. Although CD4 T cell-mediated cytotoxicity has been reported in multiple viral infections, their characteristics and the factors regulating their generation are unclear, in part due to a lack of a signature marker. We show in this article that, in mice, NKG2C/E identifies the ThCTL that develop in the lung during influenza A virus infection. ThCTL express the NKG2X/CD94 complex, in particular the NKG2C/E isoforms. NKG2C/E+ ThCTL are part of the lung CD4 effector population, and they mediate influenza A virus-specific cytotoxic activity. The phenotype of NKG2C/E+ ThCTL indicates they are highly activated effectors expressing high levels of binding to P-selectin, T-bet, and Blimp-1, and that more of them secrete IFN-γ and readily degranulate than non-ThCTL. ThCTL also express more cytotoxicity-associated genes including perforin and granzymes, and fewer genes associated with recirculation and memory. They are found only at the site of infection and not in other peripheral sites. These data suggest ThCTL are marked by the expression of NKG2C/E and represent a unique CD4 effector population specialized for cytotoxicity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Virus de la Influenza A , Subfamília C de Receptores Similares a Lectina de Células NK/análisis , Infecciones por Orthomyxoviridae/inmunología , Animales , Biomarcadores/análisis , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/clasificación , Interferón gamma/biosíntesis , Pulmón/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Factores de Transcripción/análisisRESUMEN
Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3+ Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4+ T cells that accompany the differentiation of AhR-Tr1 cells during the CD4+ T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling. Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25+ CTLA4+ GITR+ on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology.
Asunto(s)
Diferenciación Celular/inmunología , Interleucina-2/biosíntesis , Receptores de Hidrocarburo de Aril/inmunología , Linfocitos T Reguladores/inmunología , Aloinjertos , Animales , Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/inmunología , Mucosa Intestinal/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
BACKGROUND: Aspergillus fumigatus-induced allergic airway disease has been shown to involve conidial germination in vivo, but the immunological mechanisms remain uncharacterized. OBJECTIVE: A subchronic murine exposure model was used to examine the immunological mediators that are regulated in response to either culturable or nonculturable A fumigatus conidia. METHODS: Female B6C3F1/N mice were repeatedly dosed via inhalation with 1 × 105 viable or heat-inactivated conidia (HIC), twice per week for 13 weeks (26 exposures). Control mice inhaled high-efficiency particulate arrestor-filtered air. The influence of A fumigatus conidial germination on the pulmonary immunopathological outcomes was evaluated by flow cytometry analysis of cellular infiltration in the airways, assessment of lung messenger RNA expression, quantitative proteomics, and histopathology of whole lung tissue. RESULTS: Repeated inhalation of viable conidia, but not HIC, resulted in allergic inflammation marked by vascular remodeling, extensive eosinophilia, and accumulation of alternatively activated macrophages (AAMs) in the murine airways. More specifically, mice that inhaled viable conidia resulted in a mixed TH1 and TH2 (IL-13) cytokine response. Recruitment of eosinophils corresponded with increased Ccl11 transcripts. Furthermore, genes associated with M2 or alternatively activated macrophage polarization (eg, Arg1, Chil3, and Retnla) were significantly up-regulated in viable A fumigatus-exposed mice. In mice inhaling HIC, CD4+ T cells expressing IFN-γ (TH1) dominated the lymphocytic infiltration. Quantitative proteomics of the lung revealed metabolic reprogramming accompanied by mitochondrial dysfunction and endoplasmic reticulum stress stimulated by oxidative stress from repetitive microbial insult. CONCLUSION: Our studies demonstrate that A fumigatus conidial viability in vivo is critical to the immunopathological presentation of chronic fungal allergic disease.
Asunto(s)
Alérgenos/inmunología , Antígenos Fúngicos/inmunología , Aspergilosis/inmunología , Aspergillus fumigatus/fisiología , Hipersensibilidad/inmunología , Esporas Fúngicas/inmunología , Células Th2/inmunología , Administración por Inhalación , Animales , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Eosinofilia , Femenino , Humanos , Interleucina-13/metabolismo , Activación de Macrófagos , RatonesRESUMEN
Over the last decade, the known spectrum of CD4(+) T-cell effector subsets has become much broader, and it has become clear that there are multiple dimensions by which subsets with a particular cytokine commitment can be further defined, including their stage of differentiation, their location, and, most importantly, their ability to carry out discrete functions. Here, we focus on our studies that highlight the synergy among discrete subsets, especially those defined by helper and cytotoxic function, in mediating viral protection, and on distinctions between CD4(+) T-cell effectors located in spleen, draining lymph node, and in tissue sites of infection. What emerges is a surprising multiplicity of CD4(+) T-cell functions that indicate a large arsenal of mechanisms by which CD4(+) T cells act to combat viruses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/virología , Virosis/inmunología , Virus/inmunología , Animales , Citocinas/inmunología , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/metabolismo , Factores de Transcripción/metabolismo , Virosis/genética , Virosis/metabolismoRESUMEN
N-Butylbenzene sulfonamide (NBBS) is a commonly used plasticizer found in numerous products. Due to its extensive use, lack of adequate toxicological data, and suspicion of toxicity based on the presence of structural alerts, it was nominated to the National Toxicology Program for comprehensive toxicological testing. The purpose of this study was to evaluate the potential for hypersensitivity and immune suppression following dermal exposure to NBBS using a murine model. NBBS tested negative in a combined irritancy/local lymph node assay (LLNA), classifying it as nonirritating and nonsensitizing. To estimate the immunosuppressive potential of NBBS, assays that assessed immunotoxicity were performed, including the immumnoglobulin (Ig) M response to T-cell-dependent antigen sheep red blood cells (SRBC), using the plaque-forming cell (PFC) assay and immune cell phenotyping. After a 28-d treatment with NBBS, mice exposed to the lowest concentration (25% NBBS) showed a significant increase in IgM-producing B cells in the spleen. No marked changes were identified in immune cell markers in the lymph node. In contrast to body weight, a significant elevation in kidney and liver weight was observed following dermal exposure to all concentrations of NBBS. These results demonstrate that dermal exposure to NBBS, other than liver and kidney toxicity, did not apparently induce immunotoxicity in a murine model.
Asunto(s)
Plastificantes/toxicidad , Sulfonamidas/toxicidad , Administración Cutánea , Animales , Femenino , Inmunoglobulina G/inmunología , Inmunosupresores/farmacología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ovinos , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Pruebas de ToxicidadRESUMEN
Spot welding is used in the automotive and aircraft industries, where high-speed, repetitive welding is needed to join thin sections of metal. Epoxy adhesives are applied as sealers to the metal seams. Pulmonary function abnormalities and airway irritation have been reported in spot welders, but no animal toxicology studies exist. Therefore, the goal of this study was to investigate vascular, immune and lung toxicity measures after exposure to these metal fumes in an animal model. Male Sprague-Dawley rats were exposed by inhalation to 25 mg/m³ to either mild-steel spot welding aerosols with sparking (high metal, HM) or without sparking (low metal, LM) for 4 h/d for 3, 8 and 13 d. Shams were exposed to filtered air. Bronchoalveolar lavage (BAL), lung gene expression and ex vivo BAL cell challenge were performed to assess lung toxicity. Lung resistance (R(L)) was evaluated before and after challenge with inhaled methacholine (MCh). Functional assessment of the vascular endothelium in isolated rat tail arteries and leukocyte differentiation in the spleen and lymph nodes via flow cytometry was also done. Immediately after exposure, baseline R(L) was significantly elevated in the LM spot welding aerosols, but returned to control level by 24 h postexposure. Airway reactivity to MCh was unaffected. Lung inflammation and cytotoxicity were mild and transient. Lung epithelial permeability was significantly increased after 3 and 8 d, but not after 13 d of exposure to the HM aerosol. HM aerosols also caused vascular endothelial dysfunction and increased CD4+, CD8+ and B cells in the spleen. Only LM aerosols caused increased IL-6 and MCP-1 levels compared with sham after ex vivo LPS stimulation in BAL macrophages. Acute inhalation of mild-steel spot welding fumes at occupationally relevant concentrations may act as an irritant as evidenced by the increased R(L) and result in endothelial dysfunction, but otherwise had minor effects on the lung.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Endotelio Vascular/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Pulmón/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Vasculitis/inducido químicamente , Soldadura , Adhesivos/química , Aerosoles , Animales , Células Cultivadas , Endotelio Vascular/inmunología , Endotelio Vascular/fisiopatología , Incendios , Hematopoyesis Extramedular/efectos de los fármacos , Inmunidad Mucosa/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/patología , Pulmón/irrigación sanguínea , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Masculino , Ratas Sprague-Dawley , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Organismos Libres de Patógenos Específicos , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Acero/química , Pruebas de Toxicidad Aguda , Vasculitis/inmunología , Vasculitis/patología , Vasculitis/fisiopatología , Soldadura/métodosRESUMEN
CD4 T cells that acquire cytotoxic phenotype and function have been repeatedly identified in humans, mice, and other species in response to many diverse pathogens. Since CD4 cytotoxic T cells are able to recognize antigenic determinants unique from those recognized by the parallel CD8 cytotoxic T cells, they can potentially contribute additional immune surveillance and direct effector function by lysing infected or malignant cells. Here, we briefly review much of what is known about the generation of cytotoxic CD4 T cells and describe our current understanding of their role in antiviral immunity. Furthering our understanding of the many roles of CD4 T cells during an anti-viral response is important for developing effective vaccine strategies that promote long-lasting protective immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Granzimas/química , Antígenos de Histocompatibilidad Clase II/química , Humanos , Sistema Inmunológico , Proteína 1 de la Membrana Asociada a los Lisosomas/química , Ratones , Modelos Biológicos , FenotipoRESUMEN
Triclosan is an antimicrobial chemical used in healthcare settings that can be absorbed through the skin. Exposure to triclosan has been positively associated with food and aeroallergy and asthma exacerbation in humans and, although not directly sensitizing, has been demonstrated to augment the allergic response in a mouse model of asthma. The skin barrier and microbiome are thought to play important roles in regulating inflammation and allergy and disruptions may contribute to development of allergic disease. To investigate potential connections of the skin barrier and microbiome with immune responses to triclosan, SKH1 mice were exposed dermally to triclosan (0.5-2%) or vehicle for up to 7 consecutive days. Exposure to 2% triclosan for 5-7 days on the skin was shown to increase transepidermal water loss levels. Seven days of dermal exposure to triclosan decreased filaggrin 2 and keratin 10 expression, but increased filaggrin and keratin 14 protein along with the danger signal S100a8 and interleukin-4. Dermal exposure to triclosan for 7 days also altered the alpha and beta diversity of the skin and gut microbiome. Specifically, dermal triclosan exposure increased the relative abundance of the Firmicutes family, Lachnospiraceae on the skin but decreased the abundance of Firmicutes family, Ruminococcaceae in the gut. Collectively, these results demonstrate that repeated dermal exposure to the antimicrobial chemical triclosan alters the skin barrier integrity and microbiome in mice, suggesting that these changes may contribute to the increase in allergic immune responses following dermal exposure to triclosan.
Asunto(s)
Antiinfecciosos , Microbiota , Triclosán , Animales , Inmunidad , Ratones , Piel , Triclosán/toxicidadRESUMEN
Although the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through binding and activation of the aryl hydrocarbon receptor (AhR), the subsequent biochemical and molecular changes that confer immune suppression are not well understood. Mice exposed to TCDD during an acute B6-into-B6D2F1 graft-vs-host response do not develop disease, and recently this has been shown to correlate with the generation of CD4(+) T cells that express CD25 and demonstrate in vitro suppressive function. The purpose of this study was to further characterize these CD4(+) cells (TCDD-CD4(+) cells) by comparing and contrasting them with both natural regulatory CD4(+) T cells (T-regs) and vehicle-treated cells. Cellular anergy, suppressive functions, and cytokine production were examined. We found that TCDD-CD4(+) cells actively proliferate in response to various stimuli but suppress IL-2 production and the proliferation of effector T cells. Like natural T-regs, TCDD-CD4(+) cells do not produce IL-2 and their suppressive function is contact dependent but abrogated by costimulation through glucocorticoid-induced TNFR (GITR). TCDD-CD4(+) cells also secrete significant amounts of IL-10 in response to both polyclonal and alloantigen stimuli. Several genes were significantly up-regulated in TCDD-CD4(+) cells including TGF-beta3, Blimp-1, and granzyme B, as well as genes associated with the IL12-Rb2 signaling pathway. TCDD-CD4(+) cells demonstrated an increased responsiveness to IL-12 as indicated by the phosphorylation levels of STAT4. Only 2% of TCDD-CD4(+) cells express Foxp3, suggesting that the AhR does not rely on Foxp3 for suppressive activity. The generation of CD4(+) cells with regulatory function mediated through activation of the AhR by TCDD may represent a novel pathway for the induction of T-regs.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Dibenzodioxinas Policloradas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Enfermedad Aguda , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Factores de Transcripción Forkhead/biosíntesis , Regulación de la Expresión Génica/inmunología , Reacción Injerto-Huésped/efectos de los fármacos , Reacción Injerto-Huésped/inmunología , Inmunosupresores/administración & dosificación , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Dibenzodioxinas Policloradas/administración & dosificación , Receptores de Hidrocarburo de Aril/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Healthcare workers concurrently may be at a higher risk of developing respiratory infections and allergic disease, such as asthma, than the general public. Increased incidence of allergic diseases is thought to be caused, in part, due to occupational exposure to chemicals that induce or augment Th2 immune responses. However, whether exposure to these chemical antimicrobials can influence immune responses to respiratory pathogens is unknown. Here, we use a BALB/c murine model to test if the Th2-promoting antimicrobial chemical triclosan influences immune responses to influenza A virus. Mice were dermally exposed to 2% triclosan for 7 days prior to infection with a sub-lethal dose of mouse adapted PR8 A(H1N1) virus (50 pfu); triclosan exposure continued until 10 days post infection (dpi). Infected mice exposed to triclosan did not show an increase in morbidity or mortality, and viral titers were unchanged. Assessment of T cell responses at 10 dpi showed a decrease in the number of total and activated (CD44hi) CD4+ and CD8+ T cells at the site of infection (BAL and lung) in triclosan exposed mice compared to controls. Influenza-specific CD4+ and CD8+ T cells were assessed using MHCI and MHCII tetramers, with reduced populations, although not reaching statistical significance at these sites following triclosan exposure. Reductions in the Th1 transcription factor T-bet were seen in both activated and tetramer+ CD4+ and CD8+ T cells in the lungs of triclosan exposed infected mice, indicating reduced Th1 polarization and providing a potential mechanism for numerical reduction in T cells. Overall, these results indicate that the immune environment induced by triclosan exposure has the potential to influence the developing immune response to a respiratory viral infection and may have implications for healthcare workers who may be at an increased risk for developing infectious diseases.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Personal de Salud , Gripe Humana/inmunología , Exposición Profesional/efectos adversos , Células TH1/efectos de los fármacos , Triclosán/efectos adversos , Administración Tópica , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Células TH1/inmunología , Triclosán/administración & dosificaciónRESUMEN
The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfα, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (TH2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.
Asunto(s)
Antiinfecciosos/administración & dosificación , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Hipersensibilidad/inmunología , Piel/inmunología , Receptor Toll-Like 4/metabolismo , Triclosán/administración & dosificación , Administración Tópica , Alérgenos/efectos adversos , Animales , Antiinfecciosos/efectos adversos , Anticuerpos Bloqueadores/administración & dosificación , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Innata , Inmunomodulación , Ratones , Ratones Endogámicos BALB C , Transducción de Señal , Células Th2/inmunología , Triclosán/efectos adversosRESUMEN
Antisense oligomers have been shown to be effective tools for inhibiting gene expression in a highly specific manner. This technology has proven to be invaluable for determining gene function in conventional molecular and cellular studies. However, the promise of an antisense-based drug technology, suggested by antiviral efficacy shown nearly 25 years ago, is just now coming of age. Since then, numerous antisense approaches have been shown to be effective in animal models against numerous viruses and some tumors. Not surprisingly, antisense agents targeting these diseases are taking the lead in human clinical trials and FDA approval. Although comparatively smaller in scope, approaches for modulating immune responses to treat Crohn's disease, diabetes, multiple sclerosis and transplant rejection appear to be the next burgeoning phase of development in antisense therapy.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Enfermedades Autoinmunes/genética , Supervivencia de Injerto/genética , Humanos , Oligonucleótidos Antisentido/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Triclosan is an antimicrobial chemical commonly used occupationally and by the general public. Using select immune function assays, the purpose of these studies was to evaluate the immunotoxicity of triclosan following dermal exposure using a murine model. Triclosan was not identified to be a sensitizer in the murine local lymph node assay (LLNA) when tested at concentrations ranging from 0.75-3.0%. Following a 28-day exposure, triclosan produced a significant increase in liver weight at concentrations of ≥ 1.5%. Exposure to the high dose (3.0%) also produced a significant increase in spleen weights and number of platelets. The absolute number of B-cells, T-cells, dendritic cells and NK cells were significantly increased in the skin draining lymph node, but not the spleen. An increase in the frequency of dendritic cells was also observed in the lymph node following exposure to 3.0% triclosan. The IgM antibody response to sheep red blood cells (SRBC) was significantly increased at 0.75% - but not at the higher concentrations - in the spleen and serum. These results demonstrate that dermal exposure to triclosan induces stimulation of the immune system in a murine model and raise concerns about potential human exposure.
Asunto(s)
Linfocitos B/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Bazo/inmunología , Linfocitos T/inmunología , Triclosán/efectos adversos , Administración Tópica , Animales , Linfocitos B/patología , Células Dendríticas/patología , Femenino , Humanos , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos BALB C , Bazo/patología , Linfocitos T/patología , Triclosán/farmacologíaRESUMEN
Didecyldimethylammonium chloride (DDAC) is a dialkyl-quaternary ammonium compound that is used in numerous products for its bactericidal, virucidal and fungicidal properties. There have been clinical reports of immediate and delayed hypersensitivity reactions in exposed individuals; however, the sensitization potential of DDAC has not been thoroughly investigated. The purpose of these studies was to evaluate the irritancy and sensitization potential of DDAC following dermal exposure in a murine model. DDAC induced significant irritancy (0.5 and 1%), evaluated by ear swelling in female Balb/c mice. Initial evaluation of the sensitization potential was conducted using the local lymph node assay (LLNA) at concentrations ranging from 0.0625-1%. A concentration-dependent increase in lymphocyte proliferation was observed with a calculated EC3 value of 0.17%. Dermal exposure to DDAC did not induce increased production of IgE as evaluated by phenotypic analysis of draining lymph node B-cells (IgE (+) B220(+)) and measurement of total serum IgE levels. Additional phenotypic analyses revealed significant and dose-responsive increases in the absolute number of B-cells, CD4 (+) T-cells, CD8 (+) T-cells and dendritic cells in the draining lymph nodes, along with significant increases in the percentage of B-cells (0.25% and 1% DDAC) at Day 10 following 4 days of dermal exposure. There was also a significant and dose-responsive increase in the number of activated CD44 (+) CD4 (+) and CD8 (+) T-cells and CD86 (+) B-cells and dendritic cells following exposure to all concentrations of DDAC. These results demonstrate the potential for development of irritation and hypersensitivity responses to DDAC following dermal exposure and raise concerns about the use of this chemical and other quaternary ammonium compounds that may elicit similar effects.
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Linfocitos B/efectos de los fármacos , Hipersensibilidad a las Drogas/inmunología , Hipersensibilidad Tardía/inmunología , Compuestos de Amonio Cuaternario/efectos adversos , Piel/inmunología , Linfocitos T/efectos de los fármacos , Administración Tópica , Animales , Linfocitos B/inmunología , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Inmunización , Inmunoglobulina A/metabolismo , Inmunoglobulina E/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Compuestos de Amonio Cuaternario/uso terapéutico , Piel/patología , Linfocitos T/inmunologíaRESUMEN
Toluene diisocyanate (TDI) is a potent low molecular weight chemical sensitizer and a leading cause of chemical-induced occupational asthma. The regulatory potential of microRNAs (miRNAs) has been recognized in a variety of disease states, including allergic disease; however, the roles of miRNAs in chemical sensitization are largely unknown. In a previous work, increased expression of multiple miRNAs during TDI sensitization was observed and several putative mRNA targets identified for these miRNAs were directly related to regulatory T-cell (Treg) differentiation and function including Foxp3 and Runx3. In this work, we show that miR-210 expression is increased in the mouse draining lymph node (dLN) and Treg subsets following dermal TDI sensitization. Alterations in dLN mRNA and protein expression of Treg related genes/putative miR-210 targets (foxp3, runx3, ctla4, and cd25) were observed at multiple time points following TDI exposure and in ex vivo systems. A Treg suppression assay, including a miR-210 mimic, was utilized to investigate the suppressive ability of Tregs. Cells derived from TDI sensitized mice treated with miR-210 mimic had less expression of miR-210 compared to the acetone control suggesting other factors, such as additional miRNAs, might be involved in the regulation of the functional capabilities of these cells. These novel findings indicate that miR-210 may have an inhibitory role in Treg function during TDI sensitization. Because the functional roles of miRNAs have not been previously elucidated in a model of chemical sensitization, these data contribute to the understanding of the potential immunologic mechanisms of chemical induced allergic disease.
RESUMEN
Toluene diisocyanate (TDI) is a leading cause of chemical-induced occupational asthma which impacts workers in a variety of industries worldwide. Recently, the robust regulatory potential of regulatory T cells (Tregs) has become apparent, including their functional role in the regulation of allergic disease; however, their function in TDI-induced sensitization has not been explored. To elucidate the kinetics, phenotype, and function of Tregs during TDI sensitization, BALB/c mice were dermally exposed (on each ear) to a single application of TDI (0.5-4% v/v) or acetone vehicle and endpoints were evaluated via RT-PCR and flow cytometry. The draining lymph node (dLN) Treg population expanded significantly 4, 7, and 9 days after single 4% TDI exposure. This population was identified using a variety of surface and intracellular markers and was found to be phenotypically heterogeneous based on increased expression of markers including CD103, CCR6, CTLA4, ICOS, and Neuropilin-1 during TDI sensitization. Tregs isolated from TDI-sensitized mice were significantly more suppressive compared with their control counterparts, further supporting a functional role for Tregs during TDI sensitization. Last, Tregs were depleted prior to TDI sensitization and an intensified sensitization response was observed. Collectively, these data indicate that Tregs exhibit a functional role during TDI sensitization. Because the role of Tregs in TDI sensitization has not been previously elucidated, these data contribute to the understanding of the immunologic mechanisms of chemical induced allergic disease.
Asunto(s)
Proliferación Celular , Dermatitis Alérgica por Contacto/inmunología , Activación de Linfocitos , Piel/inmunología , Linfocitos T Reguladores/inmunología , 2,4-Diisocianato de Tolueno , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Células Cultivadas , Dermatitis Alérgica por Contacto/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Cadenas alfa de Integrinas/inmunología , Cadenas alfa de Integrinas/metabolismo , Cinética , Ratones Endogámicos BALB C , Neuropilina-1/inmunología , Neuropilina-1/metabolismo , Fenotipo , Receptores CCR6/inmunología , Receptores CCR6/metabolismo , Piel/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Triclosan is an antimicrobial chemical incorporated into many personal, medical and household products. Approximately, 75% of the U.S. population has detectable levels of triclosan in their urine, and although it is not typically considered a contact sensitizer, recent studies have begun to link triclosan exposure with augmented allergic disease. We examined the effects of dermal triclosan exposure on the skin and lymph nodes of mice and in a human skin model to identify mechanisms for augmenting allergic responses. Triclosan (0%-3%) was applied topically at 24-h intervals to the ear pinnae of OVA-sensitized BALB/c mice. Skin and draining lymph nodes were evaluated for cellular responses and cytokine expression over time. The effects of triclosan (0%-0.75%) on cytokine expression in a human skin tissue model were also examined. Exposure to triclosan increased the expression of TSLP, IL-1ß, and TNF-α in the skin with concomitant decreases in IL-25, IL-33, and IL-1α. Similar changes in TSLP, IL1B, and IL33 expression occurred in human skin. Topical application of triclosan also increased draining lymph node cellularity consisting of activated CD86(+)GL-7(+) B cells, CD80(+)CD86(+) dendritic cells, GATA-3(+)OX-40(+)IL-4(+)IL-13(+) Th2 cells and IL-17 A(+) CD4 T cells. In vivo antibody blockade of TSLP reduced skin irritation, IL-1ß expression, lymph node cellularity, and Th2 responses augmented by triclosan. Repeated dermal exposure to triclosan induces TSLP expression in skin tissue as a potential mechanism for augmenting allergic responses.
Asunto(s)
Antiinfecciosos Locales/toxicidad , Citocinas/biosíntesis , Dermatitis Alérgica por Contacto/patología , Células del Estroma/metabolismo , Células Th2/efectos de los fármacos , Células Th2/inmunología , Triclosán/toxicidad , Inmunidad Adaptativa/efectos de los fármacos , Administración Tópica , Animales , Dermatitis Alérgica por Contacto/inmunología , Humanos , Técnicas In Vitro , Ganglios Linfáticos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Células del Estroma/efectos de los fármacos , Linfopoyetina del Estroma TímicoRESUMEN
Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.
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Autoinmunidad/genética , Antígeno CTLA-4/genética , Diabetes Mellitus Tipo 1/genética , Susceptibilidad a Enfermedades/inmunología , Oligonucleótidos Antisentido/genética , Abatacept , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Adhesión Celular , Proliferación Celular , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Exones , Regulación de la Expresión Génica , Inmunoconjugados/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/inmunología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Índice de Severidad de la Enfermedad , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Antisense technologies are widely used for the inhibition of gene expression. Although traditionally the AUG start codon of the open reading frame is targeted to disrupt ribosome assembly and initiation, an emerging approach is targeting sequences to disrupt pre-mRNA splicing. The primary advantage to using this approach is a positive read-out for an antisense effect through detection of a novel splice product, but additional benefit can be found in generating a novel splice product with altered functional properties. The antisense compounds used here are phosphorodiamidate morpholino oligomers conjugated to an arginine-rich cell penetrating peptide (P-PMO). We describe a five-step process for selecting the best candidate antisense compound for altering IL-12Rb2 expression including (1) detecting mRNA splice products by RT-PCR, (2) measuring protein expression, (3) evaluating protein function, (4) checking cellular viability, and (5) validating efficacy of the final candidate compound. The significance of targeting exons composed of a number of base pairs divisible by 3 is also discussed. The five steps described here for selecting the best candidate P-PMO to alter IL-12Rb2 expression should be applied for designing and screening antisense compounds for other gene targets.
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Bioensayo , Péptidos de Penetración Celular/metabolismo , Subunidad beta 2 del Receptor de Interleucina-12/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Morfolinas/farmacología , Oligonucleótidos Antisentido/farmacología , Precursores del ARN/antagonistas & inhibidores , Linfocitos T/metabolismo , Animales , Arginina/química , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Expresión Génica/efectos de los fármacos , Interferón gamma/genética , Interferón gamma/metabolismo , Subunidad beta 2 del Receptor de Interleucina-12/genética , Subunidad beta 2 del Receptor de Interleucina-12/metabolismo , Ratones , Ratones Endogámicos , Morfolinas/química , Morfolinas/metabolismo , Morfolinos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , Sistemas de Lectura Abierta , Empalme del ARN/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/metabolismo , Linfocitos T/citología , Transcripción Genética/efectos de los fármacosRESUMEN
The immune toxicity of the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin, has been studied for over 35 years but only recently has the profound immune suppression induced by TCDD exposure been linked to induction of regulatory T cells (Tregs). The effects of TCDD are mediated through its binding to the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. The subsequent AHR-dependent effects on immune responses are determined by the cell types involved, their activation status, and the type of antigenic stimulus. Collectively, studies indicate that TCDD inhibits CD4+ T cell differentiation into T helper (Th)1, Th2, and Th17 effector cells, while inducing Foxp3-negative and/or preserving Foxp3+ Tregs. Although it is not yet clear how activation of AHR by TCDD induces Tregs, there is a potential therapeutic role for alternative AHR ligands in the treatment of immune-mediated disorders.