RESUMEN
The cellular basis of the apparent aggressiveness in lung cancer is poorly understood but likely associated with functional or molecular features of disseminated cancer cells (DCCs). DCCs from epithelial cancers are mostly detected by antibodies directed against histogenetic markers such as cytokeratin or EpCAM. It has been argued that marker-negative metastatic founder cells might escape detection. We therefore used ex vivo sphere formation for functional detection of candidate metastasis founders. We generated cell suspensions from 199 LN samples of 131 lung cancer patients and placed them into non-adherent cell culture. Sphere formation was associated with detection of DCCs using EpCAM immunocytology and with significantly poorer prognosis. The prognostic impact of sphere formation was strongly associated with high numbers of EpCAM-positive DCCs and aberrant genotypes of expanded spheres. We also noted sphere formation in patients with no evidence of lymphatic spread, however such spheres showed infrequent expression of signature genes associated with spheres from EpCAM-positive samples and displayed neither typical lung cancer mutations (KRAS, TP53, ERBB1) nor copy number variations, but might be linked to disease progression >5 years post curative surgery. We conclude that EpCAM identifies relevant disease-driving DCCs, that such cells can be expanded for model generation and that further research is needed to clarify the functional and prognostic role of rare EpCAM-negative sphere forming cells.
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Moléculas de Adhesión Celular , Neoplasias Pulmonares , Humanos , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Variaciones en el Número de Copia de ADN , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ganglios Linfáticos/patologíaRESUMEN
BACKGROUND: Cancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies. METHODS: To identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs. RESULTS: The screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB) nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF-NF-κB axis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis. CONCLUSION: Our data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.
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FN-kappa B , Factor de Necrosis Tumoral alfa , Apoptosis , Humanos , FN-kappa B/metabolismo , Fosforilación , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cell lineage analysis aims to uncover the developmental history of an organism back to its cell of origin. Recently, novel in vivo methods utilizing genome editing enabled important insights into the cell lineages of animals. In contrast, human cell lineage remains restricted to retrospective approaches, which still lack resolution and cost-efficient solutions. Here, we demonstrate a scalable platform based on short tandem repeats targeted by duplex molecular inversion probes. With this human cell lineage tracing method, we accurately reproduced a known lineage of DU145 cells and reconstructed lineages of healthy and metastatic single cells from a melanoma patient who matched the anatomical reference while adding further refinements. This platform allowed us to faithfully recapitulate lineages of developmental tissue formation in healthy cells. In summary, our lineage discovery platform can profile informative somatic mutations efficiently and provides solid lineage reconstructions even in challenging low-mutation-rate healthy single cells.
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Edición Génica , Repeticiones de Microsatélite , Animales , Humanos , Linaje de la Célula/genética , Estudios Retrospectivos , MutaciónRESUMEN
Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.
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Interleucina-6/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Transducción de Señal , Médula Ósea/patología , Mama/citología , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Receptor gp130 de Citocinas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Interleucina-6/genética , Mutación , Metástasis de la Neoplasia/genética , Receptores de Interleucina-6/deficiencia , Receptores de Interleucina-6/metabolismo , Células del Estroma/metabolismo , Microambiente TumoralRESUMEN
Robust anti-tumor immunity requires innate as well as adaptive immune responses. We have shown that plasmacytoid dendritic cells develop killer cell-like activity in melanoma cell cocultures after exposure to the infectious but replication-deficient herpes simplex virus 1 (HSV-1) d106S. To combine this innate effect with an enhanced adaptive immune response, the gene encoding human MelanA/MART-1 was inserted into HSV-1 d106S via homologous recombination to increase direct expression of this tumor antigen. Infection of Vero cells using this recombinant virus confirmed MelanA expression by Western blotting, flow cytometry, and immunofluorescence. HSV-1 d106S-MelanA induced expression of the transgene in fibroblast and melanoma cell lines not naturally expressing MelanA. Infection of a melanoma cell line with CRISPR-Cas9-mediated knockout of MelanA confirmed de novo expression of the transgene in the viral context. Dependent on MelanA expression, infected fibroblast and melanoma cell lines induced degranulation of HLA-matched MelanA-specific CD8+ T cells, followed by killing of infected cells. To study infection of immune cells, we exposed peripheral blood mononuclear cells and in vitro-differentiated macrophages to the parental HSV-1 d106S, resulting in expression of the transgene GFP in CD11c+ cells and macrophages. These data provide evidence that the application of MelanA-encoding HSV-1 d106S could enhance adaptive immune responses and re-direct MelanA-specific CD8+ T cells to tumor lesions, which have escaped adaptive immune responses via downregulation of their tumor antigen. Hence, HSV-1 d106S-MelanA harbors the potential to induce innate immune responses in conjunction with adaptive anti-tumor responses by CD8+ T cells, which should be evaluated in further studies.
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Expresión Génica , Vectores Genéticos/genética , Herpesvirus Humano 1 , Antígeno MART-1/genética , Virus Oncolíticos , Transgenes , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Fibroblastos/metabolismo , Marcación de Gen , Ingeniería Genética , Herpesvirus Humano 1/genética , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Melanoma , Virus Oncolíticos/genética , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.
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Histona Demetilasas/genética , Histona Demetilasas con Dominio de Jumonji/genética , Melanoma/genética , Animales , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Ratones Desnudos , Regiones Promotoras Genéticas/genéticaRESUMEN
Mouse models indicate that metastatic dissemination occurs extremely early; however, the timing in human cancers is unknown. We therefore determined the time point of metastatic seeding relative to tumour thickness and genomic alterations in melanoma. Here, we find that lymphatic dissemination occurs shortly after dermal invasion of the primary lesion at a median thickness of ~0.5 mm and that typical driver changes, including BRAF mutation and gained or lost regions comprising genes like MET or CDKNA2, are acquired within the lymph node at the time of colony formation. These changes define a colonisation signature that was linked to xenograft formation in immunodeficient mice and death from melanoma. Thus, melanoma cells leave primary tumours early and evolve at different sites in parallel. We propose a model of metastatic melanoma dormancy, evolution and colonisation that will inform direct monitoring of adjuvant therapy targets.
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Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Piel/metabolismo , Animales , Línea Celular Tumoral , Hibridación Genómica Comparativa/métodos , Femenino , GTP Fosfohidrolasas/genética , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Melanoma/patología , Proteínas de la Membrana/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas Proto-Oncogénicas p21(ras)/genética , Piel/patología , Neoplasias Cutáneas/patología , Trasplante HeterólogoRESUMEN
INTRODUCTION: Complete lymph node dissection (CLND) after a positive sentinel node (SN) biopsy provides important prognostic information in melanoma patients but has been questioned for therapeutic use recently. We explored whether quantification of the tumour spread to SNs may replace histopathology of non-sentinel nodes (NSNs) for staging purposes. PATIENTS AND METHODS: We quantified melanoma spread in SNs and NSNs in 128 patients undergoing CLND for a positive SN. In addition to routine histopathology, one-half of each of all 1496 SNs and NSNs was disaggregated into a single cell suspension and stained immunocytochemically to determine the number of melanoma cells per 106 lymph node cells, i.e. the disseminated cancer cell density (DCCD). RESULTS: We uncovered melanoma spread to NSNs in the majority of patients; however, the tumour load and the proportion of positive nodes were significantly lower in NSNs than in SNs. The relation between SN and NSN spread could be described by a mathematical function with DCCDNSN = DCCDSNc/101-c (c = 0.69; 95% confidence interval [CI]: 0.62-0.76). At a median follow-up of 67 months, multivariable Cox regression analyses revealed that DCCDSN (p = 0.02; HR 1.34, 95% CI: 1.05-1.71) and the total number of pathologically positive nodes (p = 0.02; HR 1.53, 95% CI: 1.07-2.22) were significant risk factors after controlling for age, gender, thickness of melanoma and ulceration status. A prognostic model based on DCCDSN and melanoma thickness predicted outcome as accurately as a model including pathological information of both SNs and NSNs. CONCLUSION: The assessment of DCCDSN renders CLND for staging purposes unnecessary.
Asunto(s)
Ganglios Linfáticos/patología , Melanoma/secundario , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Estimación de Kaplan-Meier , Escisión del Ganglio Linfático , Ganglios Linfáticos/cirugía , Metástasis Linfática , Masculino , Melanoma/mortalidad , Melanoma/cirugía , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Factores de Riesgo , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/cirugía , Adulto JovenRESUMEN
Accumulating data suggest that metastatic dissemination often occurs early during tumour formation, but the mechanisms of early metastatic spread have not yet been addressed. Here, by studying metastasis in a HER2-driven mouse breast cancer model, we show that progesterone-induced signalling triggers migration of cancer cells from early lesions shortly after HER2 activation, but promotes proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by increased HER2 expression and tumour-cell density involving microRNA-mediated progesterone receptor downregulation, and was reversible. Cells from early, low-density lesions displayed more stemness features, migrated more and founded more metastases than cells from dense, advanced tumours. Notably, we found that at least 80% of metastases were derived from early disseminated cancer cells. Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for human metastatic dissemination.
RESUMEN
Bone is the most frequent site of metastasis in prostate cancer and patients with bone metastases are deemed incurable. Targeting prostate cancer cells that disseminated to the bone marrow before surgery and before metastatic outgrowth may therefore prevent lethal metastasis. This prompted us to directly analyze the transcriptome of disseminated cancer cells (DCC) isolated from patients with nonmetastatic (UICC stage M0) prostate cancer. We screened 105 bone marrow samples of patients with M0-stage prostate cancer and 18 bone marrow samples of patients without malignancy for the presence of EpCAM(+) single cells. In total, we isolated 270 cells from both groups by micromanipulation and globally amplified their mRNA. We used targeted transcriptional profiling to unambiguously identify DCCs for subsequent in-depth analysis. Transcriptomes of all cells were examined for the expression of EPCAM, KRT8, KRT18, KRT19, KRT14, KRT6a, KRT5, KLK3 (PSA), MAGEA2, MAGEA4, PTPRC (CD45), CD33, CD34, CD19, GYPC, SCL4A1 (band 3), and HBA2. Using these transcripts, we found it impossible to reliably identify true DCCs. We then applied combined genome and transcriptome analysis of single cells and found that EpCAM(+) cells from controls expressed transcripts thought to be epithelial-specific, whereas true DCCs may express hematopoietic transcripts. These results point to an unexpected transcriptome plasticity of epithelial cancer cells in bone marrow and question common transcriptional criteria to identify DCCs.
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Médula Ósea/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Próstata/genética , Transcriptoma/genética , Adulto , Anciano , Antígenos de Neoplasias/genética , Médula Ósea/patología , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Moléculas de Adhesión Celular/genética , Molécula de Adhesión Celular Epitelial , Genoma Humano , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/patología , ARN Mensajero/biosíntesisRESUMEN
Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.
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Trasplante de Células Madre Hematopoyéticas , Animales , Antígenos CD34/metabolismo , Médula Ósea/inmunología , Médula Ósea/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/patología , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patologíaRESUMEN
Toll-like receptor (TLR) mediated signaling induces pro-inflammatory responses and can both suppress and exacerbate allergic responses in the airways. The aim of our study was to directly compare the efficacy of different TLR agonists in inhibiting or exacerbating the development of Th2-mediated responses in the airways and investigate if the suppressive effects were associated with increased pro-inflammatory responses. Mice were immunized on day 0, 14 and 21 by intraperitoneal injection of ovalbumin/alum and exposed to ovalbumin aerosol on day 26 and 27. TLR2, TLR3, TLR4, TLR7 and TLR9 agonists (0.001, 0.01, 0.1, or 1 mg/kg) were administered intratracheally 1 h before each allergen exposure. Both the TLR7 and TLR9 agonists dose dependently reduced airway eosinophilia, while the TLR3 agonist only reduced airway eosinophilia at a dose of 1.0 mg/kg. The TLR2 and TLR4 agonists potentiated eosinophilia. All TLR agonists enhanced neutrophil numbers at doses as low as 0.01 mg/kg, in particular TLR2 and TLR4 agonists. TLR7 and TLR9 agonists also significantly reduced IL-4 and IL-5 levels and all TLR agonists, with the exception of TLR7, enhanced the amount IL-1ß, IL-6, and TNF-α detected in the whole lung lavage. Only application of TLR9 agonist induced detectable levels of IL-10 in the lung. Suppressive effects of the TLR agonists were not dependent upon IFN-γ and IL-10 or associated with increased numbers of Foxp3(+)CD4(+) Tr cells in the lavage fluid. Airway resistance was reduced significantly only when TLR7 agonist was administered. When applied therapeutically 2 days after allergen exposure, all TLR agonists, except TLR2, similarly reduced airway eosinophilia and IL-4 levels. Taken together our results show that TLR7 agonists had the strongest anti-asthmatic effects with the lowest pro-inflammatory potential, suggesting that activating TLR7 may have the greatest potential to treat allergic disorders in humans.
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Inflamación/etiología , Interleucina-10/genética , Receptores Toll-Like/agonistas , Resistencia de las Vías Respiratorias/inmunología , Animales , Asma/tratamiento farmacológico , Asma/inmunología , Relación Dosis-Respuesta a Droga , Eosinofilia/inmunología , Femenino , Inflamación/inmunología , Glicoproteínas de Membrana/agonistas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Ovalbúmina/inmunología , Factores de Tiempo , Receptor Toll-Like 7/agonistasRESUMEN
Eosinophils play a major role in the development and severity of asthma. Robust and rapid preclinical animal models are desirable to profile novel therapeutics inhibiting the influx of eosinophils into the airways. To develop a rapid, airway eosinophil recruitment model in the rat, Brown-Norway (BN) rats were immunised with ovalbumin (OVA)/alum on day 0, 1 and 2 and challenged with OVA aerosol on day 5 and 6. On day 7 bronchoalveolar lavage fluid (BALF) was analysed for eosinophil numbers, eosinophil peroxidase (EPO) activity and cytokines. Lung sections were also examined. The immunised animals showed a strong selective influx of eosinophils into the airways correlating with enhanced EPO activity, Interleukin (IL-4), IL-5 and monocytes chemo attractant protein levels in the BALF in comparison to sham-sensitised rats. In addition the immunised rats developed goblet cell metaplasia in the lung and showed OVA specific IgG1 and IgE levels in the serum but no airway hyperreactivity after metacholine challenge. Airway inflammation was suppressed by applying the steroids Budesonide (intra tracheally) and Prednisolone (per orally), Roflumilast a phosphodiesterase-4 inhibitor, and the H1 receptor antagonists Epinastine and Ketotifen. Montelukast, a Leukotriene receptor antagonist and Chromoglycate, a mast cell stabiliser, had no effect in this model. In summary, in this novel preclinical rat model therapeutics expected to inhibit the development of airway eosinophilia can rapidly be tested.
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Antiinflamatorios/farmacología , Asma/fisiopatología , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Compuestos de Alumbre , Aminopiridinas/farmacología , Animales , Asma/tratamiento farmacológico , Benzamidas/farmacología , Líquido del Lavado Bronquioalveolar , Budesonida/farmacología , Ciclopropanos/farmacología , Dibenzazepinas/farmacología , Antagonistas de los Receptores Histamínicos H1 , Imidazoles/farmacología , Cetotifen/farmacología , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina , Inhibidores de Fosfodiesterasa/farmacología , Prednisolona/farmacología , Ratas , Ratas Endogámicas BNRESUMEN
Several mechanisms are in place to neutralize autoimmune CD8 T cells by tolerance induction. Developing self-specific CD8 T cells are eliminated in the thymus by Ag-presenting epithelial and dendritic cells (DCs). However, CD8 T cells escaping thymic central tolerance can also be inactivated by tolerance mechanisms in peripheral organs. In contrast to DCs, the role of B cells in generating CD8 T cell tolerance is not well-characterized. To investigate this question in more detail, we transcriptionally targeted Ag to B cells using B cell-specific retroviral vectors in vivo. Although Ag expression could be detected in B cells of thymus, lymph nodes, and spleen, B cells were unable to induce central tolerance of CD8 thymocytes. In contrast, in peripheral organs, we could identify clonal deletion and functional inhibition (anergy) of CD8 T cells as tolerance-inducing mechanisms. Although Ag expressed by B cells was acquired and cross-presented by DCs, B cells were also sufficient to tolerize CD8 T cells directly. These findings suggest exploitation of B cells for Ag-specific immunotherapy of CD8 T cell-mediated autoimmune diseases.
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Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos , Antígenos CD19/genética , Vectores Genéticos , Humanos , Ratones , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/inmunología , Retroviridae/genética , Timo/inmunología , Transcripción GenéticaRESUMEN
RATIONALE: Epidemiological studies suggest that infections with helminths protect from the development of asthma. Supporting this view is our published finding that infection with Nippostrongylus brasiliensis decreased ovalbumin-induced Th2 responses in the lung of mice. OBJECTIVES: To evaluate if N. brasiliensis excretory-secretory products also prevent the development of asthma. METHODS: Mice were immunized with ovalbumin/alum intraperitoneally in the absence or presence of helminthic products and then challenged intranasally with ovalbumin. Six days later, we analyzed if the mice developed Th2 responses in the lung. MAIN RESULTS: The application of the helminthic products together with ovalbumin/alum during the sensitization period totally inhibited the development of eosinophilia and goblet cell metaplasia in the airways and also strongly reduced the development of airway hyperreactivity. Allergen-specific IgG1 and IgE serum levels were also strongly reduced. These findings correlated with decreased levels of IL-4 and IL-5 in the airways in product-treated animals. The suppressive effects on the development of allergic responses were independent of the presence of Toll-like receptors 2 and 4, IFN-gamma, and most important, IL-10. Interestingly, suppression was still observed when the helminthic products were heated or treated with proteinase K. Paradoxically, we found that strong helminth product-specific Th2 responses were induced in parallel with the inhibition of ovalbumin-specific responses. CONCLUSION: Our results suggest that helminths suppress the development of asthma by secreting substances that modulate allergic responses without affecting the generation of helminth-specific Th2 immunity. The identification of these products may lead to the design of novel therapeutic intervention strategies for the treatment of asthma.
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Helmintiasis Animal/inmunología , Nippostrongylus/inmunología , Hipersensibilidad Respiratoria/inmunología , Animales , Pruebas de Provocación Bronquial , Endopeptidasa K/farmacología , Femenino , Células Caliciformes/patología , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Metaplasia/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Eosinofilia Pulmonar/inmunología , Células Th2/inmunologíaRESUMEN
The only disease-modifying treatment that is available for allergic patients is allergen-specific immunotherapy. Two competing application forms are used: subcutaneous immunotherapy, which has been used for > 90 years, and a relatively new immunotherapy where the allergen is applied sublingually. Numerous studies have shown efficacy for subcutaneous immunotherapy and have identified possible mechanisms that are responsible for the observed reduction in allergic responses. In contrast, the efficacy of sublingual immunotherapy has not been documented to the same degree and the responsible immunological mechanisms have not yet been clearly defined. This review focuses on the published clinical and experimental data on sublingual immunotherapy and points at possible mechanisms of how sublingual immunotherapy may differ from subcutaneous immunotherapy in its mode of action, and also discusses the potential advantages and pit falls of both therapies.