RESUMEN
The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Mycobacterium tuberculosis/inmunología , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/inmunología , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Animales , Antibacterianos/metabolismo , Células de la Médula Ósea/citología , Citocinas/inmunología , Citocinas/metabolismo , Retroalimentación Fisiológica , Humanos , Ligandos , Activación de Macrófagos , Ratones , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Fenazinas/metabolismo , Pigmentos Biológicos/química , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Factores de Virulencia/química , Factores de Virulencia/metabolismoRESUMEN
Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti-Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin-binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double-knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.
Asunto(s)
Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Pulmón/microbiología , Sindecano-4/inmunología , Tuberculosis Pulmonar/inmunología , Células A549 , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Adhesión Bacteriana/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Humanos , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Sindecano-1/deficiencia , Sindecano-1/genética , Sindecano-1/inmunología , Sindecano-4/antagonistas & inhibidores , Sindecano-4/deficiencia , Sindecano-4/genética , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
BACKGROUND: Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. RESULTS: The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 µg and less) and has a sound reproducibility. CONCLUSIONS: Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Macrófagos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/análisis , Transcriptoma , Cartilla de ADN , Perfilación de la Expresión Génica/instrumentación , Humanos , Nylons/química , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , ARN Mensajero/biosíntesis , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αß T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/farmacología , Citotoxicidad Inmunológica , Receptores Inmunológicos/antagonistas & inhibidores , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Voluntarios Sanos , Humanos , Inmunoglobulina G/metabolismo , Depleción Linfocítica , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
BACKGROUND: During HIV infection and/or antiretroviral therapy (ART), monocytes and macrophages exhibit a wide range of dysfunctions which contribute significantly to HIV pathogenesis and therapy-associated complications. Nevertheless, the molecular components which contribute to these dysfunctions remain elusive. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling on monocytes from patients in different stages of HIV infection and/or ART to further characterise these dysfunctions. RESULTS: Processes involved in apoptosis, cell cycle, lipid metabolism, proteasome function, protein trafficking and transcriptional regulation were identified as areas of monocyte dysfunction during HIV infection. Individual genes potentially contributing to these monocyte dysfunctions included several novel factors. One of these is the adipocytokine NAMPT/visfatin, which we show to be capable of inhibiting HIV at an early step in its life cycle. Roughly half of all genes identified were restored to control levels under ART, while the others represented a persistent dysregulation. Additionally, several candidate biomarkers (in particular CCL1 and CYP2C19) for the development of the abacavir hypersensitivity reaction were suggested. CONCLUSIONS: Previously described areas of monocyte dysfunction during HIV infection were confirmed, and novel themes were identified. Furthermore, individual genes associated with these dysfunctions and with ART-associated disorders were pinpointed. These genes form a useful basis for further functional studies concerning the contribution of monocytes/macrophages to HIV pathogenesis. One such gene, NAMPT/visfatin, represents a possible novel restriction factor for HIV.
Asunto(s)
Perfilación de la Expresión Génica , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/patogenicidad , Interacciones Huésped-Patógeno , Monocitos/virología , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Infecciones por VIH/tratamiento farmacológico , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFNγ producing Th1 and Tc1 cells after sapM::Tn BCG vaccination. These findings corroborate that a live attenuated vaccine that affords improved long-term survival upon TB infection can be obtained by a mutation that further attenuates BCG. These findings suggest that an analysis of the effectiveness of innate immune control of the vaccine bacteria could be instructive also for other live attenuated TB vaccines that are currently under development, and encourage further studies of SapM mutation as a strategy in developing a more protective live attenuated TB vaccine.
Asunto(s)
Fosfatasa Ácida/genética , Vacuna BCG/efectos adversos , Evaluación Preclínica de Medicamentos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Mutación , Mycobacterium bovis/patogenicidad , Factores de Virulencia/genética , Animales , Vacuna BCG/genética , Femenino , Interferón gamma/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Mycobacterium bovis/enzimología , Mycobacterium bovis/genética , Linfocitos T/inmunologíaRESUMEN
UNLABELLED: The risk for cardiovascular morbidity and mortality is increased in chronic kidney disease; in this process micro-inflammation plays an essential role. Responsible mechanisms remain to a large extent unidentified. In this pilot study transcriptome analysis of peripheral blood monocytes was used to identify in an unprejudiced manner which factors could be discriminative for cardiovascular disease in patients with chronic kidney disease on hemodialysis. Forty gender- and age-matched, non-diabetic, non-smoking subjects with CRP < 20 mg/L were recruited: 9 healthy controls, 11 patients with eGFR > 60 mL/min/1.73m2 and a history of cardiovascular event (CVE), 10 patients with chronic kidney disease stage 5 on hemodialysis without previous cardiovascular event (CKD5HD) and 10 with a previous cardiovascular event (CKD5HD/CVE). Monocytes were isolated and their mRNA was submitted to focused transcriptome analysis using a macroarray platform containing ca. 700 genes associated with macrophage functional capacity. The macroarray data indicated 9 genes (8 upregulated and 1 downregulated) with a significant differential expression in CKD5HD/CVE vs. CVE alone, after excluding genes differentially expressed in CKD5HD vs. CONTROL: For FCGR3A (CD16) and CX3CR1 (chemokine receptor) the upregulation vs. control and vs. CVE could be confirmed by quantitative RT-PCR for all CKD5HD patients. Furthermore, CX3CR1 relative expression on monocytes correlated with CRP. Flow cytometric analysis of purified monocytes confirmed a significant increase in the percentage of CD16 positive monocytes in all CKD5HD patients vs. control and CVE. The present study indicates the importance of a specific pro-inflammatory monocyte subpopulation, positive for CD16 and the co-expressed chemokine receptor, CX3CR1, discriminative for CKD5HD patients.
Asunto(s)
Monocitos/metabolismo , Receptores de Quimiocina/metabolismo , Receptores de IgG/metabolismo , Insuficiencia Renal Crónica/metabolismo , Transcriptoma , Anciano , Receptor 1 de Quimiocinas CX3C , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Receptores de Quimiocina/genética , Receptores de IgG/genética , Diálisis Renal , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/terapiaRESUMEN
Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy--which we call GlycoDelete--that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.
Asunto(s)
Polisacáridos/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Animales , Glicosilación , Humanos , Ratones , Polisacáridos/química , Polisacáridos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
Asunto(s)
Fosfatasa Ácida/genética , Vacuna BCG/inmunología , Proteínas Bacterianas/genética , Mycobacterium bovis/enzimología , Mycobacterium tuberculosis/inmunología , Eliminación de Secuencia , Tuberculosis Pulmonar/prevención & control , Fosfatasa Ácida/inmunología , Animales , Vacuna BCG/administración & dosificación , Vacuna BCG/genética , Proteínas Bacterianas/inmunología , Citocinas/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/fisiología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Studies in mice have advanced our understanding of the contribution of monocytes to inflammation and immunity. However, the lack of a straightforward and reliable method for the isolation of mouse blood monocytes remains a hurdle. Here we describe a fast and easy method for isolating monocytes from mouse blood based on immuno-magnetic labeling. By negative selection we were able to isolate enriched fractions (>50% purity) of unlabeled monocytes. A subsequent positive selection step resulted in purities of > or =90% viable and functional monocytes. This method is of general use for studying mouse monocyte biology.