RESUMEN
Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.
Asunto(s)
Enfermedades de los Bovinos , Infecciones por Escherichia coli , Mastitis Bovina , Femenino , Bovinos , Animales , Escherichia coli/genética , Leche/microbiología , Glándulas Mamarias Animales/microbiología , Perfilación de la Expresión Génica/veterinaria , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Células Epiteliales/microbiología , Mastitis Bovina/microbiología , Enfermedades de los Bovinos/metabolismoRESUMEN
Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.
Asunto(s)
Proteínas Aviares/metabolismo , Subgrupos de Linfocitos B/inmunología , Linfoma de Burkitt/inmunología , Elementos de Facilitación Genéticos/genética , Inmunoglobulinas/metabolismo , ARN Polimerasa II/metabolismo , Animales , Diversidad de Anticuerpos , Proteínas Aviares/genética , Linfoma de Burkitt/genética , Pollos , Citidina Desaminasa/genética , Humanos , Inmunoglobulinas/genética , Activación de Linfocitos , Mutagénesis Sitio-Dirigida , Mutación/genética , ARN Polimerasa II/genética , Hipermutación Somática de Inmunoglobulina , Transcripción GenéticaRESUMEN
The transcription factor Bach2 is required for germinal center formation, somatic hypermutation (SHM), and class-switch recombination (CSR) of immunoglobulins. SHM and CSR are initiated by activation-induced cytidine deaminase (AID) which has potential to induce human B cell lymphoma. To understand the role of Bach2 in AID-mediated immunoglobulin gene diversification processes, we established a BACH2-deficient DT40 B cell line. We show that in addition to allowing SHM, Bach2 drives immunoglobulin gene conversion (GCV), another AID-dependent antibody gene diversification process. We demonstrate that Bach2 promotes GCV by increasing the expression of AID. Importantly, we found that the regulation of AID is independent of Blimp-1 and that BACH2-deficient cells have altered expression of several genes regulating AID expression, stability and function. Furthermore, re-expression of BACH2 or AID in Bach2KO cells restored the SHM and GCV defects. These results demonstrate that Bach2 has a previously unappreciated role in the production of high-affinity antibodies.
Asunto(s)
Linfocitos B/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Citidina Desaminasa/metabolismo , Conversión Génica , Genes de Inmunoglobulinas , Hipermutación Somática de Inmunoglobulina , Factores de Transcripción/genética , Animales , Linfocitos B/metabolismo , Diferenciación Celular , Pollos , Regulación de la Expresión Génica , Cambio de Clase de Inmunoglobulina , Factores de Transcripción/inmunologíaRESUMEN
The differentiation of human primary T helper 1 (Th1) cells from naïve precursor cells is regulated by a complex, interrelated signaling network. The identification of factors regulating the early steps of Th1 cell polarization can provide important insight in the development of therapeutics for many inflammatory and autoimmune diseases. The serine/threonine-specific proviral integration site for Moloney murine leukemia virus (PIM) kinases PIM1 and PIM2 have been implicated in the cytokine-dependent proliferation and survival of lymphocytes. We have established that the third member of this family, PIM3, is also expressed in human primary Th cells and identified a new function for the entire PIM kinase family in T lymphocytes. Although PIM kinases are expressed more in Th1 than Th2 cells, we demonstrate here that these kinases positively influence Th1 cell differentiation. Our RNA interference results from human primary Th cells also suggest that PIM kinases promote the production of IFNγ, the hallmark cytokine produced by Th1 cells. Consistent with this, they also seem to be important for the up-regulation of the critical Th1-driving factor, T box expressed in T cells (T-BET), and the IL-12/STAT4 signaling pathway during the early Th1 differentiation process. In summary, we have identified PIM kinases as new regulators of human primary Th1 cell differentiation, thus providing new insights into the mechanisms controlling the selective development of human Th cell subsets.
Asunto(s)
Diferenciación Celular , Virus de la Leucemia Murina de Moloney/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Provirus/fisiología , Células TH1/citología , Células TH1/enzimología , Integración Viral/fisiología , Animales , Diferenciación Celular/genética , Polaridad Celular/genética , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Recién Nacido , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Interleucina-12/metabolismo , Factor de Transcripción STAT4/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Integración Viral/genéticaRESUMEN
Merkel cell carcinoma (MCC) is an aggressive skin cancer, which is frequently caused by Merkel cell polyomavirus (MCPyV). Mutations of MCPyV tumor (T) antigens are major pathologic events of virus-positive (MCPyV+) MCCs, but their source is unclear. Activation-induced cytidine deaminase (AID)/APOBEC family cytidine deaminases contribute to antiviral immunity by mutating viral genomes and are potential carcinogenic mutators. We studied the contribution of AID/APOBEC cytidine deaminases to MCPyV large T (LT) truncation events. The MCPyV LT area in MCCs was enriched with cytosine-targeting mutations, and a strong APOBEC3 mutation signature was observed in MCC sequences. AICDA and APOBEC3 expression were detected in the Finnish MCC sample cohort, and LT expression correlated with APOBEC3H and APOBEC3G. Marginal but statistically significant somatic hypermutation targeting activity was detected in the MCPyV regulatory region. Our results suggest that APOBEC3 cytidine deaminases are a plausible cause of the LT truncating mutations in MCPyV+ MCC, while the role of AID in MCC carcinogenesis is unlikely. Significance: We uncover APOBEC3 mutation signature in MCPyV LT that reveals the likely cause of mutations underlying MCPyV+ MCC. We further reveal an expression pattern of APOBECs in a large Finnish MCC sample cohort. Thus, the findings presented here suggest a molecular mechanism underlying an aggressive carcinoma with poor prognosis.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Humanos , Carcinoma de Células de Merkel/genética , Poliomavirus de Células de Merkel/genética , Antígenos Virales de Tumores/genética , Infecciones por Polyomavirus/genética , Neoplasias Cutáneas/genética , Carcinogénesis/genética , Mutación , Citidina , Desaminasas APOBEC/genéticaRESUMEN
In this study experimental mouse model for Chlamydia pneumoniae infection was used to elucidate the nature of immune response developing during primary and secondary infection. First we examined the mononuclear cells from different lymphoid organs in BALB/c mice during C. pneumoniae infection and detected a strong lymphocyte influx into mediastinal lymph nodes (MLN). To further characterize the C. pneumoniae induced immune response the gene expression profiles of MLN derived lymphocytes was studied. To identify genes characteristic for reinfection we compared gene expression profiles during reinfection and primary infection and found 148 genes to be differentially regulated in CD19+ cells, 7 in CD4+ cells and 12 in CD8+ cells. A panel of these genes was selected to be confirmed by real-time RT-PCR. Genes related to interferon signaling like Ifit1, Ifit3, Gbp2, Irf7 and Usp18 were found to be upregulated when reinfection was compared to primary infection. In our study we were able to identify 8 genes that were differentially expressed between reinfection and primary infection in lymphocytes. These novel gene expression signatures provide new insights and clues to the nature of protective immunity established during experimental C. pneumoniae immunity.
Asunto(s)
Infecciones por Chlamydia/genética , Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/inmunología , Perfilación de la Expresión Génica , Linfocitos/inmunología , Animales , Células Cultivadas , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The early differentiation of T helper (Th) cells is a tightly controlled and finely balanced process, which involves several factors including cytokines, transcription factors and co-stimulatory molecules. Recent studies have shown that in addition to the regulation of apoptosis, caspase activity is also needed for Th cell proliferation and activation and it might play a role in Th cell differentiation. The isoforms of the cellular FLICE inhibitory protein (c-FLIP) are regulators of CASPASE-8 activity and the short isoform, c-FLIPS, has been shown to be up-regulated by IL-4, the Th2 driving cytokine. In this work, we have studied the expression and functional role of three c-FLIP isoforms during the early Th cell differentiation. Only two of the isoforms, c-FLIPS and c-FLIPL, were detected at the protein level although c-FLIPR was expressed at the mRNA level. The knockdown of c-FLIPL led to enhanced Th1 differentiation and elevated IL-4 production by Th2 cells, whereas the knockdown of c-FLIPS diminished GATA3 expression and IL-4 production by Th2 cells. In summary, our results provide new insight into the role of c-FLIP proteins in the early differentiation of human Th cells.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Células Th2/inmunología , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proliferación Celular/fisiología , Cartilla de ADN/genética , Citometría de Flujo , Factor de Transcripción GATA3/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-4/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th2/metabolismoRESUMEN
Selective activation of T helper (Th) cell subsets plays an important role in immune response to pathogens as well as in the pathogenesis of human allergy and inflammatory diseases. Th1 cells along with the recently discovered Th17 cells play a role in the pathogenesis of autoimmune diseases. Th2 cytokines lead to series of inflammatory processes characteristic for asthma and other atopic diseases. To understand the pathogenesis of immune-mediated diseases it is crucial to dissect pathways and regulatory networks leading to the development of distinct Th subsets. Such knowledge may lead to better strategies for developing diagnostics and therapies for these diseases. The differentiation of Th1, Th2, and Th17 effector cells is driven by signals originating from T cell and costimulatory receptors as well as cytokines in the surroundings of activated naive T helper cells. There are several proteins involved in the regulation of this differentiation process. Most of the data on T helper cell differentiation have been acquired using mouse. In this review, we have summarized what is known about human T helper differentiation. In addition, selected differences between human and mouse will be discussed.