Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen

Effect of mannan and laminarin on cytokine secretion by murine dendritic cells infected with <i>P. brasiliensis</i>.

<p>Murine bone marrow-derived DCs were incubated with mannan (Man) or laminarin (Lam) at 100 and 200 µg/ml, respectively, for 30 min. Subsequently, <i>P. brasiliensis</i> yeast cells (Pb) were added to DCs at a ratio of 1∶1, and the co-culture was incubated for 6 h. Culture supernatants were harvested and secreted protein levels were measured using ELISA. Data are reported as the mean ± standard deviation. * P<0.05 compared to the DCs+Pb group.</p

Selected genes up-regulated in murine dendritic cells after 6 h of infection with <i>P. brasiliensis</i>.

<p>*cDNA clones were obtained from the Soares mouse thymus 2NbMT normalized library, available from the IMAGE Consortium (<a href="http://image.hudsonalpha.org/" target="_blank">http://image.hudsonalpha.org/</a>).</p

Quantification of cytokine secretion by murine dendritic cells infected with <i>P. brasiliensis</i>.

<p>BALB/c bone marrow-derived DCs were infected with live <i>P. brasiliensis</i> (Pb) yeast cells (1∶1 ratio of yeast to DCs). Culture supernatants were harvested after 6 h, and secreted protein levels were measured using ELISA. Data are reported as the mean ± standard deviation. * P<0.05.</p

Relative quantification of dectin-1 and mannose receptor transcripts in dendritic cells infected with <i>P. brasiliensis</i>.

<p>BALB/c bone marrow-derived DCs were cultured for 6 h with or without <i>P. brasiliensis</i> yeast cells (Pb). Then total RNA was extracted from the DCs and used in qRT-PCR assays. Fold change values were determined after each gene was normalized to the constitutively expressed <i>RPS9</i> gene using the comparative threshold method. Data are reported as the mean ± standard deviation. * P<0.05.</p

Selected genes down-regulated in murine dendritic cells after 6 h of infection with <i>P. brasiliensis</i>.

<p>*cDNA clones were obtained from the Soares mouse thymus 2NbMT normalized library, available at the IMAGE Consortium (<a href="http://image.hudsonalpha.org/" target="_blank">http://image.hudsonalpha.org/</a>).</p

Real-time PCR validation of microarray data.

<p>*Fold-change values were determined after normalization to <i>Rps9</i> using the comparative threshold method. Values indicate mean fold change ± SD of two independent experiments.</p><p>NM: not significantly modulated, as shown by microarray.</p

Effect of mannan and laminarin on the accumulation of selected immune-related transcripts in dendritic cells infected with <i>P. brasiliensis</i>.

<p>Murine bone marrow-derived DCs were incubated with mannan (Man) or laminarin (Lam) at 100 and 200 µg/ml, respectively, for 30 min. Subsequently, <i>P. brasiliensis</i> yeast cells (Pb) were added to DCs at a ratio of 1∶1, and the co-culture was incubated for 6 h. Total RNA was extracted from DCs and used in qRT-PCR assays. Fold-change values were determined after each gene was normalized to <i>RPS9</i> using the comparative threshold method. Data are reported as the mean ± standard deviation. * P<0.05 compared to the DCs+Pb/DCs group.</p

Transcriptional response of murine macrophages upon infection with opsonized Paracoccidioides brasiliensis yeast cells

Paracoccidioides brasiliensis is the etiologic agent of the Paracoccidioidomycosis the most common systemic mycosis in Latin America. Little is known about the regulation of genes involved in the innate immune host response to P. brasiliensis. We therefore examined the kinetic profile of gene expression of peritoneal macrophage infected with P. brasiliensis. Total RNA from macrophages at 6, 24 and 48 h was extracted, hybridized onto nylon membranes and analyzed. An increase in the transcription of a number of pro-inflammatory molecules encoding membrane proteins, metalloproteases, involved in adhesion and phagocytosis, are described. We observed also the differential expression of genes whose products may cause apoptotic events induced at 24 h. In addition, considering the simultaneous analyses of differential gene expression for the pathogen reported before by our group, at six hours post infection, we propose a model at molecular level for the P. brasiliensis-macrophage early interaction. In this regard, P. brasiliensis regulates genes specially related to stress and macrophages, at the same time point, up-regulate genes related to inflammation and phagocytosis, probably as an effort to counteract infection by the fungus. (c) 2007 Elsevier Masson SAS. All fights reserved

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in <it>Paracoccidioides brasiliensis </it>mycelium and yeast cells

<p>Abstract</p> <p>Background</p> <p>Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus <it>Paracoccidioides brasiliensis </it>and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both <it>in silico </it>EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above.</p> <p>Results</p> <p>In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were <it>hex </it>(encoding for a hexagonal peroxisome protein), <it>bgl </it>(encoding for a 1,3-β-glucosidase) in mycelium cells; and <it>ags </it>(an α-1,3-glucan synthase), <it>cda </it>(a chitin deacetylase) and <it>vrp </it>(a verprolin) in yeast cells; (ii) ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – <it>isc </it>and <it>ktp</it>, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (<it>pct</it>) in yeast. Also, several enzymes from the cysteine <it>de novo </it>biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase.</p> <p>Conclusion</p> <p>Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.</p