Murine dendritic cells transcriptional modulation upon Paracoccidioides brasiliensis infection

Abstract

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.

Figure 1. Quantification of cytokine secretion by murine dendritic cells infected with P. brasiliensis.

BALB/c bone marrow-derived DCs were infected with live P. brasiliensis (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.

Figure 2. Relative quantification of dectin-1 and mannose receptor transcripts in dendritic cells infected with P. brasiliensis.

BALB/c bone marrow-derived DCs were cultured for 6 h with or without P. brasiliensis 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 RPS9 gene using the comparative threshold method. Data are reported as the mean ± standard deviation. * P<0.05.

Figure 3. Effect of mannan and laminarin on the accumulation of selected immune-related transcripts in dendritic cells infected with P. brasiliensis.

Murine bone marrow-derived DCs were incubated with mannan (Man) or laminarin (Lam) at 100 and 200 µg/ml, respectively, for 30 min. Subsequently, P. brasiliensis 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 RPS9 using the comparative threshold method. Data are reported as the mean ± standard deviation. * P<0.05 compared to the DCs+Pb/DCs group.

Figure 4. Effect of mannan and laminarin on cytokine secretion by murine dendritic cells infected with P. brasiliensis.

Murine bone marrow-derived DCs were incubated with mannan (Man) or laminarin (Lam) at 100 and 200 µg/ml, respectively, for 30 min. Subsequently, P. brasiliensis 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.