Functional Genomics of Innate Host Defense Molecules in Normal Human Monocytes in Response toAspergillus fumigatus

Abstract

Aspergillus fumigatus induces the release of innate immune-related molecules from phagocytic cells early in the course of infection. Little is known, however, about the complex expression profiles of the multiple genes involved in this response. We therefore investigated the kinetics of early gene expression in human monocytes (HMCs) infected with conidia of A. fumigatus using DNA microarray analysis. Total RNA from HMCs at 0, 2, 4, and 6 h was extracted, linearly amplified, hybridized onto Affymetrix HG133 Plus 2.0 gene chips, and analyzed with an Affymetrix scanner. Changes in gene expression were calculated as a ratio of those expressed by infected versus control HMCs. Aspergillus fumigatus induced differential regulation of expression in 1,827 genes (P < 0.05). Genes encoding cytokines and chemokines involved in host defense against A. fumigatus, including interleukin-1␤ (IL-1␤), IL-8, CXCL2, CCL4, CCL3, and CCL20, as well as the opsonin long pentraxin 3, were up-regulated during the first 2 to 6 h, coinciding with an increase in phagocytosis. Simultaneously, genes encoding CD14, ficolin1, and MARCO were down-regulated, and genes encoding IL-10 and matrix metalloproteinase 1 were up-regulated. Up-regulation of the genes encoding heat shock proteins 40 and 110 and connexins 26 and 30 may point to novel molecules whose role in the pathogenesis of aspergillosis has not been previously reported. Verification of the transcriptional profiling was obtained for selected genes by reverse transcription-PCR and enzyme immunoassay. Thus, A. fumigatus conidia induced a coordinated expression of genes important in host defense and immunomodulation.Invasive aspergillosis is an important cause of morbidity and mortality among immunocompromised patients. Risk factors for developing aspergillosis include aplastic anemia (46), hematopoietic stem cell transplantation (32), solid organ transplantation (44), and phagocytic deficiencies, such as chronic granulomatous disease and human immunodeficiency virus infection (40,43). Aspergillus fumigatus is the species most commonly identified as the cause of invasive aspergillosis.Monocytes/macrophages play a pivotal role in normal host defense against A. fumigatus. The innate host response against this pathogen is mediated by release of immune-related molecules from phagocytic cells. However, little is known about the complex expression kinetic profiles of the multiple genes mediating the initial immune response to A. fumigatus. Thus, we hypothesized that conidia of A. fumigatus would induce reciprocal changes in expression of genes encoding products related to innate immunity that may provide insight into early host-parasite interaction.In this study, we used microarray analysis to elucidate the initial kinetics of the transcriptional response of genes encoding innate host defense molecules in normal human monocytes after in vitro stimulation/infection with A. fumigatus conidia. We report herein the changes in expression of genes whose products are involved in the initial innate immun...

Gene Expression Profiling of Polymorphonuclear Leukocytes Treated with the Culture Filtrate of Aspergillus fumigatus and Gliotoxin

“…Among such genes, up-regulation of mRNAs for IL1B and CCL4 are well documented in neutrophils treated with LPS (20,32). IL1B is an important proinflammatory cytokine and its up-regulation was reported in monocytes co-incubated with A. fumigatus conidia (8). Upregulation of mRNAs for chemokine (C-C motif) ligand 5 (CCL5) and CX3CR1 was also identified in this study, but these genes have not previously been recognized to be of relevance in neutrophils.…”

“…As genes for TLR1 were reportedly down-regulated in monocytes affected by A. fumigatus conidia during the early 6-hr time-course stimulation (8), its significance in response to A. fumigatus has become of interest. In our study TLR1 was down-regulated by 1 hr stimulation with gliotoxin.…”

Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549

“…Overexpression of chemokines and of chemokine receptor genes (IL-8 gene, CCL4, CXCL20 and CXCL2) has been described as early as 2 h in human monocyte models using cDNA microarray analysis (Cortez et al, 2006). In our study, we were unable to detect any significant increase before 8 h. If conidia ingestion were the triggering event, an inflammatory response would have occurred for both fungal species because the rate of internalized conidia was similar for both species: around 30-40 %, as reported previously (Wasylnka & Moore, 2002).…”

“…Most authors stress the important roles that alveolar macrophages and neutrophils play in controlling infection (Romani, 2004;Walsh et al, 2005). The release of innate immune-related molecules from professional phagocytic cells in the course of Aspergillus fumigatus infection has been widely studied (Meier et al, 2003;Pylkkanen et al, 2004;Cortez et al, 2006). However, the respiratory epithelium is the first tissue that inhaled conidia encounter, and it probably participates in the efficient coordinated response against IPA.…”

Insights into the pathobiology of Paracoccidioides brasiliensis from transcriptome analysis—advances and perspectives

“…In recent years, several approaches such as cDNA microarrays, cDNA representational difference analysis (RDA) and serial analysis of gene expression (SAGE) have been developed to identify general profiles of gene expression [32,33]. Specifically, the cDNA microarray technology has been applied to the analysis of bacterial and fungal interactions with phagocytes, thus identifying key effectors of pathogen virulence and host defence [32,[34][35][36].…”