Functional genomics of innate host defense molecules in normal human monocytes in response to Aspergillus 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-1beta (IL-1beta), 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.

Figures

FIG. 1.

Kinetics of phagocytosis of A. fumigatus conidia. Results are expressed as mean ± standard error % phagocytosis (number of HMCs ingesting ≥1 conidia of A. fumigatus per 100 counted cells) and as mean ± standard error phagocytic index (average number of conidia of A. fumigatus per phagocytosing HMC).

FIG. 2.

Heat map of gene expression profile in HMCs exposed to conidia of A. fumigatus. The results show K-Means clustering of 1,827 genes significantly differentially expressed following the maximal up-regulation (red) or down-regulation (green) over time. Cluster 1, maximal up-regulation at 2 h; cluster 2, maximal up-regulation at 4 h; cluster 3, maximal up-regulation at 6 h; cluster 4, maximal down-regulation at 2 h; cluster 5, maximal down-regulation at 4 h; cluster 6, maximal down-regulation at 6 h; cluster 7, up-regulation over 2 to 6 h.

FIG. 3.

Expression of phagocytosis- and endocytosis-related genes. (A) Mean expression ratio (log2) kinetics of genes involved in phagocytosis in HMCs in response to conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: MARCKS (myristoylated alanine-rich protein kinase C substrate), CDC42EP3 (Rho GTPase binding 3), CD14 (cluster designation antigen 14), CORO1A (coronin 1A; also known as TACO); FCN1 (M-ficolin precursor). (B) Mean expression ratio (log2) kinetics of genes involved in endocytosis in HMCs in response to conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: EHD1 (EH domain-containing 1), SH3BP4 (SH3-domain binding protein 4), and RAB20 (RAB20 member of RAS oncogene family).

FIG. 4.

Expression of cytokine- and cytokine receptor-encoding genes. (A) Mean expression ratio (log2) kinetics of genes encoding cytokines in HMCs in response to A. fumigatus conidia (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: IL1β (interleukin-1β), IL10 (interleukin-10), CSF2 (granulocyte-macrophage colony-stimulator factor). (B) Mean expression ratio (log2) kinetics of genes encoding cytokine receptors in HMCs in response to conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: TRAF1 (tumor necrosis factor receptor-associated factor 1), IRAK2 (interleukin-1 receptor-associated kinase 2), CSF1R (colony-stimulating factor 1 receptor; formerly McDonough feline sarcoma viral [v-fms] oncogene homolog; CD115), and SMAD6 (SMAD; mothers against DPP homolog 6 [Drosophila]).

FIG. 5.

Expression of chemokine and chemokine receptor genes. Mean expression ratio (log2) kinetics of genes encoding chemokines and chemokine receptors in HMCs in response to conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: CCL3/MIP1α (chemokine C-C motif ligand 3), CCL4/MIP1β (chemokine C-C motif ligand 4), CCL20/MIP3α (chemokine C-C motif ligand 20), CXCL2/Groβ (chemokine C-X-C motif ligand 2), CCL5/RANTES (regulated upon activation, normally T-cell expressed and secreted), CCR3 (chemokine C-C motif receptor 3), and IL8 (interleukin-8).

FIG. 6.

Expression of prostaglandin-related genes. Mean expression ratio (log2) kinetics of genes encoding prostaglandin synthesis molecules and receptors in HMCs in response to A. fumigatus conidia (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: COX2 (prostaglandin G/H synthase and cyclooxygenase), PTGER1 (prostaglandin E receptor 1), PTGER2 (prostaglandin E receptor 2), and PTGER4 (prostaglandin E receptor 4).

FIG. 7.

Expression of oxidative response genes. Mean expression ratio (log2) kinetics of genes encoding oxidative stress response molecules in HMCs in response to conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: SOD2 (superoxide dismutase 2), DUSP1 (dual-specificity phosphatase 1), GCH1 (GTP cyclohydrolase 1), CAT (catalase), GPX3 (glutathione peroxidase 3), PRDX5 (peroxiredoxin 5), and TXNRD2 (thioredoxin reductase 2).

FIG. 8.

Expression of genes encoding pattern recognition receptors and matrix metalloproteinases. (A) Mean expression ratio (log2) kinetics of genes encoding pattern recognition receptors in HMCs infected with conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: PTX3 (long pentraxin 3), LGALS1 (galectin 1), and LGALS9 (galectin 9). (B) Mean expression ratio (log2) kinetics of genes encoding matrix metalloproteinases in HMCs infected with conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: ADAMTS2 (a desintegrin-like metalloproteinase with thrombospondin type 1 motif 2) and MMP1 (matrix metalloproteinase 1; also known as interstitial collagenase).

FIG. 9.

Expression of genes encoding connexins and heat shock proteins. (A) Mean expression ratio (log2) kinetics of genes encoding connexins in HMCs infected with conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: GJB6 (gap junction protein B6, connexin 30) and GJB2 (gap junction protein B2, connexin 26). (B) Mean expression ratio (log2) kinetics of genes encoding heat shock proteins in HMCs infected with conidia of A. fumigatus (P ≤ 0.05 for peak change compared to T0). Results for the following genes are shown: DNAJB4 (heat shock protein 40 kDa). HSPH1 (heat shock protein 110 kDa).

FIG. 10.

Genomic expression and protein secretion of selected genes in HMCs in response to A. fumigatus conidia. (A) Mean expression ratio (log2) kinetics of genes encoding CXCL8/IL-8 and simultaneous determination of IL-8 (end product) in supernatants of HMCs infected with conidia of A. fumigatus. (B) Mean expression ratio (log2) kinetics of genes encoding CCL5/RANTES and simultaneous determination of RANTES protein in supernatants of HMCs infected with conidia of A. fumigatus. (C) Mean expression ratio (log2) kinetics of genes encoding CCL4/MIP1β and simultaneous determination of CCL4/MIP1β (end product) in supernatants of HMCs infected with conidia of A. fumigatus. (D) Mean expression ratio (log2) kinetics of genes encoding matrix metalloproteinase-1 (MMP-1) and simultaneous determination of MMP-1 (end product) in supernatants of HMCs infected with conidia of A. fumigatus.

FIG. 11.

Model of early gene expression of innate host defense molecules in normal human monocytes in response to conidia of A. fumigatus. The functional genomic response of normal human monocytes against A. fumigatus may fit a dichotomous model that is modulated by genes encoding immunoregulatory proteins. The dichotomous response involves genes coding for proteins mediating host defense and organism death or organism survival and host invasion. The latter may be induced by the organism or may be the result of immunomodulation causing down-regulation of host response. The third arm depicted in this model represents the complex and multifaceted immunoregulatory component of monocytes that may affect both arms of the reciprocal response elicited by the organism. The outcome of survival or death of A. fumigatus may be determined by specific host derangements that alter this finely regulated response. Results for the following genes are shown: IL1β (interleukin-1β); CCL3, CCL4, and CCL20 (C-C chemokine ligands 3, 4, and 20); CXCL2 (C-X-C chemokine ligand 2); IL8 (interleukin-8); PTX3 (pentraxin 3); SOD2 (dismutase 2); CAT (catalase); CD14 (cluster designation 14); CORO1A (coronin 1A); FCN1 (ficolin 1 precursor); IL10 (interleukin-10); CCL5/RANTES (C-C chemokine ligand 5); MARCO (macrophage receptor with collagenous structure); LMLN (leishmanolysin-like [metallopeptidase M8 family]); ADAMTS2 (desintegrin-like and metalloprotease [reprolysin type] with thrombospondin type 1 motif 2); MMP1 (matrix metalloproteinase 1); CSF1R (colony-stimulating factor receptor 1); PTGER1, PTGER2, and PTGER4 (prostaglandin E receptors 1, 2, and 4), COX2 (inducible cyclooxygenase 2); GAL1 and GAL9 (galectins 1 and 9); HSP40 and HSP110 (heat shock proteins 40 and 110 kDa); and GJB2 and GJB6 (connexins 26 and 30).