Comparative analysis of gene expression changes mediated by individual constituents of hemozoin

Abstract

Plasmodium protozoa, the source of malarial infections, catabolize large quantities of hemoglobin during an intraerythrocytic phase. During this process, free heme is detoxified through biomineralization into an insoluble heme aggregate, hemozoin (Hz). In its native state, Hz is associated with a variety of lipid peroxidation products including 4-hydroxy-2-nonenal (HNE). In the present study, gene expression profiles were used to compare responses to two of the individual components of Hz in a model macrophage cell line. LPS-stimulated RAW 264.7 cells were exposed to HNE and the synthetic form of Hz, beta-hematin (BH), for 6 or 24 h. Microarray analysis identified alterations in gene expression induced by exposure to HNE and opsonized BH (fold change, > or = 1.8; p value, < or = 0.01). Patterns of gene expression were compared to changes induced by an opsonized control latex bead challenge in LPS-stimulated cells and revealed that the BH response was predominantly phagocytic. Ingenuity Pathway Analysis demonstrated that HNE mediated a short-term oxidative stress response and had a prolonged effect on the expression of genes associated with categories of "Cell Cycle", "Cellular Assembly and Organization", "DNA Replication, Recombination, and Repair", and "Cellular Development". Comparisons of expression changes caused by BH and HNE with those observed during malarial infection suggest that BH and HNE are involved in inflammatory response modulation, altered NF-kappaB signal transduction, extracellular matrix (ECM) degradation, and dyserythropoiesis. HNE exposure led to several significant steady-state expression changes including repressed chemokine (C-C motif) ligand 5 (Ccl5), indicative of dyserythropoiesis, and a severe matrix metalloproteinase 9 (Mmp9)/tissue inhibitor of metalloproteinase 1 (Timp1) imbalance in favor of ECM proteolysis.

Figure 1

Overlapping genes with significant differential expression mediated by BH and HNE. Venn diagrams show the intersection of genes that were altered by 0.1 mg/mL BH with those altered by either latex bead or 35 µM HNE treatment. Numbers represent statistically significant (p ≤ 0.01) genes up- or down-regulated ≥1.8-fold relative to LPS stimulated cells at 24 h. (a) down-regulated genes identified at 6 h, (b) up-regulated genes identified at 6 h, (c) down-regulated genes identified at 24 h, (d) up-regulated genes identified at 24 h.

Figure 2

Quantitative real-time RT-PCR validation of microarray results. RAW 264.7 cells were stimulated with 0.1 µg/mL LPS and untreated or treated (A) for 6 h or (B) for 24 h with either 35 µM HNE (black bars) or 0.1 mg/mL BH (white bars). Fold-changes (treated, stimulated cells relative to stimulated controls) are shown (X̄ ± 99% confidence interval for quadruplicate measurements of n = 3 biological replicates). Abbreviations: chemokine (C-C motif) ligand (Ccl); colony stimulating factor (Csf); tissue inhibitor of metalloproteinase 1 (Timp1); matrix metalloproteinase 9 (Mmp9); interleukin 1 (Il1); tumor necrosis factor (Tnf); nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon (Nfkbie); inhibitor of kappaB kinase, epsilon (Ikbke); and FBJ osteosarcoma oncogene (Fos).

Figure 3

ELISA validation of microarray results. Equivalent numbers of cells (4 × 10⁶/well) were stimulated with LPS (1 µg/mL) in the absence or presence of 35 µM HNE for 24 h. CSF3 (colony stimulating factor 3 (granulocyte)) and MMP-9 (matrix metalloproteinase 9) released into the culture medium were analyzed by ELISA. Fold changes (HNE-treated stimulated cells relative to stimulated controls) are shown (X̄ ± SD for triplicate measurements, representative of three independent experiments).

Figure 4

Ingenuity canonical pathway analysis. Differentially expressed genes were mapped to the Ingenuity Canonical Pathway library to identify significantly altered canonical signaling pathways. (a) ‘IL-10 Signaling’ and (b) ‘Role of BRCA1 in DNA Damage Response’ were influenced by 35 µM HNE at 24 h and 6 h, respectively. Genes or gene products are represented as nodes and the intensity of the node color indicates the degree of up- (red) or down- (green) regulation.