Synthetic CpG oligonucleotides induce a genetic profile ameliorating murine myocardial I/R injury

Abstract

We previously demonstrated that pre-conditioning with CpG oligonucleotide (ODN) 1668 induces quick up-regulation of gene expression 3 hours post-murine myocardial ischaemia/reperfusion (I/R) injury, terminating inflammatory processes that sustain I/R injury. Now, performing comprehensive microarray and biocomputational analyses, we sought to further enlighten the "black box" beyond these first 3 hours. C57BL/6 mice were pretreated with either CpG 1668 or with control ODN 1612, respectively. Sixteen hours later, myocardial ischaemia was induced for 1 hour in a closed-chest model, followed by reperfusion for 24 hours. RNA was extracted from hearts, and labelled cDNA was hybridized to gene microarrays. Data analysis was performed with BRB ArrayTools and Ingenuity Pathway Analysis. Functional groups mediating restoration of cellular integrity were among the top up-regulated categories. Genes known to influence cardiomyocyte survival were strongly induced 24 hours post-I/R. In contrast, proinflammatory pathways were down-regulated. Interleukin-10, an upstream regulator, suppressed specifically selected proinflammatory target genes at 24 hours compared to 3 hours post-I/R. The IL1 complex is supposed to be one regulator of a network increasing cardiovascular angiogenesis. The up-regulation of numerous protective pathways and the suppression of proinflammatory activity are supposed to be the genetic correlate of the cardioprotective effects of CpG 1668 pre-conditioning.

Keywords: 1668; CpG oligonucleotide; IL-10; ischaemia/reperfusion injury; microarray analysis; myocardial infarction; pre-conditioning.

Figure 1

Time course of experimental interventions and assessments. Nonlinear scale for better identification of various interventions. ODN = CpG/control oligodeoxynucleotide, LAD = left anterior descending artery

Figure 2

Prolonged up‐regulation of defined pathways 3 h towards 24 h post‐I/R is a CpG‐mediated effect. Figure shows heat map for up‐regulated pathways in control and CpG ODN‐treated animals, 3 and 24 h post‐I/R. Hierarchical clustering according to IPA definition. Heat map colour‐coded according to the z‐score (see 2 section)

Figure 3

Genes predicted to increase cardiovascular angiogenesis are strongly up‐regulated by CpG pre‐conditioning 24 h post‐I/R. (A) Network analysis of CpG ODN‐induced activation of angiogenic genes. Ingenuity Pathway Analysis was used to identify the network of regulatory genes (open labels) contributing to CpG ODN‐induced activation. (B) IL1 complex is one regulator of angiogenesis 24 h post‐I/R. Figure shows network of genes significantly induced by IL1 complex. These genes are known to increase cardiovascular angiogenesis (see also Figure 3A). Asterisks indicate that fold change values of replicates have been averaged

Figure 4

CpG‐mediated IL‐10 activity effectively down‐regulates target genes (3 h vs 24 h). Figure shows target genes of IL‐10 network in CpG‐treated animals. While 3 h post‐I/R, all genes were strongly up‐regulated, most genes experience down‐regulation towards 24 h post‐I/R. Asterisks indicate that fold change values of replicates have been averaged