Transcriptome analysis revealed unique genes as targets for the anti-inflammatory action of activated protein C in human macrophages

Abstract

Background: Activated protein C (APC) has been introduced as a therapeutic agent for treatment of patients with severe sepsis due to its unique anticoagulant and anti-inflammatory properties in the vascular system. In this study we investigated novel targets for the anti-inflammatory action of APC in human macrophages.

Methods: Using a genome-wide approach, effects of APC on the expression profile in inflammatory activated human macrophages were analyzed.

Results: We identified, for the first time, genes that are specifically regulated by APC under inflammatory conditions, such as chromatin binding protein 4B (CHMP4B) and p300/CBP-associated factor (PCAF), thus indicating a role of APC in the epigenetic control of gene transcription. A functional assay showed the influence of APC in the acetyltransferase/deacetylase activity of nuclear extracts from inflamed macrophages.

Conclusion: Our data sheds new light on APC targets in inflammation and opens new lines of investigation that may be explored in order to further elucidate its unique molecule properties.

Figure 1. Functional clustering of genes regulated by LPS/INFγ.

The gene expression profile of macrophages treated with LPS/INFγ compared with untreated control was analyzed with Rosetta Biosoftware . Genes showing at least a 2.0 fold change were selected and characterized according to their GO biological process in the GeneGO software and compared with the classification of all genes present in the array in order to find which functional categories were significantly (p<0.05) over- or underrepresented in the genes downregulated (A) or upregulated (B) by LPS/INFγ. The blue bars represent the genes regulated by LPS/INFγ and the orange bars represent all genes represented in the microarray.

Figure 2. Regulation of inflammatory mediators by APC.

(A) Fold induction of IL1β, IL8, MCP1 and MIP1 β mRNA expression in LPS/INFγ stimulated macrophages treated with and without APC for 8 hr. Changes in mRNA expression were normalized to changes in GAPDH expression and are expressed as mean ± SD from at least three independent experiments. *p<0.05 (B) Concentrations of IL1β, IL8, MCP1 and MIP1 β secreted by macrophages. Cells were stimulated with LPS/INFγ in the absence or presence of APC. Control cells were untreated. Cytokines were measured in cell culture supernatants collected 12 hr after treatment using the Bio-Plex Human Cytokine Multiplex Assay on the Bio-Plex 2200 platform. Cytokine concentrations in treated cells were normalized to the concentrations in control cells and are presented as mean ± SEM from three independent experiments. *p<0.05.

Figure 3. Functional clustering of genes regulated by APC in inflamed macrophages

. The gene expression profile of macrophages treated with LPS/INFγ vs LPS/INFγ/APC was analyzed with Rosetta Biosoftware. Genes showing at least a 2.0 fold change were selected and characterized according to their GO biological process in the GeneGO software and compared with the classification of all genes present in the array in order to find which functional categories were significantly (p<0.05) over or under-represented in the genes downregulated (A) or upregulated (B) by APC. The blue bars represent the genes regulated by LPS/INFγ and the orange bars represent all genes present in the array.

Figure 4. Statistical analysis of genes exclusively affected by APC in inflamed macrophages.

In a first step of data analysis, fold changes (up or down regulated) of gene expression were calculated using the re-ratio function of Rosetta Biosoftware that allows direct comparison between two samples (LPS/INFγ and LPS/INFγ/APC) that were both hybridized against a common reference (untreated control). All sequences with a calculated fold change of more than 2.0 (p<0.01) were subjected to one-way ANOVA factorial analysis, compared with the significantly regulated sequences (fold change >2, p value<0.01) by LPS/INFγ. The ANOVA plot displays each gene (dot) in relation to its p-value (y axis) and the ranked sequence number (x-axis). Effects are considered significant at a p-value <0.01. Significantly regulated genes are plotted below the dashed red line. Highlighted genes, represented in red, were confirmed by real-time PCR (bar graphs) and when possible (antibody availability) by Western Blot.

Figure 5. APC alters the histone acetyltransferase/deacetylase activity balance in inflamed macrophages.

(A) Colorimetric determination of HAT activity in nuclear extracts of macrophages treated with LPS/INFγ with and without APC for 45 min (left) or 8 hr (right). Data are represented as mean ± SEM from three independent experiments. *p<0.05. (B) Colorimetric determination of HDAC activity in nuclear extracts of macrophages treated with LPS/INFγ with and without APC for 45 min (left) or 8 h (right). Data are represented as mean ± SEM from three independent experiments. *p<0.05.