Creating a "pro-survival" phenotype through epigenetic modulation

Abstract

Background: We demonstrated recently that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improved survival in a rodent model of lipopolysaccharide (LPS)-induced endotoxic shock. The precise mechanisms, however, have not been well-defined. The aim of this study was to investigate the impact of SAHA treatment on gene expression profiles at an early stage of shock.

Methods: Male C57BL/6J mice were treated with or without SAHA (50 mg/kg, IP), followed by a lethal dose of LPS (20 mg/kg, IP) and a second dose of SAHA. Lungs of the animals (LPS and SAHA+LPS groups; n = 3 per group) were harvested 3 hours post-LPS insult. Sham mice (no LPS and no SAHA) served as controls. RNA was isolated from the tissues and gene expression was analyzed using Affymatrix microarray (23,000 genes). A lower confidence bound of fold change was determined for comparison of LPS versus SAHA + LPS, and genes with a lower confidence bound of >2 were considered to be differentially expressed. Reverse transcriptase polymerase chain reaction, Western blotting, and tissue staining were performed to verify the key changes. Network graphs were used to determine gene interaction and biologic relevance.

Results: The expression of many genes known to be involved in septic pathophysiology changed after the LPS insult. Interestingly, a number of genes not implicated previously in the septic response were also altered. SAHA treatment attenuated expression of several key genes involved in inflammation. It also decreased neutrophil infiltration in lungs and histologic evidence of acute lung injury. Further analysis confirmed genes engaged in the cellular and humoral arms of the innate immune system were specifically inhibited by SAHA. Gene network analysis identified numerous molecules for the potential development of targeted therapies.

Conclusion: Administration of SAHA in a rodent model of LPS shock rapidly modulates gene transcription, with an attenuation of inflammatory mediators derived from both arms (cellular and humoral) of the innate immune system. This may be a novel mechanism responsible for the survival advantage seen with SAHA treatment.

Figure 1. Hierarchical clustering of inflammatory response genes in LPS insult and SAHA treatment

Each row represents a cDNA on the Affymetrix chip and each column represents an individual lung mRNA sample. Red represents over-expressed genes, and blue represents under-expressed genes. Sham: no LPS and no SAHA treated animals; LPS: lipopolysaccharide (LPS) treated animals; SAHA: suberoylanilide hydroxamic acid (SAHA) plus LPS treated animals.

Figure 2. Real-time PCR evaluations of genes identified as differentially expressed between LPS insult and SAHA + LPS groups

(A) A heat map showing seven of the genes in nine lung tissue samples are differentially expressed between LPS-induced septic shock and SAHA treatment groups. (B) Real-time PCR showing expression of TNFR1, TNFR2, TLR2, PTX3, MyD88, TRAF6 and CCL3 genes is significantly different between LPS-induced septic shock and SAHA treatment groups. The symbol * indicates that a value significantly (p<0.05) differs from LPS group. RQ, real-time quantitative PCR for relative mRNA expression levels (fold changes from sham).

Figure 2. Real-time PCR evaluations of genes identified as differentially expressed between LPS insult and SAHA + LPS groups

(A) A heat map showing seven of the genes in nine lung tissue samples are differentially expressed between LPS-induced septic shock and SAHA treatment groups. (B) Real-time PCR showing expression of TNFR1, TNFR2, TLR2, PTX3, MyD88, TRAF6 and CCL3 genes is significantly different between LPS-induced septic shock and SAHA treatment groups. The symbol * indicates that a value significantly (p<0.05) differs from LPS group. RQ, real-time quantitative PCR for relative mRNA expression levels (fold changes from sham).

Figure 3. SAHA inhibits inflammatory infiltration into lungs of mice during the progression of LPS-induced septic shock

(A). Representative slides of lung sections (n = 3) from animal groups of sham, LPS, and SAHA + LPS. The pathologic changes were examined by hematoxylin and eosin (H&E) staining and light microscopy in the lung tissues of mice treated with or without SAHA at 24 h after LPS injection. The markedly increased infiltration of inflammatory cells such as neutrophils was observed in the lungs of mice from the LPS group, which was attenuated by SAHA treatment. Scale bars represent 100 μm. (B). Myeloperoxidase (MPO) activity was assessed for neutrophil infiltration in a lung of mice treated with or without SAHA in LPS insult. The activity was analyzed by quantifying MPO levels in lung tissues. Values represent the means ± SD (n=3). The symbol * indicates that a value significantly (p<0.05) differs from LPS group.

Figure 3. SAHA inhibits inflammatory infiltration into lungs of mice during the progression of LPS-induced septic shock

(A). Representative slides of lung sections (n = 3) from animal groups of sham, LPS, and SAHA + LPS. The pathologic changes were examined by hematoxylin and eosin (H&E) staining and light microscopy in the lung tissues of mice treated with or without SAHA at 24 h after LPS injection. The markedly increased infiltration of inflammatory cells such as neutrophils was observed in the lungs of mice from the LPS group, which was attenuated by SAHA treatment. Scale bars represent 100 μm. (B). Myeloperoxidase (MPO) activity was assessed for neutrophil infiltration in a lung of mice treated with or without SAHA in LPS insult. The activity was analyzed by quantifying MPO levels in lung tissues. Values represent the means ± SD (n=3). The symbol * indicates that a value significantly (p<0.05) differs from LPS group.

Figure 4. SAHA decreases expression of PTX3 in lungs of mice during the progression of LPS-induced endotoxic shock

Whole tissue lysate of mouse lung from sham, LPS and LPS+SAHA groups at different time points after LPS insult was subjected to western blotting with anti-PTX3 and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as means±SD (n=3). The symbol * indicates that a value significantly (p < 0.002) differs from the LPS group. Note: all time points (hours) in this study use LPS injection as the reference point (time zero).

Figure 5. SAHA decreases protein levels of PTX3 in circulation during the progression of LPS-induced endotoxic shock

Serum levels of PTX3 protein were analyzed by ELISA (described in materials and methods). All analyses were performed in triplicate. The error bar indicates standard errors of means (n=3). The symbol * indicates that a value significantly (p < 0.001) differs from the LPS group.

Figure 6. Protein interaction map of the selected differentially expressed genes

The VisAnt tool was utilized for mining and visualizing possible protein-protein interaction of the seven selected inflammatory response genes in septic shock and SAHA treatment. Green nodes represent genes that were differentially expressed between the LPS and SAHA treatment groups. Pink nodes represent genes that are possibly involved in the interaction. Symbol – in green nodes indicates that gene expression is inhibited by SAHA treatment.