Effect of cystathionine-gamma-lyase gene silencing with siRNA on inflammation in acute pancreatitis
Hydrogen sulfide (H2S) is an endogenous inflammatory mediator produced by the activity of cystathionine γ–lyase (CSE) in mammals. Macrophages are a key element of the immune system and play a crucial role in inflammation. To determine the role of H2S and macrophages in inflammation, the expression of CSE in the murine macrophage cell line and human primary macrophages was investigated. The results showed that H2S is produced by the activity of CSE in macrophages. Acute pancreatitis is an inflammatory disorder in the pancreas which develops a cascade of immunological events and results in the activation of inflammatory cells including macrophages. After confirming that H2S is produced by the activity of CSE in macrophages the primary aim was to demonstrate the importance of hydrogen sulfide in the activation of macrophages by inhibiting CSE expression in macrophages during inflammation. Previous studies using pharmacological inhibition of CSE with (DL-propargylglycine) PAG produced conflicting results that may be due to the lack of specificity of this pharmacological agent. To overcome this problem, specific small interfering RNA (siRNA) molecules were used to silence CSE and inhibit the production of H2S by this enzyme in both in vitro and in vivo experiments. SiRNA treatment reduced the levels of CSE mRNA and protein in LPS-activated macrophages compared with controls and non-transfected cells. Furthermore, the levels of pro-inflammatory cytokines by LPS activated macrophages were significantly lower in siRNA transfected cells compared to untransfected controls. For example, increased levels of TNF-α (ng/ml) were observed by LPS-treated cells (11.22 ± 3.340SD) compared to control cells (0.03165 ± 0.00998SD) while there was a significant reduction (One-way ANOVA, p<0.01) in the levels of TNF-α in the siRNA transfected cells (7.782 ± 2.289SD). However, the production levels of NO by the transfected cells was higher, suggesting that CSE activity had an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages and its activity has an inhibitory effect on NO production by these cells. The mechanism by which H2S acts as an inflammatory mediator in LPS-induced macrophages (transfected with anti CSE siRNA) was investigated further. LPS-induced activation of transcription factor nuclear factor-κB (NF-κB) was reduced compared with untransfected cells and phosphorylation and activation of mitogen-activated protein kinase (ERK) 1/2 increased in LPS-induced macrophages. Phosphorylation of ERK in LPS-induced RAW 264.7 cells reached a peak at 30 minutes after activation. Silencing the CSE gene by siRNA reduced phosphorylation and activation of ERK1/2 in LPS-induced RAW 264.7 cells. These findings suggest that siRNA reduces the inflammatory effects of CSE through the ERK-NF-κB signalling pathway. To determine the role of common signalling pathway in biosynthesis of CSE in human primary macrophages, specific inhibitors were used to block NF-κB, ERK, p38 and JNK. Inhibition of NF-κB, ERK and JNK resulted in reduced levels of CSE expression in these cells but inhibition of p38 did not reduce the CSE expression in macrophages. To investigate the role of CSE expressed by macrophages, an in vivo mouse model of caerulein-induced acute pancreatitis was employed. SiRNA was injected into the tail vein to transfect blood monocytes. Higher levels of the pro-inflammatory cytokines TNF-α, IL-6, IL-1β and MIP-2 were found in the pancreas and the lung, as well as increased activity of pancreatic and lung MPO and plasma amylase levels were observed following caerulein-induced acute pancreatitis in mice. The siRNA treatment attenuated inflammation in the pancreas and lungs of mice following acute pancreatitis, as shown by MPO activity, plasma amylase activity and histology. Caerulein treatment increased pancreatic MPO activity (fold increase over saline group) (16.95 ± 3.22SD), but administration of siRNA before caerulein treatment reduced MPO activity significantly (3.97 ± 2.29SD) (One-way ANOVA, p<0.0001). There was also significantly reduced lung MPO activity following administration of siRNA. Caerulein treatment increased lung MPO activity (fold increased over saline group) (3.71 ± 0.84SD) while siRNA administration prior to caerulein treatment reduced lung MPO activity significantly (0.84 ± 0.38SD) (One-way ANOVA, p<0.0001). SiRNA treatment also reduced levels of pro-inflammatory mediators including IL-6, MCP-1, TNF-α, and IL-1β in the pancreas and the lung. These results were supported by histological experiments of the pancreas and the lung which demonstrated reduced infiltration of inflammatory leukocytes following silencing of CSE expression in macrophages compared to the mice without siRNA treatment. These findings indicate a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in acute pancreatitis and suggest a therapeutic role for siRNA to specifically silence CSE. The results from this thesis showed that CSE silencing with siRNA reduced macrophage activation and this has resulted in attenuation of inflammation in acute pancreatitis.
Advisor: Bahatia, Madhav; Chambers, Stephen
Degree Name: Doctor of Philosophy
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: cystathionine-gamma-lyase; Hydrogen sulfide; Acute pancreatitis; Inflammation; siRNA; Macrophages
Research Type: Thesis