Musc Surp 2011 1
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Sepsis is an inflammatory response to infection that causes 750,000 cases and 250,000 deaths annually in the US. The document studies how macrophages contribute to sepsis and the role of Gαi2 proteins. It is hypothesized that overexpressing Gαi2 in RAW 264.7 cells will suppress toll-like receptor induced cytokine production and signaling pathways. Preliminary data showed that Gαi2 overexpression reduced tumor necrosis factor alpha, interleukin-6, and ERK1/2 activation in response to bacterial stimuli, supporting the hypothesis. Further experiments are needed to fully understand how Gαi2 regulates macrophage response during sepsis.
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Musc Surp 2011 1
- 1. Danielle Johnson Baylor University and Texas State University Mentor: Dr. James A. Cook, Ph.D Department of Neuroscience Storm Eye Institute
- 4. PAM P mRNA TLR2 ERK TNF & IL-6 YD88 TLR4 LPS
- 5. PAM P mRNA TLR2 ERK TNF & IL-6 YD88 TLR4 LPS Ga γ β
- 8. Experiments Lentiviral over expression of Gαi 2 protein Western Blot of phosphoactive ERK 1/2 10ng/mL LPS or 1 μ g/mL Pam3CSK4 RAW 264.7 cells transfected with the control vector ELISA to measure TNF and IL-6 concentration
- 9. Results The over expression of Gαi 2 protein decrease LPS and Pam3CKS4 induced TNF α Production expression of Gαi 2 protein decrease LPS and Pam3CKS4 induced TNF α *p<0.05 LPS and Pam vs control # p<0.05 compared to vector control LPS N=3
- 11. Results con’d The Effect of G α i2 over expression on ERK Activation N=1
Editor's Notes
- Sepsis and Septic shock is the systemic host response to micro-organisms in existing sterile tissues (Silva, et al 2008) and is characterized by multiple organ failure. A cascade of signaling pathways of micro-organisms lead to the Systemic Inflammatory Response Syndrome. Sepsis and Septic shock occurs in more than 750,000 patients in the United States each year, and of those patients 250,000 patients die. Despite the increasing knowledge of sepsis and septic shock the mortality and morbidity rates are still very high.
- Sepsis is caused by the over production of inflammatory mediators by M ϕ leading to systemic inflammatory response syndrome or SIRS.
- M φ and other inflammatory cells are activated by specific toll-like receptors when stimulated by microbial products. Two ligands- Pam 3CSK4 and lipopolysaccharides (LPS) interact with TLR2 and TLR4 respectively. Activation of TLRs leads to a signaling cascade such as extracellular receptor kinase (ERK 1/2). Activation also leads to the expression of inflammatory genes such as TNF α and IL-6.
- In addition to the tradition signaling proteins activated by TLRs, there is evidence that heterotrimeric G protein may regulate TLRs.
- These observations prompted our hypothesis that the over expression of Gαi2 proteins in RAW 264.7 cells suppress TLR induced inflammatory cytokine production and signal transduction pathways.
- RAW 264.7 cells were permanently transfected with V5 antibody and a control vector to over express their respective gene using the lentivirus. β-galactosidase was used as a vector control. Blasticidin was used to select for only the RAW cells that expressed β-galactosidase and Gαi 2 .. Transfected RAW 264.7 cells were harvested from the culture dishes, counted to determine the number of cells and diluted to 2X10 5 cells/mL. The control and Gαi 2 protein were treated with 10ng/mL of LPS and 1μg of PAM3CSK4 (Invivogen) and placed into the incubator.. The samples were analyzed by ELISA to determine TNF α and IL-6 concentration. A Western Blot was run to ensure over expression of Gαi 2 protein in the cell cultures and to confirm ERK activation.
- We next determined the affect of G α i2 over expression of TNF α and IL-6 in raw cells induced by LPS and Pam cystine. the RAW Gαi 2 cells exhibiting a decrease in TNF α production (27±5% and 25±1% respectively, p<0.05) and also in IL-6 production (39±0.4% and 52±3% respectively, p<0.05) in response to the stimulation of LPS and Pam3CSK4.
- the RAW Gαi 2 cells exhibiting a decrease in TNF α production (27±5% and 25±1% respectively, p<0.05) and also in IL-6 production (39±0.4% and 52±3% respectively, p<0.05) in response to the stimulation of LPS and Pam3CSK4.
- Cytokine induction occurs in response to TLR signaling pathways. Therefore we analyzed the effect of LPS and Pam 3CKS4 on TLR induced ERK activation by western blot of phosphoactive ERK1/2. The over expression of Gαi 2 protein suppresses LPS and Pam3CSK4 and induced ERK 1/2 activation. With the phosphorylation of the Gαi 2 cells and the stimulation of LPS and Pam3CSK4 over segmented intervals of time of 2 minutes, 5 minutes, 10 minutes, 30 minutes and 60 minutes. ERK 1/2 activation for the control vector exhibited a rapid increase within the segmented intervals of time, whereas the ERK 1/2 activation in the over expression of Gαi 2 exhibited a rapid decline. There is preliminary data that suggest the over expression of G α i2 in Raw cells suppresses TLR induced ERK activation.
- Transfection of G α i2 was confirmed in Raw cells. G α i2 raw cells compared to vector control raw cells exhibited reduced TNFα and IL-6 when stimulated with the Pam3CKS4 and LPS. G α i2 raw cells exhibited reduced activation of ERK 1/2 compared to the vector control raw cells in response to LPS and Pam3CKS4 stimulation
- Our data support our hypothesis that G α i2 over expression in RAW 264.7 cells suppresses TLR induced inflammatory cytokine production and signaling.