musc surp 2011 1
TRANSCRIPT
Danielle Johnson Baylor University and Texas State UniversityMentor: Dr. James A. Cook, Ph.D Department of NeuroscienceStorm Eye Institute
What is Sepsis?
Sepsis is an inflammatory response of the immune system to an infection from bacteria or its components
Annually affects 750,000 patients Main cause of death in the Intensive
Care Facilities Mortality rate >28% in the United States
(250,000 deaths)
Macrophages and Sepsis
I m m u n e c e ll
a c t iva t io n
PAM
P
mRNA
TLR2
ERK
TNF & IL-6
ΜYD88
TLR4 LPS
PAM
P
mRNA
TLR2
ERK
TNF & IL-6
ΜYD88
TLR4LPS
γβGa
• Gi proteins co-immunoprecipitate with TLR receptor proteins •LPS directly stimulates Gαi protein activation in RAW 264.7 murine macrophages •Inhibitors of Gi protein augment TLR induced inflammatory cytokine production•Gαi2 (-/-) knock out mice are highly suceptible to lethal endotoxemia and polymicrobial sepsis
Hypothesis
The over expression of Gαi2 proteins in RAW 264.7 cells will suppress TLR induced inflammatory cytokine production and signal transduction pathways.
Experiments
Lentiviral over expression of Gαi 2 protein
Western Blot of phosphoactive ERK 1/2
10ng/mL LPS or 1μg/mL Pam3CSK4
RAW 264.7 cells transfected with the control vector
ELISA to measure TNF and IL-6 concentration
ResultsThe over expression of Gαi2 protein decrease LPS and Pam3CKS4 induced TNFα Production expression of Gαi2
protein decrease LPS and Pam3CKS4 induced TNFα
*p<0.05 LPS and Pam vs control
# p<0.05 compared to vector control LPSN=3
Results con’d
The over expression of Gαi2 protein decrease LPS and Pam3CKS4 induced IL-6
*p<0.05 LPS and Pam vs control
# p<0.05 compared to vector control LPS
N=3
Results con’d
The Effect of Gαi2 over expression on ERK Activation
N=1
Summary • Gαi2 RAW cells compared to vector control
RAWcells exhibited reduced TNFα and IL-6 when stimulated with the Pam3CKS4 and LPS.
• Preliminary data suggests that Gαi2 RAW cells exhibited reduced activation of ERK 1/2 compared to the vector control cells in response to LPS stimulation
Conclusion
Our data support our hypothesis that Gαi2 over expression in RAW 264.7 cells suppresses TLR induced inflammatory cytokine production and signaling.
Acknowledgements
Special thanks to: Dr. James A. Cook Dr. Hongkuan Fan Dr. Cynthia Wright-PI Sarah Ashton Pengfei Li Rocky Wong- Lab Partner Grant 1R254HL092611-01 Ms. Debbie Shoemaker