musc surp 2011 1

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