Bioengineering Bacterial Derived Immunomodulants: a Novel IBD Therapeutic Approach

1. Bioengineering Bacterial Derived Immunomodulants: a Novel IBD Therapeutic Approach Julie Champion, PhD Department of Chemical Engineering Georgia Institute of Technology Atlanta GA, USA Andrew S. Neish, MD Department of Pathology Emory University School of Medicine Atlanta GA, USA

2. Disclosures • Nothing to disclose

3. Acute intestinal inflammation • Intraepithelial and luminal accumulation of neutrophils • Enterocyte apoptosis/injury • Attendant loss of epithelial barrier integrity • Results from activation of signaling pathways with upregulation of proinflammatory effector molecules TNF MAMPs TLRs TNF-R TAK MKK IKK JNK NF-kB INNATE IMMUNITY APOPTOSIS

4. Observation • Salmonella and other gut pathogens have evolved protein effectors to suppress host defensive responses to further their life cycles • Certain pathogens can invade and persist within host cells without excessive pro-apoptotic and proinflammatory signaling pathways Hypothesis • Can these evolutionarily honed mechanisms be characterized, isolated and exploited therapeutically?

5. Background TNF MAMPs TLRs TNF-R • Salmonella AvrA is a member of an class of bacterial effectors (acetyltransferases) involved in host-pathogen interactions • AvrA allows innate immune suppression without cell death, consistent with its role facilitating the lifestyle of an intracellular pathogen • AvrA expression in a living animal is non toxic but suppresses inflammatory and apoptotic responses TAK MKK IKK AvrA JNK NF-kB APOPTOSIS INNATE IMMUNITY

6. Add ethanol, Mucous crosslinker layer Epithelium inflamed healthy Magnified crosslinked AvrA nanoparticle AvrA solution Particles cross mucous layer, enter cells & disassociate, k AvrA blocks JNK/NF - B & reduces inflammation Goal :Use chemical engineering/nanotechnology systems to study exploit the anti-inflammatory mechanisms of the Salmonella effector protein AvrA on intestinal inflammation Desolvation process forms protein aggregates Crosslinker stabilizes nanoparticles Intracellular conditions disassociate nanoparticles

7. Nanoparticle Fabrication and Characterization • AvrA expressed in E. coli (Western) • Uniform nanoparticles • Scanning electron microscopy, light scattering Peak at 108 nm

8. Nanoparticle Disassociation • Particles incubated in 1mM GSH • Measure soluble protein • Particles abruptly disassociated after 30 min • Particles incubated with T84 monolayers • Evaluate with immunoblot

9. Cellular uptake of AvrA nanoparticles eGFP nanoparticles Soluble AvrA AvrA nanoparticles eGFP Nanoparticles Soluble eGFP

10. Mucosal uptake of AvrA nanoparticles b-catenin AvrA-eGFP (NP) Normal Mouse colon (40x) F4/80 AvrA-eGFP NP Lamina propria in DSS colitis(40x)

11. AvrA particles mediate the expected immunosuppressive activity in vitro AvrA-GST AvrAnano (1 ug) +TNFa Mock TNFa Mock - + + 5 15 30 45 60 5 15 30 45 60 (min) TNFa 15 min P-JNK P-JNK b-actin IkBa Soluble AvrA suppresses JNK activation in cultured cells AvrA nanoparticles suppress JNK activation in T84 model epithelia IL-8 (pg/ml) AvrA nanoparticles suppress IL-8 secretion in T84 model epithelia

12. Therapeutic effect of AvrA nanoparticles in model inflammation Murine zymosan peritonitis model

13. Therapeutic effect of AvrA nanoparticles in model colitis

14. Future Directions • Development of further in vivo models • Adaptive immunity • Surface modification • Pipeline: Other bacteria proteins, targeting strategies

15. Acknowledgments Julie Champion, PhD Department of Chemical Engineering Georgia Institute of Technology Atlanta GA, USA • Neish Lab • Huixia Wu • Kai Zheng • Chuck Parkos • Ronen Sumagin • Supported by CCFA and the Rainin Foundation