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

Disclosures Nothing to disclose

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 MKK JNK NF-  B TAK TNF-R TNFMAMPs IKK TLRs INNATE IMMUNITY APOPTOSIS

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 Observation Hypothesis Can these evolutionarily honed mechanisms be characterized, isolated and exploited therapeutically?

Background 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 MKK JNK NF-  B TAK TNF-R TNFMAMPs IKK TLRs INNATE IMMUNITYAPOPTOSIS AvrA

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 AvrAsolution Add ethanol, crosslinker Magnifiedcrosslinked AvrAnanoparticle Mucous layer Epithelium inflamed healthy Particlescross mucous layer, enter cells & disassociate, AvrAblocksJNK/NF-  B&reduces inflammation

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

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

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

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

AvrA particles mediate the expected immunosuppressive activity in vitro P-JNK IBIB Mock Mock (min) TNF  AvrA nano (1 ug) +TNF  P-JNK  -actin IL-8 (pg/ml) AvrA-GST TNF  15 min Soluble AvrA suppresses JNK activation in cultured cells AvrA nanoparticles suppress JNK activation in T84 model epithelia AvrA nanoparticles suppress IL-8 secretion in T84 model epithelia

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

Therapeutic effect of AvrA nanoparticles in model colitis

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

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