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

TNF MAMPs

IKK

TLRs

INNATE IMMUNITYAPOPTOSIS

• 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

TNF MAMPs

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

AvrA solution

Add ethanol, crosslinker

Magnified crosslinkedAvrA nanoparticle

Mucous layer

Epitheliuminflamedhealthy

Particles cross mucous layer, enter cells & disassociate, AvrA blocks JNK/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 nanoparticlesSoluble AvrA

eGFP Nanoparticles Soluble eGFP

Cellular uptake of AvrA nanoparticles

Normal Mouse colon (40x)

-cateninAvrA-eGFP (NP)

Mucosal uptake of AvrA nanoparticles

F4/80AvrA-eGFP NP

Lamina propria in DSS colitis(40x)

AvrA particles mediate the expected immunosuppressive activity in vitro

P-JNK

IB

Mo

ck

5 15 30 45 60

Mo

ck

5 15 30 45 60 (min)

TNFAvrAnano (1 ug)+TNF

P-JNK

-actin

IL-8

(p

g/m

l)

- + + A

vrA

-G

ST

TNF15 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