1. Chronic NOD2 induced Autophagy and Bacterial killing is dependent on Metallothionein mediated Zinc accumulation Amit Lahiri and Clara Abraham Yale University School of Medicine

2. DISCLOSURES Nothing to Disclose

3. NOD2, a microbial recognition receptor, confers the highest disease risk in CD Stimulation of NOD2 in macrophages by its ligand muramyl dipeptide (MDP),a component of peptidoglycan Acute: results in the signaling and production of various cytokines Chronic: downregulates cytokine production upon subsequent stimulation with pattern recognition receptor (PRR) Enhances bacterial killing capacity Background Information

4. The intestine is an environment of chronic bacterial product exposure, resulting in chronic stimulation of pattern recognition receptors including NOD2 Intestinal macrophages: reduced cytokine production to PRR stimulation increased bactericidal activity Human MDM MDP 48h Assess mechanisms of enhanced bacterial killing MDM: Monocyte derived macrophages What are the mechanisms through which chronic NOD2 stimulation enhances microbial clearance? Therefore, the chronic NOD2 stimulation that occurs in the intestinal environment leads to outcomes consistent with those observed in intestinal macrophages

5. Metallothioneins (MT) are upregulated in human macrophages after MDP treatment Expression of other MT isoforms are similarly upregulated Metallothioneins (MTs) belong to a superfamily of intracellular metal-binding proteins, in particular Zinc, regulates intracellular zinc levels Since MTs regulate intracellular zinc, is intracellular zinc modulated with chronic NOD2 stimulation? M: MDP RNA expression Zinc deficiency: increases severity of experimental colitis Zinc supplementation: attenuates experimental models of colitis (Journal of nutrition, 2011)

6. NT MDP (48h) Chronic NOD2 stimulation increases intracellular zinc and zinc is required for the enhanced bacterial killing Do MTs directly contribute to the enhanced bacterial killing after chronic NOD2 stimulation ? MDP +TPEN TPEN:Tetrakis-(2-Pyridylmethyl)ethylenediamine MDM +/- MDP(48h) + S. typhimurium Gentamycin protection assay

7. Does MTF-1 knockdown impair the enhanced bacterial clearance with chronic NOD2 stimulation? MTF-1 regulate MT expression in human macrophages Due to multiple MT isoforms and their redundancy in human macrophages  wished to target all MT isoforms simultaneously. A common transcription factor metal-response element binding transcription factor-1 (MTF-1) in mice cells regulates MT expression MTF-1 knockout mice: embryonic lethal (PNAS, 1991: JBC 1996). MTF-1 knockdown: fail to increase the zinc up-regulation after chronic NOD2 stimulation

8. We observe similar findings for other bacteria including Staphylococcus and adhesive invasive E.coli Through what mechanism does MT-induced zinc enhance bacterial clearance with chronic NOD2 stimulation? Autophagy? MTs are required for the enhanced bacterial killing after chronic NOD2 stimulation

9. MTs regulate bacterial killing via zinc-mediated autophagy Inducing autophagy through an independent pathway using rapamycin rescues the impaired bacterial killing under MTF-1/MT knockdown conditions

10. Conclusion and acknowledgements This work was supported by CCFA research fellowship award to AL and NIH to CA Thank you MT: Metallothionein