Chronic NOD2 induced Autophagy and Bacterial killing is dependent on Metallothionein mediated Zinc

accumulation

Amit Lahiri and Clara Abraham

Yale University School of Medicine

DISCLOSURES

Nothing to Disclose

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

Background Information 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

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

RN

A

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RN

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Zinc deficiency: increases severity of experimental colitisZinc supplementation: attenuates experimental models of colitis (Journal of nutrition, 2011)

NTMDP (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 +TPENTPEN:Tetrakis-(2-Pyridylmethyl)ethylenediamine

MDM +/- MDP(48h)

+ S. typhimurium

Gentamycin protection assay

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-regulationafter chronic NOD2 stimulation

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

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

Conclusion and acknowledgements

This work was supported byCCFA research fellowship award to AL and NIH to

CA

Thank you

MT: Metallothionein