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
Background Information


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


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
Therefore, the chronic NOD2 stimulation that occurs in the
intestinal environment leads to outcomes consistent with those
observed in intestinal macrophages
What are the mechanisms through which chronic NOD2 stimulation
enhances microbial clearance?
Human
MDM
MDP 48h
Assess mechanisms of
enhanced bacterial killing
MDM: Monocyte derived macrophages
RNA
expression
RNA
expression
RNA
expression
Metallothioneins (MT) are upregulated in human
macrophages after MDP treatment
M: MDP
•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
Zinc deficiency: increases severity of experimental colitis
Zinc supplementation: attenuates experimental models of colitis
(Journal of nutrition, 2011)
Since MTs regulate intracellular zinc, is intracellular zinc
modulated with chronic NOD2 stimulation?
Chronic NOD2 stimulation increases intracellular zinc and
zinc is required for the enhanced bacterial killing
NT
MDP (48h)
MDP +TPEN
TPEN:Tetrakis-(2-Pyridylmethyl)ethylenediamine
+ S. typhimurium
MDM
+/MDP(48h)
Gentamycin protection assay
Do MTs directly contribute to the enhanced bacterial killing after
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
Does MTF-1 knockdown impair the enhanced bacterial clearance
with chronic NOD2 stimulation?
MTs are required for the enhanced bacterial
killing after 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 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
MT: Metallothionein
This work was supported by
CCFA research fellowship award to AL and NIH to CA
Thank you