Proceedings of the 10th International Colloquium on Paratuberculosis

#209 Murine macrophages carrying an insertional mutation at residue 2939 <strong>of</strong> Nod2 show a
proinflammatory response to Mycobacterium avium subsp. paratuberculosis and its cell
wall components
Elise Angele Lamont, Sayed Raza Ali, Michael Karin, Srinand Sreevatsan,University <strong>of</strong> Minnesota, USA;
UC San Diego, USA
Genetic linkage studies have identified <strong>the</strong> Nod2 as a Crohn’s Disease (CD) susceptibility locus <strong>of</strong> which
Nod2 3020insC is <strong>the</strong> most common mutation. Several studies suggest that Nod2 3020insC may result in increased
expression <strong>of</strong> proinflammatory cytokines and mediators <strong>of</strong> apoptosis in response to bacteria. We hypo<strong>the</strong>size
that Mycobacterium avium subsp. paratuberculosis (MAP) signals through Nod2 3020insC to initiate an aberrant
inflammatory response, which is prolonged and enhanced via cross-talk with Toll-like receptors (TLRs) 2 and
4. We investigated Nod2 intact and Nod2 2939insC , <strong>the</strong> murine equivalent <strong>of</strong> human Nod2 3020insC , macrophages in
response to MAP (strain K-10) and MAP cell wall glycolipids at early time points (10 min., 30 min., 60 min., and
120 min.). Gene and cytokine expression pr<strong>of</strong>iles were determined using Quantitative Real-Time PCR (QT-RT-
PCR) and ELISA, respectively. Transcriptional analyses show an 80,000-fold increase <strong>of</strong> Nod2, TLR4, and beta
defensin-1 in K-10 stimulated Nod2 2939insC macrophages. Nfkbib and Ticam2, which are involved in NF-κβ and
TLR stimulation, were also elevated in MAP treatment at 2 hr. Previous studies have shown that optimization
<strong>of</strong> beta-defensins rely on Nod2 expression, which in turn are capable <strong>of</strong> acting as TLR4 ligands. Cytokine data
show rapid and unique responses to K-10, mannosylated lipoarabinomannan (ManLAM) and lipomannan (LM).
LM induced proinflammatory responses as shown by upregulation <strong>of</strong> IL-1α and IL-6, while ManLAM induces an
IL-10 anti-inflammatory response at 2hr. These data suggest that initial signaling with MAP occurs via Nod2,
which may act as a central signaling receptor that connects TLRs 2 and 4 in a sustained circuit. Establishment
<strong>of</strong> a Nod2 signaling circuit is <strong>the</strong> first step in elucidating a mechanistic role <strong>of</strong> MAP involvement in CD onset.
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