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Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

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#209 Murine macrophages carrying an inserti<strong>on</strong>al mutati<strong>on</strong> at residue 2939 <str<strong>on</strong>g>of</str<strong>on</strong>g> Nod2 show a<br />

proinflammatory resp<strong>on</strong>se to Mycobacterium avium subsp. paratuberculosis and its cell<br />

wall comp<strong>on</strong>ents<br />

Elise Angele Lam<strong>on</strong>t, Sayed Raza Ali, Michael Karin, Srinand Sreevatsan,University <str<strong>on</strong>g>of</str<strong>on</strong>g> Minnesota, USA;<br />

UC San Diego, USA<br />

Genetic linkage studies have identified <str<strong>on</strong>g>the</str<strong>on</strong>g> Nod2 as a Crohn’s Disease (CD) susceptibility locus <str<strong>on</strong>g>of</str<strong>on</strong>g> which<br />

Nod2 3020insC is <str<strong>on</strong>g>the</str<strong>on</strong>g> most comm<strong>on</strong> mutati<strong>on</strong>. Several studies suggest that Nod2 3020insC may result in increased<br />

expressi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> proinflammatory cytokines and mediators <str<strong>on</strong>g>of</str<strong>on</strong>g> apoptosis in resp<strong>on</strong>se to bacteria. We hypo<str<strong>on</strong>g>the</str<strong>on</strong>g>size<br />

that Mycobacterium avium subsp. paratuberculosis (MAP) signals through Nod2 3020insC to initiate an aberrant<br />

inflammatory resp<strong>on</strong>se, which is prol<strong>on</strong>ged and enhanced via cross-talk with Toll-like receptors (TLRs) 2 and<br />

4. We investigated Nod2 intact and Nod2 2939insC , <str<strong>on</strong>g>the</str<strong>on</strong>g> murine equivalent <str<strong>on</strong>g>of</str<strong>on</strong>g> human Nod2 3020insC , macrophages in<br />

resp<strong>on</strong>se to MAP (strain K-10) and MAP cell wall glycolipids at early time points (10 min., 30 min., 60 min., and<br />

120 min.). Gene and cytokine expressi<strong>on</strong> pr<str<strong>on</strong>g>of</str<strong>on</strong>g>iles were determined using Quantitative Real-Time PCR (QT-RT-<br />

PCR) and ELISA, respectively. Transcripti<strong>on</strong>al analyses show an 80,000-fold increase <str<strong>on</strong>g>of</str<strong>on</strong>g> Nod2, TLR4, and beta<br />

defensin-1 in K-10 stimulated Nod2 2939insC macrophages. Nfkbib and Ticam2, which are involved in NF-κβ and<br />

TLR stimulati<strong>on</strong>, were also elevated in MAP treatment at 2 hr. Previous studies have shown that optimizati<strong>on</strong><br />

<str<strong>on</strong>g>of</str<strong>on</strong>g> beta-defensins rely <strong>on</strong> Nod2 expressi<strong>on</strong>, which in turn are capable <str<strong>on</strong>g>of</str<strong>on</strong>g> acting as TLR4 ligands. Cytokine data<br />

show rapid and unique resp<strong>on</strong>ses to K-10, mannosylated lipoarabinomannan (ManLAM) and lipomannan (LM).<br />

LM induced proinflammatory resp<strong>on</strong>ses as shown by upregulati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> IL-1α and IL-6, while ManLAM induces an<br />

IL-10 anti-inflammatory resp<strong>on</strong>se at 2hr. These data suggest that initial signaling with MAP occurs via Nod2,<br />

which may act as a central signaling receptor that c<strong>on</strong>nects TLRs 2 and 4 in a sustained circuit. Establishment<br />

<str<strong>on</strong>g>of</str<strong>on</strong>g> a Nod2 signaling circuit is <str<strong>on</strong>g>the</str<strong>on</strong>g> first step in elucidating a mechanistic role <str<strong>on</strong>g>of</str<strong>on</strong>g> MAP involvement in CD <strong>on</strong>set.<br />

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