NOD1, but not NOD2 agonists induce nosii and vasoplegia via a TLR2 and TLR4 independent pathway

Neil Cartwright, David Van Heel†, Shaun McMaster, Mark Paul-Clark, Timothy W Evans, Jane A Mitchell. Department of Critical Care, National Heart and Lung Institute, Imperial College, London. †Department of Gastroenterology, Hammersmith campus, Imperial College, London

Gram positive and Gram negative bacteria are sensed in blood vessels via Toll like receptors (TLR) 4 and 2 respectively. However, whole bacteria also contain ligands for another type of pathogen recognition receptors (PRR), the nucleotide-binding oligomerisation domain (NOD) receptors. There are two NOD PRRs, NOD1 and NOD2. These cytosolic proteins recognise specific elements of peptidoglycan, a structural component in bacterial cell walls. TLR4 and TLR2 ligands in bacteria activate blood vessels to induce nitric oxide synthase (NOS)II which leads to an inability of the vessels to contract. However, the potential for NOD ligands to affect vascular function or NOSII induction has not previously been addressed.

In the current study we investigated the effects of the NOD1 agonist FK565, or the NOD2 agonist muramyl dipeptide (MDP-Lys18), on murine aortae, isolated rat vascular smooth muscle cells and murine J774 macrophages using standard techniques. C57BK/6, TLR2-/- or TLR4-/- mice (28-31g) were sacrificed and aortae removed, cut into rings 2mm long and cultured for 24h in DMEM alone (control) or with DMEM containing FK565 (10nM) or MDP-Lys 18(1 μM). Cultured aortic rings were mounted on a DMT wire myograph in physiological saline solution containing L-arginine (10-4M) at 37 ° C bubbled with 5%CO2, 95%O2 (PSS). Phenylephrine (PE) was added cumulatively (10-8–3x10-6 M) and isometric force was measured. Tension was calculated from vessel length. The vessels were then washed with PSS and incubated with L-NAME (10-3M) for 30 minutes before PE induced responses were repeated. All results were analysed using a paired two-way ANOVA. p<0.05 was considered significant. Cells were treated in 96-well plates with the agonists for 24 hours.

MDP-Lys18 had no significant effect on contractility of vessels to PE (n=6, p=0.35). By contrast, FK565 reduced the ability of vessels to contract in response to PE (Emax for control 2.51±0.09, plus FK565 0.97±0.17 N/m; n=6, p<0.0001), and effect which was reduced by NOS inhibitor L-NAME (1mM; 2.07±0.48 N/m; n=6, p<0.0001). The ability of FK565 to induce hyporeactivity persisted in vessels from TLR2 -/- (control 1.96±24, plus FK565 1.32±0.10 N/m) or TLR4 -/- mice (control 2.44±0.16, plus FK565 0.67±0.22 N/m). FK565 activated vascular smooth muscle cells to express NOSII and release nitrite (control <2 m M; plus FK565, 25±2 μM). By contrast FK565 had no effect on nitrite release by macrophages.

These results demonstrate that NOD1, but not NOD2 activation is linked to NOSII induction in vessels, but not in the macrophage, a divergence in how vessels (compared to immune cells) respond to pathogens. These novel observations have implications for our understanding of sepsis and other types of cardiovascular disease, in which infection is a recognised risk factor.

This work was kindly supported by the British Heart Foundation.