037P Brighton
Winter Meeting December 2007



Relative roles of TLR4 vs NOD1 in the sensing of E. coli by vascular smooth muscle cells and macrophages: identification of distinct signalling pathways


Laura Moreno, Lucy K. Bailey, Neil Cartwright, Prakash Kachhala, Kazuhiro Ito, Shaun K. McMaster, Mark Paul-Clark, Jane A. Mitchell
NHLI. Imperial College, London, United Kingdom


Gram negative bacteria contain pathogen associated molecular patterns (PAMPs) specific for the pattern recognition receptors (PRRs), Toll like receptor (TLR) 4 and NOD1 (Mitchell et al., 2007). LPS specifically activates TLR4 whilst peptidoglycan products activate NOD1. We have recently shown that activation of NOD1 by the specific agonist FK565 in vascular smooth muscle cells (VSMCs) results in a profound induction of NOSII and shock (Cartwright et al. 2007). In the current study we have compared responses in macrophages and VSMCs to whole E.coli, LPS and FK565 for induction of NOSII activity. We have gone on to use a number of pharmacological inhibitors to characterise the pathway by which NOSII activity is induced following TLR4 or NOD1 receptor activation. Rat VSMCs and murine macrophages (J774) were cultured using standard techniques. Cells were stimulated with heat-killed E. coli (108 CFU/ml) LPS (1μg/ml) or FK565 (10nM) for 24 hours before NOSII activity was assessed by the accumulation of nitrite using the Griess reaction (Cartwright et al., 2007).

Table 1: Effect of signalling pathway inhibitors in the accumulation of nitrite induced by TLR4 and NOD1 activation in VSMC vs macrophages. The responses were expressed as a percentage of values obtained in control experiments (first row shows actual nitrite values). Abbreviations: TSA, Trichostatin A; -----, not analyzed. Results were analyzed using one sample t test and * denotes p<0.05. n=3-15.
Vascular smooth muscle J774 macrophages
w/o E. coli LPS FK565 w/o E. coli LPS FK565
Control (μM) 0 18.1±2 5.6±1.2 18.8±3 0 42.2±3 38.2±2.5 0
Control (100%) 0 100 100 100 0 100 100 -----
Z-VAD-fmk (10) 0 103.9±9 ----- 109±6 0 19.8±4* 24.7±9* -----
SC-514 (10) 0 66.7±4* ----- 37.3.±9* 0 39.3±7* 65.6±2* -----
Gö6976 (0.1) 0 98.2±3 ----- 103.4±3 0 ----- 15.73±6* -----
Gö6983 (0.1) 0 88.5±2* ----- 96.4±3 0 ----- 99.96±4 -----
Dexamethasone(1) 0 ----- ----- 0±0* 0 37.3±4* 44.1±5* -----
TSA (0.1) ----- 186±2* ----- 467±21* ----- 0±0* 0±0 * -----



Induction of NOSII activity by E. coli was mimicked in VSMCs by the NOD1 agonist FK565 and in macrophages by the TLR4 agonist LPS (Table 1). The pan-caspase inhibitor, z-VAD-fmk inhibited responses in macrophages but not VSMCs. The IKK2 inhibitor, SC-514, inhibited responses in both cell types. The PKC inhibitor Gö6976 (blocks α, β, and μ) but not Gö6983 (blocks α, β, γ, δ, ζ and λ) inhibited activity in macrophages, but not VSMCs, suggesting a role for PKC μ. The corticosteroid dexamethasone inhibited activity in both cell types. The HDAC inhibitor Trichostatin A (TSA) increased activity in VSMC but blocked it in macrophages. Our findings suggest a selectively role of TLR4 and NOD1 in VSMCs and macrophages and that the signalling pathways of these two PRRs are separate, but converge at the level of (NF)-κB-dependent for NOSII induction



Cartwright N. et al. (2007) Am J Respir Crit Care Med 175:595-603.
Mitchell J.A. et al. (2007) J Endocrinol. 2007 193:323-30.


Funded by a Marie Curie EU Fellowship, the British Lung Foundation and the British Heart Foundation