Evidence for TLR3 and TLR2/4 cross-talk during viral and bacterial infections

Carmel Duffin, Mark Paul-Clarke, Shaun McMaster, Helen Stride, Shiranee Sriskandan and Jane A. Mitchell. Cardiothoracic Pharmacology, UCCM, NHLI, Royal Brompton Hospital, Imperial College London Dovehouse Street, London SW3 6LY.

Lung epithelial cells form an interface between the external environment and the internal milieu and therefore are ideally situated to recognise pathogens and initiate innate host defence mechanisms. The innate immune system is stimulated through the activation of Toll-like receptors (TLRs), each recognising distinct conserved pathogen-associated molecular patterns to initiate an appropriate anti-microbial effector response, e.g. cytokine production. Human lung epithelial cells express TLRs, including TLR2 and 4 which sense Gram positive and Gram negative bacteria respectively and TLR3 which is activated by viruses. Lung inflammation is often induced by pathogen insult and bacterial and viral infections can co-exist. Indeed, viral influenza can occur together with secondary bacterial bronchopneumonia, which is associated with a higher mortality. Thus, in this study, we have used human lung epithelial cells (A549) in culture and measured the neutrophil chemokine CXCL-8 as an indicator of activation to address the effects of co-stimulation of cells with viral and bacterial ligands. A549s were cultured to confluence using standard techniques in 96 well culture plates (Mitchell et al., 1997). Cells were stimulated with increasing concentrations of the viral TLR3 ligand, Poly I:C (double-stranded RNA analogue; 1 to 1000 µg/ml) alone or in combination with sub-threshold concentrations of the TLR2/1 ligand Pam3CSK4 (10ng/ml), the TLR2/6 ligand FSL-1 (10ng/ml) or the TLR4 ligand LPS (1 µg/ml). Media was then removed and the CXCL-8 levels were determined using ELISA

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Figure 1: CXCL-8 release from A549 cells over 24 hours in response to Poly I:C ( µg/ml) alone or in combination with sub-threshold concentrations of Pam3CSK4 (A), FSL-1 (B) and LPS (C). Data is expressed as mean ± S.E. mean for n=4.

Poly I:C caused a concentration dependent increase in CXCL-8 release form A549 cells up to a concentration of 100 µg/ml, which was reduced at a concentration of 1000 µg/ml (this reduction coincided with a decrease in cell viability. Co-administration of sub-threshold concentrations of Pam3CSK4, FSL-1 or LPS synergised with Poly I:C in the release of CXCL-8 (Figure 1). Endotoxin-free bacterial DNA (TLR9 ligand) had no direct affect on CXCL-8 release (basal release, 439.9 ± 4.7pg/ml: plus 10 µg/ml bacteria DNA, 469.3 ± 46.9pg/ml). These results show that viral TLR3 ligands activate human lung epithelial cells directly and that a significant synergy exists between TLR3 and TLR2 and TLR4 in these cells. This may explain the heightened inflammation and poorer prognosis observed in patients with primary viral infection together with secondary bacterial colonisation.

Mitchell JA, Saunders M, Barnes PJ, Newton R, Belvisi MG. Sodium salicylate inhibits cyclo-oxygenase-2 activity independently of transcription factor (nuclear factor kappaB) activation:role of arachidonic acid. Mol Pharmacol. 1997 51(6):907-12.