Differential effects of Gram‐positive versus Gram‐negative bacteria on NOSII and TNFα in macrophages: role of TLRs in synergy between the two

Gram-negative and Gram-positive bacteria are sensed by Toll-like receptor (TLR)4 and TLR2, respectively. TLR4 recruits MyD88 and TRIF, whereas TLR2 recruits MyD88 without TRIF. NOSII and TNFα are central genes in innate immunity and are thought to be differentially regulated by the MyD88 versus TRIF signalling pathways. Here, we have used Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and highly selective TLR ligands to establish the precise relationship between TLR2, TLR1, TLR6 and TLR4 for NOSII versus TNFα induction. In murine macrophages at 24 h, E. coli or LPS (TLR4) induced NO and TNFα release. In contrast, S. aureus (TLR2/TLR1/TLR6) or Pam3CSK4 (TLR2/TLR1), or FSL-1 and LTA (TLR2/TLR6) induced TNFα without an effect on NO. At later time points (48–72 h), S. aureus induced NO release. The ability of S. aureus, but not E. coli or LPS, to induce NO release was inhibited by anti-TNFα-binding antibodies. At 24 h, LPS synergised with TLR2 ligands to induce NO release and NOSII protein expression. LPS also induced the expression of TLR2 gene expression without affecting levels of TLR4. Using cells from TLR2−/− or TLR4−/− mice, the ability of LPS to synergise with S. aureus or Pam3CSK4 was found to be dependent on both TLR2 and TLR4. These observations are the first to clearly delineate the role of separately activating TLR2 and TLR4 in the induction of NOSII and TNFα genes compared with their coinduction when both receptor pathways are activated. Keywords: Toll-like receptors, nitric oxide, TNF, LPS, PAMPs, purrs, bacteria Introduction Pathogens have, within their structure, ‘pathogen-associated molecular patterns' (PAMPs), which are ligands for ‘pattern recognition receptors' (PRRs) expressed on immune cells (Moynagh, 2005). Gram-negative bacteria and LPS activate, and are therefore sensed by, the PRR Toll-like receptor (TLR)4. Gram-positive bacteria or fungi activate TLR2. TLR2 forms heterodimers with either TLR1 or TLR6. There are two well-defined TLR signalling pathways mediated, respectively, by MyD88 (together with MAL) or TRIF (together with TRAM) adapter proteins. TLR4 activation results in the recruitment of both MyD88 and TRIF, whereas TLR2 activation results in the recruitment of MyD88 and not TRIF. MyD88 and TRIF are thought to orchestrate separate gene arms because of temporal differences in how they activate NF-κB (Toshchakov et al., 2002). Nitric oxide synthase II (NOSII) and TNFα are key genes in innate immunity, which are thought to be regulated by separate arms of the MyD88–TRIF adapter protein pathway (Vogel et al., 2003). However, the potential for TLR2 ligands to induce NOSII in macrophages, and the precise need or nature of a priming agent, has not been fully addressed. We have recently shown that in vascular smooth muscle, which may be different to macrophages, NOSII is induced by the TLR2 ligands Staphylococcus aureus and MALP-2 (Cartwright et al., 2005). Thus, the precise nature of how TLR4 versus TLR2 may regulate NOSII and TNFα is not clear. Moreover, the potential for TLR ligands to interact and modulate NOSII versus TNFα has not been fully addressed. Thus, in the current study, we have used murine macrophages as a model to study the responses of whole heat-killed bacteria and selective bacterial TLR ligands on NOSII and TNFα induction. Some of these observations have been published in abstract form (Anandarajah et al., 2003; Belcher et al., 2003).