Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide During Cellular Invasion by Live Salmonella typhimurium Is a Critical But Not Exclusive Event Leading to Macrophage Responses

Abstract

Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4−/− (TLR-4−/−) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-α. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-κB is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial invasion caused translocation of NF-κB, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-α is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae.

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes

“…11 Finally, it has been shown that LPS is the main PAMP mediating Salmonellainduced production of tumor necrosis factor-a and nitric oxide by infected macrophages. 12 Knowing the importance of Tlr4 in the mouse model of Salmonella infection, we are interested in investigating how changes in the level of expression of Tlr4 impact on the mouse response to this pathogen. To address this question, we previously generated four lines of Tlr4 transgenic mice carrying various copy numbers of this gene on a C57BL10/ScNCr (B10/ScNCr) Tlr4 null background.…”

Toll-Like Receptor 4 Dependence of Innate and Adaptive Immunity to Salmonella: Importance of the Kupffer Cell Network

“…3B) was ineffective in controlling the infection. Although the lack of TLR4-dependent iNOS stimulation (25,26) can be compensated by the addition of IFN-␥ in vitro ( Fig. 2A), this necessitates a costly delay in macrophage activation until the arrival of cytokine-producing cells in vivo.…”

IFN-γ Enhances Production of Nitric Oxide from Macrophages via a Mechanism That Depends on Nucleotide Oligomerization Domain-2

“…These nod-2 Ϫ/Ϫ mice are phenotypically similar to those generated and described by Kobayashi et al (9). Primary BMM were isolated from the femur and tibia of mice killed by cervical dislocation and cultured in RPMI 1640, 10% FCS, 5% horse serum, 2 mM Lglutamine, 1 mM sodium pyruvate, 10 g/ml gentamicin, and 20% supernatant taken from L929 cells (28). Before the experiments, cells were plated onto 96-well plates at a plating density of 2 ϫ 10 5 /well.…”