UBC Theses and Dissertations
Investigation of novel signals induced by lipopolysaccharide in innate immune cells Duncan, Sherie Kristina
Macrophages play key roles in innate immune responses. They phagocytose and destroy bacteria, and are activated by various microbial products, recognized through a variety of pattern recognition receptors, including toll-like receptors (TLRs). Engagement of TLRs triggers various intracellular signals, leading to the release of mediators that help orchestrate both the innate and adaptive immune responses. Deficiency in the ability of the host to detect and respond to bacterial products leads to repeated or chronic infections, whereas excessive responses to bacterial products can lead to septic shock and death. Lipopolysaccharide (LPS) is a major constituent of the cell wall of gram-negative bacteria and a chief contributor to the development of septic shock in infections with gram-negative bacteria. The current paradigm is that all biological responses to LPS from Escherichia coli depend on TLR4. Here, we refute this paradigm and present evidence demonstrating that alternate receptors, in addition to TLR4, transduce signals from LPS in macrophages. Using ultra-pure preparations of LPS, we found that activation of p21Ras and phosphorylation of Akt and Src family kinase targets such as Cbl, Pyk2, Vav and Syk occurred even in absence of TLR4, whereas MAPK activation was completely dependent on TLR4. CD14 and class A (SR-AI/II) and class B (CD36) scavenger receptor members were implicated in TLR4-independent signalling, indicating that these LPS-binding proteins play a greater role in pathogen recognition than previously appreciated. We identified downstream pathways mediated by TLR4-independent signalling that include promotion of cellular viability, changes in cell morphology and induction of gene expression, most notably Il1a and Il1b expression. We also observed changes in leukocyte distribution in TLR4-deficient animals following LPS treatment, confirming that TLR4-independent pathways also function in vivo. We observed that TLR4 signalling negatively regulated the activation of p21Ras and Cbl, indicating that the TLR4-independent and TLR4-dependent pathways influence one another. Furthermore, we found that the negative effect of TLR signalling on p21Ras activation required MAPK activity and also affected cytokine signalling. We propose that the TLR4-independent pathway provides signals that are critical for the development of a global response to infection and may represent a general mechanism for sensing pathogens.
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