The control of Toll-like receptor (TLR)-mediated neutrophil activation

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A white blood cell, the neutrophil, is a principal defence against infection. Its recruitment to, and activation within, tissues results in the removal of microbes and survival of the host. Unfortunately, inappropriate neutrophil activation also occurs in many lung diseases, contributing to tissue damage, and ultimately ill health or even death. This project will develop a deeper understanding of the ways in which neutrophils are activated by microbial products, which are sensed by the neutrophil using proteins called Toll-like receptors (TLRs).
We propose that neutrophils recruited into tissue will show altered patterns of TLR responses, which will affect their tissue positioning and activation. Our first set of studies will determine patterns of neutrophil responses in tissue cells compared with those in the peripheral blood, using a range of established and novel assays of neutrophil activation. We will subsequently investigate the molecular mechanisms underpinning altered cell phenotype at the level of intracellular signalling. We will determine how TLR signalling affects subsequent cell migration and activation driven by other neutrophil activators, principally chemoattractants.
We hypothesise that neutrophil responses to TLR agonists will be determined by trafficking of the TLR to the cell membrane and specific intracellular compartments, and will explore this in primary neutrophils in collaboration with Prof Smythe (Sheffield University). We will determine if activated TLRs can recycle to the cell membrane, and if control of recycling provides further mechanisms to regulate neutrophil responses. In parallel, we will determine the molecular pathways resulting in TLR internalisation and reexpression in cell lines. We propose that the box 3 region of the intracellular TIR domain may be involved in trafficking of receptors, and we will use strategies involving receptor mutagenesis and construction of chimaeric proteins to determine the importance of these regions in receptor function.
Finally, we hypothesise that neutrophils become rapidly unresponsive to TLR agonists, which we propose is part of a mechanism preventing their excessive activation at inflammatory sites. We have developed novel preliminary data describing the cellular and molecular mechanisms causing neutrophils to become unresponsive, and will study these systems in more detail to determine if they represent therapeutic targets controlling neutrophil activation. We will investigate whether other inflammatory products also serve to limit neutrophil inflammation by inhibiting TLR activation in these cells.
These studies will identify new ways of targeting neutrophilic activation in inflammatory disease with wide-ranging therapeutic consequences for respiratory diseases including asthma and COPD.