The Mechanisms of TLR-Mediated Regulation of Neutrophil Survival
Lead Research Organisation:
University of Sheffield
Department Name: Medicine and Biomedical Science
Abstract
There is a large group of lung diseases that are both common and hard to treat. Their unifying feature is that they are all caused by the over-activation of a white blood cell called the neutrophil. In general, neutrophils are hugely important. They are very numerous in the blood, and are mobilised very quickly to sites of infection, where they hunt and destroy bacteria, fungi, and some viruses. Their ability to kill bugs comes from carrying a range of toxic proteins which they deliberately release onto the microbes. When the neutrophil?s job is done, it naturally dies and is removed by cells specialised in clearing up (like macrophages). The death of the neutrophil is highly regulated in a process called apoptosis, and is designed so that the neutrophil doesn?t spill any of its toxic antimicrobial compounds into healthy or healing tissues. However, if the neutrophil lives too long, or doesn?t get cleared properly when it dies, spillage of these toxic compounds can cause major tissue damage. This is thought to be an important contributor to a wide range of diseases, for example chronic obstructive pulmonary disease (COPD, commonly caused by smoking).
We think we?ve worked out how neutrophil survival might be regulated at sites of infection. This survival response depends upon a clever series of orchestrated signals that keep the neutrophil alive for just the right length of time. We also think that, in diseases like COPD, these survival signals are doing too much. Our work plans on studying these systems in much more detail. We want to look at normal neutrophils, and neutrophils taken from the lungs during inflammation. To study these pathways in the test tube, we plan to make neutrophils from stem cells that we have manipulated to be deficient in specific proteins. Putting together these approaches, many of which are really new and technically very exciting, we think we?ll identify some genuinely new potential drug targets that might revolutionise the treatment of lung disease.
We think we?ve worked out how neutrophil survival might be regulated at sites of infection. This survival response depends upon a clever series of orchestrated signals that keep the neutrophil alive for just the right length of time. We also think that, in diseases like COPD, these survival signals are doing too much. Our work plans on studying these systems in much more detail. We want to look at normal neutrophils, and neutrophils taken from the lungs during inflammation. To study these pathways in the test tube, we plan to make neutrophils from stem cells that we have manipulated to be deficient in specific proteins. Putting together these approaches, many of which are really new and technically very exciting, we think we?ll identify some genuinely new potential drug targets that might revolutionise the treatment of lung disease.
Technical Summary
The neutrophil is our most numerous phagocyte and a principal line of antimicrobial defence, but where activated inappropriately, neutrophilic inflammation is a major cause of airways disease and is very difficult to treat.
Neutrophils are short-lived cells, whose death by apoptosis allows resolution of inflammation once infection has been removed. We have shown that neutrophil lifespan is extended through activation of a major group of pathogen and damage recognition receptors, the TLRs. At sites of inflammation, infectious stimuli and signals released from damaged tissues activate TLRs to cause a delay in apoptosis that facilitates clearance of infections.
We have now shown that TLR-mediated neutrophil survival is a two-stage process, regulated by independent signalling pathways. First, direct activation of TLRs on the neutrophil results in transient induction of neutrophil survival. Secondly, activation of TLRs on other cells present at sites of inflammation, such as monocytes, releases survival signals that cause prolonged neutrophil survival. These indirect survival signals act on neutrophil cAMP signalling, and may explain why drugs acting to raise cAMP can fail to control neutrophilic inflammation.
We propose that the handover of regulation of neutrophil survival to external control allows for the withdrawal of survival stimuli and facilitates resolution of injury when danger is passed.
We hypothesise that the targeting of these pathways would restore normal rates of neutrophil apoptosis in inflammatory diseases.
We propose to study, in vitro and in vivo, both components of the regulation of neutrophil survival. First, we will determine how the direct infection signal (mediated by activation of TLR4) is limited in its efficacy, since this may be dysregulated in disease, and represent a new therapeutic target. Secondly, we will use a combination of microarray and proteomic approaches to determine how cAMP-mediated signalling causes neutrophil survival. We will challenge human subjects with inhaled endotoxin, and explore in vivo how these direct and indirect survival signals contribute to the regulation of neutrophilic airway inflammation. Neutrophils are hard to genetically modify, so when we identify new targets involved in the regulation of these survival pathways, we will knock down their function in neutrophils derived from bone marrow and embryonic stem cells, using methodology established in our groups. These studies will identify new pathways, and hence therapeutic targets, regulating neutrophil apoptosis in man, and target their function using novel approaches to the study of neutrophil biology.
Neutrophils are short-lived cells, whose death by apoptosis allows resolution of inflammation once infection has been removed. We have shown that neutrophil lifespan is extended through activation of a major group of pathogen and damage recognition receptors, the TLRs. At sites of inflammation, infectious stimuli and signals released from damaged tissues activate TLRs to cause a delay in apoptosis that facilitates clearance of infections.
We have now shown that TLR-mediated neutrophil survival is a two-stage process, regulated by independent signalling pathways. First, direct activation of TLRs on the neutrophil results in transient induction of neutrophil survival. Secondly, activation of TLRs on other cells present at sites of inflammation, such as monocytes, releases survival signals that cause prolonged neutrophil survival. These indirect survival signals act on neutrophil cAMP signalling, and may explain why drugs acting to raise cAMP can fail to control neutrophilic inflammation.
We propose that the handover of regulation of neutrophil survival to external control allows for the withdrawal of survival stimuli and facilitates resolution of injury when danger is passed.
We hypothesise that the targeting of these pathways would restore normal rates of neutrophil apoptosis in inflammatory diseases.
We propose to study, in vitro and in vivo, both components of the regulation of neutrophil survival. First, we will determine how the direct infection signal (mediated by activation of TLR4) is limited in its efficacy, since this may be dysregulated in disease, and represent a new therapeutic target. Secondly, we will use a combination of microarray and proteomic approaches to determine how cAMP-mediated signalling causes neutrophil survival. We will challenge human subjects with inhaled endotoxin, and explore in vivo how these direct and indirect survival signals contribute to the regulation of neutrophilic airway inflammation. Neutrophils are hard to genetically modify, so when we identify new targets involved in the regulation of these survival pathways, we will knock down their function in neutrophils derived from bone marrow and embryonic stem cells, using methodology established in our groups. These studies will identify new pathways, and hence therapeutic targets, regulating neutrophil apoptosis in man, and target their function using novel approaches to the study of neutrophil biology.
