MICA: Key mechanisms underlying inhaled GM-CSF's enhancement of phagocytosis and bacterial clearance by human alveolar macrophages.
Lead Research Organisation:
Newcastle University
Department Name: Translational and Clinical Res Institute
Abstract
Despite the widespread availability of antibiotics, infection deep in the lung (pneumonia) remains an important cause of death in older people, as well as in critically ill patients in intensive care units.
Our lungs contain a population of cells that sit in the air pockets of the lung and sense the presence of bugs. These cells are called alveolar macrophages (AMs). When AMs sense bugs in the lung, they kill them and if necessary send signals to the wider immune system for more help. Our study wishes to find out if AMs work less well as we get older, or when we are critically ill, as this may explain why we become susceptible to pneumonia. We know that a natural chemical in the body, called granulocyte-macrophage colony-stimulating factor (GM-CSF), maintains the health of our AMs.
A further aim of our study is to determine whether giving GM-CSF as an inhaled drug, directly into the lung, might boost the function of AMs. If it does, this would provide the impetus for further research to see if GM-CSF could be given to people at very high risk of pneumonia (for example those in intensive care units), to prevent infection. This may have great advantages, because GM-CSF is not an antibiotic. The very heavy use of antibiotics in intensive care units has led to the emergence of "superbugs" that are not killed by antibiotics, and there is an urgent need to develop safe treatments that might boost immune cells such as AMs, instead of relying entirely on antibiotics.
Nearly all of our information on how GM-CSF improves the function of AMs comes from studies in mice. We need to understand better how human AMs kill bugs, we need to know if this goes wrong as we get older or become critically unwell, and we need to know if GM-CSF can improve things. These issues have driven the design of our study.
We shall ask 20 young volunteers (aged 18-30) and 20 older volunteers (60 or over) to come to the hospital on three days. On day one and day two they will inhale GM-CSF or a placebo, but neither they nor the research team will know which they inhaled. Each session will last about an hour. On day three they will come back for a telescope test of the lungs (bronchoscopy), where they are closely monitored and fluid is instilled into the a small area of the lung and gently sucked back. The fluid sucked back contains millions of AMs. The test lasts about 20 minutes and the person rests in hospital afterwards for a few hours before going home. Separately, a group of critically ill patients in the ICU will have the same protocol, i.e. GM-CSF or placebo on days 1 and 2, and bronchoscopy on day 3. In a final variation, the young volunteers will come back at least one month later, and have the same procedures done again, except that if they received GM-CSF first time round they will receive placebo the second time, and vice versa.
We can take the AMs to the lab and study how well they eat bugs. We can block the function of specific molecules in the AMs and if this prevents the anti-bug effects we can infer that these molecules must be important for AM function. This way we shall build up a profile of the key molecules at the surface of the AM ("receptors") or inside the AM. Once we have the results we can "unblind" ourselves to find out who had GM-CSF and who had placebo. In this way we can piece together the answers to our questions - how do human AMs get rid of bugs? Do ageing and critical illness reduce the function of AMs and, if so, how? Does GM-CSF restore good function to AMs?
The study will generate entirely new information on the function of human AMs. If GM-CSF is safe and boosts AM function we shall take this information forward to work out if inhaled GM-CSF can effectively and safely prevent pneumonia in patients who are at highest risk.
Our lungs contain a population of cells that sit in the air pockets of the lung and sense the presence of bugs. These cells are called alveolar macrophages (AMs). When AMs sense bugs in the lung, they kill them and if necessary send signals to the wider immune system for more help. Our study wishes to find out if AMs work less well as we get older, or when we are critically ill, as this may explain why we become susceptible to pneumonia. We know that a natural chemical in the body, called granulocyte-macrophage colony-stimulating factor (GM-CSF), maintains the health of our AMs.
A further aim of our study is to determine whether giving GM-CSF as an inhaled drug, directly into the lung, might boost the function of AMs. If it does, this would provide the impetus for further research to see if GM-CSF could be given to people at very high risk of pneumonia (for example those in intensive care units), to prevent infection. This may have great advantages, because GM-CSF is not an antibiotic. The very heavy use of antibiotics in intensive care units has led to the emergence of "superbugs" that are not killed by antibiotics, and there is an urgent need to develop safe treatments that might boost immune cells such as AMs, instead of relying entirely on antibiotics.
Nearly all of our information on how GM-CSF improves the function of AMs comes from studies in mice. We need to understand better how human AMs kill bugs, we need to know if this goes wrong as we get older or become critically unwell, and we need to know if GM-CSF can improve things. These issues have driven the design of our study.
We shall ask 20 young volunteers (aged 18-30) and 20 older volunteers (60 or over) to come to the hospital on three days. On day one and day two they will inhale GM-CSF or a placebo, but neither they nor the research team will know which they inhaled. Each session will last about an hour. On day three they will come back for a telescope test of the lungs (bronchoscopy), where they are closely monitored and fluid is instilled into the a small area of the lung and gently sucked back. The fluid sucked back contains millions of AMs. The test lasts about 20 minutes and the person rests in hospital afterwards for a few hours before going home. Separately, a group of critically ill patients in the ICU will have the same protocol, i.e. GM-CSF or placebo on days 1 and 2, and bronchoscopy on day 3. In a final variation, the young volunteers will come back at least one month later, and have the same procedures done again, except that if they received GM-CSF first time round they will receive placebo the second time, and vice versa.
We can take the AMs to the lab and study how well they eat bugs. We can block the function of specific molecules in the AMs and if this prevents the anti-bug effects we can infer that these molecules must be important for AM function. This way we shall build up a profile of the key molecules at the surface of the AM ("receptors") or inside the AM. Once we have the results we can "unblind" ourselves to find out who had GM-CSF and who had placebo. In this way we can piece together the answers to our questions - how do human AMs get rid of bugs? Do ageing and critical illness reduce the function of AMs and, if so, how? Does GM-CSF restore good function to AMs?
The study will generate entirely new information on the function of human AMs. If GM-CSF is safe and boosts AM function we shall take this information forward to work out if inhaled GM-CSF can effectively and safely prevent pneumonia in patients who are at highest risk.
Technical Summary
Pneumonia is a major cause of death in people who are older or critically ill. Non-antibiotic-based strategies are urgently required to improve pneumonia outcomes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is the key regulator of maturation and function of the lung's resident patrolling phagocytes, alveolar macrophages (AMs). However, pivotal data were derived in mice, and mechanistic data are lacking for human AMs. Inhaled GM-CSF has potential to enhance clearance of pathogens by AMs. Defining the underlying mechanisms would provide data necessary to progress inhaled GM-CSF to clinical trials evaluating prevention or treatment of pneumonia in patients at highest risk. We shall test the hypotheses that a high ratio of Rac1/RhoA (key small Rho GTPases) promotes effective human AM phagocytosis, that in ageing and critical illness a predominance of RhoA activation impairs AM phagocytosis, and that GM-CSF restores the Rac1/RhoA ratio and efficient phagocytosis.
We shall deliver recombinant human GM-CSF (250 microgrammes) or placebo by nebulisation in a randomised, double-blind study, on each of two consecutive days in three groups: young healthy volunteers; older healthy volunteers; and critically ill patients intubated and mechanically ventilated and at high risk of pneumonia. The next day AMs will be retrieved by bronchoalveolar lavage (BAL). In total, 80 BALs will be performed in subjects receiving GM-CSF or placebo. Phagocytosis and bacterial killing by AMs will be assessed using Gram-positive pathogens, Gram-negative pathogens and zymosan. The ability to clear cellular debris (efferocytosis) will be assessed by feeding autologous apoptotic neutrophils to AMs. Blockade of specific AM phagocytic receptors, selective inhibition of key Rho GTPases, and assessment of transcription factors implicated in AM phagocytosis will characterise phagocytic mechanisms in the presence or absence of GM-CSF.
We shall deliver recombinant human GM-CSF (250 microgrammes) or placebo by nebulisation in a randomised, double-blind study, on each of two consecutive days in three groups: young healthy volunteers; older healthy volunteers; and critically ill patients intubated and mechanically ventilated and at high risk of pneumonia. The next day AMs will be retrieved by bronchoalveolar lavage (BAL). In total, 80 BALs will be performed in subjects receiving GM-CSF or placebo. Phagocytosis and bacterial killing by AMs will be assessed using Gram-positive pathogens, Gram-negative pathogens and zymosan. The ability to clear cellular debris (efferocytosis) will be assessed by feeding autologous apoptotic neutrophils to AMs. Blockade of specific AM phagocytic receptors, selective inhibition of key Rho GTPases, and assessment of transcription factors implicated in AM phagocytosis will characterise phagocytic mechanisms in the presence or absence of GM-CSF.
