Alpha-1 Antitrypsin therapy modulates the neutrophil membrane proteome and secretome
RCSI researchers identify that neutrophils in patients with alpha-1 antitrypsin deficiency have an altered membrane protein profile that can be modified by alpha-1 antitrypsin augmentation therapy, potentially improving lung function.
Alpha-1 anti-trypsin deficiency (AATD) is a genetic condition characterised by emphysematous lung changes with progression to chronic obstructive airway disease. Alpha-1 anti-trypsin (AAT) functions as a protease inhibitor that protects lung elastin tissue from the action of elastase, while a deficiency permits unopposed neutrophil elastase activity, resulting in lung alveolar destruction.
The modern treatment of AATD is focused on augmentation of circulating plasma levels of AAT in addition to lifelong avoidance of smoking. The RAPID trial demonstrated that AATD lung density loss was significantly less in patients with AATD treated by AAT augmentation compared to placebo controls. Extension of the RAPID-RCT in an open-label extension study (RAPID-OLE) demonstrated continued efficacy of AAT in slowing disease progression over 4 years of treatment.
The disordered neutrophil activity associated with AATD is understood to be related to loss of the anti-inflammatory properties of AAT. In a study available here, Professor Noel G. McElvaney and Dr Emer Reeves of the Irish Centre for Genetic Lung Disease at RCSI University of Medicine & Health Sciences present the results of a proteomic analysis of the neutrophil plasma membrane from AATD individuals.
This study highlights that neutrophils in patients with AATD have an altered membrane protein expression and primary granule exocytosis pattern compared to cells from COPD patients without AATD. This difference may be corrected by AAT augmentation therapy.
Authors recruited participants to represent healthy controls, obstructive and non-obstructive AATD and non-AATD COPD (Chronic Obstructive Pulmonary Disease).
Healthy control volunteers were required to have normal serum AAT levels. Obstructive AATD and non-obstructive AATD were defined by lung function testing and characteristic changes in lung CT imaging. Non-AATD COPD individuals were required to be non-smokers, have confirmed airway obstruction based on lung function testing and normal serum AAT. Lung function testing was used to match AATD and non-AATD COPD individuals for comparison. The final study group was AATD individuals receiving AAT augmentation with 60mg/kg body weight by weekly infusion.
Authors first established that 15 proteins had differential expression in the plasma membrane of neutrophils from individuals with obstructive AATD compared with non-AATD COPD individuals. Eight of these proteins were constituents of neutrophil granules and, most notably, an abundance of myeloperoxidase (MPO) and bactericidal/permeability-increasing protein (BPI) was found in AATD individuals.
Secondly, authors compared the process of neutrophil degranulation between healthy controls, obstructive AATD, non-obstructive AATD and non-AATD COPD individuals.
Following TNF-based neutrophil stimulation, significantly increased levels of degranulated MPO and degranulated neutrophil elastase was detected in obstructive AATD individuals compared to non-obstructive AATD individuals or healthy controls.
The mechanism of neutrophil degranulation in AATD was explored by investigating Rac2 protein, known to have a central role in granule exocytosis. Active Rac2 was increased in obstructive AATD individuals compared with healthy controls. Use of a RAC inhibitor decreased degranulation of MPO and neutrophil elastase confirming the degranulation mechanism. In-vitro treatment of neutrophils with AAT decreased the levels of MPO released from stimulated neutrophils, indicating an inhibitory effect of AAT on neutrophil degranulation.
Neutrophil elastase is known to increase reactive oxygen species. Neutrophils from obstructive AATD individuals produced significantly higher levels of superoxide anions in response to neutrophil elastase stimulation compared to non-AATD COPD patients or healthy controls. Administration of AAT inhibited the generation of superoxide anions by neutrophils in obstructive AATD individuals.
Proteomic analysis was performed on the plasma membrane of neutrophils collected in AATD individuals treated with AAT augmentation therapy. 66 intrinsic neutrophil proteins were found to be differentially expressed between day 0 pre-therapy and day 2 post-therapy with higher expression in 65 of 66 proteins prior to AAT therapy. These proteins were characterised by a gene ontology cluster analysis. Analysis revealed that the differentially expressed proteins were largely involved in neutrophil exocytosis.
These findings were confirmed by western blot of neutrophil membrane proteins at day 2 post-therapy compared to day 0 pre-treatment. A significant decrease in MPO expression was observed in the neutrophil membrane at day 2 post-AAT therapy. TNF-based stimulation of Day 0 and day 2 neutrophils was likewise performed and significantly reduced level of neutrophil degranulation was observed at day 2 post-therapy.
Finally, the neutrophil plasma membrane proteome of non-AATD COPD patients was compared with the proteome of AATD patients post AAT therapy. Only one protein was different between the two groups, suggesting that AAT therapy reverts neutrophils to a condition akin to non-AATD COPD.
In summary, these experiments have demonstrated that circulating neutrophils in AATD have disordered function with high levels of degranulation. Degranulation of MPO and neutrophil elastase may be linked to the pathological alveolar destruction associated with the disease.
Neutrophil elastase promotes oxygen free radical release from AATD neutrophils, contributing to alveolar injury. The mechanism of degranulation in AATD is based on activation of the Rac2 protein.
Treatment with AAT corrected disordered neutrophil degranulation in vitro and this finding was confirmed by an in-vivo study of AATD patients receiving AAT therapy. The profile of protein expression in circulating neutrophil membranes in AATD was altered by AAT therapy to resemble that of a non-AATD COPD neutrophil.
This study identifies a regulatory role for AAT on circulating neutrophil function and demonstrates correction of disordered neutrophil function through the administration of AAT. Most importantly, these effects were identified in a clinical setting, in patients receiving standard dosing of AAT.
Treatment benefit through the slowing of lung density loss has been previously identified by a clinical RCT with open label extension. This study now highlights the vital role of AAT augmentation therapy by revealing mechanisms for AAT in regulating neutrophil biology and improving lung function.
Journal Article Information:
α1 Antitrypsin therapy modulates the neutrophil membrane proteome and secretome
European Respiratory Journal 2020 55: 1901678
https://doi.org/10.1183/13993003.01678–2019
