Blocking a pro-inflammatory protein complex, broadly known as the neutrophil inflammasome, represents a new way of treating chronic Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients, a proof-of-concept study demonstrated.
A hallmark of CF is chronic infections by the bacteria P. aeruginosa. Lipopolysaccharide (LPS), a major component in the bacteria’s outer membrane, stimulates an immune response that results in the production of the pro-inflammatory protein interleukin-1β (IL-1β).
Specific adaptations of P. aeruginosa in the CF airway are responsible for bacterial persistence by stimulating excessive production of IL-1β. This leads to increased mucus secretions, and triggers the release of more pro-inflammatory proteins, impairing the immune system’s ability to clear the bacterial infection.
The IL-1β protein is created in two stages. The first stage is the synthesis of an inactive precursor, called pro-IL-1β; in the subsequent second stage, the inactive pro-IL-1β converts to an active form, IL-1β.
Activation of IL-1β is achieved by the enzyme caspase-1, which is part of a large, multi-protein complex called the NLRP3 inflammasome (NLRP3 stands for nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors, pyrin domain-containing protein 3).
In response to infection and other danger signals, the proteins of the NLRP3 inflammasome assemble to facilitate the conversion to active IL-1β from available pro-IL-1β, stimulating an inflammatory response.
In the presence of LPS, many immune cells increase the synthesis of pro-IL-1β by altering their metabolism (immunometabolism). They shift to a less efficient way of producing energy (called the Warburg effect), which in turn generates metabolic by-products that trigger pro-IL-1β synthesis. With additional stimulation of the inflammasome by LPS linked to P. aeruginosa in the lungs of CF patients, the IL-1β pro-inflammatory response is intensified.
An increase in both pro-IL-1β by metabolic changes, and IL-1β by NLRP3 inflammasome activation has been reported in immune cells such as macrophages, monocytes, and dendritic cells.
But in white blood cells called neutrophils, the primary type of immune cell present in patients’ lungs with P. aeruginosa infection, this process remains understudied.
Researchers collected airway secretions (bronchoalveolar lavage [BAL] and sputum samples) from 16 CF patients to examine if secretions and isolated neutrophil cells contribute to the inflammatory response by overproducing pro-IL-1β and IL-1β.
The team also analyzed secretions and isolated cells from 12 healthy people (controls), and six CF patients who had undergone a double-lung transplant.
Results showed that in BAL secretions from CF patients, IL-1β levels were significantly higher compared to controls and transplant patients. Consistently, 71% of the immune cells present in the secretions were neutrophils. Researchers then demonstrated that the increase in lung IL-1β levels correlated with the percentage of neutrophils seen.
They also found correlations between IL-1β levels in CF BAL and sputum samples, and two clinical markers — FEV1 (the maximal amount of air one can forcefully exhale in one second), and CF-ABLE score (a score taking into account age, body mass index or BMI, lung function, and number of exacerbations in the last three months) — demonstrating that IL-1β is a clinically relevant indicator of neutrophil-associated inflammation in CF.
To assess if neutrophils in the blood also altered their metabolism to synthesize more pro-IL-1β, researchers analyzed circulating neutrophils for an enzyme that regulates the change in metabolism, called pyruvate kinase (PKM2). In neutrophils from the blood of CF patients, PKM2 and its activated form were significantly increased. Higher levels of pro-IL-1β were also observed in these cells.
Interestingly, low levels of LPS were found in blood from CF patients, while higher LPS levels were found in CF BAL secretions. CF BAL samples also had higher levels of caspase-1 activity compared to CF blood samples.
Researchers then analyzed neutrophils from both CF and healthy controls by treating them with a small molecule inhibitor of the NLRP3 inflammasome, called MCC950. Results showed lower IL-1β production in MCC950-treated cells in a dose-dependent manner in both cell types.
The team then tested the clinical potential of NLRP3 inflammasome inhibition in two mouse models (an in vivo, or living organism, experiment).
First, healthy mice were treated with MCC950 before and after LPS administration. Pre- and post-MCC950 treatments significantly reduced IL-1β in BAL samples from these mice, demonstrating both a prophylactic (preventative) and a therapeutic effect.
When the experiment was repeated in CF-specific mice infected with P. aeruginosa, results showed that both MCC950 treatments resulted in a 92% reduction in IL-1β in the lung compared to untreated mice; a significant reduction in the numbers of P. aeruginosa bacteria and in airway inflammation were also observed.
Altogether, the results show how low levels of LPS from an P. aeruginosa infection can circulate in the blood and alter the metabolism of neutrophils, priming them with pro-IL-1β before they migrate to the lungs. Once in the lungs, higher levels of LPS can activate the NLRP3 inflammasome to convert pro-IL-1β to active IL-1β, causing sustained inflammation.
Importantly, blocking the NLRP3 inflammasome significantly lowered IL-1β levels and lessened its inflammatory effects in mouse models.
“This study identifies a new anti-inflammatory therapeutic strategy for cystic fibrosis (CF). We show for the first time that alterations in CF neutrophil immunometabolism drive pro-inflammatory cytokine release by these cells, an effect that correlates closely with clinical outcomes and can be abrogated by a specific small molecule inhibitor of the NLRP3 inflammasome,” the researchers wrote.