Control the Platelets, Control the Disease: A Novel Cystic Fibrosis Hypothesis

Cystic fibrosis (CF) is a life-threatening disease associated with a viscous mucous that predisposes CF patients to chronic neutrophilic lung inflammation, often leading to respiratory failure. In a recent article published in the Journal of Clinical Investigation, Ortiz-Munoz et al. demonstrate that platelets regulate the hyper-inflammatory response and suggest that they may be to key to controlling the inflammation in CF patients.[1]

Cystic Fibrosis is a lethal genetic disease

Cystic Fibrosis (CF) is the most common life-limiting Mendelian autosomal recessive disease in Caucasians.[2, 3] It affects over 70,000 individuals worldwide and is characterized by dysregulated ion transport that results in production of an unusually thick mucus that covers the afflicted organs.[2–4] Gelatinous mucous leads to reduced mucociliary clearance, which predisposes CF patients to bronchiectasis, respiratory and small airway obstruction, progressive airway impairment, pancreatic insufficiency & malabsorption (associated with pancreatic tissue lysis), biliary cirrhosis, diabetes mellitus, and infertility.[3, 5] In the lungs, the pathophysiology of CF leads to respiratory failure in ~85% of the patients.[6] With substantial changes in CF specialized care, significant improvements in CF patient survival have been achieved. What was once considered a fatal disease of children, now sees the greater percentage of its patients live well into adulthood. In 2017, adults made up 53.5 percent of the CF population, compared with 29.8 percent in 1987.[6] Despite years of dedicated research and groundbreaking improvements in clinical care, CF remains a constant battle for those affected as it takes a toll on several physiological systems.

At a molecular level, the majority of CF patients have a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cAMP-activated, ATP-gated chloride and bicarbonate channel, that plays a central role in the development of CF.[7] CFTR is functionally coupled to other channels such as the epithelial sodium channel (ENaC) and the canonical transient receptor potential channel-6 (TRPC6).[2, 8, 9] Together these channels regulate the electrophysiological properties of mucosal epithelium. There are over 2000 documented mutations in CTFR that cause a range of phenotypes from reduced conductance to no or greatly reduced expression on the cell surface.[3, 10] The most common mutation, F508del, causes protein misfolding leading to 85% of expressed protein being degraded in the proteasome. [3, 10] Mutations that disrupt CFTR reduce chloride release resulting in a disruption of epithelial fluid transport in the lungs, pancreas and other organs. Chloride transport disruption results in increased viscosity of mucous and decreases mucociliary clearance of foreign substances. Viscid mucous renders CF patients highly susceptible to opportunistic microbial colonization, excessive inflammation, and the eventual remodeling of mucosal surfaces, causing decrease or loss of organ function.[2, 3, 7]

From a clinical point of view, chronic opportunistic bacterial infections characterize CF. By the age of three, CF patients have significant mucous obstruction, and neutrophil-driven pulmonary inflammation.[5, 11] These processes set the stage for deficient pathogenic clearance, and a conducive environment for the progression of pathological processes such as bronchiectasis. [7] The exact link between the mechanisms by which the CFTR mutation and aberrant inflammation exacerbate CF has evaded researchers for decades and has undermined therapeutic management of CF.

CF Treatment Options:

Recent therapeutic developments have focused on CFTR modulators and have led to the development of several clinically approved drugs. Potentiators, such as Ivacaftor (IVA), enhance chloride conductance of CFTR at the epithelial cell membrane. Correctors, such as Lumacaftor (LUM), Tezacaftor (TEZ), Elexacaftor (ELX) improve folding and trafficking of F508del CTFR mutations. Read-through agents, such as Ataluren, amplify CFTR translation allowing ribosomes to ignore mutations that cause premature termination of transcription.[12] IVA treatment in patients with gating mutations that reduce the channel conductance showed an increase in lung function of >10% in 1 s forced expiratory volume (FEV1) and a 55% reduction in the frequency of pulmonary exacerbations.[13] In clinical trials treating F508del CF patients with the ELX/TEZ/IVA combination, an improved lung function by 10 – 14% FEV, and reduced exacerbations by 63% were observed.[14, 15] Read-through agents, however, have shown limited evidence of clinical efficacy to date.[16] Even with the great improvements that have been made, many CF patients are still faced with the requirement of lung transplant before the age of 18.[17]

Chronic Inflammation in CF Patients

Critical to the loss of lung function is the chronic airway inflammation experienced by affected individuals. Cumulative evidence suggests a paradigm in which CFTR deficiency in the immune cells of CF patients leads to an atypical immune response (hyperinflammation) and decreased clearance of microorganisms.[18, 19] It has been demonstrated that lung epithelium of CF individuals displays increased Nf-κb signaling and an associated increased secretion of cytokines such as Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the neutrophil chemoattractant IL-8.[20–22] Consistent with raised levels of IL-8, basal levels of pulmonary neutrophils are increased in CF patients.[3, 11, 23, 24] The thickened mucous layer and reduced mucociliary removal of bacteria result in chronic lung infections and eventual colonization of the lungs by opportunistic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacian.[2, 3, 19] Inflammatory macrophages and neutrophils are recruited to the lung epithelia to fight these infections.

Interestingly, neutrophils express CFTR and it has been shown in several models that the neutrophils in CF patients have diminished ability to eliminate pathogens.[19, 25, 26] CF neutrophils were demonstrated to have reduced NADPH oxidase activity associated with reduced ClO⁻ production.[25] Therefore, instead of an efficient innate immune response, we have an increased influx of neutrophils with reduced ability to clear bacteria. The pathological consequences of hyperinflammation combined with decreased pathogen clearance is a remodeling of the epithelial layer and fibrosis of the lungs, leading to untimely death for many CF patients.[2, 3]

As immune modulation of cells containing the CFTR gene becomes more feasible, it is becoming increasingly relevant to elucidate the mechanisms coupling inflammatory cells to CF progression.

While neutrophils are obvious culprits for driving CF-related excessive inflammation, evidence is mounting that their anuclear counterparts, platelets, may play a pivotal role in the hyperinflammatory syndrome. Mattoscio et. al demonstrated that human platelets have a functional CFTR in their plasma membrane and that platelets from CF patients produce ~40% less of the anti-inflammatory lipoxin A₄ compared to healthy controls.[27] In studies using F508del mice, intertracheal lipopolysaccharide led to increased inflammation and caused severe thrombocytopenia compared to wild type mice.[28] Treatment of the F508del mice with aspirin (blocks platelet activation), or blockade of platelet activating factor or PSGL-1 (mediates platelet neutrophil interactions), significantly reduced the inflammation seen in the F508del mice.[28] Furthermore, studies by Vemana and Karim et al have established that TRPC6 mediates receptor-operated calcium entry (ROCE) and platelet activation downstream of thromboxane A₂ and other G_q-coupled receptors.[29] Previous studies demonstrated that TRPC6 and CFTR are functionally and reciprocally coupled within a molecular complex in human airway epithelial cells, and because this functional coupling is lost in CF cells, the TRPC6-dependent Ca⁺⁺ influx is abnormal.[8] Thus, the question arises, “do mutations in the CFTR affect TRPC6 in platelets?” Also, “how important are platelets to the hyperinflammatory syndrome in CF patients”?

Platelets Regulate Lung Injury in CF Patients

In their article, Ortiz-Munoz & M.A Yu et.al tackle these very questions.[1] They use an amalgamation of in vitro and in vivo experiments to elegantly dissect the functions of the CFTR and TRPC6 receptors in platelets and subsequently delineate a role for platelets in inflammation driven by CFTR dysfunction.

The authors begin by confirming that murine platelets, like in humans, have functional CFTR and subsequently demonstrate the importance of CFTR in platelets to lung injury in their model.[1] Using either a lipopolysaccharide (LPS) or P. aeruginosa intratracheal challenge, they show that these mice display augmented lung injury as defined by a strikingly increased neutrophilic inflammation, neutrophil extracellular trap formation, lung barrier disruption, increased platelet-neutrophil aggregates, and impaired bacterial clearance. In what is probably the key experiment, they use floxed CTFR mice (CTFR^fl/fl) to create lineage-specific deletions of CTFR in neutrophils, the myeloid lineage (monocytes, mature macrophages, and granulocytes), and in platelets (CF-PF4-Cre). Each of these strains was challenged with intertracheal LPS and evaluated for lung injury. Surprisingly, in the mice in which CTFR was deleted in neutrophils or the myeloid lineage, the lung injury was not significantly different from CTFR^fl/fl controls. However, the CF-PF4-Cre mice phenocopied the global CTFR deletion. The CF-PF4-Cre mice also demonstrated a similar degree of lung injury after P. aeruginosa challenge. This suggests that neutrophils do not independently initiate a hyperinflammatory state in CF patients. These findings are consistent with previous evidence demonstrating that CF patients have an increased activation status of circulating platelets and increased levels of platelet-neutrophil aggregates. [1, 30]

The authors subsequently identify a key role for TRPC6 as the mechanism behind the increased platelet activation in the absence of CFTR. Because CFTR and TRPC6 are reciprocally linked, they evaluated the effect of pharmacological TRPC6 inhibition after thrombin activation using the platelet activation marker P-selectin (CD62). They demonstrated that inhibition of TRPC6 reverses the hyper-CD62 expression after CFTR pharmacological blockade, in CFTR^−/− mice, and in CF-PF4-Cre mice. Genetic deletion of TRPC6 supported these findings in crosses with CFTR^−/− mice. Accordingly, CFTR^−/− mice showed increased calcium entry as measured by Indo-1 peak median fluorescent intensity (MFI) compared to WT or TRPC6^−/− mice. These differences were diminished by pharmacologic inhibition of TRPC6.[1] In human subjects, they were able to demonstrate that platelets from healthy individuals were hyperreactive after pharmacological CFTR inhibition and that this effect is reversed by TRPC6 inhibition. Finally, the authors challenged the CFTR^−/−/TRPC6^−/− mice with LPS or P. aeruginosa and demonstrated that the TRPC6 mutation reverses the lung injury of the CFTR^−/− mice. These findings directly implicate an association between dysfunctional CFTR and an increase in TRPC6 activity, suggesting that the downregulation of CFTR channels initiates the upregulation of TFPC6, augmenting calcium entry and platelet activation, which precedes the hyperinflammatory response in CF patients.

Hopes for the future treatment of CF

While life expectancy has improved, current treatments for CF are neither preventive nor curative. Ideally, gene-addition therapies are the only approach with the immediate potential to prevent CF lung disease. Lysophosphatidylcholine gene-addition has already been validated in multiple animal models.[12, 31] Nevertheless, before airway gene therapy can be translated to the clinic some of the key tasks involve ensuring long-term efficacy, the safety of Lysophosphatidylcholine delivery in a CF lung, ability to re-dose and boost CFTR gene expression levels, and the ability to translate these techniques into human-sized lungs.[31]

Many CF patients ultimately require a lung transplant to prolong life.[32] Lung transplantation is a complex, high risk procedure with more candidates than available donors. Moreover, lung transplant is not a cure for CF lung disease because the defective CFTR gene is found in several cell types of the body, with the exception of the newly transplanted lungs. There remains a lot of controversy when considering the long-term benefits. Lung transplant is considered a potentially life-saving therapy for the end-stage lung disease of cystic fibrosis, however it has demonstrated only a limited improvement on survival.[17, 33]

A conventional approach to the treatment of CF is directed at the downstream consequences of CF: mucous build up and infection. The use of mucolytic agents (N-acetyl-l-cysteine, Dornase alfa), airway clearance (physiotherapy and exercise), airway surface rehydration strategies (hypertonic saline, mannitol), nonsteroidal anti-inflammatory agents (ibuprofen) and antibiotics (targeting P. Aeruginosa, S. Aureus, etc) have drastically improved the management and prognosis of CF.[12] Nonetheless, clinicians are still faced with a vast array of adverse effects associated with conventional approaches, timely treatment and eradication of early infections in CF patients, management of acute exacerbations, suppression of chronic infection and the management of resistant strains of opportunist microorganisms (including MRSA). Clinical trials have had conflicting outcomes using standard conventional approaches with a plethora of challenges to be faced by both the clinician and patient.[12]

Consequently, the last decade of research has been focused upstream, targeting the mechanism of development rather than the outcome of disease.[24] The insight provided by Ortiz-Munoz & M.A Yu et.al on the role of CFTR and TRPC6 in the pathogenesis of hyperinflammation in CF may lead to novel therapeutic approaches targeting TRPC6 in platelets.[1] The severe lung injury induced by years of chronic inflammation is a major cause of death in CF patients. While there are caveats to the current study, such as those found with using mouse models, the prospect of targeting TRPC6 in the platelets of CF patients holds promise of extending the life span beyond the 5 years expected from lung transplant with minimal invasive procedures.[1, 24, 32]