Hypothesis paperRethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation
Introduction
Cystic fibrosis (CF) is a genetic disease afflicting nearly 30,000 persons in the United States and Canada, and an estimated 250,000 persons worldwide. The mutated gene is recessive, and there are hundreds of genetic mutations that can produce cystic fibrosis. The mutations all cause the ion channel created by the cystic fibrosis transmembrane conductance regulator (CFTR) protein to be either defective or absent altogether. More specifically, this protein creates an organic anion efflux channel in the cell membrane, permeant to chloride and other larger organic anions, such as reduced glutathione [1], [2]. CF patients typically die of respiratory failure due to profound lung injury secondary to chronic inflammation and chronic pathogen colonization of the lung, although several exocrine organs are negatively affected, including the pancreas and liver. In countries where these patients receive optimal care, average survival has risen to approximately 30 years.
Though the cause of CF pathology is understood to be the mutation of the CFTR protein, it has been difficult to trace the exact mechanisms by which the pathology arises and progresses from the mutation. For example, even when not facing pathogen challenge, inflammation is present in the youngest infants [3], [4], [5], [6], and inflammatory mediators and cytokine markers appear to be constitutively elevated in CF patients [7], [8], [9], [10], [11], [12]. When pathogens do begin to challenge the system, neutrophil infiltration in CF is especially high in response [13], and very young CF patients have been found to clear bacteria even without antibiotic intervention [14]. Over time, however, that capability decreases, and despite continuing high neutrophil infiltration rates, bacteria are no longer cleared. By age 2, most CF patients are consistently culturing at least one pathogen [15], [16]. Indeed, lung cells expressing the mutant CFTR fail to undergo apoptosis in response to infection with Pseudomonas aeruginosa [17]. In many pulmonary diseases involving inflammation due to pathogen challenge, exhaled NO of patients is elevated [18], [19], [20], [21], [22]. In CF, exhaled NO is not elevated [19], [22], [23], [24], [25], [26], [27]. In pulmonary conditions involving high oxidant stress, extracellular levels of reduced glutathione (GSH) increase [28], [29], [30]. In CF, a systemic deficiency of extracellular GSH develops and progresses over time [31], [32], [33].
How are all of these puzzling phenomena related to a defective CFTR channel? We believe that abnormal transport of GSH caused by CFTR mutation, recently demonstrated by two research teams [1], [2], is significantly related to the pathological puzzles mentioned above, as well as other aspects of cystic fibrosis disease. As will be detailed in a later section, CFTR mutation results in significantly diminished efflux of cellularly produced GSH into the extracellular milieu from certain cells without redundant channels to effect such efflux. The focus of this article will be on the role of this phenomenon in the pathogenesis of cystic fibrosis. We hypothesize that this transport abnormality will result in (i) normal levels of total glutathione in epithelia (those without anion channels redundant to the CFTR at the apical surface; however, cytosolic GSH:GSSG ratio may be diminished in these cells), (ii) a chronic and progressive extracellular deficit of GSH, and (iii) a similar deficit in cells with redundant anion channels, such as leukocytes, lymphocytes, erythrocytes, and hepatocytes. But perhaps most importantly in a theoretical sense, this transport abnormality is directly caused by the CFTR mutation, providing at least part of the missing linkage between the genetics and the pathology of CF. To explain this hypothesis, we must first step back and understand the importance of GSH in lung defense.
Section snippets
GSH and lung defense
Reduced glutathione (GSH) is a ubiquitous tripeptide produced by plants and animals alike from the amino acids glutamine, glycine, and cysteine (with cysteine being the rate-limiting constituent). Its sulfur-hydrogen, or thiol, group is a potent reducing agent, and GSH can be considered the one of the body’s most important water-soluble antioxidants.
Antioxidant defenses in humans are comprised of both enzymatic and nonenzymatic defenses; some defenses operate intracellularly and others have
Associations of GSH deficiency
Given that GSH is one of the organic anions whose efflux depends on a functioning CFTR channel (or a channel redundant to the CFTR), we would expect CF persons to manifest a progressive systemic extracellular deficiency of GSH, with profound deficits in areas heavily exposed to ROS, such as the lung. This is, in fact, the case, as we will detail in a later section. We would also hypothesize that cells with anion channels redundant to the CFTR, such as leukocytes, lymphocytes, erythrocytes, and
Impaired antioxidant capability
When a key antioxidant such as GSH is deficient in the extracellular compartment and in RACP cells, direct damage from oxidants increases greatly. In addition, glutathione deficiency in leukocytes (a type of RACP cell) has been shown to cause increased release of hydrogen peroxide [49]. Oxidant damage to lung epithelial cells from a diminished extracellular antioxidant screen reduces lung function, causing fibrosis and permitting greater adhesion of bacteria [35], [36], [50], [51], [52]. When
Impaired mucolysis
Extracellular GSH deficiency impairs mucolysis, as GSH facilitates cleavage of disulfide bonds in mucus, in much the same type of mucokinetic activity demonstrated by NAC [135]. Increased viscoelasticity of mucus further inhibits ciliary beating, and also allows for increased opportunity for bacterial colonization of the lung [136], [137], [138], [139], [140]. Interestingly, GSH depletion has also been shown to provoke mucin secretion by tracheal epithelial cells [141], and increased NFκB due
Abnormal immune response
Though GSH is well known for its antioxidant properties, and somewhat less well known for its mucolytic capability, it is not as widely known for the key role it plays in immune system regulation. Because RACP cells include leukocytes and lymphocytes, the literature in this area should be pertinent to the case of CF. The following discussion is not comprehensive, but rather serves to outline the broad parameters of the topic.
First, as noted in the introduction, the redox status of GSH is the
Decreased nitric oxide production and availability
NO, an important factor in cell signaling, pathogen killing, and smooth muscle relaxation, is profoundly affected by GSH system function. In the extracellular compartment, GSH deficiency will result in less extracellularly produced GSNO (S-nitrosoglutathione, which is produced both extracellularly and intracellularly), an important source of NO. In RACP cells (and perhaps non-RACP cells if depressed intracellular GSH:GSSG ratios were present in them), a variety of noteworthy consequences
Observation: the list of effects of GSH deficiency resembles the list of key pathological events in CF
For those familiar with CF pathology, the previous inventory of known effects of GSH deficiency should have seemed very familiar. For those unfamiliar with CF, we present Table 1, which identifies which known associations of GSH deficiency have also been found to be present in CF disease. We are not suggesting that GSH deficiency, but not other negative effects of CFTR mutation, produce each of the following pathological consequences. (To give but one example, pancreatic insufficiency in most
Hypothesis: GSH deficiency in CF is a primary, not a secondary effect of CFTR mutation, and plays a critical role in the pathogenesis of CF
A question has arisen over whether GSH deficiency in CF is merely a by-product of increased oxidant stress and pathogen burden [313]. Recent research allows us to assert that GSH deficiency in CF, though it may be aggravated over time by higher oxidant stress in CF, is nevertheless caused in the first place by the CFTR mutation itself. For this hypothesis to be valid, certain empirical phenomena should be present. We will examine two such expected phenomena and assess the evidence confirming
CF cells without redundant anion transport channels at the apical surface will have markedly impaired GSH efflux
This impairment will lead to extracellular GSH deficiency, which should start out small and be progressive over time. To underscore that the CFTR mutation is at work, all other components of the GSH system should be functional in CF. Evidence:
Puzzles solved
We do not claim that glutathione system dysfunction is the only initiator of pathology in CF. Neither do we suggest that such dysfunction is the primary cause of CF pathology. We merely assert that it is an important part of the pathological picture in CF, sometimes causing certain pathological effects, sometimes aggravating pathology caused by other factors. A diagram summarizing this hypothesis can be found in Fig. 2. In the absence of all desirable empirical evidence, strong warrant for a
Therapeutic implications
Should this theoretical framework be validated by further empirical research, therapeutic implications present themselves. These therapies would not be cures, as only genetic therapy holds that promise, and these therapies could not substitute for other useful pharmacologic therapies, such as DHA supplementation [97]. Though our focus here is on exogenous GSH augmentation, it should be noted that any intervention that increases anion transport would also positively affect the GSH system
Intravenous administration
Intravenous GSH, with the GSH as a sodium salt, has been used to treat chemical or radiation poisoning, as well as to treat diabetes and Parkinson’s disease [73], [348]. Intravenous GSH has been shown to raise not only blood levels of GSH, but also ELF GSH [340].
Oral administration
The oral ingestion of GSH has often been overlooked as an effective route of augmentation of extracellular GSH for a number of reasons. First, the unique CFTR-derived problem of GSH efflux is not present in other diseases, so simple provision of additional cysteine is sufficient to increase GSH levels in those other diseases. The second reason centers around the dispute over whether GSH is cleaved or destroyed in the digestive tract, or whether GSH can be taken up intact from the duodenum and
Inhalation
There have been eight in vivo studies of inhaled GSH, one a murine study and the other seven human studies [340], [341], [342], [343], [344], [345], [346], [347]. Human subjects ranged in age from 4 years old on up to mature adulthood. Only one small in vivo study of seven subjects has specifically examined CF patients [347]. This study found that certain inflammatory markers significantly decreased after 3 d use of inhaled reduced glutathione. In addition, one in vitro study using CF sputum
Conclusion
New research suggests that the CFTR defect associated with cystic fibrosis also causes abnormal GSH transport in non-RACP cells. Thus, the genetically defective CFTR appears to establish the foundation of a progressive GSH deficiency in the extracellular milieu and in RACP cells, including immune system cells. A diminished extracellular antioxidant shield, increased mucus viscoelasticity, and exaggerated inflammation coupled with ineffective immune response result. As inflammation becomes
Acknowledgements
The author would like to thank Milton G. Smith, M.D., for his assistance. This paper is dedicated to the memory of Richard Andrew Young.
References (358)
- et al.
Early bacteriologic, immunologic, and clinical courses of young infants with cystic fibrosis identified by neonatal screening
J. Pediatr.
(1991) - et al.
Nitric oxide synthase activity is elevated in inflammatory lung disease in humans
Eur. J. Pharmacol.
(1995) - et al.
Nasal and exhaled nitric oxide is reduced in adult patients with cystic fibrosis and does not correlate with cystic fibrosis genotype
Chest
(2000) Glutathionein defence of the lung
Food Chem. Toxicol.
(1999)- et al.
Systemic and pulmonary oxidative stress in idiopathic pulmonary fibrosis
Free Radic. Biol. Med.
(1999) - et al.
Glutathione deficiency and human immunodeficiency virus infection
Lancet
(1992) - et al.
Alveolar glutathione fluid decreases in asymptomatic HIV-seropositive subjects over time
Chest
(1997) - et al.
Chrysolite-mediated imbalance in the glutathione redox system in the development of pulmonary injury
Toxicol. Lett.
(1999) - et al.
Buthionine sulfoximine-induced glutathione depletion. Its effect on antioxidants, lipid peroxidation and calcium homeostasis in the lung
Biochem. Pharmacol.
(1995) - et al.
Mitochondrial glutathione depletion in alcoholic liver disease
Alcohol
(1993)
Effect of liver cirrhosis and age on the glutathione concentration in the plasma, erythrocytes, and gastric mucosa of man
Free Radic. Biol. Med.
Role of glutathione and oxidative stress in phalloidin-induced cholestasis
J. Hepatol.
Glutathione depletion alters hepatocellular high-energy phosphate metabolism
J. Surg. Res.
Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes
Brain Res.
Toxicity of low levels of methylglyoxaldepletion of blood glutathione and adverse effect on glucose tolerance in mice
Toxicol. Lett.
Modulation of oxidant status by meloxicam in experimentally induced arthritis
Pharmacol. Res.
Phenytoin-induced glutathione depletion in rat peripheral nerve
Free Radic. Biol. Med.
Replenishment of glutathione levels improves mucosal function in experimental acute colitis
Lab. Invest.
Gastric mucosal damage in experimental diabetes in ratsrole of endogenous glutathione
Gastroenterology
Role of lipid peroxidation and antioxidants in gastric mucosal injury induced by hypoxanthine-xanthine oxidase system in rats
Free Radic. Biol. Med.
Low levels of glutathione in endoscopic biopsies of patients with Crohn’s colitisthe role of malnutrition
Clin. Nutr.
Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells
Arch. Biochem. Biophys.
Glutathione-dependent factors and inhibition of rat liver microsomal lipid peroxidation
Free Radic. Biol. Med.
Macrophage glutathione content and glutathione peroxidase activity are inversely related to cell-mediated oxidation of LDLin vitro and in vivo studies
Free Radic. Biol. Med.
Cytotoxicity and apoptosis produced by arachidonic acid in Hep G2 cells overexpressing human cytochrome P4502E1
J. Biol. Chem.
Oxidant-protease interaction in the lungprospects for antioxidant therapy
Chest
Glutathione permeability of CFTR
Am. J. Physiol.
Abnormal glutathione transport in cystic fibrosis airway epithelia
Am. J. Physiol.
Early pulmonary inflammation in infants with cystic fibrosis
Am. J. Respir. Crit. Care Med.
The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis
Pediatr. Pulmonol.
Current understanding of the inflammatory process in cystic fibrosisonset and etiology
Pediatr. Pulmnol.
Early inflammation is a hallmark of proximal and distal human CF fetal airway xenografts
Pediatr. Pulmonol. Suppl.
Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients
Am. J. Respir. Crit. Care Med.
Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis
J. Infect. Dis.
Distinct sputum cytokine profiles in cystic fibrosis and other chronic inflammatory airway diseases
Eur. Respir. J.
Activation of NFKB in CF and normal respiratory epithelial cells
Pediatr. Pulmonol. Suppl.
Effects of CFTR mutations on epithelial cytokine expression
Pediatr. Pulmonol. Suppl.
Constitutive secretion of IL-8 by cystic fibrosis airway epithelial cells is suppressed by functional CFTR
Pediatr. Pulmonol. Suppl.
Bronchoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation
Am. J. Respir. Crit. Care Med.
Acquisition of Pseudomonas aeruginosa in young infants
Pediatr. Pulmonol. Suppl.
CF microbiology in young children
Pediatr. Pulmonol. Suppl.
Defective apoptosis of lung cells expressing mutant CFTR after infection with Pseudomonas aeruginosa
Pediatr. Pulmonol. Suppl.
Exhaled nitric oxide in paediatric asthma and cystic fibrosis
Arch. Dis. Child.
Increased nitric oxide in the exhaled air of normal human subjects with upper respiratory tract infections
Eur. Respir. J.
Elevated levels of exhaled nitric oxide in bronchiectasis
Am. J. Respir. Crit. Care Med.
Flow-dependency of exhaled nitric oxide in children with asthma and cystic fibrosis
Eur. Respir. J.
Decreased concentration of exhaled nitric oxide (NO) in patients with cystic fibrosis
Pediatr. Pulmonol.
Reduced upper airway nitric oxide in cystic fibrosis
Arch. Dis. Child.
Airway nitric oxide in asthmatic children and patients with cystic fibrosis
Eur. Respir. J.
Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide
Thorax
Cited by (146)
Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas
2022, Advances in Redox ResearchInvestigation of thiolysis of 4-substituted SBD derivatives and rational design of a GSH-selective fluorescent probe
2021, Organic and Biomolecular ChemistryA bifunctional fluorescent probe for simultaneous detection of GSH and H<inf>2</inf>S<inf>n</inf> (n > 1) from different channels with long-wavelength emission
2021, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCase report: Three adult brothers with cystic fibrosis (delF508-delF508) maintain unusually preserved clinical profile in the absence of standard CF care
2021, Respiratory Medicine Case Reports