Hypothesis paper
Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation

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Abstract

Though the cause of cystic fibrosis (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. Recent research findings have noted that the CFTR channel is not only permeant to chloride anions, but other, larger organic anions, including reduced glutathione (GSH). This explains the longstanding finding of extracellular GSH deficit and dramatically reduced extracellular GSH:GSSG (glutathione disulfide) ratio found to be chronic and progressive in CF patients. Given the vital role of GSH as an antioxidant, a mucolytic, and a regulator of inflammation, immune response, and cell viability via its redox status in the human body, it is reasonable to hypothesize that this condition plays some role in the pathogenesis of CF. This hypothesis is advanced by comparing the literature on pathological phenomena associated with GSH deficiency to the literature documenting CF pathology, with striking similarities noted. Several puzzling hallmarks of CF pathology, including reduced exhaled NO, exaggerated inflammation with decreased immunocompetence, increased mucus viscoelasticity, and lack of appropriate apoptosis by infected epithelial cells, are better understood when abnormal GSH transport from epithelia (those without anion channels redundant to the CFTR at the apical surface) is added as an additional explanatory factor. Such epithelia should have normal levels of total glutathione (though perhaps with diminished GSH:GSSG ratio in the cytosol), but impaired GSH transport due to CFTR mutation should lead to progressive extracellular deficit of both total glutathione and GSH, and, hypothetically, GSH:GSSG ratio alteration or even total glutathione deficit in cells with redundant anion channels, such as leukocytes, lymphocytes, erythrocytes, and hepatocytes. Therapeutic implications, including alternative methods of GSH augmentation, are discussed.

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.

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