High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis

Abstract

Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high oxidant producers, airway neutrophils contribute largely to the systemic redox imbalance seen in CF. In turn, this chronic and profound imbalance can impact circulating neutrophils before their migration into airways. Indeed, in 18 CF patients with stable disease, blood neutrophils were readily deficient in the pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls). In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently, neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as was the number of airway neutrophils actively releasing elastase-rich granules (P = 0.005), as measured by flow cytometry. Pulmonary function measures were not improved, as expected with short-term treatment. After excluding data from subjects without baseline airway inflammation, positive treatment effects were more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

Fig. 1.

The basal GSH deficiency in blood neutrophils from stable CF patients is ameliorated by short-term high-dose oral NAC. Blood neutrophils from 18 CF patients in stable condition were assessed at baseline for fluorescent GSB (shown in median fluorescence intensity, MFI), revealing a significant decrease compared with 9 healthy controls. CF patients were reassessed after 4-week treatment with high-dose oral NAC, showing a significantly increase in GSB MFI. Individual data are shown in box plot (median line in box delimited by 25th and 75th quantiles, ± 1.5 × interquartile range, delimited by whiskers; see Materials and Methods).

Fig. 2.

Short-term high-dose oral NAC decreases neutrophil count in CF airways. Airway neutrophil count (expressed in absolute count) follows a logarithmic distribution in CF patients, reflecting the self-amplifying characteristic of CF airway inflammation. Upon NAC treatment, airway neutrophil count was markedly reduced in patients, even more so when the three patients with baseline airway neutrophil count in the normal range were excluded from the analysis (Table 1). Identical results were obtained when neutrophil count was expressed in relative numbers per unit of sputum volume or weight. Data are shown as box plots (see Materials and Methods).

Fig. 3.

Short-term high-dose oral NAC decreases elastase activity in CF airways. Elastase activity (measured by a specific enzymatic assay; see Materials and Methods) is the best predictor of CF pulmonary function (data not shown). Baseline airway elastase activity was below the measurable range in 9 of 9 healthy controls and only 1 of 18 CF patients included in our study. Upon NAC treatment, 2 of 18 patients were now below the measurable range, and elastase activity was significantly decreased in the patient group as a whole. Data are shown as box plots (see Materials and Methods).

Fig. 4.

Short-term high-dose oral NAC decreases the overactive release of elastase-rich granules by live CF airway neutrophils, not at the single-cell level, but in aggregate. (A) Live nonapoptotic neutrophils from CF compared with healthy airways show an overactive baseline release of elastase-rich primary granules, reflected by a markedly increased percentage of cells with high surface expression of CD63 (marker specific for primary granules, expressed at the surface upon granule fusion with the plasma membrane). High-dose oral NAC, in this short-term trial, did not inhibit this cellular defect. (B) The physiologically relevant compound index of elastase-rich granule release (percentage CD63^hi neutrophils × airway neutrophil count) was significantly decreased by NAC treatment. Data are shown as box plots (see Materials and Methods).

Fig. 5.

A biphasic dysregulation model for CF airway inflammatory disease. We propose that inflammatory disease in CF occurs in two distinct steps, which may be targeted by different treatments. The first step, featuring the abnormal recruitment of neutrophils from the systemic circulation to the airways, may be efficiently countered by high-dose oral NAC treatment. The second step in this self-amplifying inflammatory process is the functional dysregulation of neutrophils once they have reached CF airways. We have demonstrated in a related study (R.T., R.B.M., C.K.C., Leonore A. Herzenberg, and Leonard A. Herzenberg, unpublished work) that this dysfunctional regulation features not only an abnormal death and postnecrotic leakage of neutrophil by-products, as generally assumed, but also an upstream overactive release of elastase-rich granules by live nonapoptotic airway neutrophils. High-dose oral NAC in the long term or other drugs may target this defect.