Evaluation of thiol-based antioxidant therapeutics in cystic fibrosis sputum: Focus on myeloperoxidase

Abstract

Neutrophil-dependent reactions catalysed by myeloperoxidase (MPO) are thought to play important roles in the pulmonary pathobiology of cystic fibrosis (CF). Aerosolized thiol antioxidants such as glutathione (GSH) and N-acetylcysteine (NAC) are currently being utilized as therapeutics to modify CF respiratory tract oxidative processes. This study hypothesized that MPO in CF airway lining fluids may be a target of such therapeutics. MPO activity in sputum from 21 adult CF patients was found to be inversely associated with lung function (FEV(1)). In contrast, systemic inflammation (assessed by plasma C-reactive protein) was not correlated with lung function. Ex vivo studies revealed that GSH and NAC effectively scavenged N-chloramines in sputum and inhibited sputum MPO activity with potency exquisitely dependent upon MPO activity levels. Detailed kinetic analyses revealed that NAC and GSH inhibit MPO by distinct mechanisms. Activation of the key pro-inflammatory transcription factor NF-κB in cultured HBE1 cells was inhibited by GSH. The findings reveal that MPO activity and its reactive products represent useful predictors of the doses of inhaled thiol antioxidants required to ameliorate airway oxidative stress and inflammation in CF patients and provide mechanistic insight into the antioxidative/anti-inflammatory mechanisms of action of GSH and NAC when administered into the CF lung.

Figure 1. Sputum MPO but not plasma CRP levels in CF patients correlate with lung function

(A) An inverse correlation between CF sputum MPO levels and FEV₁ (% predicted) (n=21, r=−0.59, P=0.004) was observed. (B) No correlation was observed between plasma CRP levels and FEV₁ (% predicted) (n=17, r=0.033, P=0.9). Spearmann’s correlation coefficient was used to measure correlation coefficient between samples.

Figure 2. Sputum MPO levels in CF patients

CF patients had varied levels of sputum MPO levels and were categorized into low, moderate and high groups comprising 27%, 33% and 39% of total patients. Values are presented as mean ± SEM.

Figure 3. Correlation between sputum MPO and N-chloramine levels in CF patients

No correlation was observed between sputum MPO and chloramine levels (n=9, r=0.0005). Spearmann’s correlation coefficient was used to measure correlation coefficient between samples.

Figure 4. Inhibition of CF sputum MPO activity by GSH and NAC as represented by IC₅₀ values

CF sputum was incubated with different concentrations of antioxidants and MPO-activity measured as described in Materials and Methods. MPO levels were determined from the standard graph using purified human MPO. The measurements were compared to untreated samples which served as controls. Antioxidant IC₅₀ values of individual sputum samples were plotted against respective MPO levels. The figure suggests that the higher the MPO levels, the more the antioxidant needed for MPO inhibition.

Figure 5. GSH and NAC inhibit MPO by distinct mechanisms

Double reciprocal plot of initial rate of MPO-catalyzed oxidation of TMB vs. its concentration in the presence of (A) 0, 0.2, 0.4, 0.6 or 0.8 μM GSH and (B) 0, 0.4, 0.5, 0.6, 0.7 or 0.8 μM NAC. Inhibition of MPO-catalyzed oxidation of TMB by GSH was observed to occur by competitive mode, whereas NAC showed a non-competitive mode of inhibition as demonstrated by data from reciprocal plots of velocity of reaction (1/V) against TMB concentrations (1/[TMB]).

Figure 6. GSH and NAC scavenge N-chloramines in CF sputum

Sol-phase of CF sputum was incubated with/without 2 μM of GSH or NAC for 30 minutes and chloramine levels measured as described in Materials and Methods. N-Chloramine levels were measured by a standard curve using hypochlorous acid. Fig 6A and B shows sputum N-chloramine levels before and after treatment with GSH or NAC, respectively. GSH treatment lowered N-chloramine levels by 62 ± 11 %, whereas NAC treatment lowered N-chloramine levels by 80 ± 11%.

Figure 7. GSH inhibits CF sputum-induced NF-κB activation

HBE1 cells were starved of growth factors and treated with CF sputum (sol-phase) (10 μl/ml of media). Nuclear and cytosolic protein fractions were extracted at 0, 30 and 60 minutes. NF-κB activation was analysed by western blot assay. (A) Nuclear translocation of NF-κB subunit, p65 and degradation of cytosolic IκB-α suggesting CF sputum (sol-phase) mediated NF-κB activation. β-actin and p84 served as loading controls for cytosolic and nuclear protein fractions respectively. (B) Nuclear translocation of p65 in TNF-α treated (20 μg/ml) HBE1 cells which served as controls. (C) HBE1 cells when treated with CF sputum (sol-phase) along with GSH (1 mM) did not show p65 nuclear translocation suggesting inhibition of NF-κB activation. The blots shown are a representative of 3 independent experiments. Densitometric analysis was performed for each blot, and the quantitative data illustrated in graphs to the right of the figure.

Figure 8. Proposed mechanisms by which GSH and NAC inhibit MPO-dependent reactions in the CF airway

(A) Scavenging of N-chloramines. (B) Scavenging of organic radicals produced by MPO (non-competitive inhibition). (C) GSH-dependent inhibition of substrate binding to MPO (competitive inhibition).