The lactoperoxidase system links anion transport to host defense in cystic fibrosis

Abstract

Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN-) to function and epithelia use CFTR to concentrate SCN- at the apical surface. To test whether CFTR mutations result in impaired LPO-mediated host defense, CF epithelial SCN- transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN- in the apical compartment. The lower CF [SCN-] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO-mediated defense and cannot be compensated by LPO or H2O2 upregulation.

Figure 1. Thiocyanate transport in CF airway epithelium is defective

Panel A. ¹⁴C-SCN⁻ (70 μM) was added to the basolateral media of non-CF and CF cultures for 18 – 24 h and stable unstimulated transport rates measured as described previously [17]. Non-CF cultures showed a 4-fold higher rate of transport using 18 from three non-CF and three CF individuals (n= 18, p < 0.0001). Panel B. ¹⁴C-SCN⁻ (70 μM) was added to the basolateral media of cultures for 18 – 24 h. The ¹⁴C -SCN⁻ accumulated on the apical surface was collected in a PBS wash and measured by liquid scintillation counting (n = 18 cultures each from three non-CF and three CF, p < 0.007).

Figure 2. PKA stimulation increases thiocyanate transport in non-CF but not in CF airway epithelia

To initiate the experiments, apical surfaces of the cultures incubated overnight in ¹⁴C-SCN⁻ were rapidly washed three times. Additional PBS aliquots were placed on the apical surface for sequential 2 min washes and ¹⁴C-SCN⁻ was determined by liquid scintillation counting. cAMP stimulation of SCN⁻ efflux was demonstrated by adding dibutyryl cAMP and forskolin to the PBS washes (18 min, indicated by the bar at top). The stimulated transport was inhibited by addition of glibenclamide (28 min) to washes containing dibutyryl cAMP and forskolin. SCN⁻ flux is expressed in nMoles of SCN⁻/h normalized to 1 cm² filter area. In the CF culture, SCN⁻ transport was greatly diminished and not stimulated by cAMP. Bars at the top of graphs indicate the presence of PBS alone, dibutyryl cAMP and forskolin (PKAst), or dibutyryl cAMP, forskolin and glibenclamide (Glib). Panels A–C show single experiments comparing pairs of cultures obtained from three different CF patients and three different non-CF patients.

Figure 3. Thiocyanate-mediated bacterial killing in ALI culture secretions

Non-CF (solid bars, n = 6 cultures on separate days) and CF (open bars, n = 4 cultures on separate days) ALI cultures were incubated in the presence or absence of SCN⁻ in the basolateral media. Apical surfaces were washed with PBS and combined for assay of antibacterial activity as described in Methods. Assays were performed with no additions (panel A) or with LPO and H₂O₂ added to the washes (panel B). LPO and H₂O₂ did not increase the antibacterial activity of the washes in the absence of SCN⁻, however in the presence of basolateral SCN⁻ (panel C), only non-CF culture washes showed antibacterial activity. Addition of SCN⁻ to washes of CF cultures restored the antibacterial activity and increased the activity of non-CF culture washes (panel D).

Figure 4. LPO activity varies with thiocyanate concentration and with pH

An arithmetic model of LPO activity using the kinetic constants published by Pruitt et al. [31] calculated the expected LPO activity with [SCN⁻] between 0.0001 M and 0.01 M (left panel) at pH 7.0, pH 6.5 and pH 6.0. The right panel shows an expanded view between 0 and 0.5 mM (0.4 mM being the concentration found in human airway secretions).

Figure 5. Defective thiocyanate transport predicts a loss of LPO antibacterial activity

Computational modeling of LPO enzymatic activity in airway surface liquid shows predicted changes in response to altered conditions in CF airways, i.e. decreased volume and decreased SCN⁻ transport rates. Initial conditions (shown in Table 1) were chosen based on published values, except [H₂O₂] that was arbitrarily set to zero. The H₂O₂ production rate was set to balance SCN⁻ transport rates in normal cultures. Integration steps were 0.1 sec. Shown below are values of non-CF (solid line) and CF (dotted line) conditions. Changes in [SCN⁻], (panel A) and [H₂O₂], (panel B) include consumption by LPO catalysis and appearance due to production or transport. Differentiation of OSCN⁻ product concentration with respect to time is shown in panel C. After 300 sec, LPO concentration was reset to a level 10 fold higher than the CF initial [LPO] to mimic possible upregulation of the enzyme concentration in response to lower SCN⁻ transport. Despite a higher initial CF [LPO] to accommodate decreased CF ASL volume, computation showed that SCN⁻ could not be replaced fast enough given the transport defect in CF. An increase in [LPO] (300 sec) was unable to restore LPO reaction rate in CF due to faster consumption of SCN⁻. The model predicts a loss of antibacterial activity in CF airways that cannot be compensated by increased LPO or H₂O₂.