Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa

Abstract

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.

Figure 1. CFTR mRNA expression in human in vitro differentiated macrophages from non-CF donors.

Panel A: Mean relative quantitiy (RQ) of CFTR mRNA in monocytes (CD14⁺) and monocyte derived macrophages (MDM) calibrated versus the H441 cells (low control); each symbol represents a single donor. Panel B: CFTR mRNA in MDM (mean RQ) calibrated versus parental monocytes. Each bar represents a single donor, whiskers above and below are the RQ-max and RQ-min, respectively.

Figure 2. Confocal microscopy of the cellular localization of CFTR in control and F508del human in vitro differentiated macrophages.

(A) Localization of CFTR in macrophages from non-CF (top row) and del508F homozygous CF individuals (bottom row). Permeabilized macrophages were stained with the polyclonal anti-CFTR antibody (H-182) and with Alexa Fluor 488-conjugated secondary antibody. Nuclei were counterstained with DAPI. Scale bars = 20 micron. Isotype negative controls are shown in the insets. (B) Permeabilized macrophages were stained with the anti-CFTR and the anti-LAMP1 antibodies; the secondary antibodies were Alexa Fluor-594 and Alexa Fluor-488 conjugated F(ab)2 IgG. Scale bar = 10 micron.

Figure 3. Whole-cell patch clamp of cAMP-evoked Cl⁻ current in human peripheral macrophages.

(A) representative currents in basal condition (basal) or in response to the administration the cAMP-containing cocktail (cAMP), recorded on voltage-clamped macrophages. cAMP-evoked currents were blocked in the presence of the specific inhibitor CFTR_inh-172 (10 µM), added to the bath solution (cAMP/CFTR_inh-172). Currents were recorded in 200 ms voltage steps from −110 to +110 mV with 10 mV increments from a holding potential of −40 mV. (B) Averaged current/voltage relationship in basal conditions (basal, n = 9), in the presence of a cAMP-containing cocktail (cAMP, n = 16) or cAMP-containing cocktail plus 10 µM CFTR_inh-172 (cAMP/CFTR_inh-172, n = 7). (C) Current densities obtained at +110 mV in the three described experimental conditions, in parentheses the number of recorded cells are shown. Data reported in panel (B) and (C) are means±S.E.

Figure 4. Surviving bacteria within human macrophages.

Percentage of intracellular live bacteria rescued from P. aeruginosa infected macrophages two (2 hr) and four (4 hr) hours after infection. 100% refers to bacteria recovered at the end of infection (t0). Samples: non-CF, healthy donor macrophages (N = 12); CF, macrophages from cystic fibrosis patients (N = 15). Each symbol represents a single individual, the line is the median percentage of live bacteria.

Figure 5. Summary data of live intracellular bacteria.

The percentage of surviving bacteria two (t2) and four (t4) hours after infection with respect to live bacteria recovered at the end of infection (t0). Data are expressed as box plots representing, the 25 and 75 percentiles, median, minimal and maximal values. Statistical analysis: non-CF vs CF, P = 0,7697 and P = 0,0359 two and four hours after infection, respectively.