If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, OH, United StatesDivision of Pulmonary Medicine, Nationwide Children's Hospital, Columbus, OH, United States
There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes.
Methods
Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds.
Results
AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells.
Conclusions
The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.
Cystic fibrosis (CF) is a systemic disorder characterized by recurrent sino-pulmonary infections which leads to chronic morbidity and increased mortality [
]. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause mucus stasis and impairments in immunologic function that enable the development of persistent bacterial infections [
]. Multi-drug resistant (MDR) bacteria remain a major cause of chronic morbidity and mortality in patients with CF due to the inability of CF patients to clear these chronic infections and the resultant repeated courses of inhaled and systemic antibiotics. Despite an increase in the number of MDR pathogens in CF, few new antibiotics are under development due to the high costs associated with this process. Therefore, there remains an urgent need for novel therapeutics to replace or synergize with current antibiotics in the treatment of MDR bacteria in CF.
Recent studies suggest that poor bacterial clearance in CF is due to a combination of immunological defects and bacterial resistance mechanisms [
Human cystic fibrosis macrophages have defective calcium-dependent protein kinase C activation of the NADPH oxidase, an effect augmented by burkholderia cenocepacia.
]. Autophagy is an example of an immunological mechanism that is defective in CF. In normal cells functional autophagy results in lysosomal degradation of ingested bacteria and augments innate responses to intracellular pathogens, but autophagy is reduced in CF macrophages and epithelial cells and results in increased bacterial load and proteostasis [
]. AR-12 is the lead molecule of a compound series with pleiotropic effects that include apoptosis and autophagy induction in various malignant cells and is active against intracellular pathogens such as Francisella tularensis and Salmonella enterica [
]. Our lab and others have demonstrated the potential of autophagy-based therapies in the treatment of antibiotic-resistant CF lung infections such as those caused by Burkholderia cenocepacia, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus fumigatus, and Non-Tuberculous Mycobacterium [
], which may in part explain its heightened virulence in CF. Therefore, we aimed to test the effects of AR-12 and its derivatives against MDR pathogens such as B. cenocepacia in CF. We hypothesized that AR-12 or a derivative would increase bacterial killing in CF phagocytes via enhanced autophagy.
2. Material and methods
2.1 Ethics statement
Human subject recruitment was approved by the Institutional Review Board at Nationwide Children's Hospital. Study subjects performed written consent for procedures if of legal age, and children provided written assent and a parent or guardian of any child participant provided informed consent on their behalf.
2.2 Reagents
AR compounds were obtained from the Ohio State University ARNO Therapeutics drug discovery program and used at concentrations ranging from 1 to 5 μM for all experiments. The structure and dosing of these compounds was previously described [
]. CFTR modulators VX-809 and VX-661 were used at 5 μM (Selleck Chem).
2.3 Bacterial killing assay
Two hundred μl of 0.1 optical density bacteria in Luria Bertani (LB) broth were added to 96 well plates with or without desired treatments. The control wells contained LB media only. Optical density at 600 nm was measured on a Synergy H1 Hybrid Reader spectrophotometer (Biotech Instruments) over 24 h with shaking every 15 min at 37 °C. Antibiotics used included gentamicin 50 μg/ml (Invitrogen, 3560), meropenem trihydrate 50 μg/ml (Sigma, M2574), ciprofloxacin 16 μg/ml (Sigma, 17,850), ceftazidime hydrate 100 μg/ml (Sigma, C3809), linezolid 40 μg/ml (Sigma, PZ0014) and penicillin/streptomycin 1% (Life Technologies, 15,140,122).
2.4 Bacterial strains/phagocyte infection
Macrophages were infected with a RFP-expressing B. cenocepacia strain k56–2 and/or a methicillin-resistant Staphylococcus aureus (MRSA) isolate obtained from a CF patient's sputum. The B. cenocepacia strain is representative of an epidemic clinical strain from the ET12 lineage [
]. Neutrophils were infected with a MDRP. aeruginosa isolate obtained from a CF patient's sputum. Bacteria were reproducibly grown in LB media over 24 h. Colony forming unit (CFU) analysis was performed as previously described [
]. Antibiotics (ceftazidime, gentamicin, ciprofloxacin, meropenem, and linezolid) were used at concentrations as listed for the direct bacterial killing assay. Recovered bacteria were quantified by plating serial dilutions on LB agar plates and analyzed for CFUs.
2.5 Phagocyte isolation
CF patients homozygous for the F508del mutation and non-CF healthy controls donated heparinized blood samples. Subjects were excluded if using chronic immunosuppressants, CFTR modulators, or had a history of transplantation. Peripheral monocytes were separated from whole blood using Lymphocyte Separation Medium (Corning, 25–072-CV). Isolated monocytes were re-suspended in RPMI (Gibco, 22,400–089) plus 10% human AB serum (Corning, 35–060-Cl) and differentiated for 5 days at 37 °C into monocyte-derived macrophages (MDMs) [
Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors.
]. MDMs were then placed in a monolayer culture, and infected at bacterial multiplicity of infection (MOI) of 10. CF neutrophils were isolated from human blood and purified by negative selection. Briefly, blood was transferred to 50 ml conical tube and 50 μl of an antibody cocktail added per ml of blood (Stemcell technologies, Vancouver, BC, Canada), plus 50 μl of magnetic beads per ml of blood. The sample was incubated 5 min at RT, then PBS-EDTA was added up to 50 ml, the tube was placed in the magnet for 10 min, the supernatant was transferred to a new tube and magnetic beads were added at the same amount of the previous step. The sample was placed 5 min in the magnet and the supernatant was collected. The cells were centrifuged at 1600 rpm, and re-suspended in 1 ml of HBSS plus 1% of FBS before further experimentation.
2.6 Macrophage and epithelial cell viability/apoptosis
MDMs were plated at a density of 2 × 106/ml in 12 well plates. MDMs were left uninfected or infected with B. cenocepacia at a MOI of 5. AR-13 was added to indicated wells at a concentration of 5 μM. Viability assay was performed by flow cytometry and fluorescence-activated cell sorting analysis using APC Annexin V (Biolegend, 640,920) and DAPI (Molecular Probes, D1306). MDMs were detached by Accutase solution, collected, washed, and re-suspended in 100 μl of Annexin V Binding Buffer (Biolegend 422,201), and then stained with 5 μl of Annexin V and 0.4 μg/ml DAPI for 15 min at room temperature in the dark. The percentages of viable and apoptotic cells was assessed using flow cytometry (BD LSR 11 Flow Cytometer; BD Bioscience). Cytotoxicity of AR compounds on airway epithelial cells was evaluated by measuring lactate dehydrogenase (LDH) release. LDH assay was performed using TOX-7 kit (Sigma-Aldrich, Saint Louis, MO) following the manufacturer's protocol. Briefly, reactions were measured at 490 nm absorbance and percentage of cell death was compared to the positive control designated 100% cell death obtained with 1% Triton X-100 as previously described [
Cells were recovered from the nasal respiratory epithelium of patients homozygous for the F508del mutation via gentle brushing with a cytology brush. Cells are removed from the brush by agitation and cultured on irradiated fibroblast feeder layers in medium as recently published [
]. At 80% confluence, passage 1 epithelial cells are recovered by double trypsinization and re-plated on collagen-coated transwells in proliferation medium [
]. At confluence, the cultures are transitioned to Pneumacult (Stem Cells, Inc) medium. Cultures are allowed to differentiate for 21 days and are then treated with vehicle or drug.
2.8 Transepithelial short-circuit currents
Differentiated nasal cell cultures on transwells were mounted in Ussing chambers and short-circuit currents (Isc) were recorded as previously described [
]. Briefly, Ringer's buffer (115 mM NaCl, 25 mM NaHCO3, 2.4 KH2PO4, 1.24 K2HPO4, 1.2 CaCl2, 1.2 mM MgCl2, and 10 mM d-glucose; pH 7.4) was added in the basal compartment of the Ussing chamber, whereas low chloride (1.2 mM NaCl and 115 mM Na gluconate replacing 115 mM NaCl) was added to the apical chamber. Bath solutions were vigorously stirred and gassed with 5% CO2. Solutions and chambers were maintained at 37 °C. Amiloride (100 μM) was first added to inhibit the epithelial sodium channel ENaC. Forskolin (10 μM) was then added to the apical chamber to evaluate cAMP-stimulated currents. The specific CFTR inhibitor Inh-172 (10 μM) was added to the apical side at the end of the experiment to block CFTR channels.
2.9 Immunoblotting
MDMs were infected at a MOI of 10 for immunoblotting experiments. After removal of cell supernatants and washing with PBS, plated cells were lysed in lysis buffer (1M HEPES, 1M MgCl2, 0.5M EGTA, 1M KCL, and 1% NP-40) with protease inhibitor (Roche Applied Science, 10-519-978-001). Nasal epithelial cell cultures were washed with Hams/F12 at 4 degC for 20 min on ice. The transwell membrane was then excised and immersed in 100 ul lysis buffer. Protein was recovered by two freeze/thaw cycles. Then, 30 μg of protein was separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were immunoblotted for calreticulin (Enzo Life Sciences, ADI-SPA-600-F), LC3 (Sigma, L8918- 200), Beclin-1 (Abcam, ab55878), p62 (Sigma, P0067-200), CFTR (R&D Systems, MAB 25031) and GAPDH (Santa Cruz Biotechnology). Protein bands were detected with HRP-conjugated secondary antibodies and visualization performed using enhanced chemiluminescence (ECL) reagents (Life Sciences, RPN2106).
2.10 Microscopy
One million macrophages were cultured on 12 mm glass cover slips in 24-well tissue culture plates and infected synchronously with B. cenocepacia at an MOI of 2 (an MOI of 10 renders counting of bacteria in untreated CF MDMs difficult due to clumping). Macrophage nuclei were stained blue with DAPI. LC3 was detected with LC3 antibody (Abgent, AP1805a) followed by fluorescent secondary antibodies (Molecular Probes, A11008). Microscopy was performed using an Axiovert 200M inverted epifluorescence microscope equipped with the Apotome attachment for improved fluorescence resolution and an Axiocam MRM CCD camera (Carl Zeiss Inc., Thornwood, NY). Five independent fields with at least 10 macrophages were scored for each condition. All experiments were performed in triplicate.
2.11 Statistical analysis
Statistical analyses were performed using GraphPad Prism software (version 6.1). Two sample t-tests were used for independent sample comparisons. Statistical significance was defined as a p value <.05. Age and gender matched healthy controls were used for comparison.
3. Results
3.1 AR-13 inhibits growth of CF pathogens
In order to determine if AR-12 or its related derivatives inhibit growth of drug-resistantB. cenocepacia, we determined bacterial viability in LB media alone. AR-12 and 2 derivative compounds were added to bacterial cultures for 24 h and bacterial growth assessed. Only the AR-13 derivative (N-{4-[5-(Phenanthren-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] phenyl}sulfuric diamide) demonstrated a significant inhibition of bacterial growth compared to LB-only wells (Fig 1A, B ). Due to this discovery, we then assessed for synergistic killing activity of AR-13 with antibiotics against B. cenocepacia. Antibiotics did not enhance direct killing of drug-resistantB. cenocepacia in combination with AR-13 (Fig. 1C). Next, AR-13's ability to inhibit growth of other common CF pathogens that can develop antibiotic resistance such as MRSA and P. aeruginosa was assessed. AR-13 demonstrated synergistic growth inhibition with penicillin/streptomycin and gentamicin against MRSA (Fig 1D, E). Because synergistic growth inhibition was present, a dose-response assay was performed and demonstrated the greatest efficacy of AR-13 against MRSA at 5 μM (Fig. 1F), similar to established AR-12 dosing. AR-13 did not demonstrate direct growth inhibition of P. aeruginosa alone, or in combination with antibiotics (Fig 1G, H).
Fig. 1AR-13 directly inhibits B. cenocepacia and MRSA growth. A) Screening bacterial growth inhibition assay of AR-12 (5 μM) and derivatives (5 μM) against B. cenocepacia (Bc) in media alone. Optical density was measured over 24 h, representative images shown for 3 analyses/group. B) End-point assay analysis of bacterial density at 24 h for Fig. 1A. C) End-point analysis of bacterial density at 24 h of B. cenocepacia with antibiotics alone or antibiotics plus AR-13. Optical density measured over 24 h, n = 3. D) Bacterial inhibition assay of CF clinical Methicillin-resistant Staphylococcus aureus (MRSA) isolate with antibiotics alone or antibiotics plus AR-13. Optical density measured over 24 h, representative images shown for 3 analyses/group. E) End-point analysis at 24 h of 1D. F) End-point analysis of bacterial density of MRSA in response to dose titration of AR-13 at 24 h, n = 3. G) Bacterial inhibition assay of CF Pseudomonas aeruginosa isolate with antibiotics alone or antibiotics plus AR-13. Optical density measured over 24 h, representative image presented, n = 3. H) End-point analysis at 24 h of 1G. “*” = P value <.05, “**” = P value <.01, “***” = P value <.001. Antibiotics for Fig. 1 included gentamicin 50 μg/ml (gent), meropenem trihydrate 50 μg/ml (mero), ciprofloxacin 16 μg/ml (cipro), ceftazidime hydrate 100 μg/ml (ceftaz), linezolid 40 μg/ml, and penicillin/streptomycin 1% (pen/strep). Statistical significance for Fig. 1 comparisons made by unpaired t-test.
]. Therefore, we determined the impact of AR-13 on phagocyte-mediated bacterial killing. First, a CFU assay was performed for B. cenocepacia growth in human CF and non-CF MDMs. There was no difference in bacterial growth with the addition of AR-13 in CF or non-CF MDMs (Fig. 2A ), or with antibiotics alone in CF (Fig. 2B). However, AR-13 demonstrated significant synergistic decreases in B. cenocepacia growth in CF MDMs when combined with multiple broad-spectrum gram-negative antibiotics including ceftazidime, ciprofloxacin, gentamicin, and meropenem (Fig. 2B). Similar findings were observed in non-CF MDMs (Fig. S1A). A dose-response assay demonstrated the greatest efficacy of AR-13 against B. cenocepacia in CF MDMs at 5 μM (Fig. 2C), again similar to established AR-12 dosing.
Fig. 2AR-13 enhances CF phagocyte-mediated killing of drug-resistant pathogens. A) Human CF and non-CF monocyte-derived macrophages (MDMs) were infected with B. cenocepacia (Bc) ± treatment with AR-13, n = 3. CFUs were assayed at 24 h. B) 24 h CFU assay of human CF MDMs infected with Bc and treated with antibiotics alone or antibiotics plus AR-13, n = 3. C) CFU assay of human CF MDMs infected with Bc in response to dose titration of AR-13 at 24 h, n = 3. D) 24 h CFU assay of human CF and non-CF MDMs infected with Methicillin-resistant Staphylococcus aureus (MRSA) ± treatment with AR-13, n = 3. E) 24 h CFU assay of human MDMs infected with MRSA and treated with antibiotics alone or antibiotics plus AR-13, n = 3. F) 24 h CFU assay of human CF and non-CF neutrophils (PMNs) infected with P. aeruginosa (Pa), n = 3. G) 24 h CFU assay of human CF neutrophils infected with P. aeruginosa and treated with antibiotics alone or antibiotics plus AR-13, n = 3. “*” = P value <.05, “**” = P value <.01, “***” = P value <.001. Antibiotics for Fig. 2 included gentamicin 50 μg/ml (gent), meropenem trihydrate 50 μg/ml (mero), ciprofloxacin 16 μg/ml (cipro), ceftazidime hydrate 100 μg/ml (ceftaz), linezolid 40 μg/ml, and penicillin/streptomycin 1% (pen/strep). Statistical significance for Fig. 2 comparisons made by unpaired t-test.
Next, the impact of AR-13 treatment on MRSA growth in human MDMs was assessed. AR-13 decreased MRSA bacterial load in CF MDMs (Fig. 2D, 99% reduction, log scale), but had no additional synergistic effect with antibiotics in CF (Fig. 2E) or non-CF MDMs (Fig. S1B). We then examined the impact of AR-13 on neutrophil-mediated killing of P. aeruginosa because of the known neutrophil role in Pseudomonas killing in CF [
Harnessing neutrophil survival mechanisms during chronic infection by pseudomonas aeruginosa: Novel therapeutic targets to dampen inflammation in cystic fibrosis.
]. Both CF (71% reduction) and non-CF (70% reduction) human neutrophils demonstrated decreased P. aeruginosa growth with AR-13 treatment (Fig. 2F, log scale). Further synergistic decreases in growth were not noted when CF (Fig. 2G) or non-CF (Fig. S1C) neutrophils were treated in combination with AR-13 and ciprofloxacin, gentamicin, or meropenem, although an overall decreased trend was noted for ciprofloxacin and gentamicin in both CF and non-CF neutrophils.
Finally, we assessed macrophage viability and apoptosis in the presence or absence of AR-13 to ensure that the observed increase in bacterial killing was not due to changes in cell stability. CF and non-CF MDMs were treated with AR-13 alone or in combination with B. cenocepacia infection. CF and non-CF MDMs demonstrated similar patterns of apoptosis (Fig. S2A). The percentage of apoptotic MDMs was highest during B. cenocepacia infection in both groups, with slight improvement during infection plus AR-13 treatment (Fig. S2A). There was no difference between MDMs at baseline or with AR-13 treatment alone for either CF or non-CF MDMs (Fig. S2A).
3.3 AR-13 enhances autophagy in CF macrophages
AR-13's parent compound, AR-12, has known autophagy-inducing properties [
]. Therefore, we examined AR-13's ability to induce autophagy in CF macrophages. Microtubule-associated proteins 1A/1B light chain 3A (LC3) is often used to monitor autophagosome formation [
]. During functional autophagy cytosolic LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-II, which is mobilized to the autophagosomal membrane. We analyzed the conversion of LC3-I to LC3-II, as well as the expression of a key autophagy regulating protein, Beclin-1. Western blotting demonstrated an increase in LC3-II/LC3-I expression in CF macrophages treated with AR-13 during infection in comparison to infection or treatment alone (Figs 3A, B ). The addition of bafilomycin (Figs 3A, B) or 3-MA (Figs S3A, B) to block autophagy reduced this effect. Beclin-1 expression was reduced at baseline and during infection in CF, but increased with AR-13 treatment during infection. We also measured expression of the autophagy cargo marker SQSTM1/p62, but p62 expression was unchanged throughout all CF and non-CF conditions.
Fig. 3AR-13 increases macrophage autophagy. A) LC3, p62, and beclin-1 Western blotting of CF and non-CF MDMs infected with B. cenocepacia (Bc) for 2 h with or without treatment with AR-13 or AR-13 and bafilomycin. Calreticulin is presented as a loading control. Representative images of n = 3. B) Densitometry analysis of 3A for non-CF and CF MDMs. “*” = P value <.05, “**” = P value <.01, “***” = P value <.001. Statistical significance for Fig. 3 comparisons made by unpaired t-test.
Fluorescent microscopy was then performed to corroborate Western blot findings of increased autophagosome formation (LC3-II/LC3-I conversion). CF macrophages demonstrated a 20.3% increase (55.6% total co-localization) in B. cenocepacia co-localization with LC3 after treatment with AR-13 compared to untreated CF macrophages (Fig. 4, p value = .05). This was similar to the percentage of co-localizationof B. cenocepacia with LC3 in non-CF macrophages treated with AR-13 (55.1%, p value 0.97). These results provide further evidence that AR-13 increases autophagy, similar to its parent compound AR-12 [
Fig. 4AR-13 increases autophagosome formation. A) Photomicrographs of CF and non-CF MDMs infected with B. cenocepacia (Bc) for 24 h ± treatment with AR-13. The macrophage nucleus is stained blue with DAPI, Bc is shown in red, LC3 is shown in green, and bacteria co-localized with LC3 are yellow in the merged image. Examples of co-localization are noted by white arrows. n = 3. B) Summed quantification of bacterial co-localization with LC3. Bacterial co-localization with LC3 was determined by scoring the percentage of co-localized bacteria for each experimental condition in five independent fields with at least 10 macrophages. This was performed for at least 3 individual patients per condition. Unpaired t-tests were then used to compare the means for each condition. “*” = P value <.05, “**” = P value <.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4 AR-13 increases autophagy and CFTR expression and function in CF epithelial cells
Airway epithelial cells are a major host defense against pulmonary bacterial infections. CF epithelial cells have decreased CFTR function and autophagy, which contribute to host pathology [
Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on DeltaF508 cystic fibrosis transmembrane conductance regulator.
]. We exposed well-differentiated human CF airway epithelial cells derived from nasal brushings of patients homozygous for the F508del mutation to B. cenocepacia and treated with a vehicle control (DMSO), AR-12, or AR-13 for 24 h and examined CFTR and LC3 expression. AR-13 treatment alone increased both CFTR and LC3-II expression in epithelial cells compared to untreated cells or those treated with AR-12 (Fig. 5A, B , replicate blots in Fig. S4). Collectively our MDM and epithelial studies indicate that AR-13 can increase autophagy in multiple human cell types. Treatment of epithelial cells with AR-12 or AR-13 in combination with CFTR correctors VX-809 or VX-661 did not alter expression of mature CFTR, although there was a trend towards an increase with VX-661 plus AR-13 (Fig. 5C, replicate blots in Fig. S5, VX-661 shown). We then measured CFTR-dependent currents in airway epithelial cells in response to AR-12 or AR-13 with or without CFTR correctors (VX-809, VX-661). AR-13, but not AR-12 increased CFTR function (Fig. 5D). AR-13 acted synergistically with CFTR correctors to increase CFTR-mediated currents. The greatest synergy was seen with AR-13 in combination with VX-661 (Fig. 5D, VX-661 shown). CFTR-mediated currents from CF epithelial cells treated with VX-661/AR-13 remained lower than non-CF currents (Fig. 5D). Examples of CFTR-dependent current tracings for the experiments are displayed in Fig. S6A. Amiloride-sensitive currents were also responsive to AR-13 in combination with VX-661 (Fig. S6B, S6C). Lastly, we used the LDH assay to determine if AR compounds altered epithelial cell viability. As observed for MDMs, AR treatment did not alter epithelial cell viability. There was no difference in CF epithelial cell LDH release in response to AR-13 compared to no treatment (Fig. S2B).
Fig. 5AR-13 increases autophagy and CFTR function in human CF airway epithelial cells. A) Well-differentiated human CF F508del/F508del airway epithelial cells were derived from nasal brushings. At passage 2 the cells were differentiated using the air-liquid-interface method. On differentiation day 21, the well differentiated cells were treated with DMSO, AR-12, or AR-13 for 48 h. LC3 and CFTR were assayed by Western blotting. Representative images of n = 4. B) Corresponding densitometry for 5A normalized to calreticulin or GAPDH for all replicates. C) Well-differentiated human CF F508del/F508del airway epithelial cells were treated with VX-661 for 48 h ± AR-12 or AR-13 and Western blotting performed for CFTR. Corresponding densitometry of all replicates normalized to GAPDH. Blots shown in Fig. S5. D) CFTR-mediated short-circuit currents were measured using Ussing chambers on human well-differentiated non-CF and CF F508del/F508del airway epithelial cells. Cells were treated with AR-12, AR-13, VX-661, or a combination of two of the compounds. n = 3–6. ΔIsc represents forskolin-induced change in current. “*” = P value <.05, “**” = P value <.01, “***” = P value <.001. Statistical significance for Fig. 5 comparisons made by unpaired t-test.
There remain no known effective treatments for B. cenocepacia and other MDR infections in patients with CF due to intrinsic bacterial resistance mechanisms and defective host killing that involves macrophages. Additionally, MDR pathogens from the Burkholderia cepacia complex are becoming increasingly recognized causative agents of nosocomial acquired infections in patients without CF [
]. The lack of effective Burkholderia treatments combined with increased nosocomial Burkholderia infections indicate an urgent need for development of new strategies to eradicate these pathogens. Importantly, herein we demonstrate the effectiveness of a novel compound, AR-13, which works in synergy with antibiotics to kill B. cenocepacia, improve macrophage and epithelial cell autophagy, and increase CFTR function.
The recent role of CFTR modulators to improve outcomes for most patients has been clearly demonstrated [
Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis.
Restoring cystic fibrosis transmembrane conductance regulator function reduces airway bacteria and inflammation in people with cystic fibrosis and chronic lung infections.
]. AR-13 demonstrated not only an ability to increase bacterial killing in CF phagocytes independent of CFTR modulators, but also an improvement in airway epithelial cell CFTR function, either alone or in combination with a promising CFTR modulator, VX-661. Although this study was limited to patients homozygous for F508del mutations, our data suggest that AR-13 could decrease bacterial burden and improve overall CFTR function across CFTR genotypes.
Anti-microbial therapeutic development in CF is arduous due to a variety of technical-, host-, and pathogen-dependent factors. We noted AR-13's impact on bacterial killing was dependent on the presence or absence of host phagocytes. These findings underscore the importance of testing novel therapeutic targets in human models. In particular, human phagocytes such as macrophages may provide a protective habitat for pathogens during replication. Altered host defenses may render some new agents ineffective, or in the case of AR-13 and B. cenocepacia, AR-13 may be needed in combination with existing therapeutics to provide an effective treatment regimen. Additionally, human phagocytes can behave differently compared to animal models, which further highlight the need to test novel compounds with human specimens/cells.
In addition to the aforementioned CF-specific benefits of AR-13, the robust autophagy induction properties of AR-13 (similar to its parent compound AR-12 [
]), suggests that it may be of benefit in other disorders of autophagy dysfunction, or disorders whereby bacteria persist due to dysfunctional autophagy. Similar to CF, patients with chronic granulomatous disease (CGD) also have defective autophagy and suffer from infections with B. cenocepacia [
]. The utility of AR-13 in such disorders remains to be determined.
In summary, AR-13 in combination with antibiotics has synergistic effects on reducing antibiotic-resistant B. cenocepacia, P. aeruginosa, and MRSA burden in CF phagocytes and is associated with increased autophagy and CFTR expression. AR-13 holds promise as a potential new therapeutic option for patients who are acutely or chronically infected with B. cenocepacia and other resistant organisms, for which there are no currently available therapies.
Conflict of interest statement
BK has served on a Vertex Cystic Fibrosis Advisory Board. There are no other relevant conflicts of interest.
Acknowledgements
The authors thank ARNO therapeutics for their kind donation of AR-12 and related derivatives to study. We thank Ky Huang for his assistance with AR-13 handling and Dr. Miguel Valvano for his contribution of B. cenocepacia isolates. This work was supported by the OSU ARNO Therapeutics drug discovery program and Cystic Fibrosis Foundation Research Grants (SDR).
Human cystic fibrosis macrophages have defective calcium-dependent protein kinase C activation of the NADPH oxidase, an effect augmented by burkholderia cenocepacia.
Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors.
Harnessing neutrophil survival mechanisms during chronic infection by pseudomonas aeruginosa: Novel therapeutic targets to dampen inflammation in cystic fibrosis.
Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on DeltaF508 cystic fibrosis transmembrane conductance regulator.
Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis.
Restoring cystic fibrosis transmembrane conductance regulator function reduces airway bacteria and inflammation in people with cystic fibrosis and chronic lung infections.
30869-5&id=gr1.jpg){kind=link}
30869-5&id=gr2.jpg){kind=link}
30869-5&id=gr3.jpg){kind=link}
30869-5&id=gr4.jpg){kind=link}
30869-5&id=gr5.jpg){kind=link}