IFN-γ Stimulates Autophagy-Mediated Clearance of <i>Burkholderia cenocepacia</i> in Human Cystic Fibrosis Macrophages

<div><p><i>Burkholderia cenocepacia</i> is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), survives intracellularly in macrophages, and uniquely causes systemic infections in CF. Autophagy is a physiologic process that involves engulfing non-functional organelles and proteins and delivering them for lysosomal degradation, but also plays a role in eliminating intracellular pathogens, including <i>B. cenocepacia</i>. Autophagy is defective in CF but can be stimulated in murine CF models leading to increased clearance of <i>B. cenocepacia,</i> but little is known about autophagy stimulation in human CF macrophages. IFN-γ activates macrophages and increases antigen presentation while also inducing autophagy in macrophages. We therefore, hypothesized that treatment with IFN-γ would increase autophagy and macrophage activation in patients with CF. Peripheral blood monocyte derived macrophages (MDMs) were obtained from CF and non-CF donors and subsequently infected with <i>B. cenocepacia</i>. Basal serum levels of IFN-γ were similar between CF and non-CF patients, however after <i>B. cenocepacia</i> infection there is deficient IFN-γ production in CF MDMs. IFN-γ treated CF MDMs demonstrate increased co-localization with the autophagy molecule p62, increased autophagosome formation, and increased trafficking to lysosomes compared to untreated CF MDMs. Electron microscopy confirmed IFN-γ promotes double membrane vacuole formation around bacteria in CF MDMs, while only single membrane vacuoles form in untreated CF cells. Bacterial burden is significantly reduced in autophagy stimulated CF MDMs, comparable to non-CF levels. IL-1β production is decreased in CF MDMs after IFN-γ treatment. Together, these results demonstrate that IFN-γ promotes autophagy-mediated clearance of <i>B. cenocepacia</i> in human CF macrophages.</p></div

IFN-γ is deficiently produced in CF PBMCs in response to <i>B. cenocepacia.</i>

<p>1A) IFN-γ levels from the serum of 14 non-CF and 14 CF patients at the time of blood donation, p = 0.056, n = 14 subjects for each condition, Mann-Whitney testing. 1B) IFN-γ production from PBMC 24 hour cell supernatants from CF and non-CF patients. NT represents uninfected cells, and k56 were infected with <i>B. cenocepacia</i> k56-2. n = 8 subjects for NT and 10 subjects k56, Mann-Whitney testing. 1C) Immunoblot for IFN-receptor 1 from cell lysates of MDMs infected with k56-2+/− treatment with IFN-γ. 1D) Immunoblot for IFN-receptor β from cell lysates of MDMs infected with k56-2+/− treatment with IFN-γ.</p

IL-1β is decreased with IFN-γ treatment in CF.

<p>IL-1β levels in macrophage supernatants after infection with k56-2 and either a 4 hour treatment with IFN-γ or a control diluent (7A) or a 24 hour IFN-γ treatment (7B), n = 7 subjects, Mann-Whitney analysis. 7C) IL-10 levels in macrophage supernatants after infection with k56-2 and a 24 hour treatment with IFN-γ or a control diluent, n = 7 subjects, Mann-Whitney analysis. Significant differences are noted.</p

Autophagic flux is decreased in CF.

<p>3A) Representative immunoblot of <u>4 </u>hour treatment protein lysates for LC3-1 and LC3-2 from non-CF and CF macrophages, with corresponding summed band densitometries normalized to loading control. All lanes are marked with a “+” if the corresponding treatments were added: <i>B. cenocepacia</i> infection (k56-2), autophagy stimulators (IFN-y, Rapamycin) and autophagy inhibitors (Bafilomycin, 3-MA). Inhibitors were added one hour prior to infection. Autophagy stimulators were added one hour after infection for a total of 4 hours of treatment. Data is representative of 3 independent experiments. 3B) Representative immunoblot of <u>24 hour</u> treatment protein lysates for LC3-1 and LC3-2 from non-CF and CF macrophages with corresponding summed band densitometries normalized to loading control. All lanes are marked with a “+” if the corresponding treatments were added: <i>B. cenocepacia</i> infection (k56-2), autophagy stimulators (IFN-y, Rapamycin) and autophagy inhibitors (Bafilomycin, 3-MA). Inhibitors were added one hour prior to infection. Autophagy stimulators were added one hour after infection for a total of 24 hours of treatment. Data is representative of 3 independent experiments.</p

Patient Demographics.

<p>Patient Demographics.</p

Cell death is decreased with autophagy stimulation in CF.

<p>Percent LDH release from MDM supernatants treated with autophagy stimulators IFN-γ or Rapamycin for 4 hours (8A) or 24 hours (8B), n = at least 6 subjects per condition, Mann-Whitney testing. 8c) Percent of macrophages deemed viable per naphthol staining after a 24 hour infection, n = 2.</p

IFN-γ stimulates double-membrane autophagosome formation.

<p>5A) Electron microscopy of non-CF macrophage infected with k56-2 only for 24 hours. White arrow indicates double membrane formation indicative of autophagosomes. 5B) EM of non-CF macrophage treated with IFN-γ for 24 hours. 5C) EM of CF macrophage infected with k56-2 only. Black arrow indicates single membrane vacuole. 5D) EM of CF macrophage treated with IFN-γ for 24 hours. White arrow indicates double membrane formation. Pictures are marked with 500 nm marker.</p

IFN-γ increases <i>B. cenocepacia</i> co-localization with p62 and decreases p62 accumulation in CF.

<p>2A) Confocal microscopy for non-CF and CF macrophages infected with m-RFP expressing k56-2. IFN-y or rapamycin treatment was administered after 1 hour of infection for a 24 hour treatment period. p62 is stained green, and macrophage nuclei are stained blue with DAPI. Co-localization of bacteria with p62 is noted in yellow in the bottom panel. 2B) The percentage of bacterial co-localization with p62 was scored for over 100 macrophages per condition, n = 5 subjects per condition, Mann-Whitney testing. 2C) Immunoblot for non-CF and CF macrophages demonstrating p62 accumulation in CF with reduction during IFN-y therapy, representative of 5 subjects. Immunoblot of beclin-1 levels for non-CF and CF macrophages from cell lysates of control (NT) and MDMs infected with k56-2+/− treatment with IFN-γ, n = 4.</p

IFN-γ increases autophagosome formation.

<p>4A) Confocal microscopy for non-CF and CF macrophages infected with m-RFP expressing k56-2. IFN-y treatment was administered after 1 hour of infection for a 4 hour treatment period. LC3 is stained green, and macrophage nuclei are stained blue with DAPI. Co-localization of bacteria with LC3 is noted in yellow in the bottom panel as noted by white arrows. 4B) 24 hour IFN-γ and rapamycin treatments similar to 4A. 4C) Summary of scored bacteria per 100 macrophages from individual subjects for the confocal microscopy experiments from 4B, n = 17 subjects, with 2 replicates per subject, unpaired t-test. 4D) The percentage of bacterial co-localization with LC3 was scored for over 100 macrophages per condition from 4B, n = 17 subjects, with 2 replicates per subject, unpaired t-test. 4E) CFU counts for non-CF and CF macrophages infected with MHK1 for 24 hours (n = 6).</p

Proposed model of LL-37 induced mutagenesis.

<p>At sub-inhibitory concentrations, LL-37 can penetrate <i>P. aeruginosa</i> cells and enter the bacterial cytosol, where LL-37 dimers then bind to DNA. DNA binding by LL-37 then promotes DinB-dependent replication, which potentiates mutations in <i>mucA</i> leading to mucoid conversion. Alginate overproducing bacteria are then protected from lethal concentrations of LL-37 and mucoid variants are selected for and persist in CF.</p