Autophagy stimulation by rapamycin suppresses lung inflammation and infection byBurkholderia cenocepaciain a model of cystic fibrosis

Abstract

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

“…Notably, B. cenocepacia is also cleared by autophagosomes in healthy macrophages [5], but the role of CASP4 in intracellular trafficking of this bacterium is not known. In order to determine whether CASP4 is involved in the restriction of B. cenocepacia , we infected bone marrow-derived macrophages from C57BL/6 wild-type (WT) and casp4 −/- mice and examined intracellular bacterial growth.…”

“…Because functional autophagy is important to clear intracellular B. cenocepacia in WT macrophages and epithelial cells [5], we next determined if the interaction between B. cenocepacia and the cellular autophagy system is affected by the lack of CASP4. We quantified the colocalization of B. cenocepacia with the autophagy marker LC3 in macrophages, and found fewer colocalization events at 0.5 h and 2 h post infection in the absence of CASP4 compared to WT cells (Figure 3a and b).…”

“…The transcription of genes coding for major autophagy proteins is downregulated upon B. cenocepacia infection in both WT and CFTR∆F508 (CF) macrophages, suggesting that B. cenocepacia actively interferes with the autophagy pathway [5]. Nevertheless, WT macrophages successfully restrict B. cenocepacia growth, as functional autophagy machinery is restored in WT but not in CF cells [5,41].…”

“…The breakdown of damaged organelles and proteins via autophagy leads to the recycling of nutrients and sustains cellular energy levels. In addition, autophagy targets various intracellular pathogens such as Mycobacterium tuberculosis [1], Salmonella enterica serovar Typhimurium [2], Legionella pneumophila [3], Streptococcus pyogenes [4], and Burkholderia cenocepacia [5]. This bacteria-specific autophagic process, designated xenophagy, promotes bacterial clearance and prevents cellular escape.…”

“…Therefore, it can be concluded that defective autophagy promotes survival of B. cenocepacia in professional phagocytes. However, pharmacological stimulation of autophagy prior to infection restores bacterial delivery to acidic lysosomal compartments, thereby improving degradation of B. cenocepacia within murine CF macrophages [5]. Human macrophages derived either from patients with CF or chronic granulomatous disease also show improved restriction of B. cenocepacia after treatment with the autophagy-inducing agent rapamycin [13,14].…”