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Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia . The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Badr, Eltobgy, Krause, Hamilton, Estfanous, Daily, Abu Khweek, Hegazi, Anne, Carafice, Robledo-Avila, Saqr, Zhang, Bonfield, Gavrilin, Partida-Sanchez, Seveau, Cormet-Boyaka and Amer.)

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

Burkholderia cenocepacia, the causative agent of cepacia syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B. cenocepacia have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B. cenocepacia infection. Infection led to early signaling events such as activation of Erk, Akt, and NF-κB. Further, TNFα, IL-6, IL-8, and IL-1β were all significantly induced upon infection, but no IL-1β was detected in the supernatants. Because caspase-1 is required for IL-1β processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1β following B. cenocepacia infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1β secretion.

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.

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1β) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Autophagy is a highly regulated, biological process that provides energy during periods of stress and starvation. This conserved process also acts as a defense mechanism and clears microbes from the host cell. Autophagy is impaired in Cystic Fibrosis (CF) patients and CF mice, as their cells exhibit low expression levels of essential autophagy molecules. The genetic disorder in CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for a chloride channel. CF patients are particularly prone to infection by pathogens that are otherwise cleared by autophagy in healthy immune cells including Burkholderia cenocepacia (B. cenocepacia). The objective of this study is to determine the mechanism underlying weak autophagic activity in CF macrophages and find therapeutic targets to correct it. Using reduced representation bisulfite sequencing (RRBS) to determine DNA methylation profile, we found that the promoter regions of Atg12 in CF macrophages are significantly more methylated than in the wild-type (WT) immune cells, accompanied by low protein expression. The natural product epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. Accordingly, EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy and preventing dissemination. In addition, EGCG improved the function of CFTR protein. Altogether, utilizing RRBS for the first time in the CF field revealed a previously unrecognized mechanism for reduced autophagic activity in CF. Our data also offers a mechanism by which EGCG exerts its positive effects in CF. • The promoter of Atg12 in Cystic Fibrosis macrophages is significantly more methylated than in the wild-type immune cells • Epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. • EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy. • EGCG improved the function of CFTR protein [ABSTRACT FROM AUTHOR]