Defective immunometabolism pathways in cystic fibrosis macrophages.

BACKGROUND: Mitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages. METHODS: Mitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients. RESULTS: We have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia. CONCLUSIONS: The mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.

Peroxisome Proliferator-activated Receptors as Potential Targets for Carcinogenic Activity of Polychlorinated Biphenyls: A Computational Perspective.

BACKGROUND: Polychlorinated biphenyls (PCBs) are ubiquitous environment-contaminating synthetic chemicals that have been associated with increased risk of hepatic cancer, melanoma, non-Hodgkin lymphoma and cancer of many other body organs. Structural binding analyses of PCB 77 and PCB 118 with peroxisome proliferator-activated receptors (PPARα, PPARß/δ and PPARγ) was performed to predict the association of PCBs with potential disruption of PPAR signaling pathways. MATERIALS AND METHODS: The crystal structures of human PPARα, PPARß/δ and PPARγ were obtained from the Protein Data Bank. Structures of PCB 77 and PCB 118 were obtained from PubChem database. Docking was performed using glide (Schrodinger) induced fit docking (IFD) module. RESULTS: The PCB 77 and PCB 118 interacted with PPARα, PPARß/δ and PPARγ showing an overlapping of 40-58% interacting amino acid residues with synthetic co-complex agonists of the three PPARs. The binding affinity was higher for PCB 118 than for PCB 77 during docking interactions with each of the three PPARs. CONCLUSION: The consistent commonality of interacting residues for PCB 77 and PCB 118 with co-complex synthetic agonists of the PPARs together with good binding affinity suggested that the PPAR signaling pathway is a potential target for toxicologic activity of PCBs.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery.

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis.

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-1ß. 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.

Asc-dependent and independent mechanisms contribute to restriction of legionella pneumophila infection in murine macrophages.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase(-1-/-) and Nlrc4(-/-) allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc(-/-) macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.

Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes.

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.

Replication of Legionella Pneumophila in Human Cells: Why are We Susceptible?

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious and often fatal form of pneumonia. The susceptibility to L. pneumophila arises from the ability of this intracellular pathogen to multiply in human alveolar macrophages and monocytes. L. pneumophila also replicates in several professional and non-professional phagocytic human-derived cell lines. With the exception of the A/J mouse strain, most mice strains are restrictive, thus they do not support L. pneumophila replication. Mice lacking the NOD-like receptor Nlrc4 or caspase-1 are also susceptible to L. pneumophila. On the other hand, in the susceptible human hosts, L. pneumophila utilizes several strategies to ensure intracellular replication and protect itself against the host immune system. Most of these strategies converge to prevent the fusion of the L. pneumophila phagosome with the lysosome, inhibiting host cell apoptosis, activating survival pathways, and sequestering essential nutrients for replication and pathogenesis. In this review, we summarize survival mechanisms employed by L. pneumophila to maintain its replication in human cells. In addition, we highlight different human-derived cell lines that support the multiplication of this intracellular bacterium. Therefore, these in vitro models can be applicable and are reproducible when investigating L. pneumophila/phagocyte interactions at the molecular and cellular levels in the human host.

Functional quorum sensing systems affect biofilm formation and protein expression in Yersinia pestis.

Gram-negative bacteria predominantly use two types of quorum sensing (QS) systems--LuxI-LuxR, responsible for synthesis of N-acylhomoserine lactones (AHL or AI-1 signal molecule), and LuxS, which makes furanones (AI-2 signal molecule). We showed that LuxS and two LuxI-LuxR (YtbIR and YpsIR) systems are functional in Y. pestis. Four different AHL molecules were detected in Y. pestis extracts using TLC bioassays. Our data suggest that YtbIR is responsible for the production of long chain AHLs. Confocal laser scanning microscopy showed that biofilm formation is decreased in an ytbIR ypsIR luxS mutant. Two-dimensional gel electrophoresis revealed altered levels of protein expression in a Y. pestis triple QS mutant at 26 degrees C and 37 degrees C.