An Autophagic Flux Probe that Releases an Internal Control.

Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.

Gallic acid promotes macrophage phagosome acidification and phagolysosome formation by activating NLRP3/mTOR signaling pathway.

INTRODUCTION: Gallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). METHODS: A cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. RESULTS: Our results demonstrated that GA at 50 µM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. CONCLUSIONS: In summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.

The helminth product ES-62 protects against septic shock via Toll-like receptor 4-dependent autophagosomal degradation of the adaptor MyD88.

Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.

Small molecule probes for targeting autophagy.

Autophagy is implicated in a wide range of (patho)physiological processes including maintenance of cellular homeostasis, neurodegenerative disorders, aging and cancer. As such, small molecule autophagy modulators are in great demand, both for their ability to act as tools to better understand this essential process and as potential therapeutics. Despite substantial advances in the field, major challenges remain in the development and comprehensive characterization of probes that are specific to autophagy. In this Review, we discuss recent developments in autophagy-modulating small molecules, including the specific challenges faced in the development of activators and inhibitors, and recommend guidelines for their use. Finally, we discuss the potential to hijack the process for targeted protein degradation, an area of great importance in chemical biology and drug discovery.

Granulocyte macrophage-colony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival.

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⁺ channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.

Mitophagy is involved in the mitochondrial dysfunction of vitrified porcine oocytes.

Mitochondrial dysfunction is considered a crucial factor aggravating oocyte viability after vitrification-warming. To clarify the role of mitophagy in mitochondrial extinction of vitrified porcine oocytes, mitochondrial function, ultrastructural characteristics, mitochondria-lysosomes colocalization, and mitophagic proteins were detected with or without chloroquine (CQ) treatment. The results showed that vitrification caused mitochondrial dysfunction, including increasing reactive oxygen species production, decreasing mitochondrial membrane potential, and mitochondrial DNA copy number. Damaged mitochondrial cristae and mitophagosomes were observed in vitrified oocytes. A highly fused fluorescence distribution of mitochondria and lysosomes was also observed. In the detection of mitophagic flux, mitophagy was demonstrated as increasing fluorescence aggregation of microtubule-associated protein light chain 3B (LC3B), enhanced colocalization between LC3B, and voltage-dependent anion channels 1 (VDAC1), and upregulated LC3B-II/I protein expression ratio. CQ inhibited the degradation of mitophagosomes in vitrified oocytes, manifested as decreased mitochondria-lysosomes colocalization, increased fluorescence fraction of VDAC1 overlapping LC3B, increased LC3B-II/I protein expression ratio, and p62 accumulation. The inhibition of mitophagosomes degradation by CQ aggravated mitochondrial dysfunction, including increased oxidative damage, reduced mitochondrial function, and further led to loss of oocyte viability and developmental potentiality. In conclusion, mitophagy is involved in the regulation of mitochondrial function during porcine oocyte vitrification.

TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation.

Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1ß, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1ß activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.

Iron overload inhibits late stage autophagic flux leading to insulin resistance.

Iron overload, a common clinical occurrence, is implicated in the metabolic syndrome although the contributing pathophysiological mechanisms are not fully defined. We show that prolonged iron overload results in an autophagy defect associated with accumulation of dysfunctional autolysosomes and loss of free lysosomes in skeletal muscle. These autophagy defects contribute to impaired insulin-stimulated glucose uptake and insulin signaling. Mechanistically, we show that iron overload leads to a decrease in Akt-mediated repression of tuberous sclerosis complex (TSC2) and Rheb-mediated mTORC1 activation on autolysosomes, thereby inhibiting autophagic-lysosome regeneration. Constitutive activation of mTORC1 or iron withdrawal replenishes lysosomal pools via increased mTORC1-UVRAG signaling, which restores insulin sensitivity. Induction of iron overload via intravenous iron-dextran delivery in mice also results in insulin resistance accompanied by abnormal autophagosome accumulation, lysosomal loss, and decreased mTORC1-UVRAG signaling in muscle. Collectively, our results show that chronic iron overload leads to a profound autophagy defect through mTORC1-UVRAG inhibition and provides new mechanistic insight into metabolic syndrome-associated insulin resistance.

Extracellular vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing.

Extracellular vesicles (EVs) have been shown to carry microbial components and function in the host defense against infections. In this study, we demonstrate that Mycobacterium tuberculosis (M.tb) RNA is delivered into macrophage-derived EVs through an M.tb SecA2-dependent pathway and that EVs released from M.tb-infected macrophages stimulate a host RIG-I/MAVS/TBK1/IRF3 RNA sensing pathway, leading to type I interferon production in recipient cells. These EVs also promote, in a RIG-I/MAVS-dependent manner, the maturation of M.tb-containing phagosomes through a noncanonical LC3 pathway, leading to increased bacterial killing. Moreover, treatment of M.tb-infected macrophages or mice with a combination of moxifloxacin and EVs, isolated from M.tb-infected macrophages, significantly lowered bacterial burden relative to either treatment alone. We hypothesize that EVs, which are preferentially removed by macrophages in vivo, can be combined with effective antibiotics as a novel approach to treat drug-resistant TB.

Rac1-dependent phagocytosis of apoptotic cells by oral squamous cell carcinoma cells: A possible driving force for tumor progression.

Apoptotic cell death frequently occurs in human cancer tissues including oral squamous cell carcinoma (SCC), wherein apoptotic tumor cells are phagocytosed not only by macrophages but also by neighboring tumor cells. We previously reported that the engulfment of apoptotic SCC cells by neighboring SCC cells frequently occurs at the invading front. Therefore, we hypothesized that the phagocytosis of these apoptotic cells by tumor cells contributes to disease progression. Herein, using cultured oral SCC cells, we aimed to confirm whether tumor cells actually phagocytose apoptotic cells and to examine whether cellular activities are regulated by the phagocytosis of apoptotic cells. Co-culture experiments showed that living cells could ingest apoptotic cells into phagolysosomes. NSC23766, an inhibitor of Rac1, which is a key regulator of phagocytic cup formation in professional phagocytes, dramatically suppressed the phagocytosis of apoptotic cells by living cells. Additionally, cell migration and the secretion of DKK1, a tumor-promoting protein, were enhanced by co-culture with apoptotic cells, whereas NSC23766 inhibited these effects. These results show that tumor cells can actively phagocytose apoptotic neighbors in a Rac1-dependent manner and that such activity increases their migration. The regulation of apoptotic cell phagocytosis thus represents new directions for therapeutic intervention for oral cancer.

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

Spatiotemporal Changes of the Phagosomal Proteome in Dendritic Cells in Response to LPS Stimulation.

Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. The phagosomal proteome of DCs is only partially studied and is highly dynamic as it changes during phagosome maturation, when phagosomes sequentially interact with endosomes and lysosomes. In addition, the activation status of the phagocyte can modulate the phagosomal composition and is able to shape phagosomal functions.In this study, we determined spatiotemporal changes of the proteome of DC phagosomes during their maturation and compared resting and lipopolysaccharide (LPS)-stimulated bone marrow-derived DCs by label-free, quantitative mass spectrometry. Ovalbumin-coupled latex beads were used as phagocytosis model system and revealed that LPS-treated DCs show decreased recruitment of proteins involved in phagosome maturation, such as subunits of the vacuolar proton ATPase, cathepsin B, D, S, and RAB7. In contrast, those phagosomes were characterized by an increased recruitment of proteins involved in antigen cross-presentation, e.g. different subunits of MHC I molecules, the proteasome and tapasin, confirming the observed increase in cross-presentation efficacy in those cells. Further, several proteins were identified that were not previously associated with phagosomal functions. Hierarchical clustering of phagosomal proteins demonstrated that their acquisition to DC phagosomes is not only dependent on the duration of phagosome maturation but also on the activation state of DCs. Thus, our study provides a comprehensive overview of how DCs alter their phagosome composition in response to LPS, which has profound impact on the initiation of efficient immune responses.

Docosahexaenoic acid impacts macrophage phenotype subsets and phagolysosomal membrane permeability with particle exposure.

Inhalation of particles results in pulmonary inflammation; however, treatments are currently lacking. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid shown to exhibit anti-inflammatory capabilities. The impact of DHA on particle-induced inflammation is unclear; therefore, the aim of this study was to examine the hypothesis that DHA downregulates macrophage inflammatory responses by altering phagolysosomal membrane permeability (LMP) and shifting macrophage phenotype. Isolated Balb/c alveolar macrophages (AM) were polarized into M1, M2a, M2b, or M2c phenotypes in vitro, treated with DHA, and exposed to a multi-walled carbon nanotube (MWNCT) or crystalline silica (SiO2). Results showed minimal cytotoxicity, robust effects for silica particle uptake, and LMP differences between phenotypes. Docosahexaenoic acid prevented these effects to the greatest extent in M2c phenotype. To determine if DHA affected inflammation similarly in vivo, Balb/c mice were placed on a control or 1% DHA diet for 3 weeks, instilled with the same particles, and assessed 24 hr following instillation. Data demonstrated that in contrast to in vitro findings, DHA increased pulmonary inflammation and LMP. These results suggest that pulmonary responses in vivo may not necessarily be predicted from single-cell responses in vitro.

The influence of coconut oil on the growth, immune, and antioxidative responses and the intestinal digestive enzymes and histomorphometry features of Nile tilapia (Oreochromis niloticus).

The trials of finding non-conventional and alternative aquafeed ingredients are increasing. In this sense, this study evaluated the influence of coconut oil on the growth, feed utilization, immune, and antioxidative responses of Nile tilapia. Five test diets were formulated by mixing coconut oil with the other ingredients at 0, 1, 2, 3, and 4% of the total ration and presented for tilapia for 60 successive days. The final weight, SGR, weight gain (WG), and feed intake were superior in fish delivered 2% of coconut oil (P < 0.05). Concurrently, fish that received 2% coconut oil had lower FCR and higher PER than fish of the control and 4% groups (P < 0.05). Higher lipase activity was observed in fish of 2% and 3% levels than the remaining groups (P < 0.05). Besides, the amylase and protease activities of fish in 1%, 2%, and 3% groups were higher than the 0% level (P < 0.05). The total blood cholesterol, RBCs, and PCV showed higher values in Nile tilapia fed 2% and 3% coconut oil (P < 0.05). The lysozyme and phagocytic activities were higher in fish fed 2% and 3% levels than the control (P < 0.05), while the phagocytic index in 2% and 3% levels was higher than 0% and 4% levels. Furthermore, SOD and CAT were higher in fish fed 1%, 2%, and 3% than fish fed 0% and 4% levels while GSH was higher in fish of 1%, 2%, and 3% than fish fed 0% level (P < 0.05). However, the MDA level was markedly lower in fish fed 25, 3%, and 4% coconut oil than the 0% level (P < 0.05). The intestine's histological structure in all groups appeared normal, forming of intestinal villi projecting from the intestinal wall. Also, the structure of the hepatopancreas had a normal architecture in all groups. To sum up, the inclusion of coconut oil at 2 to 3% is recommended as a replacer for fish oil in Nile tilapia diets.

Lipid Rafts Assemble Dynein Ensembles.

New work by Rai et al. identifies a novel mechanism regulating phagosome transport in cells: the clustering of dynein motors into lipid microdomains, leading to enhanced unidirectional motility. Clustering may be especially important for dynein, a motor that works most efficiently in teams.

Transcriptome changes of Takifugu obscurus liver after acute exposure to the oxygenated-PAH 9,10-phenanthrenequione.

Contamination with polycyclic aromatic hydrocarbons (PAHs) causes noticeable ecological problems in aquatic ecosystems. 9,10-Phenanthrenequione (9,10-PQ) is an oxidized PAH and is highly toxic to aquatic animals. However, the effects of 9,10-PQ on the molecular metabolism of fish remain largely unknown. In this study, Takifugu obscurus juveniles were acutely exposed to 44.30 µg/L 9,10-PQ for 3 days. The transcriptome profile changes in their livers were compared between the 9,10-PQ treatment group and the control using T. rubripes as the reference genome. The results identified 22,414 genes in our transcriptome. Among them, 767 genes were differentially expressed after exposure to 9,10-PQ, which enriched 16 KEGG pathways. Among them, the glycolysis, phagosome, and FOXO signaling pathways were significantly activated in 9,10-PQ treatment compared with the control. These data indicate that 9,10-PQ increased the glycolysis capacity to produce more energy for resistance and harmed immune function. Moreover, several genes related to tumorigenesis were significantly upregulated in response to 9,10-PQ, displaying the carcinogenic toxicity of 9,10-PQ to T. obscurus. Genes in steroid biosynthesis pathways were downregulated in the 9,10-PQ treatment group, suggesting interference with the endocrine system. Overall, these findings provide information to help evaluate the environmental risks that oxygenated-PAHs present to T. obscurus.

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.

Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52-MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

Inflammasome-Independent Leukotriene B4 Production Drives Crystalline Silica-Induced Sterile Inflammation.

Silicosis is a lung inflammatory disease caused by chronic exposure to crystalline silica (CS). Leukotriene B4 (LTB4) plays an important role in neutrophilic inflammation, which drives silicosis and promotes lung cancer. In this study, we examined the mechanisms involved in CS-induced inflammatory pathways. Phagocytosis of CS particles is essential for the production of LTB4 and IL-1ß in mouse macrophages, mast cells, and neutrophils. Phagosomes enclosing CS particles trigger the assembly of lipidosome in the cytoplasm, which is likely the primary source of CS-induced LTB4 production. Activation of the JNK pathway is essential for both CS-induced LTB4 and IL-1ß production. Studies with bafilomycin-A1- and NLRP3-deficient mice revealed that LTB4 synthesis in the lipidosome is independent of inflammasome activation. Small interfering RNA knockdown and confocal microscopy studies showed that GTPases Rab5c, Rab40c along with JNK1 are essential for lipidosome formation and LTB4 production. BI-78D3, a JNK inhibitor, abrogated CS-induced neutrophilic inflammation in vivo in an air pouch model. These results highlight an inflammasome-independent and JNK activation-dependent lipidosome pathway as a regulator of LTB4 synthesis and CS-induced sterile inflammation.

A potential bio-control agent from baical skullcap root against listeriosis via the inhibition of sortase A and listeriolysin O.

Listeria monocytogenes (LM) is a classical model intracellular pathogen and the leading cause of listeriosis, which has long been a global public health issue. The successful infection of LM is related to a series of virulence factors, such as the transpeptidase enzyme sortase A (SrtA) and listeriolysin O (LLO), which are crucial for bacterial internalization and escape from phagosomes respectively. It is speculated that targeting multiple virulence factors may be due to a synergistic effect on listeriosis therapy. In this study, an active flavonoids component of Scutellaria baicalensis Georgi, baicalein, was found to potently block both listerial SrtA catalyzed activity and LLO hemolytic activity within 16 µg/mL. After pretreatment with baicalein, 86.30 (±11.35) % of LM failed to associate with Caco-2 cells compared to the LM without preincubation (regarded as 100% internalization). Furthermore, baicalein addition may aid in bacterial degradation and clearance in macrophagocytes. During a 5 h observation, LM in cells incubated with baicalein showed significantly decreased vacuole escapes and sluggish endocellular growth. In addition, baicalein directly prevented LM-induced cells injury and mice fatality (survival rate from 10.00% to 54.55% in 4 days post-intraperitoneal injection). Taken together, as an antagonist against SrtA and LLO, baicalein can be further developed into a biotherapeutic agent for listeriosis.

Melatonin enhances autophagy and decreases apoptosis induced by nanosilica in RAW264.7 cells.

Melatonin is one of the main hormones that regulate biological rhythms and have immunomodulation, anti-inflammatory, and antioxidation functions. In this study, we aimed to explore the effect of melatonin on the autophagy, apoptosis, and inflammatory reaction of macrophages (RAW264.7 cells) stimulated by nanosilica. SiO2 (100 mg/mL, 10-20 nm) was used to stimulate RAW264.7 cells at different time points (0, 2, 4, 8, 12, and 24 hr). Melatonin (200 µM) was added to SiO2 -stimulated macrophages at 12 hr. Beclin-1, LC3, Bax, Bcl-2, and Caspase-3 were examined with western blotting. Flow cytometry was used to detect apoptosis. The levels of TNF-α, IL-1ß, and IL-18 were detected by ELISA. The level of TNF-α in the supernatant of SiO2 -stimulated cells gradually increased with time but decreased following melatonin administration. In contrast, the expression of IL-1ß and IL-18 increased after melatonin treatment. LC3 and Bax signaling pathways were activated in SiO2 -stimulated RAW264.7 cells, showing elevated expression of LC3 and reduced expression of Bax in the melatonin-treated cells. GFP-LC3 puncta were significantly increased in SiO2 -stimulated RAW264.7 cells and decreased in melatonin-treated cells. The apoptotic rate in SiO2 -stimulated RAW264.7 cells increased with time and decreased after melatonin treatment, and the number of phagosomes increased with the stimulation of nanosilica and the treatment of melatonin. Melatonin might promote autophagy and inhibit apoptosis as well as inflammatory responses of RAW264.7 cells stimulated by nanosilica. © 2019 IUBMB Life, 2019.