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Infection and Immunity Jun 2024The human-specific bacterial pathogen group A (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous...
The human-specific bacterial pathogen group A (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.
Topics: Streptococcus pyogenes; Humans; Macrophages; Lysosomes; Streptolysins; Bacterial Proteins; Phagosomes; THP-1 Cells; Phagocytosis; Streptococcal Infections; Cathepsin B; Hydrogen-Ion Concentration
PubMed: 38722166
DOI: 10.1128/iai.00141-24 -
Biochemical and Biophysical Research... Jul 2024Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining... (Review)
Review
Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining cellular homeostasis. It involves the formation of a membraned structure called the autophagosome, which carries cargo that includes toxic protein aggregates and dysfunctional organelles to the lysosome for degradation and recycling. Autophagy is primarily considered and studied as a cell-autonomous mechanism. However, recent studies have illuminated an underappreciated facet of autophagy, i.e., non-autonomously regulated autophagy. Non-autonomously regulated autophagy involves the degradation of autophagic components, including organelles, cargo, and signaling molecules, and is induced in neighboring cells by signals from primary adjacent or distant cells/tissues/organs. This review provides insight into the complex molecular mechanisms governing non-autonomously regulated autophagy, highlighting the dynamic interplay between cells within tissue/organ or distinct cell types in different tissues/organs. Emphasis is placed on modes of intercellular communication that include secreted molecules, including microRNAs, and their regulatory roles in orchestrating this phenomenon. Furthermore, we explore the multidimensional roles of non-autonomously regulated autophagy in various physiological contexts, spanning tissue development and aging, as well as its importance in diverse pathological conditions, including cancer and neurodegeneration. By studying the complexities of non-autonomously regulated autophagy, we hope to gain insights into the sophisticated intercellular dynamics within multicellular organisms, including mammals. These studies will uncover novel avenues for therapeutic intervention to modulate intercellular autophagic pathways in altered human physiology.
Topics: Humans; Autophagy; Animals; Cell Communication; MicroRNAs; Neoplasms; Signal Transduction; Autophagosomes
PubMed: 38701555
DOI: 10.1016/j.bbrc.2024.150024 -
Medicine May 2024Hepatocellular carcinoma (HCC) is one of the most common malignant tumors globally and often develops on the foundation of chronic liver disease or cirrhosis. Cirrhosis... (Observational Study)
Observational Study
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors globally and often develops on the foundation of chronic liver disease or cirrhosis. Cirrhosis is a clinically prevalent chronic progressive liver disease characterized by diffuse liver damage resulting from long-term or repeated actions of 1 or more etiological factors. However, the impact of CENPF and nuclear division cycle 80 (NDC80) genes on rehabilitation nursing of HCC and cirrhosis remains unclear. HCC and cirrhosis datasets GSE63898 and GSE89377 profile files were downloaded from the gene expression omnibus database generated on platforms GPL13667 and GPL6947, respectively. Differentially expressed genes (DEGs) screening, weighted gene co-expression network analysis (WGCNA), construction and analysis of protein-protein interaction (PPI) networks, functional enrichment analysis, gene set enrichment analysis (GSEA), survival analysis, immune infiltration analysis, and comparative toxicogenomics database (CTD) analysis were conducted. Gene expression heatmaps were plotted. miRNAs regulating central DEGs were selected through TargetScan. A total of 626 DEGs were identified. According to gene ontology (GO) analysis, they were primarily enriched in small molecule metabolic processes, drug metabolic processes, binding of identical proteins, and lipid metabolic processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis results indicated that the target genes were mainly enriched in metabolic pathways, phagosomes, glycine, serine, and threonine metabolism. The construction and analysis of the PPI network revealed 3 core genes (NDC80, CENPF, RRM2). Gene expression heatmaps showed that core genes (CENPF, NDC80) were highly expressed in HCC and cirrhosis samples. CTD analysis found that 2 genes (CENPF and NDC80) were associated with liver, jaundice, ascites, fever, dyspepsia, and hepatic encephalopathy. CENPF and NDC80 are highly expressed in HCC and cirrhosis, and CENPF and NDC80 might be the biomarkers of rehabilitation nursing of HCC and cirrhosis.
Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Cirrhosis; Nuclear Proteins; Protein Interaction Maps; Gene Expression Profiling; Cytoskeletal Proteins
PubMed: 38701255
DOI: 10.1097/MD.0000000000037984 -
Scientific Reports May 2024The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively,...
The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes could allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core. Indeed, our biochemical analyses revealed the overlap of binding sites for HOPS-specific VPS41 and CORVET-specific VPS8 on the shared core subunit VPS18. We found that the overexpression of CORVET-specific VPS8 or Tgfbrap1 decreased the amount of core proteins VPS11 and VPS18 that are assembled with HOPS-specific subunits VPS41 or VPS39, indicating reduced amount of assembled HOPS. In line with this, we observed the elevation of both lipidated, autophagosome-associated LC3 protein and the autophagic cargo p62 in these cells, suggesting impaired autophagosome-lysosome fusion. In contrast, overexpression of HOPS-specific VPS39 or VPS41 did not affect the level of assembled CORVET or autophagy. VPS8 or Tgfbrap1 overexpression also induced Cathepsin D accumulation, suggesting that HOPS-dependent biosynthetic delivery of lysosomal hydrolases is perturbed, too. These indicate that CORVET-specific subunit levels fine-tune HOPS assembly and activity in vivo.
Topics: Endosomes; Humans; Vesicular Transport Proteins; Lysosomes; Protein Subunits; Autophagy; Autophagosomes; HeLa Cells; Protein Binding
PubMed: 38698024
DOI: 10.1038/s41598-024-59775-0 -
Free Radical Biology & Medicine Aug 2024Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a...
Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a potential anticancer drug. Our recent report showed a favorable anticancer effect of agrimol B in HCC, however, the mechanism of action remains unclear. Here, we found agrimol B inhibits the growth and proliferation of HCC cells in vitro as well as in an HCC patient-derived xenograft (PDX) model. Notably, agrimol B drives autophagy initiation and blocks autophagosome-lysosome fusion, resulting in autophagosome accumulation and autophagy arrest in HCC cells. Mechanistically, agrimol B downregulates the protein level of NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) through caspase 3-mediated degradation, leading to mitochondrial reactive oxygen species (mROS) accumulation and autophagy arrest. NDUFS1 overexpression partially restores mROS overproduction, autophagosome accumulation, and growth inhibition induced by agrimol B, suggesting a cytotoxic role of agrimol B-induced autophagy arrest in HCC cells. Notably, agrimol B significantly enhances the sensitivity of HCC cells to sorafenib in vitro and in vivo. In conclusion, our study uncovers the anticancer mechanism of agrimol B in HCC involving the regulation of oxidative stress and autophagy, and suggests agrimol B as a potential therapeutic drug for HCC treatment.
Topics: Animals; Humans; Mice; Apoptosis; Autophagosomes; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Electron Transport Complex I; Indoles; Liver Neoplasms; Mice, Nude; Mitochondria; Reactive Oxygen Species; Sorafenib; Spiro Compounds; Xenograft Model Antitumor Assays
PubMed: 38697493
DOI: 10.1016/j.freeradbiomed.2024.04.242 -
Molecular Biology of the Cell Jul 2024Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse...
Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the eye via overexpression of resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the -induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.
Topics: Animals; Drosophila Proteins; Autophagy; Autophagic Cell Death; Drosophila melanogaster; Sodium-Hydrogen Exchangers; Hydrogen-Ion Concentration; Eye; Apoptosis; Lysosomes; Drosophila; Autophagosomes
PubMed: 38696256
DOI: 10.1091/mbc.E24-02-0058 -
Biomacromolecules May 2024Intracellular bacteria in dormant states can escape the immune response and tolerate high-dose antibiotic treatment, leading to severe infections. To overcome this...
Intracellular bacteria in dormant states can escape the immune response and tolerate high-dose antibiotic treatment, leading to severe infections. To overcome this challenge, cascade-targeted nanoplatforms that can target macrophages and intracellular bacteria, exhibiting synergetic antibiotic/reactive oxygen species (ROS)/nitric oxide (NO)/immunotherapy, were developed. These nanoplatforms were fabricated by encapsulating trehalose (Tr) and vancomycin (Van) into phosphatidylserine (PS)-coated poly[(4-allylcarbamoylphenylboric acid)--(arginine-methacrylamide)--(,'-bisacryloylcystamine)] nanoparticles (PABS), denoted as PTVP. PS on PTVP simulates a signal of "eat me" to macrophages to promote cell uptake (the first-step targeting). After the uptake, the nanoplatform in the acidic phagolysosomes could release Tr, and the exposed phenylboronic acid on the nanoplatform could target bacteria (the second-step targeting). Nanoplatforms can release Van in response to infected intracellular overexpressed glutathione (GSH) and weak acid microenvironment. l-arginine (Arg) on the nanoplatforms could be catalyzed by upregulated inducible nitric oxide synthase (iNOS) in the infected macrophages to generate nitric oxide (NO). ,'-Bisacryloylcystamine (BAC) on nanoplatforms could deplete GSH, allow the generation of ROS in macrophages, and then upregulate proinflammatory activity, leading to the reinforced antibacterial capacity. This nanoplatform possesses macrophage and bacteria-targeting antibiotic delivery, intracellular ROS, and NO generation, and pro-inflammatory activities (immunotherapy) provides a new strategy for eradicating intracellular bacterial infections.
Topics: Reactive Oxygen Species; Nitric Oxide; Anti-Bacterial Agents; Mice; Animals; RAW 264.7 Cells; Nanoparticles; Macrophages; Immunotherapy; Vancomycin; Bacterial Infections; Trehalose
PubMed: 38693753
DOI: 10.1021/acs.biomac.4c00339 -
Nature May 2024Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide. Transplanting endothelial cells (ECs) is a promising...
Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.
Topics: Animals; Humans; Male; Mice; Autophagosomes; Endothelial Cells; Energy Metabolism; Human Umbilical Vein Endothelial Cells; Ischemia; Mesenchymal Stem Cells; Mice, Nude; Mitochondria; Mitophagy; Protein Kinases; Ubiquitin-Protein Ligases; Cell- and Tissue-Based Therapy
PubMed: 38693258
DOI: 10.1038/s41586-024-07340-0 -
Frontiers in Neuroscience 2024In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of...
In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTK) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTK retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTK and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTK RPE primary cultures phagocytosed less POS , while , the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina.
PubMed: 38680449
DOI: 10.3389/fnins.2024.1256522 -
Journal of Ethnopharmacology Sep 2024Tanreqing injection (TRQ) is widely used, traditional Chinese medicine (TCM) injection used in China to treat respiratory infections. Modern pharmacological studies have...
ETHNOPHARMACOLOGICAL RELEVANCE
Tanreqing injection (TRQ) is widely used, traditional Chinese medicine (TCM) injection used in China to treat respiratory infections. Modern pharmacological studies have confirmed that TRQ can protect against influenza viruses. However, the mechanism by which TRQ inhibits influenza viruses remains unclear.
AIM OF THE STUDY
To explore the therapeutic effects and possible mechanisms of TRQ inhibition by the influenza virus.
MATERIALS AND METHODS
Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to determine the chemical composition of TRQ. Isobaric tags for relative and absolute quantification (iTRAQ) were used to define differential proteins related to TRQ inhibition of viruses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation. For experimental validation, we established an in vitro model of the influenza virus infection by infecting A549 cells with the virus. The detection of the signaling pathway was carried out through qPCR, western blotting,and immunofluorescence.
RESULTS
Fifty one components were identified using UPLC/Q-TOF MS. We confirmed the inhibitory effect of TRQ on influenza virus replication in vitro. Ninety nine differentially expressed proteins related to the inhibitory effect of TRQ were identified using iTRAQ. KEGG functional enrichment analysis showed that the TRQ may inhibit influenza virus replication by affecting autophagy. Through network analysis, 29 targets were selected as major targets, and three key targets, HSPA5, PARP1, and GAPDH, may be the TRQ targets affecting autophagy. In vitro experiments showed that TRQ inhibits influenza virus replication by interfering with the expression and localization of STX17 and VAMP8 proteins, thereby promoting the fusion of autophagosomes with lysosomes.
CONCLUSION
TRQ inhibits influenza virus replication by promoting the fusion of autophagosomes with lysosomes. We additionally established potential gene and protein targets which are affected by TRQ. Therefore, our findings provide new therapeutic targets and a foundation further studies on influenza treatment with TRQ.
Topics: Drugs, Chinese Herbal; Virus Replication; Humans; A549 Cells; Antiviral Agents; Lysosomes; Autophagosomes; Endoplasmic Reticulum Chaperone BiP; Animals; Autophagy
PubMed: 38677572
DOI: 10.1016/j.jep.2024.118159