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Frontiers in Immunology 2024Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent...
Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.
Topics: Humans; Dengue Virus; Dendritic Cells; Autophagy; Dengue; Autophagosomes; Secretory Pathway; Virus Replication; Extracellular Vesicles; Cells, Cultured
PubMed: 38863700
DOI: 10.3389/fimmu.2024.1260439 -
Frontiers in Cellular Neuroscience 2024Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties....
Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties. However, the precise mechanisms underlying the neuroprotective actions of TGF-β1 on the cortex require further investigation. In this study, we were aimed to investigate the regulatory function of TGF-β1 on neuronal autophagy and apoptosis using an primary cortical neuron trauma-injury model. LDH activity was assayed to measure cell viability, and intracellular [Ca] was measured using Fluo-4-AM in an primary cortical neuron trauma-injury model. RNA-sequencing (RNAseq), immunofluorescent staining, transmission electron microscopy (TEM), western blot and CTSD activity detection were employed. We observed significant enrichment of DEGs related to autophagy, apoptosis, and the lysosome pathway in trauma-injured cortical neurons. TEM confirmed the presence of autophagosomes as well as autophagolysosomes. Western blot revealed upregulation of autophagy-related protein light chain 3 (LC3-II/LC3-I), sequestosome 1 (SQSTM1/p62), along with apoptosis-related protein cleaved-caspase 3 in trauma-injured primary cortical neurons. Furthermore, trauma-injured cortical neurons showed an upregulation of lysosomal marker protein (LAMP1) and lysosomal enzyme mature cathepsin D (mCTSD), but a decrease in the activity of CTSD enzyme. These results indicated that apoptosis was up-regulated in trauma- injured cortical neurons at 24 h, accompanied by lysosomal dysfunction and impaired autophagic flux. Notably, TGF-β1 significantly reversed these changes. Our results suggested that TGF-β1 exerted neuroprotective effects on trauma- injured cortical neurons by reducing lysosomal dysfunction, decreasing the accumulation of autophagosomes and autophagolysosomes, and enhancing autophagic flux.
PubMed: 38863498
DOI: 10.3389/fncel.2024.1381279 -
Nature Communications Jun 2024Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour...
Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA CAFs can form immunological synapses with Foxp3 regulatory T cells (Tregs) in tumours. Notably, α-SMA CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA CAFs and Tregs in an autophagy-dependent manner.
Topics: T-Lymphocytes, Regulatory; Cancer-Associated Fibroblasts; Humans; Immunological Synapses; Animals; Tumor Microenvironment; Mice; Autophagy; Actins; Autophagy-Related Protein 5; Neoplasms; Mice, Inbred C57BL; Forkhead Transcription Factors; Female; Mice, Knockout
PubMed: 38862534
DOI: 10.1038/s41467-024-49282-1 -
Scientific Reports Jun 2024Lung cancer is the most common oncological disease worldwide, with non-small cell lung cancer accounting for approximately 85% of lung cancer cases. α-Hederin is a...
Lung cancer is the most common oncological disease worldwide, with non-small cell lung cancer accounting for approximately 85% of lung cancer cases. α-Hederin is a monodesmosidic triterpenoid saponin isolated from the leaves of Hedera helix L. or Nigella sativa and has been extensively studied for its antitumor activity against a variety of tumor cells. It has been suggested that α-Hederin is a potential regulator of autophagy and has high promise for application. However, the specific mechanism and characteristics of α-Hederin in regulating autophagy are not well understood. In this study, we confirmed the potential of α-Hederin application in lung cancer treatment and comprehensively explored the mechanism and characteristics of α-Hederin in regulating autophagy in lung cancer cells. Our results suggest that α-Hederin is an incomplete autophagy inducer that targets mTOR to activate the classical autophagic pathway, inhibits lysosomal acidification without significantly affecting the processes of autophagosome transport, lysosome biogenesis, autophagosome and lysosome fusion, and finally leads to impaired autophagic flux and triggers autophagic damage in NSCLC.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Lysosomes; Autophagy; Lung Neoplasms; Oleanolic Acid; Saponins; Cell Line, Tumor; TOR Serine-Threonine Kinases; Autophagosomes; A549 Cells
PubMed: 38858422
DOI: 10.1038/s41598-024-63348-6 -
Journal of Virology Jun 2024Hepatitis E virus (HEV) poses a global threat, which currently remains understudied in terms of host interactions. Epidermal growth factor receptor (EGFR) plays...
UNLABELLED
Hepatitis E virus (HEV) poses a global threat, which currently remains understudied in terms of host interactions. Epidermal growth factor receptor (EGFR) plays multifaceted roles in viral pathogenesis, impacting host-cell entry, viral replication, and host-defense modulation. On the one hand, EGFR signaling emerged as a major driver in innate immunity; on the other hand, a crosstalk between HEV and EGFR requires deeper analysis. We therefore aimed to dissect the receptor's involvement in the HEV life cycle. In persistently HEV-infected cells, the EGFR amount is decreased alongside with enhanced receptor internalization. As compared with the control ligand-induced EGFR, activation revealed an early receptor internalization and degradation in HEV-replicating cells, resulting in a notable EGFR signaling delay. Interestingly, inhibition or silencing of EGFR increased viral replication, extracellular and intracellular viral transcripts, and released infectious particles. The pro-viral impact of EGFR inhibition was attributed to (i) impaired expression of interferon-stimulated genes, (ii) activation of the autophagosomal system, (iii) virus-induced inhibition of lysosomal acidification, and (iv) a decrease of the cellular cholesterol level.
IMPORTANCE
This study identifies epidermal growth factor receptor (EGFR) as a novel host factor affecting hepatitis E virus (HEV): EGFR downregulation promotes viral replication, release, and evasion from the innate immune response. The discovery that EGFR inhibition favors viral spread is particularly concerning for HEV patients undergoing EGFR inhibitor treatment.
PubMed: 38856640
DOI: 10.1128/jvi.00580-24 -
Frontiers in Cell and Developmental... 2024
PubMed: 38855162
DOI: 10.3389/fcell.2024.1392810 -
BioRxiv : the Preprint Server For... May 2024Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Although lowering...
Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Although lowering mHTT by stimulating autophagy has been considered a possible therapeutic strategy, the role and competence of autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex of HD brains staged from HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins p62/SQSTM1 and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. Importantly, no blockage in autophagosome formation and autophagosome-lysosome fusion was detected, thus pinpointing autophagy substrate clearance deficits as a basis for autophagic flux declines. The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but failure at advanced stages contributes to progressive HTT build-up and potential neurotoxicity. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, may have therapeutic benefits in HD patients.
PubMed: 38854113
DOI: 10.1101/2024.05.29.596470 -
Cell Death Discovery Jun 2024Renal cell carcinoma (RCC) is a malignant tumor with high incidence in adult kidney. Long non-coding RNAs (lncRNAs) have recently been recognized as important regulators...
Renal cell carcinoma (RCC) is a malignant tumor with high incidence in adult kidney. Long non-coding RNAs (lncRNAs) have recently been recognized as important regulators in the development of RCC. However, whether lncRNA SNHG1 is associated with RCC progression remains to be elucidated. Here, the role of SNHG1 in RCC autophagy and sunitinib resistance was evaluated. Expression of SNHG1 in RCC tissues and cells was assessed using RT-qPCR. Western blot was utilized to measure the levels of autophagy-related molecules and ATG7. RNA pull-down and RIP assays were performed to confirm the molecular axis between SNHG1/PTBP1/ATG7. Cell proliferation, migration, invasion and apoptosis were analyzed by CCK-8, EdU, transwell and flow cytometry, respectively. The subcellular localization of SNHG1 was determined by an intracellular fractionation assay. The fluorescence intensity of GFP-LC3 autophagosome in RCC cells was detected. IHC staining was performed to test ATG7 expression in tumor tissues from nude mice. Here, a positive correlation of upregulated SNHG1 with poor prognosis of RCC patients was observed in RCC tissues and cells. SNHG1 knockdown suppressed tumor growth and reversed sunitinib resistance and autophagy of RCC cells. Additionally, SNHG1 was found to directly bind to PTBP1, thereby positively regulating ATG7 expression. Furthermore, we verified that SNHG1 mediated the malignant behavior of RCC cells through the PTBP1/ATG7 axis. To sum up, SNHG1 regulates RCC cell autophagy and sunitinib resistance through the PTBP1/ATG7 axis, which highlights a promising therapeutic target for RCC treatment.
PubMed: 38851811
DOI: 10.1038/s41420-024-02021-3 -
Biomedicine & Pharmacotherapy =... Jul 2024Integrated stress response (ISR) is activated to promote cell survival by maintaining the phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). We...
OBJECTIVE
Integrated stress response (ISR) is activated to promote cell survival by maintaining the phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). We investigated whether Sephin1 enhances ISR and attenuates myocardial ischemia-reperfusion (MIR) injury.
METHODS
Male C57BL/6 J mice were injected with Sephin1 (2 mg/kg,i.p.) 30 min before surgery to establish a model of MIR with 45 min ischemia and 180 min reperfusion. In vitro, the H9C2 cell line with hypoxia-reoxygenation (H/R) was used to simulate MIR. Myocardial injury was evaluated by echocardiography, histologic observation after staining with TTC and H&E and electron microscopy. ISR, autophagy and apoptosis in vivo and in vitro were evaluated by immunoblotting, immunohistochemistry, immunofluorescence, and flow cytometry, respectively. Global protein synthesis was determined using a non-radioactive SUnSET Assay based on the puromycin method. Autophinib, an autophagy-specific inhibitor, was used to investigate the correlation between autophagy and apoptosis in the presence of Sephin1.
RESULTS
In vivo, Sephin1 significantly reduced myocardial injury and improved the cardiac function in MIR mice. Sephin1 administration prolonged ISR, reduced cell apoptosis, and promoted autophagy. In vitro, Sephin1 increased the number of stress granules (SGs) and autophagic vesicles, enhanced ISR and related protein synthesis suppression, and reduced cell apoptosis. Autophinib partly reversed autophagosome formation and apoptosis in H9c2 cells.
CONCLUSIONS
Sephin1 enhances ISR and related protein synthesis suppression, ameliorates myocardial apoptosis, and promotes autophagy during MIR stress. Sephin1 could act as a noval ISR enhancer for managing acute myocardial ischemia disease.
Topics: Animals; Autophagy; Male; Myocardial Reperfusion Injury; Mice, Inbred C57BL; Mice; Apoptosis; Cell Line; Myocytes, Cardiac; Disease Models, Animal; Erythropoietin; Peptide Fragments
PubMed: 38850665
DOI: 10.1016/j.biopha.2024.116869 -
IScience Jun 2024The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded...
The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.
PubMed: 38832010
DOI: 10.1016/j.isci.2024.109810