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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 -
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 Proteome Research Jun 2024This study aimed to identify characteristic proteins in infantile epileptic spasm syndrome (IESS) patients' plasma, offering insights into potential early diagnostic...
This study aimed to identify characteristic proteins in infantile epileptic spasm syndrome (IESS) patients' plasma, offering insights into potential early diagnostic biomarkers and its underlying causes. Plasma samples were gathered from 60 patients with IESS and 40 healthy controls. Data-independent acquisition proteomic analysis was utilized to identify differentially expressed proteins (DEPs). These DEPs underwent functional annotation through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Gene set enrichment analysis (GSEA) was employed for both GO (GSEA-GO) and KEGG (GSEA-KEGG) analyses to examine the gene expression profiles. Receiver operating characteristic (ROC) curves assessed biomarkers' discriminatory capacity. A total of 124 DEPs were identified in IESS patients' plasma, mainly linked to pathways, encompassing chemokines, cytokines, and oxidative detoxification. GSEA-GO and GSEA-KEGG analyses indicated significant enrichment of genes associated with cell migration, focal adhesion, and phagosome pathways. ROC curve analysis demonstrated that the combination of PRSS1 and ACTB, PRSS3, ACTB, and PRSS1 alone exhibited AUC values exceeding 0.7. This study elucidated the significant contribution of cytokines, chemokines, oxidative detoxification, and phagosomes to the IESS pathogenesis. The combination of PRSS1 and ACTB holds promise as biomarkers for the early diagnosis of IESS.
PubMed: 38857073
DOI: 10.1021/acs.jproteome.4c00298 -
BioRxiv : the Preprint Server For... May 2024The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously...
The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously expressed, is highly abundant in cells of the innate immune system. Given the importance of central and peripheral immune cells in the development of PD, we sought to investigate the consequences of the G2019S mutation on microglial and monocyte transcriptome and function. We have generated large-scale transcriptomic profiles of isogenic human induced microglial cells (iMGLs) and patient derived monocytes carrying the G2019S mutation under baseline culture conditions and following exposure to the proinflammatory factors IFNγ and LPS. We demonstrate that the G2019S mutation exerts a profound impact on the transcriptomic profile of these myeloid cells, and describe corresponding functional differences in iMGLs. The G2019S mutation led to an upregulation in lipid metabolism and phagolysosomal pathway genes in untreated and LPS/IFNγ stimulated iMGLs, which was accompanied by an increased phagocytic capacity of myelin debris. We also identified dysregulation of cell cycle genes, with a downregulation of the E2F4 regulon. Transcriptomic characterization of human-derived monocytes carrying the G2019S mutation confirmed alteration in lipid metabolism associated genes. Altogether, these findings reveal the influence of G2019S on the dysregulation of the myeloid cell transcriptome under proinflammatory conditions.
PubMed: 38854101
DOI: 10.1101/2024.05.27.594821 -
Experimental Cell Research Jul 2024Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause...
Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of β-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.
Topics: Humans; Autophagy; Mutation; Calcium; Distal Myopathies; HSP70 Heat-Shock Proteins; Multienzyme Complexes; HEK293 Cells; Autophagosomes; India
PubMed: 38852763
DOI: 10.1016/j.yexcr.2024.114118 -
Biochemical and Biophysical Research... Sep 2024Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic...
Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.
Topics: Humans; N-Acetylglucosaminyltransferases; Autophagy; Endosomes; Down-Regulation; Lysosomes; rab7 GTP-Binding Proteins; Nutrients; rab GTP-Binding Proteins; Colon; HCT116 Cells; Autophagosomes
PubMed: 38852504
DOI: 10.1016/j.bbrc.2024.150198 -
The ISME Journal Jan 2024Amoeba-bacteria interactions are prevalent in both natural ecosystems and engineered environments. Amoebae, as essential consumers, hold significant ecological...
Amoeba-bacteria interactions are prevalent in both natural ecosystems and engineered environments. Amoebae, as essential consumers, hold significant ecological importance within ecosystems. Besides, they can establish stable symbiotic associations with bacteria. Copper plays a critical role in amoeba predation by either killing or restricting the growth of ingested bacteria in phagosomes. However, certain symbiotic bacteria have evolved mechanisms to persist within the phagosomal vacuole, evading antimicrobial defenses. Despite these insights, the impact of copper on the symbiotic relationships between amoebae and bacteria remains poorly understood. In this study, we investigated the effects of copper stress on amoebae and their symbiotic relationships with bacteria. Our findings revealed that elevated copper concentration adversely affected amoeba growth and altered cellular fate. Symbiont type significantly influenced the responses of the symbiotic relationships to copper stress. Beneficial symbionts maintained stability under copper stress, but parasitic symbionts exhibited enhanced colonization of amoebae. Furthermore, copper stress favored the transition of symbiotic relationships between amoebae and beneficial symbionts toward the host's benefit. Conversely, the pathogenic effects of parasitic symbionts on hosts were exacerbated under copper stress. This study sheds light on the intricate response mechanisms of soil amoebae and amoeba-bacteria symbiotic systems to copper stress, providing new insights into symbiotic dynamics under abiotic factors. Additionally, the results underscore the potential risks of copper accumulation in the environment for pathogen transmission and biosafety.
Topics: Copper; Symbiosis; Amoeba; Bacteria; Stress, Physiological; Bacterial Physiological Phenomena
PubMed: 38848278
DOI: 10.1093/ismejo/wrae100 -
Current Health Sciences Journal 2024Microglial cells play a pivotal role in the brain's health and operation through all stages of life and in the face of illness. The contributions of microglia during the...
Microglial cells play a pivotal role in the brain's health and operation through all stages of life and in the face of illness. The contributions of microglia during the developmental phase of the brain markedly contrast with their contributions in the brain of adults after injury. Enhancing our understanding of the pathological mechanisms that involve microglial activity in brains as they age and in cerebrovascular conditions is crucial for informing the creation of novel therapeutic approaches. In this work we provide results on microglia transcriptomics in the juvenile vs injured adult brain and its impact on adult brain regeneration after cerebral ischemia. During fetal brain development, microglia cells are involved in gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis, neurogenesis and synaptic reorganization by engulfing neuronal extensions. Within the mature, intact brain, microglial cells exhibit reduced movement of their processes in response to minimal neuronal activity, while they continuously monitor their surroundings and clear away cellular debris. Following a stroke in the adult brain, inflammation, neurodegeneration, or disruptions in neural equilibrium trigger alterations in both the genetic blueprint and the structure and roles of microglia, a state often described as "activated" microglia. Such genetic shifts include a notable increase in the pathways related to phagosomes, lysosomes, and the presentation of antigens, coupled with a rise in the expression of genes linked to cell surface receptors. We conclude that a comparison of microglia transcriptomic activity during brain development and post-stroke adult brain might provide us with new clues about how neurodegeneration occurs in the adult brain. This information could very useful to develop drugs to slow down or limit the post-stroke pathology and improve clinical outcome.
PubMed: 38846476
DOI: 10.12865/CHSJ.50.01.17 -
Fish & Shellfish Immunology Jun 2024Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to...
Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7-9 x 10 vs 2-5 x 10 cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion.
PubMed: 38844186
DOI: 10.1016/j.fsi.2024.109664 -
Frontiers in Cellular and Infection... 2024is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for in...
INTRODUCTION
is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for in the U.S. warrants further research into pathogenesis. Within the host cells, replicates in an acidic phagolysosome-like vacuole termed -containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for survival and that the Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in - infected cells remained unclear.
METHODS
We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry- to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and intracellular growth.
RESULTS
The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in -infected cells compared to mock, suggesting that increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication.
DISCUSSION
Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for intracellular survival and potentially uncovers novel host cell factors essential for pathogenesis.
Topics: Coxiella burnetii; rab GTP-Binding Proteins; Humans; Vacuoles; HeLa Cells; Endosomes; Host-Pathogen Interactions; Q Fever
PubMed: 38841112
DOI: 10.3389/fcimb.2024.1394019