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International Journal of Molecular... Jun 2024In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing...
The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
Topics: Humans; Viral Nonstructural Proteins; Virus Replication; Autophagosomes; HeLa Cells; Phlebovirus; Autophagy; Tyrosine; Tryptophan; TOR Serine-Threonine Kinases; Mutation; Amino Acid Substitution; Severe Fever with Thrombocytopenia Syndrome; Lysosomes; Nucleoproteins
PubMed: 38928101
DOI: 10.3390/ijms25126394 -
Journal For Immunotherapy of Cancer Jun 2024Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However,...
BACKGROUND
Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However, the mechanism of NET formation is not fully understood. This study posits that tumor cell-released autophagosomes (TRAPs) play a crucial role in this process.
METHODS
TRAPs were isolated from breast cancer cell lines to analyze their impact on NET formation in both human and mouse neutrophils. The study used both in vitro and in vivo models, including Toll-like receptor 4 (TLR4/) mice and engineered breast cancer cell lines. Immunofluorescence, ELISA, Western blotting, RNA sequencing, and flow cytometry were employed to dissect the signaling pathways leading to NET production and to explore their immunosuppressive effects, particularly focusing on the impact of NETs on T-cell function. The therapeutic potential of targeting TRAP-induced NETs and their immunosuppressive functions was evaluated using DNase I and αPD-L1 antibodies. Clinical relevance was assessed by correlating circulating levels of TRAPs and NETs with lung metastasis in patients with breast cancer.
RESULTS
This study showed that TRAPs induced the formation of NETs in both human and mouse neutrophils by using the high mobility group box 1 and activating the TLR4-Myd88-ERK/p38 signaling axis. More importantly, PD-L1 carried by TRAP-induced NETs inhibited T-cell function in vitro and in vivo, thereby contributing to the formation of lung premetastatic niche (PMN) immunosuppression. In contrast, KD-4T1 breast tumors with decreased circulating TRAPs in vivo reduced the formation of NETs, which in turn attenuated the immunosuppressive effects in PMN and resulted in a reduction of breast cancer pulmonary metastasis in murine models. Moreover, treatment with αPD-L1 in combination with DNase I that degraded NETs restored T-cell function and significantly reduced tumor metastasis. TRAP levels in the peripheral blood positively correlated with NET levels and lung metastasis in patients with breast cancer.
CONCLUSIONS
Our results demonstrate a novel role of TRAPs in the formation of PD-L1-decorated NETs, which may provide a new strategy for early detection and treatment of pulmonary metastasis in patients with breast cancer.
Topics: Animals; Humans; Mice; Female; Breast Neoplasms; Lung Neoplasms; Extracellular Traps; B7-H1 Antigen; Autophagosomes; T-Lymphocytes; Cell Line, Tumor
PubMed: 38926151
DOI: 10.1136/jitc-2024-009082 -
Developmental and Comparative Immunology Jun 2024To overcome bacterial invasion and infection, animals have evolved various antimicrobial effectors such as antimicrobial peptides and lysozymes. Although C. elegans is...
To overcome bacterial invasion and infection, animals have evolved various antimicrobial effectors such as antimicrobial peptides and lysozymes. Although C. elegans is exposed to a variety of microbes due to its bacterivorous lifestyle, previous work on the components of its immune system mainly based on the description of transcriptional changes during bacterial challenges. Very few effector components of its immune system have been characterized so far. To investigate the role of lysozymes in terms of antibacterial defense and digestion, we studied a member of the widely neglected family of C. elegans invertebrate lysozymes (ILYS). We focused on the so far virtually undescribed ILYS-5, which we purified from protein extracts of C. elegans tracing its peptidoglycan-degrading activity and localized the tissue expression of the gene in vivo using a translational reporter construct. We recombinantly synthesized ILYS-5 and determined the physicochemical activity optimum and the antibacterial spectrum of a lysozyme from C. elegans for the first time. With an activity optimum at low ionic strength (≤ 100 mM) and at acidic pH (≤ pH 4.0), ILYS-5 is likely to be involved in killing and digestion of bacteria within acidified phagolysosomes and acidic regions of the gut, presumably secreted by lysosome-like vesicles. This notion is supported by potent activity against various live Gram-positive and Gram-negative bacteria. Notably, members of the natural associated microbiome of C. elegans are substantially less susceptible to ILYS-5. Ablation of the ilys-5 gene resulted in reduction of lifespan and fertility when cultured on the standard food bacterium Escherichia coli OP50, whereas exposure of the ilys-5 knock-out mutant to the host-associated bacterium Pseudomonas lurida MYb11 did not have a clear effect. These findings indicate a role of ILYS-5 in immunity and nutrition and a co-evolved adaptation of host and bacteria to the mutualistic nature of their interaction.
PubMed: 38925432
DOI: 10.1016/j.dci.2024.105220 -
Microbiology Spectrum Jun 2024() as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug...
UNLABELLED
() as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular and in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone . In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections.
IMPORTANCE
Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including () and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of and in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an tuberculosis model based on infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.
PubMed: 38916320
DOI: 10.1128/spectrum.00167-24 -
International Journal of Biological... 2024Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is...
Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.
Topics: Pulmonary Artery; Animals; Cell Proliferation; Humans; Zinc; Mice; Rats; Myocytes, Smooth Muscle; Metallothionein 3; Male; Autophagosomes; Autophagy-Related Protein 5; Rats, Sprague-Dawley; Transcription Factors; Autophagy; Hypertension, Pulmonary; Mice, Inbred C57BL; DNA-Binding Proteins; Transcription Factor MTF-1; Metallothionein
PubMed: 38904023
DOI: 10.7150/ijbs.92992 -
Nature Communications Jun 2024Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require...
Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.
Topics: Arabidopsis; Autophagosomes; Endosomal Sorting Complexes Required for Transport; Arabidopsis Proteins; Calcium-Binding Proteins; Autophagy; Phosphatidylinositol Phosphates; Autophagy-Related Protein 8 Family; Vacuoles; Phase Separation
PubMed: 38898014
DOI: 10.1038/s41467-024-49485-6 -
Methods in Molecular Biology (Clifton,... 2024One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance... (Review)
Review
One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process, named phagocytosis, for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this, he gave us the basis for our modern understanding of inflammation and the innate immune response. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In this chapter, we present a general view of our current knowledge on phagocytosis performed mainly by professional phagocytes through antibody and complement receptors and discuss aspects that remain incompletely understood.
Topics: Phagocytosis; Humans; Animals; Phagosomes; Phagocytes; Signal Transduction; Immunity, Innate
PubMed: 38888769
DOI: 10.1007/978-1-0716-3890-3_3 -
The Journal of Antimicrobial... Jun 2024Q fever is a worldwide zoonosis due to Coxiella burnetii, responsible for endocarditis and endovascular infections. Since the 1990s, the combination...
Q fever is a worldwide zoonosis due to Coxiella burnetii, responsible for endocarditis and endovascular infections. Since the 1990s, the combination hydroxychloroquine + doxycycline has constituted the curative and prophylactic treatment in persistent focalized Q fever. This combination appears to have significantly reduced the treatment's duration (from 60 to 26 months), yet substantial evidence of effectiveness remains lacking. Data are mostly based on in vitro and observational studies. We conducted a literature review to assess the effectiveness of this therapy, along with potential alternatives. The proposed in vitro mechanism of action describes the inhibition of Coxiella replication by doxycycline through the restoration of its bactericidal activity (inhibited in acidic environment) by alkalinization of phagolysosome-like vacuoles with hydroxychloroquine. So far, the rarity and heterogeneous presentation of cases have made it challenging to design prospective studies with statistical power. The main studies supporting this treatment are retrospective cohorts, dating back to the 1990s-2000s. Retrospective studies from the large Dutch outbreak of Q fever (>4000 cases between 2007 and 2010) did not corroborate a clear benefit of this combination, notably in comparison with other regimens. Thus, there is still no consensus among the medical community on this issue. However insufficient the evidence, today the doxycycline + hydroxychloroquine combination remains the regimen with the largest clinical experience in the treatment of 'chronic' Q fever. Reinforcing the guidelines' level of evidence is critical. We herein propose the creation of an extensive international registry, followed by a prospective cohort or ideally a randomized controlled trial.
PubMed: 38888195
DOI: 10.1093/jac/dkae145 -
Infection and Immunity Jun 2024() is an intracellular pathogen that survives and grows in macrophages. A mechanism used by to achieve intracellular survival is to secrete effector molecules that...
() is an intracellular pathogen that survives and grows in macrophages. A mechanism used by to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PIP), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame mutants in both the attenuated bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and . Characterization of these mutants demonstrated that SapM is required for PMA in BCG and . Further, by establishing a role for SapM in PMA in BCG, and subsequently in a mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and , and that it can function independent of the known mechanism for molecules to leave the phagosome.
PubMed: 38884474
DOI: 10.1128/iai.00217-24 -
Autophagy Jun 2024Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome...
Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V domain and the membrane-embedded V domain. The V-ATPase a subunit, an integral part of the V domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species.: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.
PubMed: 38873931
DOI: 10.1080/15548627.2024.2366748