Autoimmunity and the clearance of dead cells.

To maintain organismal homeostasis, phagocytes engulf dead cells, which are recognized as dead by virtue of a characteristic "eat me" signal exposed on their surface. The dead cells are then transferred to lysosomes, where their cellular components are degraded for reuse. Inefficient engulfment of dead cells activates the immune system, causing disease such as systemic lupus erythematosus, and if the DNA of the dead cells is not properly degraded, the innate immune response becomes activated, leading to severe anemia and chronic arthritis. Here, we discuss how the endogenous components of dead cells activate the immune system through both extracellular and intracellular pathways.

The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc.

Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.

Peritumoral SPARC expression induced by exosomes from nasopharyngeal carcinoma infected Epstein-Barr virus: A poor prognostic marker.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) cells have high metastatic potential. Recent research has revealed that the interaction of between tumor cells and the surrounding stroma plays an important role in tumor invasion and metastasis. In this study, we showed the prognostic value of expression of SPARC, an extracellular matrix protein with multiple cellular functions, in normal adjacent tissues (NAT) surrounding NPC. In the immunohistochemical analysis of 51 NPC biopsy specimens, SPARC expression levels were significantly elevated in the NAT of EBER (EBV-encoded small RNA)-positive NPC compared to that in the NAT of EBER-negative NPC. Moreover, increased SPARC expression in NAT was associated with a worsening of overall survival. The enrichment analysis of RNA-seq of publicly available NPC and NAT surrounding NPC data showed that high SPARC expression in NPC was associated with epithelial mesenchymal transition promotion, and there was a dynamic change in the gene expression profile associated with interference of cellular proliferation in NAT, including SPARC expression. Furthermore, EBV-positive NPC cells induce SPARC expression in normal nasopharyngeal cells via exosomes. Induction of SPARC in cancer-surrounding NAT cells reduced intercellular adhesion in normal nasopharyngeal structures and promoted cell competition between cancer cells and normal epithelial cells. These results suggest that epithelial cells loosen their own binding with the extracellular matrix as well as stromal cells, facilitating the invasion of tumor cells into the adjacent stroma by activating cell competition. Our findings reveal a new mechanism by which EBV creates a pro-metastatic microenvironment by upregulating SPARC expression in NPC.

Extracellular vesicles promote silica nanoparticle aggregation that inhibits silica-induced cytotoxicity.

Amorphous silica has been approved as a food and pharmaceutical additive. However, its potential to enhance the carcinogenicity of epithelial cells is incontrovertible. With their expanded surface area per unit mass and distinctive cellular incorporation, nano-sized silica particles (nSPs) exhibit heightened cytotoxicity compared to micrometer-sized counterparts. The precise effect of nSPs on the generation of small extracellular vesicles (sEVs) within endosomes after cellular uptake remains unclear. In the present study, we explored the secretion of sEVs from cells and their functional implications following exposure to nSPs. Our findings demonstrate that nSP50 exposure not only induced epithelial-mesenchymal transition (EMT) but also promoted the maturation of multivesicular endosomes (MVEs) along with the secretion of sEVs in A549 cells. Inhibition of sEV secretion using GW4869 and apoptosis activator 2 exacerbated nSP50-induced EMT, indicating that sEV secretion may suppress EMT. Analysis of the function of sEV in a cell-free system revealed that co-incubation of sEVs with nSP50 led to the formation of micrometer-sized aggregates, which exhibited limited uptake efficiency within A549 cells. These results strongly suggest that the secretion of sEVs plays a protective role against the cytotoxicity attributed to nSP50 exposure.

Nanoscopic Assessment of Anti-SARS-CoV-2 Spike Neutralizing Antibody Using High-Speed AFM.

Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.

Nanopipette Fabrication Guidelines for SICM Nanoscale Imaging.

Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.

Preventing SARS-CoV-2 Infection Using Anti-spike Nanobody-IFN-ß Conjugated Exosomes.

PURPOSE: To inhibit the transmission of SARS-CoV-2, we developed engineered exosomes that were conjugated with anti-spike nanobodies and type I interferon ß (IFN-ß). We evaluated the efficacy and potency of nanobody-IFN-ß conjugated exosomes to treatment of SARS-CoV-2 infection. METHODS: Milk fat globule epidermal growth factor 8 (MFG-E8) is a glycoprotein that binds to phosphatidylserine (PS) exposed on the exosomes. We generated nanobody-IFN-ß conjugated exosomes by fusing an anti-spike nanobody and IFN-ß with MFG-E8. We used the SARS-CoV-2 pseudovirus with the spike of the D614G mutant that encodes ZsGreen to mimic the infection process of the SARS-CoV-2. The SARS-CoV-2 pseudovirus was infected with angiotensin-converting enzyme-2 (ACE2) expressing adenocarcinomic human alveolar basal epithelial cells (A549) or ACE2 expressing HEK-blue IFNα/ß cells in the presence of nanobody-IFN-ß conjugated exosomes. By assessing the expression of ZsGreen in target cells and the upregulation of interferon-stimulated genes (ISGs) in infected cells, we evaluated the anti-viral effects of nanobody-IFN-ß conjugated exosomes. RESULTS: We confirmed the anti-spike nanobody and IFN-ß expressions on the exosomes. Exosomes conjugated with nanobody-hIFN-ß inhibited the interaction between the spike protein and ACE2, thereby inhibiting the infection of host cells with SARS-CoV-2 pseudovirus. At the same time, IFN-ß was selectively delivered to SARS-CoV-2 infected cells, resulting in the upregulation of ISGs expression. CONCLUSION: Exosomes conjugated with nanobody-IFN-ß may provide potential benefits in the treatment of COVID-19 because of the cooperative anti-viral effects of the anti-spike nanobody and the IFN-ß.

Tumor-secreted proliferin-1 regulates adipogenesis and lipolysis in cachexia.

Cancer-associated cachexia (CAC) is a common syndrome in cancer patients and is characterized by loss of body weight accompanied by the atrophy of fat and skeletal muscle. Metabolic changes are a critical factor in CAC; however, the mechanisms through which tumors inhibit adipogenesis and promote lipolysis are poorly understood. To clarify these mechanisms, we investigated adipogenesis-limiting factors released by tumors in a cell culture system. We identified proliferin-1 (PLF-1), a member of the growth hormone/prolactin gene family, as a key factor secreted from certain tumors that inhibited preadipocyte maturation and promoted the lipolysis of mature adipocytes. Importantly, mice transplanted with PLF-1-depleted tumor cells were protected from fat loss due to CAC. These data show that tumor-secreted PLF-1 plays an essential role in impaired adipogenesis and accelerated lipolysis and is a potential therapeutic target against CAC.

Characterization of LILRB3 and LILRA6 allelic variants in the Japanese population.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) are encoded by members of a human multigene family, comprising 11 protein-coding genes and two pseudogenes. Among the LILRs, LILRB3 and LILRA6 show the highest homology with each other, along with high allelic and copy number variations. Therefore, it has been difficult to discriminate between them, both genetically and functionally, precluding disease association studies of LILRB3 and LILRA6. In this study, we carefully performed variant screening of LILRB3 and LILRA6 by cDNA cloning from Japanese individuals and identified four allelic lineages showing significantly high non-synonymous-to-synonymous ratios in pairwise comparisons. Furthermore, the extracellular domains of the LILRB3*JP6 and LILRA6*JP1 alleles were identical at the DNA level, suggesting that gene conversion-like events diversified LILRB3 and LILRA6. To determine the four allelic lineages from genomic DNA, we established a lineage typing method that accurately estimated the four allelic lineages in addition to specific common alleles from genomic DNA. Analysis of LILRA6 copy number variation revealed one, two, and three copies of LILRA6 in the Japanese-in-Tokyo (JPT) population. Flow cytometric analysis showed that an anti-LILRB3 antibody did not recognize the second most common lineage in the Japanese population, indicating significant amino acid differences across the allelic lineages. Taken together, our findings indicate that our lineage typing is useful for classifying the lineage-specific functions of LILRB3 and LILRA6, serving as the basis for disease association studies.

Novel properties of myoferlin in glucose metabolism via pathways involving modulation of adipose functions.

While adipose tissue is required to maintain glucose metabolism, excessive calorie intake induces obesity via mechanisms including accelerated proliferation and differentiation of preadipocytes, leading to insulin resistance. Here, we investigated the role of myoferlin (MYOF), a ferlin family protein, in regulating glucose metabolism by mainly focusing on its unknown role in adipose tissue. Whereas young MYOF knockout (KO) mice on a normal diet showed aggravated glucose tolerance and insulin sensitivity, those on a high-fat diet (HFD) showed preserved glucose tolerance with an attenuated gain of body weight, reduced visceral fat deposits, and less severe fatty liver. The Adipose MYOF expression was reduced by aging but was restored by an HFD along with the retained expression of NFAT transcription factors. Loss-of-function of MYOF in preadipocytes suppressed proliferation and differentiation into mature adipocytes along with the decreased expression of genes involved in adipogenesis. The MYOF expression in preadipocytes was reduced with differentiation. Attenuated obesity in MYOF KO mice on an HFD was also accompanied with increased oxygen consumption by an unidentified mechanism and with reduced adipose inflammation due to less inflammatory macrophages. These insights suggest that the multifunctional roles of MYOF involve the regulation of preadipocyte function and affect glucose metabolism bidirectionally depending on consumed calories.

High-Speed AFM Reveals Molecular Dynamics of Human Influenza A Hemagglutinin and Its Interaction with Exosomes.

Influenza A hemagglutinin (HA) is one of the crucial virulence factors that mediate host tropism and viral infectivity. Presently, the mechanism of the fusogenic transition of HA remains elusive. Here, we used high-speed atomic force microscopy (HS-AFM) to decipher the molecular dynamics of HA and its interaction with exosomes. Our data reveal that the native conformation of HA in the neutral buffer is ellipsoidal, and HA undergoes a conformational change in an acidic buffer. Real-time visualization of the fusogenic transition by HS-AFM suggests that the mechanism is possibly fit to the "uncaging" model, and HA intermediate appears as Y-shaped. A firm interaction between the HA and exosome in an acidic buffer indicates the insertion of a fusion peptide into the exosomal layer and subsequently destabilizes the layer, resulting in the deformation or rupture of exosomes, releasing exosomal contents. In contrast, the HA-exosome interaction is weak in a neutral buffer because the interaction is mediated by weak bonds between the HA receptor-binding site and receptors on the exosome.

Glioma-derived extracellular vesicles promote tumor progression by conveying WT1.

Glioma persists as one of the most aggressive primary tumors of the central nervous system. Glioma cells are known to communicate with tumor-associated macrophages/microglia via various cytokines to establish the tumor microenvironment. However, how extracellular vesicles (EVs), emerging regulators of cell-cell communication networks, function in this process is still elusive. We report here that glioma-derived EVs promote tumor progression by affecting microglial gene expression in an intracranial implantation glioma model mouse. The gene expression of thrombospondin-1 (Thbs1), a negative regulator of angiogenesis, was commonly downregulated in microglia after the addition of EVs isolated from different glioma cell lines, which endogenously expressed Wilms tumor-1 (WT1). Conversely, WT1-deficiency in the glioma-derived EVs significantly attenuated the Thbs1 downregulation and suppressed the tumor progression. WT1 was highly expressed in EVs obtained from the cerebrospinal fluid of human patients with malignant glioma. Our findings establish a novel model of tumor progression via EV-mediated WT1-Thbs1 intercellular regulatory pathway, which may be a future diagnostic or therapeutic target.

Myoferlin-Mediated Lysosomal Exocytosis Regulates Cytotoxicity by Phagocytes.

During inflammation, phagocytes release digestive enzymes from lysosomes to degrade harmful cells such as pathogens and tumor cells. However, the molecular mechanisms regulating this process are poorly understood. In this study, we identified myoferlin as a critical regulator of lysosomal exocytosis by mouse phagocytes. Myoferlin is a type II transmembrane protein with seven C2 domains in the cytoplasmic region. It localizes to lysosomes and mediates their fusion with the plasma membrane upon calcium stimulation. Myoferlin promotes the release of lysosomal contents, including hydrolytic enzymes, which increase cytotoxicity. These data demonstrate myoferlin's critical role in lysosomal exocytosis by phagocytes, providing novel insights into the mechanisms of inflammation-related cellular injuries.

Development of a Method That Delivers Drugs to Enveloped Viruses.

Hepatitis C virus (HCV) infection leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in 50-80% of the cases. Interferons (IFNs) and the nucleoside analog ribavirin form the basis of the treatment of this infection but are not considered sufficiently effective and cause several side effects. In this study, we developed a novel viral-specific drug delivery method. Enveloped viruses, including HCV, expose an anionic phospholipid, phosphatidylserine (PS), on their surface to mediate their binding and entry into cells for infection. To target such exposed PS on HCV, we developed a chimeric recombinant protein containing human IFN and mouse lactadherin (also known as milk fat globule epidermal growth factor 8), which binds with high affinity to PS. The IFN-lactadherin fusion protein showed a high binding affinity toward PS and HCV and consequently blocked viral replication in the infected cells more efficiently than conventional IFN. Overall, these data suggest that conjugation with lactadherin facilitates the delivery of any protein drug to PS-exposing enveloped viruses.

The Role of Exosomes/Extracellular Vesicles in Neural Signal Transduction.

Exosomes, in a broad sense extracellular vesicles (EVs), are secreted from several cells and also exist in cerebrospinal fluid (CSF); they contribute to signal transduction not only between neural cells but also among hematopoietic cells. In addition to the peripheral nervous system, the association of regeneration and EVs has also been reported in the central nervous system, for example, following a spinal cord injury. Furthermore, it has become clear that major causative factors of neurodegenerative diseases are transmitted by EVs; thus, EVs are involved in the pathogenesis of neurodegenerative diseases. In particular, we would like to outline the relationship between neurophysiology and neurological disorders centered on EV-mediated communication between neural and glial cells.

MHC class I-dressing is mediated via phosphatidylserine recognition and is enhanced by polyI:C.

In addition to cross-presentation, cross-dressing plays an important role in the induction of CD8+ T cell immunity. In the process of cross-dressing, conventional dendritic cells (DCs) acquire major histocompatibility complex class I (MHCI) from other cells and subsequently prime CD8+ T cells via the pre-formed antigen-MHCI complexes without antigen processing. However, the mechanisms underlying the cross-dressing pathway, as well as the relative contributions of cross-presentation and cross-dressing to CD8+ T cell priming are not fully understood. Here, we demonstrate that DCs rapidly acquire MHCI-containing membrane fragments from dead cells via the phosphatidylserine recognition-dependent mechanism for cross-dressing. The MHCI dressing is enhanced by a TLR3 ligand polyinosinic-polycytidylic acid (polyI:C). Further, polyI:C promotes not only cross-presentation but also cross-dressing in vivo. Taken together, these results suggest that cross-dressing as well as cross-presentation is involved in inflammatory diseases associated with cell death and type I IFN production.

Precise analysis of single small extracellular vesicles using flow cytometry.

Methods that enable specific and sensitive quantification of small extracellular vesicles (sEVs) using flow cytometry are still under development. Aggregation or adsorption of antibodies causes sub-nano sized particles or non-specific binding and largely affects the results of flow cytometric analysis of single sEVs. Comparison of control IgG and target-specific IgG is inappropriate because they have different characters. Here, we evaluate four preparation methods for flow cytometry, including ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method by using tetraspanin-deficient sEVs. The ultracentrifugation or density gradient centrifugation preparation method has large false-positive rates for tetraspanin staining. Conversely, preparation methods using SEC or the TIM4-affinity method show specific detection of single sEVs, which elucidate the roles of sEV biogenesis regulators in the generation of sEV subpopulations. The methods are also useful for the detection of rare disease-related markers, such as PD-L1. Flow cytometric analysis using SEC or the TIM4-affinity method could accelerate research into sEV biogenesis and the development of sEV-based diagnostics and therapies.

Identification of the hybrid gene LILRB5-3 by long-read sequencing and implication of its novel signaling function.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) on human chromosome 19q13.4 encode 11 immunoglobulin superfamily receptors, exhibiting genetic diversity within and between human populations. Among the LILR genes, the genomic region surrounding LILRB3 and LILRA6 has yet to be fully characterized due to their significant sequence homology, which makes it difficult to differentiate between them. To examine the LILRB3 and LILRA6 genomic region, a tool named JoGo-LILR CN Caller, which can call copy number from short-read whole genome sequencing (srWGS) data, was applied to an extensive international srWGS dataset comprising 2,504 samples. During this process, a previously unreported loss of both LILRB3 and LILRA6 was detected in three samples. Using long-read sequencing of these samples, we have discovered a novel large deletion (33,692 bp) in the LILRB3 and LILRA6 genomic regions in the Japanese population. This deletion spanned three genes, LILRB3, LILRA6, and LILRB5, resulting in LILRB3 exons 12-13 being located immediately downstream of LILRB5 exons 1-12 with the loss of LILRA6, suggesting the potential expression of a hybrid gene between LILRB5 and LILRB3 (LILRB5-3). Transcription and subsequent translation of the LILRB5-3 hybrid gene were also verified. The hybrid junction was located within the intracellular domain, resulting in an LILRB5 extracellular domain fused to a partial LILRB3 intracellular domain with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), suggesting that LILRB5-3 acquired a novel signaling function. Further application of the JoGo-LILR tool to srWGS samples suggested the presence of the LILRB5-3 hybrid gene in the CEU population. Our findings provide insight into the genetic and functional diversity of the LILR family.

A validation study for the utility of serum microRNA as a diagnostic and prognostic marker in patients with osteosarcoma.

In our previous study, osteosarcoma advanced locally, and metastasis was promoted through the secretion of large number of small extracellular vesicles, followed by suppressing osteoclastogenesis via the upregulation of microRNA (miR)-146a-5p. An additional 12 miRNAs in small extracellular vesicles were also detected ≥6× as frequently in high-grade malignancy with the capacity to metastasize as in those with a low metastatic potential. However, the utility of these 13 miRNAs for determining the prognosis or diagnosis of osteosarcoma has not been validated in the clinical setting. In the present study, the utility of these miRNAs as prognostic and diagnostic markers was therefore assessed. In total, 30 patients with osteosarcoma were retrospectively reviewed, and the survival rate was compared according to the serum miRNA levels in 27 patients treated with chemotherapy and surgery. In addition, to confirm diagnostic competency for osteosarcoma, the serum miRNA levels were compared with those in patients with other bone tumors (n=112) and healthy controls (n=275). The patients with osteosarcoma with high serum levels of several miRNAs (miR-146a-5p, miR-1260a, miR-487b-3p, miR-1260b and miR-4758-3p) exhibited an improved survival rate compared with those with low levels. In particular, patients with high serum levels of miR-1260a exhibited a significantly improved overall survival rate, metastasis-free survival rate and disease-free survival rate compared with those with low levels. Thus, serum miR-1260a may potentially be a prognostic marker for patients with osteosarcoma. Moreover, patients with osteosarcoma had higher serum miR-1261 levels than those with benign or intermediate-grade bone tumors and thus may be a potential therapeutic target, in addition to being useful for differentiating whether or not a bone tumor is high-grade. A larger investigation is required to clarify the actual utility of these miRNAs in the clinical setting.

TIM4-Affinity Methods Targeting Phosphatidylserine for Isolation or Detection of Extracellular Vesicles.

Small extracellular vesicles (SEVs) secreted from various cells are lipid bilayer vesicles, 30-150 nm in size, that carry proteins, nucleic acids, and lipids as cargos to other cells. They include exosomes, which are generated in multivesicular endosomes (MVEs) and secreted upon fusion of MVEs with plasma membranes and a part of microvesicles, which directly bud from plasma membranes. SEVs have attracted attention as diagnostic and drug discovery targets, since it has been demonstrated that SEVs are involved in the intercellular communication in many diseases and physiological phenomena such as cancer, neurodegenerative diseases, and immunity. There are five isolation methods for SEVs, which include ultracentrifugation, density gradient ultracentrifugation, polymer precipitation, affinity isolation, and size-exclusion chromatography. The affinity isolation, which isolates SEVs using magnetic beads conjugated with binding molecules such as antibodies, has the ability to isolate highly pure SEVs in character. However, the population of SEVs is limited by the binding molecules and it is difficult to elute intact SEVs from the antibody beads. In this chapter, we present a TIM4-affinity isolation method that targets phosphatidylserine (PS), a component of the SEV membrane. TIM4 binds to PS in a Ca2+-dependent manner, which enables the elution of intact SEVs from TIM4-beads in the presence of the chelating reagent ethylenediaminetetraacetic acid (EDTA). The TIM4-affinity isolation method helps overcome the disadvantages of the affinity isolation method and enables the isolation of heterogeneous SEVs at high purity. This method will facilitate the functional analysis of SEVs, development of diagnostic methods, and drug development of engineered SEVs.