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Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins; however, whether these proteins are uniformly present on each LEL, or if there are cell-type-dependent LEL subpopulations with unique protein compositions is unclear. We employed quantitative, multiplexed DNA-PAINT super-resolution imaging to examine the distribution of seven key LEL proteins (LAMP1, LAMP2, CD63, Cathepsin D, TMEM192, NPC1, and LAMTOR4). While LAMP1, LAMP2, and Cathepsin D were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type-specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts., (© 2024 Bond et al.)

A promising strategy in cancer immunotherapy is to restore or enhance the cytotoxicity of NK cells, among others, by activating the mechanism of antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies targeting tumor antigens, such as rituximab (targeting CD20), induce NK cell-mediated ADCC and have been used to treat B cell malignancies, such as non-Hodgkin lymphoma, but not always successfully. The aim of this study was to analyze the gene expression profile of the NK cells involved in the cytolytic response stimulated by rituximab. NK cells were co-cultured with rituximab-opsonized Raji cells. Sorting into responder and non-responder groups was based on the presence of CD107a, which is a degranulation marker. RNA-seq results showed that the KIT and TNFSF4 genes were strongly down-regulated in the degranulating population of NK cells (responders); this was further confirmed by qRT-PCR. Both genes encode surface proteins with cellular signaling abilities, namely c-KIT and the OX40 ligand. Consistent with our findings, c-KIT was previously reported to correlate inversely with cytokine production by activated NK cells. The significance of these findings for cancer immunotherapy seems essential, as the pharmacological inhibition of c-KIT and OX40L, or gene ablation, could be further tested for the enhancement of the anti-tumor activity of NK cells in response to rituximab.

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever. Successful entry of LASV requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus (LCMV) residue (L84 ​N, K88E, L10F, and H170S) reduced the binding affinity of LASV GP1 for LAMP1. Moreover, all mutations caused decreases in glycoprotein precursor (GPC)-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPC L84N or GPC K88E decreased sharply in multiple cell types, while L107F and H170S had only mild effects on infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order of binding affinity being L84 ​N ​> ​L107F ​> ​K88E ​> ​H170S. The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4 + cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150 Glued . We found that the mTORC1 inhibitor rapamycin enhanced p150 Glued -AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150 Glued -AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150 Glued -AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150 Glued -AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150 Glued -AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport., (© 2024. The Author(s).)

Acinetobacter baumannii (A. baumannii) causes autophagy flux disorder by degrading STX17, resulting in a serious inflammatory response. It remains unclear whether STX17 can alter the inflammatory response process by controlling autolysosome function. This study aimed to explore the role of STX17 in the regulation of pyroptosis induced by A. baumannii. Our findings indicate that overexpression of STX17 enhances autophagosome degradation, increases LAMP1 expression, reduces Cathepsin B release, and improves lysosomal function. Conversely, knockdown of STX17 suppresses autophagosome degradation, reduces LAMP1 expression, augments Cathepsin B release, and accelerates lysosomal dysfunction. In instances of A. baumannii infection, overexpression of STX17 was found to improve lysosomal function and reduce the expression of mature of GSDMD and IL-1β, along with the release of LDH, thus inhibiting pyroptosis caused by A. baumannii. Conversely, knockdown of STX17 led to increased lysosomal dysfunction and further enhanced the expression of mature of GSDMD and IL-1β, and increased the release of LDH, exacerbating pyroptosis induced by A. baumannii. These findings suggest that STX17 regulates pyroptosis induced by A. baumannii by modulating lysosomal function., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Autophagy is a major catabolic degradation and recycling process that maintains homeostasis in cells and is especially important in postmitotic neurons. We implemented a high-content phenotypic assay to discover small molecules that promote autophagic flux and completed target identification and validation studies to identify protein targets that modulate the autophagy pathway and promote neuronal health and survival. Efficient syntheses of the prioritized compounds were developed to readily access analogues of the initial hits, enabling initial structure-activity relationship studies to improve potency and preparation of a biotin-tagged pulldown probe that retains activity. This probe facilitated target identification and validation studies through pulldown and competition experiments using both an unbiased proteomics approach and western blotting to reveal Lamin A/C and LAMP1 as the protein targets of compound RH1115 . Evaluation of RH1115 in neurons revealed that this compound induces changes to LAMP1 vesicle properties and alters lysosome positioning. Dysfunction of the autophagy-lysosome pathway has been implicated in a variety of neurodegenerative diseases, including Alzheimer's disease, highlighting the value of new strategies for therapeutic modulation and the importance of small-molecule probes to facilitate the study of autophagy regulation in cultured neurons and in vivo .

Thyroid hormone (T 3 )-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T 3 on lysosomal protein expression and trafficking in detail. Our findings showed that T 3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T 3 -promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T 3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T 3 , although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T 3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T 3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interestsCheng-Chih Chang reports financial support was provided by Chang Gung Memorial Hospital, Chiayi, Taiwan. Kwang-Huei Lin reports financial support was provided by Chang Gung Memorial Hospital, Taoyuan, Taiwan. Kwang-Huei Lin reports financial support was provided by National Science and Technology Council., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. Ultimately, after particle degradation, phagosomes then fragment to reform lysosomes through phagosome resolution. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation and resolution. These changes can be assessed at the single-phagosome level by using immunofluorescence methods. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Commonly, progression of phagosomes into phagolysosomes can be determined by staining cells for Lysosomal-Associated Membrane Protein I (LAMP1) and measuring the fluorescence intensity of LAMP1 around each phagosome by microscopy or flow cytometry. However, this method can be used to detect any molecular marker for which there are compatible antibodies for immunofluorescence., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoe tm1Unc J/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients. Abbreviations: AMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.

Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a common cause of uterine infertility. We previously demonstrated that partial epithelial-mesenchymal transition (EMT) and the loss of epithelial homeostasis play a vital role in the development of endometrial fibrosis. As a pro-survival strategy in maintaining cell and tissue homeostasis, macroautophagy/autophagy, conversely, may participate in this process. However, the role of autophagy in endometrial fibrosis remains unknown. Here, we demonstrated that autophagy is defective in endometria of IUA patients, which aggravates EMT and endometrial fibrosis, and defective autophagy is related to DIO2 (iodothyronine deiodinase 2) downregulation. In endometrial epithelial cells (EECs), pharmacological inhibition of autophagy by chloroquine (CQ) promoted EEC-EMT, whereas enhanced autophagy by rapamycin extenuated this process. Mechanistically, silencing DIO2 in EECs blocked autophagic flux and promoted EMT via the MAPK/ERK-MTOR pathway. Inversely, overexpression of DIO2 or triiodothyronine (T3) treatment could restore autophagy and partly reverse EEC-EMT. Furthermore, in an IUA-like mouse model, the autophagy in endometrium was defective accompanied by EEC-EMT, and CQ could inhibit autophagy and aggravate endometrial fibrosis, whereas rapamycin or T3 treatment could improve the autophagic levels and blunt endometrial fibrosis. Together, we demonstrated that defective autophagy played an important role in EEC-EMT in IUA via the DIO2-MAPK/ERK-MTOR pathway, which provided a potential target for therapeutic implications. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AMPK: adenosine 5'-monophosphate-activated protein kinase; AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; CDH1/E-cadherin: cadherin 1; CDH2/N-cadherin: cadherin 2; CQ: chloroquine; CTSD: cathepsin D; DIO2: iodothyronine deiodinase 2; DEGs: differentially expressed genes; EECs: endometrial epithelial cells; EMT: epithelial-mesenchymal transition; FN1: fibronectin 1; IUA: intrauterine adhesions; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; T3: triiodothyronine; T4: tetraiodothyronine; TFEB: transcription factor EB; PBS: phosphate-buffered saline; TEM: transmission electron microscopy; TGFB/TGFβ: transforming growth factor beta.

TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation. Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO 2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H 2 S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H 2 S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2 + cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68 + and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = -0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H 2 S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H 2 S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H 2 S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H 2 S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1 . Conclusively, CTH-H 2 S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H 2 S may act as a warning biomarker for vulnerable plaque. Abbreviations ATG9A: autophagy related 9A; CTH: cystathionine gamma-lyase; CQ: chloroquine; HASMCs: human aortic smooth muscle cells; H 2 S: hydrogen sulfide; LAMP1: lysosomal associated membrane protein 1; LAPTM5: lysosomal protein transmembrane 5; NaHS: sodium hydrosulfide hydrate; ox-LDL: oxidized-low density lipoprotein; PPG: DL- propagylglycine; TFEB: transcription factor EB; 3-MA: 3-methyladenine; VSMCs: vascular smooth muscle cells.

Objectives: Intermittent fasting (IF) activates autophagy in cardiac muscle and pancreatic islets. We examined the effect of IF on markers of autophagy in liver and skeletal muscle in mice and in humans., Methods: Ten-wk-old C57 BL/6 J male mice were ad libitum (AL) fed a high-fat diet (HFD) or chow diet for 8 wk, before randomization to AL or IF (24-h fast, 3 non-consecutive days per week) for 8 wk (8-16 per group). Tissue was collected in the fed or 22-h fasted state. Fifty women (51 ± 2 y, 31.8 ± 4.3 kg/m 2 ) were randomly assigned to one of two IF protocols (24-hfast, 3 non-consecutive days per week) and fed at 70% (IF70) or 100% (IF100) of energy requirements for 8 wk. Vastus lateralis muscle was collected at 0800 after 12- and 24-h fasts. Microtubule-associated protein light chain 1 (Map1 lc3 b), Beclin1 (Becn1), Sequestosome 1 (Sqstm1/p62), and Lysosomal associated membrane protein 2 (Lamp2) were assessed by quantitative polymerase chain reaction and LC3, BECLIN1 and LAMP1 protein content by immunoblotting., Results: Fasting increased hepatic LC3 I protein and Map1 lc3 b mRNA levels in IF mice fed chow or HFD. LAMP1 protein and Beclin1 mRNA levels in liver were also increased by fasting, but only in chow-fed mice. IF did not activate markers of autophagy in mouse muscle. In humans, a 24-h fast increased SQSTM1. BECLIN1, SQSTM1 and LAMP2 mRNA levels were decreased in IF70 after a 12-h overnight fast ., Conclusion: Markers of autophagy in liver, but not in muscle, were elevated in response to IF in mice. In humans, autophagy markers in muscle were reduced, likely in response to weight loss., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry., Competing Interests: The authors have declared that no competing interests exist.

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Background: C1q/tumor necrosis factor-related protein 3 (CTRP3) has been reported to be a crucial regulator in myocardial infarction. Nevertheless, the potential molecular mechanism of CTRP3 in ischemia/reperfusion (I/R) injury remains largely unclear., Methods: The cell model of myocardial I/R injury was established by oxygen-glucose deprivation/reoxygenation (OGD/R) of rat cardiomyocyte H9C2. Expression of CTRP3 and lysosomal-associated membrane protein 1 (LAMP1) was detected in H9C2 cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R). H9C2 cells were transfected with overexpression plasmids of CTRP3 (pcDNA-CTRP3) and LAMP1 (pcDNA-LAMP1), or CTRP3 small interfering RNA (si-CTRP3) or/and pcDNA-LAMP1, and cell proliferation, apoptosis and oxidative stress were testified. Co-IP assay was performed to validate the relationship among CTRP3, LAMP1 and JIP2. The role of CTRP3 and LAMP1 in JIP2/JNK pathway was evaluated with Western blot assay. Furthermore, in vivo myocardial I/R injury model was constructed to investigate the effect of CTRP3., Results: Overexpression of CTRP3 and LAMP1 both significantly promoted cell proliferation, inhibited apoptosis and the production of reactive oxygen species (ROS), malondialdehyde (MAD) and cardiac troponin (cTn-I), while silencing CTRP3 exerted the opposite effects, and LAMP1 overexpression reversed the effect of silencing CTRP3 on the aspects above. CTRP3 interacted with LAMP1, and both CTRP3 and LAMP1 bound with JIP2. SP600125 (JNK inhibitor) could restore the effects of CTRP3 or LAMP1 overexpression on the expression of JIP2 and phosphorylated-JNK (p-JNK), proliferation and apoptosis. Moreover, overexpression of CTRP3 improved cardiac I/R injury in vivo ., Conclusion: CTRP3 alleviates cardiac I/R injury by elevating LAMP1 and activating JIP2/JNK signaling pathway, which may serve as a potential therapeutic target for I/R injury.

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures.

In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4 + and CD8 + T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART ( n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8 + and CD107a and IL-2 production in HIV-specific CD4 + and CD8 + T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4 + and CD8 + T cells in PWH on ART. These combinations should be further explored for an HIV cure., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: Cetuximab is used for colorectal cancer (CRC) treatment. However, the early biomarker of treatment efficacy of cetuximab has not been identified., Methods: After 1 year of cetuximab treatment, patients were divided into an effective group and an ineffective group. The interleukin-33 (IL-33) level and the distribution of lymphatic cells in patients were investigated by analyzing the peripheral blood mononuclear cells via flow cytometry analysis and ELISA. The correlation between IL-33 immunomodulatory effect and cetuximab treatment efficacy was determined through experiments in vivo and in vitro., Results: The IL-33 level in the peripheral blood was increased at 4 weeks after cetuximab administration of effective group, meanwhile, the osteopontin (OPN) was reduced. Whereas neither IL-33 level nor OPN level of ineffective patients changed. In the effective group, the number of natural killer (NK) and CD8 + T cells were increased. Moreover, CD137 and CD107a expression on NK cells were higher in the effective group compared to the ineffective group. In vitro cetuximab treatment also increased the number of NK and CD8 + T cells as well as CD137 and CD107a expression upon IL-33 stimulation. Moreover, the secretion of OPN was inhibited by IL-33 administration in cetuximab-treated PBMCs from the effective group patients. IL-33 upregulated the cytotoxicity of NK cells and inhibited tumor cells growth in the effective cetuximab treatment mice., Conclusion: Effective cetuximab treatment induced a change of IL-33 and OPN at the early stage and triggered the NK cells antitumor activity. Consequently, significantly increased IL-33 level and decreased OPN level in the peripheral blood at the early treatment are proposed as potential predictors of cetuximab treatment efficacy., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Lactoferrin (LF) was used at first as a vehicle to deliver non-soluble active compounds to the body, including the central nervous system (CNS). Nonetheless, it soon became evident that, apart from acting as a vehicle, LF itself owns active effects in the CNS. In the present study, the effects of LF are assessed both in baseline conditions, as well as to counteract methamphetamine (METH)-induced neurodegeneration by assessing cell viability, cell phenotype, mitochondrial status, and specific autophagy steps. In detail, cell integrity in baseline conditions and following METH administration was carried out by using H&E staining, Trypan blue, Fluoro Jade B, and WST-1. Western blot and immuno-fluorescence were used to assess the expression of the neurofilament marker βIII-tubulin. Mitochondria were stained using Mito Tracker Red and Green and were further detailed and quantified by using transmission electron microscopy. Autophagy markers were analyzed through immuno-fluorescence and electron microscopy. LF counteracts METH-induced degeneration. In detail, LF significantly attenuates the amount of cell loss and mitochondrial alterations produced by METH; and mitigates the dissipation of autophagy-related proteins from the autophagy compartment, which is massively induced by METH. These findings indicate a protective role of LF in the molecular mechanisms of neurodegeneration.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In mucosa such as tonsil, antibody-producing plasmocytes (PCs) lie in sub-epithelium space, which is thought to provide a suitable environment for their survival. A proliferation inducing ligand (APRIL) is one key survival factor for PCs present in this area. According to in situ staining, apical epithelial cells produced APRIL, and the secreted product had to migrate all through the stratified surface epithelium to reach basal cells. A similar process also occurred in the less-organized crypt epithelium. Tonsil epithelial cells captured secreted APRIL, thanks to their surface expression of the APRIL coreceptor, either syndecan-1 or -4 depending on their differentiation stage. In the most basal epithelial cells, secreted APRIL accumulated inside secretory lamp-1 + vesicles in a polarized manner, facing the sub-epithelium. The tonsil epithelium upregulated APRIL production by apical cells and secretion by basal cells upon Toll-like receptor stimulation. Furthermore, LPS-stimulated epithelial cells sustained in vitro PC survival in a secreted APRIL-dependent manner. Taken together, our study shows that the tonsil epithelium responds to pathogen sensing by a polarized secretion of APRIL in the sub-epithelial space, wherein PCs reside., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sturm, Quinterot, Guyot, Righini, Daamen, van Kuppevelt and Huard.)

Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The global obesity epidemic is contributing to increased prevalence of diseases fuelled by chronic inflammation, including cancer. Oesophageal adenocarcinoma (OAC) is an obesity-associated malignancy with increasing prevalence, dismal prognosis, and severely dysregulated immune processes. We previously reported that αβ T cells migrate to omentum and liver in OAC and contribute to inflammation in these tissues. Here, we assessed the tissue distribution and phenotype of gamma/delta (γδ) T cells in the blood, omentum, liver and tumour of OAC patients. Our data show that the Vδ1 and Vδ3 subsets of γδ T cells are most prevalent in omentum and liver of OAC patients. Furthermore, γδ T cells are predominantly pro-inflammatory in these tissues, and co-express IFN-γ and IL-17. Moreover, γδ T cells exhibit cytotoxic capabilities in OAC omentum and liver. This study provides the first indication that γδ T cells contribute to obesity-associated inflammation in OAC and might be exploited therapeutically., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Documenting the importance of NK cell function as a biomarker for diseases and physiologic conditions including myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), will require assays amenable to clinical implementation and standardization. Research studies typically perform NK functional assays on the day of sample collection. This pilot study was conducted to compare assay formats and specimen processing to identify those that are most tolerant of conditions required for shipping and amenable to standardization as shown by inter-assay and inter-laboratory correlation of results. We compared performance within and between assays that measure NK cell function using direct cytotoxicity [chromium-51 release (CRCA) or fluorescence (Flow Cytometry Cytotoxicity Assay, FCCA)] or an indirect surrogate marker (CD107a surface expression)]. Additional variables for within/between assay comparisons included time of testing (same day as specimen collection or next day within 24 h), specimen types [whole blood or isolated peripheral blood mononuclear cells (PBMCs)], and processing method (fresh or cryopreserved). Statistical measures included number of samples tested in assay conditions (n), medians (x͂), interquartile range (IQR), Pearson correlation coefficient (R 2 ), and correlation p-value (p). Samples came from 3 clinics and included 31 participants. Same day testing was only available for the subset of participants enrolled from the site of the laboratory performing CRCA. Results from same day CRCA testing of whole blood were considered the gold standard [n = 10, x͂=10.0%, IQR = 7.2%], and correlated well with PBMCs isolated next day [n = 26, x͂= 15.6%, IQR = 13.1%] [R 2  = 0.59, p = 0.03]. Next day CRCA results were compromised using whole blood or frozen PBMCs. Next day FCCA cytotoxicity in PBMC [n = 30, x͂=34.1%, IQR = 15.5%] correlated with same day CRCA PMBC [R 2  = 0.8, p = 0.001] and next day CRCA PMBC [R 2  = 0.5, p < 0.0001]. CD107a expression after induction by PMA and ionomycin did not correlate with other cytotoxicity measures. NK function can be measured in PBMCs isolated after overnight shipping/storage at ambient temperature and CRCA and FCCA results on this sample type are well correlated., (Copyright © 2021. Published by Elsevier B.V.)

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5 -/- or CLN6 -/- animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5 -/- animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6 -/- animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5 -/- animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5 -/- sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales., (The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi., (© 2021 Götz et al.)

Problem: Immune checkpoint molecules are receptors that can transmit inhibitory signals into cells to negatively modulate the immune response. However, their roles in NK cells during normal pregnancy remain poorly understood., Method of Study: Peripheral blood samples were collected from women during the first, second and third trimesters of pregnancy. Peripheral blood NK (pNK) cells and T cells were analyzed for the expression of the immune checkpoint molecules TIGIT and PD-1 by flow cytometry. In addition, the ability of pNK cells to secret functional molecules was also evaluated., Results: The expression of TIGIT on pNK cells increased gradually throughout pregnancy, whereas that of PD-1 showed the opposite pattern. However, on T cells, the expression of both TIGIT and PD-1 peaked during early pregnancy, and then declined gradually thereafter. Moreover, the expressions of granzyme B, IFN-γ and CD107a by pNK cells also decreased over the course of pregnancy. Compared with TIGIT- NK cells, TIGIT + NK cells possessed reduced expression of functional molecules., Conclusions: As pregnancy progressed, the levels of immune checkpoint molecules expressed on pNK cells and T cells changed and the two molecules showed different trends. Furthermore, the secretion of functional molecules from pNK cells was negatively correlated with the trend of TIGIT expression, indicating TIGIT may play an important role in modulating the functions of pNK cells during pregnancy. Further study of TIGIT expression on pNK cells may enhance our understanding of its role in maintaining maternal-fetal tolerance and provide a useful marker for predicting instability during pregnancy., (Copyright © 2020 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Thymic stromal lymphopoietin (TSLP) is released by epithelial cells following disturbed homeostasis to act as "alarmin" and driver of Th2-immunity. Aberrant TSLP expression is a hallmark of atopic diseases, including atopic dermatitis (AD). Mast cells (MCs) are overabundant in AD lesions and show signs of degranulation, but it remains unknown whether TSLP contributes to granule discharge. Degranulation of skin MCs proceeds via two major routes, i.e., FcεRI-dependent (allergic) and MRGPRX2-mediated (pseudo-allergic/neurogenic). Evidence is accumulating that MRGPRX2 may be crucial in the context of skin diseases, including eczema. The current study reveals TSLP as a novel priming factor of human skin MCs. Interestingly, TSLP selectively cooperates with MRGPRX2 to support granule discharge, while it does not impact spontaneous or FcεRI-driven exocytosis. TSLP-assisted histamine liberation triggered by compound 48/80 or Substance P, two canonical MRGPRX2 agonists, was accompanied by an increase in CD107a+ cells (a MC activation marker). The latter process was less potent, however, and detectable only at the later of two time points, suggesting TSLP may prolong opening of the granules. Mechanistically, TSLP elicited phosphorylation of STAT5 and JNK in skin MCs and the reinforced degranulation critically depended on STAT5 activity, while JNK had a contributory role. Results from pharmacological inhibition were confirmed by RNA-interference, whereby silencing of STAT5 completely abolished the priming effect of TSLP on MRGPRX2-mediated degranulation. Collectively, TSLP is the first factor to favor MRGPRX2- over FcεRI-triggered MC activation. The relevance of TSLP, MCs and MRGPRX2 to pruritis and atopic skin pathology indicates broad repercussions of the identified connection.

Natural killer (NK) cells are innate immune lymphocytes that play an important role in anti-viral and anti-tumour immune responses. Several cancer immunotherapy approaches targeting NK cells are currently in clinical or preclinical development. Here, we aimed to find food nutrients that activate NK cells and determine their usefulness as candidates for anti-cancer and anti-metastatic drugs. Resveratrol appeared to activate NK cells most effectively among the substances tested and synergistically increased IFN-γ secretion and NK cell cytotoxicity with interleukin-2 (IL-2). CD107a, NKp30, and NKG2D expression levels were upregulated on the surface of NK cells upon treatment with resveratrol in combination with IL-2 compared with treatment with IL-2 alone. Moreover, NK cell activity in human and mouse whole blood was enhanced upon treatment with resveratrol. Most importantly, administration of resveratrol effectively inhibited tumour growth and metastasis in mice. In conclusion, we suggest that resveratrol may represent a candidate anti-cancer drug that acts by activating NK cells in vivo., (The Author(s). Published by S. Karger AG, Basel.)

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes., (Published by Elsevier Inc.)

Restrictions on antimicrobials demand alternative strategies to improve broiler health, such as supplying feed additives which stimulate innate immune cells like natural killer (NK) cells. The main objective of this study was to characterize intestinal NK cells in broiler chickens during embryonic and early life and compare these to NK cells in spleen, blood and bone marrow. Also T-cell subsets were determined. The majority of intestinal NK cells expressed IL-2Rα rather than 20E5 and 5C7, and showed low level of activation. Within intestinal NK cells the activation marker CD107 was mostly expressed on IL-2Rα + cells while in spleen and blood 20E5 + NK cells primarily expressed CD107. High percentages of intestinal CD8αα + , CD8αβ + and from 2 weeks onward also gamma delta T cells were found. Taken together, we observed several intestinal NK subsets in broiler chickens. Differences in NK subsets were mostly observed between organs, rather than differences over time. Targeting these intestinal NK subsets may be a strategy to improve immune-mediated resistance in broiler chickens., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batches of APEX labeling and different APEX probes. We conducted a comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.

Natural killer (NK) cells play a key role in both antibacterial and antitumor immunity. Pseudomonas aeruginosa infection has already been reported to alter NK cell functions. We studied in vitro the effect of P. aeruginosa on NK cell cytotoxic response (CD107a membrane expression) to a lymphoma cell line. Through positive and negative cell sorting and adoptive transfer, we determined the influence of monocytes, lymphocytes, and regulatory T cells (Treg) on NK cell function during P. aeruginosa infection. We also studied the role of the activating receptor natural killer group 2D (NKG2D) in NK cell response to B221. We determined that P. aeruginosa significantly altered both cytotoxic response to B221 and NKG2D expression on NK cells in a Treg-dependent manner and that the NKG2D receptor was involved in NK cell cytotoxic response to B221. Our results also suggested that during P. aeruginosa infection, monocytes participated in Treg-mediated NK cell alteration. In conclusion, P. aeruginosa infection impairs NK cell cytotoxicity and alters antitumor immunity. These results highlight the strong interaction between bacterial infection and immunity against cancer., (Copyright © 2020 American Society for Microbiology.)

Markers associated to NK cytolytic activity are in a great need to regulate NK cell immunotherapy products. We assume that biomarkers which response to cytolysis will change their transcription, expression or secretion. To find NK-92 indicator to cytolytic activity, we have evaluated the potential markers by quantifying the expression of well-known cytotoxicity functional molecules (cytokine IFN-γ, Granzyme B, perforin, CD69 and CD107a), and explored candidate markers by a sweeping transcription picture of NK-92 using a direct cytolysis model (incubation with K562). We found that IFN-γ secretion was highly correlated to cytotoxicity of NK-92, neither Granzyme B, perforin secretion, nor CD69, CD107a positive population were upregulated by K562 stimulation. RNAseq revealed 432 genes expression changed during cytolysis, several genes (BIRC3, CSF2, VCAM1 and TNFRSF9) mRNA expression were validated by real time RT-PCR under K562 being killed or protected from being killed conditions. Results suggested IFN-γ secretion, BIRC3 and TNFRSF9 transcription in NK-92 were responsive to K562 cytolysis. In a word, our results confirmed one marker and reveal an array of novel candidate markers associated with NK-92 cytotoxicity. Further studies are greatly needed to determine the roles these new makers play in NK-92 cytolysis process., (Copyright © 2020. Published by Elsevier Ltd.)

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107a low cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107a high cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107a low cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107a high cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107a low cells are precursors of CD107a high cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest., Competing Interests: JX, YW, CH, SN, RT, YG, YT, TS, CM, WH, LC, SH, NK, KB No competing interests declared, BP BP is the inventor of perivascular stem cell related patents held by the UC Regents (Patent number: 9730965) and is on the editorial board of Stem Cells. AJ AWJ is a paid consultant for Novadip. This arrangement has been reviewed and approved by the JHU in accordance with its conflict of interest polices. AWJ receives funding for unrelated research from MTF Biologics and Novadip, and is on the editorial board of American Journal of Pathology and Bone Research. AWJ is the inventor of methods to purify CD107a progenitor cells held by the Johns Hopkins University., (© 2020, Xu et al.)

Our recent study showed a high rate of HBsAg seroconversion in inactive HBsAg carriers (IHCs) treated with pegylated IFN (PEG-IFN). To understand the immune-mediated component of the HBsAg seroconversion better, this study investigated the role of NK cells. A total of 44 IHCs were given 48 wk of PEG-IFN. Fifteen cases achieved HBsAg seroconversion (R group), whereas 29 failed (NR group). The proportion and activity (CD107α and IFN-γ production) of NK cells were measured before and during treatment. We found that the proportion of NK cells in the R group was higher than in the NR group at baseline and during PEG-IFN treatment, even when patients were matched for age, sex and treatment period. IFN- γ secretion and CD107α expression from NK cells in cases who achieved HBsAg seroconversion were significantly higher than patients matched for age, sex, HBsAg and treatment period in the NR group at baseline and during PEG-IFN treatment. We also found that in HBsAg seroconversion cases, NK cells activity increased after PEG-IFN treatment, especially before HBsAg seroconversion. These effects were not found in non-responders. In conclusion, we demonstrated that the increase of NK cells accompanied by enhanced activity during PEG-IFN treatment favoured HBsAg seroconversion for IHC, and that NK cells may play a role in HBV seroconversion.

HIV-infected older individuals may have a diminished immune response because of exhaustion/immune aging of T-cells. Therefore, we have investigated HIV-specific CD4 and CD8 T-cell responses in 100 HIV-infected patients (HIV+) who have aged on long-term antiretroviral therapy (ART) and achieved controlled viremia (mostly undetectable viral load; 92 patients with <20 to <40 HIV RNA copies/mL and 8 <60 to <100) and improved CD4 T-cell counts. We show that the median frequencies of HIV-specific CD4+ and CD8+ IFN-γ T-cells were higher in HIV+ than uninfected individuals (HIV-), including increasing levels of IFN-γproduced by CD4+ T-cells and decreasing levels by CD8+ T-cells with increasing CD4 T-cell counts in HIV+. No correlation was found between T-cell responses and varying levels of undetectable viremia. HIV-specific TNF-α made by CD8+ T-cells was higher in HIV+ than HIV-, including decreasing levels with increasing CD4 T-cell counts in HIV+. Furthermore, the CD8+ T-cell mediators, CD107a and Granzyme-B, were higher in HIV+ than HIV-, and decreased with increasing CD4 T-cell counts in HIV+. Remarkably, HIV-specific CD8 T-cells produced decreasing levels of IFN-γwith increasing age of HIV+, including decreased levels of CD107a and Granzyme-B in older HIV+. However, HIV-specific CD8+ T-cells produced increasing levels of TNF-α with increasing age of the HIV+, suggesting continued inflammation. In conclusion, HIV+ with controlled viremia on long-term ART and with higher CD4 T-cell counts showed reduced HIV-specific CD8 T-cell responses as compared to those with lower CD4 T-cell counts, and older HIV+ exhibited decreasing levels of CD8 T-cell responses with increasing age., Competing Interests: The authors have declared that no competing interests exist.

Background: Increasing evidence supports a critical role of CD8+ T-cell immunity against influenza. Activation of mucosal CD8+ T cells, particularly tissue-resident memory T (TRM) cells recognizing conserved epitopes would mediate rapid and broad protection. Matrix protein 1 (M1) is a well-conserved internal protein., Methods: We studied the capacity of modified vaccinia Ankara (MVA)-vectored vaccine expressing nucleoprotein (NP) and M1 (MVA-NP+M1) to activate M1-specific CD8+ T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue from children and adults., Results: After MVA-NP+M1 stimulation, M1 was abundantly expressed in adenotonsillar epithelial cells and B cells. MVA-NP+M1 activated a marked interferon γ-secreting T-cell response to M1 peptides. Using tetramer staining, we showed the vaccine activated a marked increase in M158-66 peptide-specific CD8+ T cells in tonsillar mononuclear cells of HLA-matched individuals. We also demonstrated MVA-NP+M1 activated a substantial increase in TRM cells exhibiting effector memory T-cell phenotype. On recall antigen recognition, M1-specific T cells rapidly undergo cytotoxic degranulation, release granzyme B and proinflammatory cytokines, leading to target cell killing., Conclusions: MVA-NP+M1 elicits a substantial M1-specific T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue, demonstrating its strong capacity to expand memory T-cell pool exhibiting effector memory T-cell phenotype, therefore offering great potential for rapid and broad protection against influenza reinfection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Oxidative stress and T h 17 cytokines are important mediators of inflammation. Treatment with beta-adrenoceptor (ADRB) antagonists (beta-blockers) is associated with induction or aggravation of psoriasis-like skin inflammation, yet the underlying mechanisms are poorly understood. Herein, we identify lysosomotropic beta-blockers as critical inducers of IL23A in human monocyte-derived Langerhans-like cells under sterile-inflammatory conditions. Cytokine release was not mediated by cAMP, suggesting the involvement of ADRB-independent pathways. NFKB/NF-κB and MAPK14/p38 activation was required for propranolol-induced IL23A secretion whereas the NLRP3 inflammasome was dispensable. MAPK14 regulated recruitment of RELB to IL23A promoter regions. Without affecting the ubiquitin-proteasome pathway, propranolol increased lysosomal pH and induced a late-stage block in macroautophagy/autophagy. Propranolol specifically induced reactive oxygen species production, which was critical for IL23A secretion, in Langerhans-like cells. Our findings provide insight into a potentially crucial immunoregulatory mechanism in cutaneous dendritic cells that may explain how lysosomotropic drugs regulate inflammatory responses., Abbreviations: ATF: activating transcription factor; DC: dendritic cell; ChIP: chromatin immunoprecipitation; gDNA: genomic DNA; IL: interleukin; LAMP1: lysosomal associated membrane protein 1; LC: Langerhans cell; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MoDC: monocyte-derived DC; MoLC: monocyte-derived Langerhans-like cell; mtDNA: mitochondrial DNA; NAC: N-acetyl-L-cysteine; NLRP3: NLR family pyrin domain containing 3; PBMC: peripheral blood mononuclear cell; PI: propidium iodide; PYCARD/ASC: PYD and CARD domain containing; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TLR: Toll-like receptor; TRAF6: TNF receptor associated factor 6; TNF: tumor necrosis factor; Ub: ubiquitin.

Cord blood platelet rich plasma (CB-PRP) derivatives have been investigated as potential therapeutic agents for the treatment of diverse conditions including ocular surface disease and skin ulcers. We have developed processes for the formulation of several CB-PRP preparations, which have different composition and attributes. Here we describe the molecular characteristics of these preparations and we make recommendations as to their most appropriate clinical application based on functional and immunomodulatory profiles. We show that incubation of adult peripheral blood mononuclear cells (PBMCs) with all three preparations dramatically reduced the production of INFγ and the expression of NKG2D and CD107a in NK, NKT, and T cells thus diminishing their activation, we propose that the likely mechanism is the high levels of soluble NKG2D ligands present in plasma. Of the three preparations we investigated, CB platelet lysate (PL) and platelet releaseate (PR) have higher concentrations of trophic and pro-angiogenic factors, CB platelet poor plasma (PPP) has the lowest concentration of all analytes measured. Based on these finding we propose that CB-PR is the most suitable raw material for skin wound patches, while CB-PL and PPP can be used to prepare eye drops for severe ocular surface pathologies and inflammatory conditions such as corneal ulcers or severe dry eye disease, respectively., (Copyright © 2020 Samarkanova, Cox, Hernandez, Rodriguez, Casaroli-Marano, Madrigal and Querol.)

The epididymis plays a significant role as a quality control organ for long-term sperm storage, maturation, and fertilizing ability and perform filtration function to eliminate abnormal or residual spermatozoa by phagocytosis. However, the role of autophagy in spermiophagy during sperm storage in turtle epididymis still needs to be studied. In this study, we reported in vivo spermiophagy via the cellular evidence of lysosome engulfment and autophagy within the principal cells during sperm storage in the turtle epididymis. Using immunofluorescence, Lysosome associated membrane protein-1 (LAMP1) and microtubule-associate protein light chain 3 (LC3) showed strong immunosignals within the apical cytoplasm of epididymal epithelia during hibernation than non-hibernation. Co-immunolabeling of LAMP1 and LC3 was strong around the phagocytosed spermatozoa in the epididymal epithelia and protein signaling of LAMP1 and LC3 was confirmed by western blotting. During hibernation, ultrastructure showed epididymal principal cells were involved in spermiophagy and characterized by the membrane's concentric layers around phagocytosed segments of spermatozoa, degenerative changes in the sperm head and lysosome direct attachment, and with the existence of cellular components related to autophagy (autophagosome, autolysosome). In conclusion, spermiophagy occurs by lysosomal engulfment and autophagic activity within the principal cells of the turtle epididymis during sperm storage.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a malignant gastrointestinal disease. The enzyme indoleamine-2,3-dioxgenase (IDO) is often overexpressed in PDAC and its downstream metabolite kynurenine has been reported to inhibit T cell activation and proliferation. Since γδ T cells are of high interest for T cell-based immunotherapy against PDAC, we studied the impact of IDO and kynurenine on γδ T cell cytotoxicity against PDAC cells., Methods: IDO expression was determined in PDAC cells by flow cytometry and Western blot analysis. PDAC cells were cocultured with γδ T cells in medium or were stimulated with phosphorylated antigens or bispecific antibody in the presence or absence of IDO inhibitors. Additionally, γδ T cells were treated with recombinant kynurenine. Read-out assays included degranulation, cytotoxicity and cytokine measurement as well as cell cycle analysis., Results: Since IDO overexpression was variable in PDAC, IDO inhibitors improved γδ T cell cytotoxicity only against some but not all PDAC cells. γδ T cell degranulation and cytotoxicity were significantly decreased after their treatment with recombinant kynurenine., Conclusions: Bispecific antibody drastically enhanced γδ T cell cytotoxicity against all PDAC cells, which can be further enhanced by IDO inhibitors against several PDAC cells, suggesting a striking heterogeneity in PDAC escape mechanisms towards γδ T cell-mediated anti-tumor response.

Deep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) is effective in treating temporal lobe epilepsy (TLE) and protects hippocampal neurons. Autophagy plays an essential role in epileptogenesis; however, the underlying effect of autophagy on ANT-DBS-mediated neuroprotection remains unclear. A monkey model of epilepsy was established by injecting kainic acid into the hippocampus and amygdala using a robot-assisted system. ANT-DBS was delivered in the chronic stage of the epileptic model and continued for 8 weeks. We found that ANT-DBS reduced the frequency of seizures and exerted neuroprotective effects via activating autophagy in hippocampal neurons. ANT-DBS increased light chain 3 (LC3) II level and co-localization of LC3 and lysosomal-associated membrane protein-1, accompanied by decreased expression of the autophagy substrate ubiquitin-binding protein p62, suggesting increased autophagosome formation. Most importantly, brain-derived neurotrophic factor (BDNF) -tropomyosin-related kinase type B (TrkB) pathway were involved in the regulation of autophagy. Both protein levels were reduced by ANT-DBS, and there was less phosphorylation of downstream regulators, extracellular signal-regulated kinase and Akt, followed by inactivation of mammalian target of rapamycin complex 1. Taken together, chronic ANT-DBS exerts neuroprotective effects on hippocampal neurons through inducing autophagy via suppressing the BDNF-TrkB pathway in a TLE monkey model.

Natural killer (NK) cells participate in early immune defenses against pathogens and tumors and play a major role as immune effector and regulatory cells. The NK cell-mediated elimination of an infected or cancerous cell is a highly regulated process that requires the formation of a cell contact, the establishment of an immunological synapse and the polarization and release of lytic granules. Additionally, the detachment of NK cells from target cells is important for NK cells to bind and kill other cells in a process called serial killing. However, very little is known about this detachment process. Here, we show that NK detachment is directly connected to the successful killing of a target cell. The inhibition of killing due to reduced NK cell cytotoxicity or increased target cell resistance results in defective detachment and prolonged contact times. This effect leads to sustained Ca 2+ flux in NK cells and the hypersecretion of proinflammatory cytokines. Linking defective cytotoxicity with enhanced cytokine secretion via reduced detachment may explain inflammatory pathologies in several diseases.