BACKGROUND: Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm.
OBJECTIVE: Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants.
METHODS: HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release.
RESULTS: We show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein.
CONCLUSIONS: These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs.
CONCLUSIONS: These findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.

The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients\' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.

Despite significant improvement in the prognosis of multiple myeloma (MM), the disease remains incurable; thus, more effective therapies are required. Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is significantly associated with drug resistance, rapid relapse, and poor prognosis. Previously, we found that 4-hydroxysalicylanilide (osalmid), a specific inhibitor of RRM2, exhibits anti-MM activity in vitro, in vivo, and in human patients; however, the mechanism remains unclear. Osalmid inhibits the translocation of RRM2 to the nucleus and stimulates autophagosome synthesis but inhibits subsequent autophagosome-lysosome fusion. We confirm that RRM2 binds to receptor-interacting protein kinase 3 (RIPK3) and reduces RIPK3, inhibiting autophagosome-lysosome fusion. Interestingly, the combination of osalmid and bafilomycin A1 (an autophagy inhibitor) depletes RIPK3 and aggravates p62 and autophagosome accumulation, leading to autophagic cell death. Combination therapy demonstrates synergistic cytotoxicity both in vitro and in vivo. Therefore, we propose that combining osalmid and bafilomycin A1(BafA1) may have clinical benefits against MM.

Podocytes are sensitive to insulin, which governs the functional and structural integrity of podocytes that are essential for proper function of the glomerular filtration barrier. Lysosomes are acidic organelles that are implicated in regulation of the insulin signaling pathway. Cathepsin D (CTPD) and lysosome-associated membrane protein 1 (LAMP1) are major lysosomal proteins that reflect the functional state of lysosomes. However, the effect of insulin on lysosome activity and role of lysosomes in the regulation of insulin-dependent glucose uptake in podocytes are unknown. Our studies showed that the short-term incubation of podocytes with insulin decreased LAMP1 and CTPD mRNA levels. Insulin and bafilomycin A1 reduced both the amounts of LAMP1 and CTPD proteins and activity of CTPD, which were associated with a decrease in the fluorescence intensity of lysosomes that were labeled with LysoTracker. Bafilomycin A1 inhibited insulin-dependent endocytosis of the insulin receptor and increased the amounts of the insulin receptor and glucose transporter 4 on the cell surface of podocytes. Bafilomycin A1 also inhibited insulin-dependent glucose uptake despite an increase in the amount of glucose transporter 4 in the plasma membrane of podocytes. These results suggest that lysosomes are signaling hubs that may be involved in the coupling of insulin signaling with the regulation of glucose uptake in podocytes. The dysregulation of this mechanism can lead to the dysfunction of podocytes and development of insulin resistance.

Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.

FeHV-1 is a member of the Herpesviridae family that is distributed worldwide and causes feline viral rhinotracheitis (FVR). Since its relationship with the autophagic process has not yet been elucidated, the aim of this work was to evaluate the autophagy mediated by FeHV-1 and to determine its proviral or antiviral role. Our data showed that autophagy is induced by FeHV-1 in a viral dose and time-dependent manner. Phenotypic changes in LC3/p62 axis (increase of LC3-II and degradation of p62) were detected from 12 h post infection using western blot and immuno-fluorescence assays. In a second step, by using late autophagy inhibitors and inducers, the possible proviral role of autophagy during FeHV-1 infection was investigating by assessing the effects of each chemical in terms of viral yield, cytotoxic effects, and expression of viral glycoproteins. Our findings suggest that late-stage autophagy inhibitors (bafilomycin and chloroquine) have a negative impact on viral replication. Interestingly, we observed an accumulation of gB, a viral protein, when cells were pretreated with bafilomycin, whereas the opposite effect was observed when an autophagy inducer was used. The importance of autophagy during FeHV-1 infection was further supported by the results obtained with ATG5 siRNA. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, and the negative impact of late autophagy inhibitors on viral replication.

UNASSIGNED: The skin barrier is the first line of defense of the body, while skin lipids play an important role in the skin permeability barrier. Lamellar bodies are also involved in maintaining the stability of the skin permeability barrier. However, the exact origin of lamellar bodies remains unclear. Recent studies have suggested that autophagy may participate in the formation of lamellar bodies.
UNASSIGNED: This study aimed to investigate the role of autophagy in the formation of lamellar bodies in keratinocytes and the regulation of keratinocyte lipids.
UNASSIGNED: Keratinocytes were incubated with autophagy inducer Rapamycin and autophagy inhibitor Bafilomycin A1. The changes in autophagy flux were detected by Western blot, and the formation of lamellar bodies was observed by transmission electron microscopy. Furthermore, the changes in keratinocytes lipidomics were detected by liquid chromatography-mass spectrometry.
UNASSIGNED: Our research showed that the autophagy inducer promoted autophagy activation and formation of lamellar bodies in keratinocytes, while the inhibitor inhibited autophagy signals and the formation of lamellar bodies in keratinocytes. In addition, the lipidomics results revealed a significant change in glycerophospholipids after autophagy induction and autophagy inhibition.
UNASSIGNED: These results demonstrate that autophagy may play an essential role in skin lipids via glycerophospholipids pathway.

Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type.

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFβ1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.