N-Glycosylation-dependent regulation of immune-specific tetraspanins CD37 and CD53.

Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different receptors on the plasma membrane. Most tetraspanins are highly glycosylated at their large extracellular loop; however, little is known about the function of tetraspanin glycosylation in immune cells. In this study we investigated the effects of glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants of CD37 and CD53 and analyzed their localization, nanoscale plasma membrane organization, and partner protein interaction capacity. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas surface expression of CD53 was unaffected by its glycosylation. Single-molecule dSTORM microscopy revealed that the nanoscale organization of CD53 was not dependent on glycosylation. CD37 interaction with its partner proteins CD53 and CD20 was affected by glycosylation in a localization-dependent way, whereas its interaction with IL-6Rα was independent of glycosylation. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45, CD20, and, to a lesser extent CD37. Together, our data show that glycosylation affects the interaction capacity of immune-specific tetraspanins CD37 and CD53, which adds another layer of regulation to immune membrane organization.

Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells.

Intracellular vesicle transport is essential for cellular homeostasis and is partially mediated by SNARE proteins. Endosomal trafficking to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of immune responses. Despite its critical importance, the specific molecular components that regulate DC cytokine secretion are poorly characterised. Galectin-9, a ß-galactoside-binding protein, has emerged as a novel cellular modulator although its exact intracellular roles in regulating (immune) cell homeostasis and vesicle transport are virtually unknown. We investigated galectin-9 function in primary human DCs and report that galectin-9 is essential for intracellular cytokine trafficking to the cell surface. Galectin-9-depleted DCs accumulate cytokine-containing vesicles in the Golgi complex that eventually undergo lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using immunoprecipitation-mass-spectrometry and identified galectin-9 was required for rerouting Vamp-3-containing endosomes upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle transport and sheds new light into the multiple roles galectins play in governing cell function.

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters.

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37.

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents.

BACKGROUND: While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics. RESULTS: To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy. CONCLUSIONS: A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.

Novel and conventional inhibitors of canonical autophagy differently affect LC3-associated phagocytosis.

In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.

Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.

Chromogranin A regulates gut permeability via the antagonistic actions of its proteolytic peptides.

AIM: A "leaky" gut barrier has been implicated in the initiation and progression of a multitude of diseases, for example, inflammatory bowel disease (IBD), irritable bowel syndrome and celiac disease. Here we show how pro-hormone Chromogranin A (CgA), produced by the enteroendocrine cells, and Catestatin (CST: hCgA352-372 ), the most abundant CgA-derived proteolytic peptide, affect the gut barrier. METHODS: Colon tissues from region-specific CST-knockout (CST-KO) mice, CgA-knockout (CgA-KO) and WT mice were analysed by immunohistochemistry, western blot, ultrastructural and flowcytometry studies. FITC-dextran assays were used to measure intestinal barrier function. Mice were supplemented with CST or CgA fragment pancreastatin (PST: CgA250-301 ). The microbial composition of cecum was determined. CgA and CST levels were measured in blood of IBD patients. RESULTS: Plasma levels of CST were elevated in IBD patients. CST-KO mice displayed (a) elongated tight, adherens junctions and desmosomes similar to IBD patients, (b) elevated expression of Claudin 2, and (c) gut inflammation. Plasma FITC-dextran measurements showed increased intestinal paracellular permeability in the CST-KO mice. This correlated with a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in various diseases. Supplementation of CST-KO mice with recombinant CST restored paracellular permeability and reversed inflammation, whereas CgA-KO mice supplementation with CST and/or PST in CgA-KO mice showed that intestinal paracellular permeability is regulated by the antagonistic roles of these two peptides: CST reduces and PST increases permeability. CONCLUSION: The pro-hormone CgA regulates the intestinal paracellular permeability. CST is both necessary and sufficient to reduce permeability and primarily acts by antagonizing PST.

Sugary Logistics Gone Wrong: Membrane Trafficking and Congenital Disorders of Glycosylation.

Glycosylation is an important post-translational modification for both intracellular and secreted proteins. For glycosylation to occur, cargo must be transported after synthesis through the different compartments of the Golgi apparatus where distinct monosaccharides are sequentially bound and trimmed, resulting in increasingly complex branched glycan structures. Of utmost importance for this process is the intraorganellar environment of the Golgi. Each Golgi compartment has a distinct pH, which is maintained by the vacuolar H+-ATPase (V-ATPase). Moreover, tethering factors such as Golgins and the conserved oligomeric Golgi (COG) complex, in concert with coatomer (COPI) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion, efficiently deliver glycosylation enzymes to the right Golgi compartment. Together, these factors maintain intra-Golgi trafficking of proteins involved in glycosylation and thereby enable proper glycosylation. However, pathogenic mutations in these factors can cause defective glycosylation and lead to diseases with a wide variety of symptoms such as liver dysfunction and skin and bone disorders. Collectively, this group of disorders is known as congenital disorders of glycosylation (CDG). Recent technological advances have enabled the robust identification of novel CDGs related to membrane trafficking components. In this review, we highlight differences and similarities between membrane trafficking-related CDGs.

Membrane trafficking as an active regulator of constitutively secreted cytokines.

Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-α, upon acute infection.

Radical Stress Is More Cytotoxic in the Nucleus than in Other Organelles.

Cells are exposed to reactive oxygen species (ROS) as a by-product of mitochondrial metabolism, especially under hypoxia. ROS are also enzymatically generated at the plasma membrane during inflammation. Radicals cause cellular damage leading to cell death, as they react indiscriminately with surrounding lipids, proteins, and nucleotides. However, ROS are also important for many physiological processes, including signaling, pathogen killing and chemotaxis. The sensitivity of cells to ROS therefore likely depends on the subcellular location of ROS production, but how this affects cell viability is poorly understood. As ROS generation consumes oxygen, and hypoxia-mediated signaling upregulates expression of antioxidant transcription factor Nrf2, it is difficult to discern hypoxic from radical stress. In this study, we developed an optogenetic toolbox for organelle-specific generation of ROS using the photosensitizer protein SuperNova which produces superoxide anion upon excitation with 590 nm light. We fused SuperNova to organelle specific localization signals to induce ROS with high precision. Selective ROS production did not affect cell viability in most organelles except for the nucleus. SuperNova is a promising tool to induce locally targeted ROS production, opening up new possibilities to investigate processes and organelles that are affected by localized ROS production.

The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation.

Antigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal "clickable" antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

SWAP70 is a universal GEF-like adaptor for tethering actin to phagosomes.

We recently identified a key role for SWAP70 as the tethering factor stabilizing F-actin filaments on the surface of phagosomes in human dendritic cells by interacting both with Rho-family GTPases and the lipid phosphatidylinositol (3,4)-bisphosphate. In this study, we aimed to investigate whether this role of SWAP70 was general among immune phagocytes. Our data reveal that SWAP70 is recruited to early phagosomes of macrophages and dendritic cells from both human and mouse. The putative inhibitor of SWAP70 sanguinarine blocked phagocytosis and F-actin polymerization, supporting a key role for SWAP70 in phagocytosis as demonstrated previously with knock-down. Moreover, SWAP70 was recently shown to sequester the F-actin severing protein cofilin and we investigated this relationship in phagocytosis. Our data show an increased activation of cellular cofilin upon siRNA knockdown of SWAP70. Finally, we explored whether SWAP70 would be recruited to the immune synapse between dendritic cells and T cells required for antigen presentation, as the formation of such synapses depends on F-actin. However, we observed that SWAP70 was depleted at immune synapses and specifically was recruited to phagosomes. Our data support an essential and specific role for SWAP70 in tethering and stabilizing F-actin to the phagosomal surface in a wide range of phagocytes.

Interleukin-6 secretion is limited by self-signaling in endosomes.

Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood. In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.

Hypoxia potentiates monocyte-derived dendritic cells for release of tumor necrosis factor α via MAP3K8.

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Endosomal and Phagosomal SNAREs.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein family is of vital importance for organelle communication. The complexing of cognate SNARE members present in both the donor and target organellar membranes drives the membrane fusion required for intracellular transport. In the endocytic route, SNARE proteins mediate trafficking between endosomes and phagosomes with other endosomes, lysosomes, the Golgi apparatus, the plasma membrane, and the endoplasmic reticulum. The goal of this review is to provide an overview of the SNAREs involved in endosomal and phagosomal trafficking. Of the 38 SNAREs present in humans, 30 have been identified at endosomes and/or phagosomes. Many of these SNAREs are targeted by viruses and intracellular pathogens, which thereby reroute intracellular transport for gaining access to nutrients, preventing their degradation, and avoiding their detection by the immune system. A fascinating picture is emerging of a complex transport network with multiple SNAREs being involved in consecutive trafficking routes.

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules.

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin-6 (IL-6) in human dendritic cells. By comparing this to the total secreted IL-6, we estimate that secretory vesicles contain about 0.5-3 IL-6 molecules, but with a large spread among cells/donors. Moreover, IL-6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL-6 production. IL-6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs.