FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Dynamin-2 controls actin remodeling for efficient complement receptor 3-mediated phagocytosis.

BACKGROUND INFORMATION: Phagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. RESULTS: Here, we reveal that Dynamin-2 is recruited concomitantly with polymerized actin at the phagocytic cup and during phagosome formation and closure. Inhibition of Dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. CONCLUSIONS: Dynamin-2 regulates the assembly of the F-actin phagocytic cup for successful CR3-mediated phagocytosis. SIGNIFICANCE: These results highlight an important role for Dynamin-2 in actin remodeling downstream of integrins.

Zika virus infection of mature neurons from immunocompetent mice generates a disease-associated microglia and a tauopathy-like phenotype in link with a delayed interferon beta response.

BACKGROUND: Zika virus (ZIKV) infection at postnatal or adult age can lead to neurological disorders associated with cognitive defects. Yet, how mature neurons respond to ZIKV remains substantially unexplored. METHODS: The impact of ZIKV infection on mature neurons and microglia was analyzed at the molecular and cellular levels, in vitro using immunocompetent primary cultured neurons and microglia, and in vivo in the brain of adult immunocompetent mice following intracranial ZIKV inoculation. We have used C57BL/6 and the genetically diverse Collaborative Cross mouse strains, displaying a broad range of susceptibility to ZIKV infection, to question the correlation between the effects induced by ZIKV infection on neurons and microglia and the in vivo susceptibility to ZIKV. RESULTS: As a result of a delayed induction of interferon beta (IFNB) expression and response, infected neurons displayed an inability to stop ZIKV replication, a trait that was further increased in neurons from susceptible mice. Alongside with an enhanced expression of ZIKV RNA, we observed in vivo, in the brain of susceptible mice, an increased level of active Iba1-expressing microglial cells occasionally engulfing neurons and displaying a gene expression profile close to the molecular signature of disease-associated microglia (DAM). In vivo as well as in vitro, only neurons and not microglial cells were identified as infected, raising the question of the mechanisms underlying microglia activation following brain ZIKV infection. Treatment of primary cultured microglia with conditioned media from ZIKV-infected neurons demonstrated that type-I interferons (IFNs-I) secreted by neurons late after infection activate non-infected microglial cells. In addition, ZIKV infection induced pathological phosphorylation of Tau (pTau) protein, a hallmark of neurodegenerative tauopathies, in vitro and in vivo with clusters of neurons displaying pTau surrounded by active microglial cells. CONCLUSIONS: We show that ZIKV-infected mature neurons display an inability to stop viral replication in link with a delayed IFNB expression and response, while signaling microglia for activation through IFNs-I secreted at late times post-infection. In the brain of ZIKV-infected susceptible mice, uninfected microglial cells adopt an active morphology and a DAM expression profile, surrounding and sometimes engulfing neurons while ZIKV-infected neurons accumulate pTau, overall reflecting a tauopathy-like phenotype.

IL4I1 binds to TMPRSS13 and competes with SARS-CoV-2 spike.

The secreted enzyme interleukin four-induced gene 1 (IL4I1) is involved in the negative control of the adaptive immune response. IL4I1 expression in human cancer is frequent and correlates with poor survival and resistance to immunotherapy. Nevertheless, its mechanism of action remains partially unknown. Here, we identified transmembrane serine protease 13 (TMPRSS13) as an immune cell-expressed surface protein that binds IL4I1. TMPRSS13 is a paralog of TMPRSS2, of which the protease activity participates in the cleavage of SARS-CoV-2 spike protein and facilitates virus induced-membrane fusion. We show that TMPRSS13 is expressed by human lymphocytes, monocytes and monocyte-derived macrophages, can cleave the spike protein and allow SARS-CoV-2 spike pseudotyped virus entry into cells. We identify regions of homology between IL4I1 and spike and demonstrate competition between the two proteins for TMPRSS13 binding. These findings may be relevant for both interfering with SARS-CoV-2 infection and limiting IL4I1-dependent immunosuppressive activity in cancer.

The NSs Protein Encoded by the Virulent Strain of Rift Valley Fever Virus Targets the Expression of Abl2 and the Actin Cytoskeleton of the Host, Affecting Cell Mobility, Cell Shape, and Cell-Cell Adhesion.

Rift Valley fever virus (RVFV) is a highly pathogenic zoonotic arbovirus endemic in many African countries and the Arabian Peninsula. Animal infections cause high rates of mortality and abortion among sheep, goats, and cattle. In humans, an estimated 1 to 2% of RVFV infections result in severe disease (encephalitis, hepatitis, or retinitis) with a high rate of lethality when associated with hemorrhagic fever. The RVFV NSs protein, which is the main virulence factor, counteracts the host innate antiviral response to favor viral replication and spread. However, the mechanisms underlying RVFV-induced cytopathic effects and the role of NSs in these alterations remain for the most part unknown. In this work, we have analyzed the effects of NSs expression on the actin cytoskeleton while conducting infections with the NSs-expressing virulent (ZH548) and attenuated (MP12) strains of RVFV and the non-NSs-expressing avirulent (ZH548ΔNSs) strain, as well as after the ectopic expression of NSs. In macrophages, fibroblasts, and hepatocytes, NSs expression prevented the upregulation of Abl2 (a major regulator of the actin cytoskeleton) expression otherwise induced by avirulent infections and identified here as part of the antiviral response. The presence of NSs was also linked to an increased mobility of ZH548-infected cells compared to ZH548ΔNSs-infected fibroblasts and to strong changes in cell morphology in nonmigrating hepatocytes, with reduction of lamellipodia, cell spreading, and dissolution of adherens junctions reminiscent of the ZH548-induced cytopathic effects observed in vivo Finally, we show evidence of the presence of NSs within long actin-rich structures associated with NSs dissemination from NSs-expressing toward non-NSs-expressing cells.IMPORTANCE Rift Valley fever virus (RVFV) is a dangerous human and animal pathogen that was ranked by the World Health Organization in 2018 as among the eight pathogens of most concern for being likely to cause wide epidemics in the near future and for which there are no, or insufficient, countermeasures. The focus of this work is to address the question of the mechanisms underlying RVFV-induced cytopathic effects that participate in RVFV pathogenicity. We demonstrate here that RVFV targets cell adhesion and the actin cytoskeleton at the transcriptional and cellular level, affecting cell mobility and inducing cell shape collapse, along with distortion of cell-cell adhesion. All these effects may participate in RVFV-induced pathogenicity, facilitate virulent RVFV dissemination, and thus constitute interesting potential targets for future development of antiviral therapeutic strategies that, in the case of RVFV, as with several other emerging arboviruses, are presently lacking.

Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked ß-N-Acetylglucosaminylation and Attenuate Inflammation in Macrophages.

Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.

SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends.

Understanding the viral-host cell interface during HIV-1 infection is a prerequisite for the development of innovative antiviral therapies. Here we show that the suppressor of G2 allele of skp1 (SUGT1) is a permissive factor for human immunodeficiency virus (HIV)-1 infection. Expression of SUGT1 increases in infected cells on human brain sections and in permissive host cells. We found that SUGT1 determines the permissiveness to infection of lymphocytes and macrophages by modulating the nuclear import of the viral genome. More importantly, SUGT1 stabilizes the microtubule plus-ends (+MTs) of host cells (through the modulation of microtubule acetylation and the formation of end-binding protein 1 (EB1) comets). This effect on microtubules favors HIV-1 retrograde trafficking and replication. SUGT1 depletion impairs the replication of HIV-1 patient primary isolates and mutant virus that is resistant to raltegravir antiretroviral agent. Altogether our results identify SUGT1 as a cellular factor involved in the post-entry steps of HIV-1 infection that may be targeted for new therapeutic approaches.

Extracellular Release of Antigen by Dendritic Cell Regurgitation Promotes B Cell Activation through NF-κB/cRel.

Dendritic cells (DCs) are professional APCs, which sample Ags in the periphery and migrate to the lymph node where they activate T cells. DCs can also present native Ag to B cells through interactions observed both in vitro and in vivo. However, the mechanisms of Ag transfer and B cell activation by DCs remain incompletely understood. In this study, we report that murine DCs are an important cell transporter of Ag from the periphery to the lymph node B cell zone and also potent inducers of B cell activation both in vivo and in vitro. Importantly, we highlight a novel extracellular mechanism of B cell activation by DCs. In this study, we demonstrate that Ag released upon DC regurgitation is sufficient to efficiently induce early B cell activation, which is BCR driven and mechanistically dependent on the nuclear accumulation of the transcription factor NF-κB/cRel. Thus, our study provides new mechanistic insights into Ag delivery and B cell activation modalities by DCs and a promising approach for targeting NF-κB/cRel pathway to modulate the DC-elicited B cell responses.

Arpin is critical for phagocytosis in macrophages and is targeted by human rhinovirus.

Human rhinovirus is a causative agent of severe exacerbations of chronic obstructive pulmonary disease (COPD). COPD is characterised by an increased number of alveolar macrophages with diminished phagocytic functions, but how rhinovirus infection affects macrophage function is still unknown. Here, we describe that human rhinovirus 16 impairs bacterial uptake and receptor-mediated phagocytosis in macrophages. The stalled phagocytic cups contain accumulated F-actin. Interestingly, we find that human rhinovirus 16 downregulates the expression of Arpin, a negative regulator of the Arp2/3 complex. Importantly, re-expression of the protein rescues defective internalisation in human rhinovirus 16-treated cells, demonstrating that Arpin is a key factor targeted to impair phagocytosis. We further show that Arpin is required for efficient uptake of multiple targets, for F-actin cup formation and for successful phagosome completion in macrophages. Interestingly, Arpin is recruited to sites of membrane extension and phagosome closure. Thus, we identify Arpin as a central actin regulator during phagocytosis that it is targeted by human rhinovirus 16, allowing the virus to perturb bacterial internalisation and phagocytosis in macrophages.

Targeting CCR5 trafficking to inhibit HIV-1 infection.

Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.

The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Dendritic cells mature to resist lamin degradation and herpes virus release.

Herpes simplex viruses bud into the nuclear membrane of infected cells. Turan et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201801151) demonstrate that mature dendritic cells control the peripheral location of lysosomes, reducing autophagic degradation of lamins and inhibiting viral release.

Observing Frustrated Phagocytosis and Phagosome Formation and Closure Using Total Internal Reflection Fluorescence Microscopy (TIRFM).

Complementary methods to observe frustrated phagocytosis and phagosome closure using total internal reflection fluorescence microscopy (TIRFM) are described here. Frustrated phagocytosis occurs when phagocytic cells are exposed to an opsonized surface and spread as if trying to engulf it, allowing for the observation of phagocytic spreading and the biochemical events that directly precede it. Phagosome formation and closure is an inherently three-dimensional process though, and cannot be studied in the "frustrated" situation. Here we describe a method to visualize with unprecedented high-resolution phagosome formation and closure in three dimensions. It allows for observation of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Defective Phagocytic Properties of HIV-Infected Macrophages: How Might They Be Implicated in the Development of Invasive Salmonella Typhimurium?

Human immunodeficiency virus type 1 (HIV-1) infects and kills T cells, profoundly damaging the host-specific immune response. The virus also integrates into memory T cells and long-lived macrophages, establishing chronic infections. HIV-1 infection impairs the functions of macrophages both in vivo and in vitro, which contributes to the development of opportunistic diseases. Non-typhoidal Salmonella enterica serovar Typhimurium has been identified as the most common cause of bacterial bloodstream infections in HIV-infected adults. In this review, we report how the functions of macrophages are impaired post HIV infection; introduce what makes invasive Salmonella Typhimurium specific for its pathogenesis; and finally, we discuss why these bacteria may be particularly adapted to the HIV-infected host.

How to build a phagosome: new concepts for an old process.

Phagocytosis is a complex and elegant mechanism requiring finely coordinated deformation and restructuring of the membrane and the underlying cytoskeleton. Here we discuss the early events of receptor clustering and engagement required for signal transduction and actin remodeling. In addition, we summarize recent studies of the mechanisms whereby the nascent phagosome seals and evolves into a degradative phagolysosome by a process that seemingly involves the autophagic machinery. These studies provided new insights of the molecular basis of this long-appreciated, essential homeostatic process.

HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger.

Human rhinovirus is frequently seen as an upper respiratory tract infection but growing evidence proves the virus can cause lower respiratory tract infections in patients with chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). In addition to airway epithelial cells, macrophages are crucial for regulating inflammatory responses to viral infections. However, the response of macrophages to HRV has not been analyzed in detail. We used in vitro monocyte-derived human macrophages to study the cytokine secretion of macrophages in response to the virus. Our results showed that macrophages were competent at responding to HRV, as a robust cytokine response was detected. However, after subsequent exposure to non-typeable Haemophilus influenzae (NTHi) or to LPS, HRV-treated macrophages secreted reduced levels of pro-inflammatory or regulatory cytokines. This "paralyzed" phenotype was not mimicked if the macrophages were pre-treated with LPS or CpG instead of the virus. These results begin to deepen our understanding into why patients with COPD show HRV-induced exacerbations and why they mount a defective response toward NTHi.

Meeting after meeting: 20 years of discoveries by the members of the Exocytosis-Endocytosis Club.

Twenty years ago, a group of French cell biologists merged two scientific clubs with the aim of bringing together researchers in the fields of Endocytosis and Exocytosis. Founded in 1997, the first annual meeting of the Exocytosis Club was held in 1998. The Endocytosis Club held quarterly meetings from its founding in 1999. The first joint annual meeting of the Exocytosis-Endocytosis Club took place in Paris in April, 2001. What started as a modest gathering of enthusiastic scientists working in the field of cell trafficking has gone from strength to strength, rapidly becoming an unmissable yearly meeting, vividly demonstrating the high quality of science performed in our community and beyond. On the occasion of the 20th meeting of our club, we want to provide historic insight into the fields of exocytosis and endocytosis, and by extension, to subcellular trafficking, highlighting how French scientists have contributed to major advances in these fields. Today, the Exocytosis-Endocytosis Club represents a vibrant and friendly community that will hold its 20th meeting at the Presqu'Ile de Giens, near Toulon in the South of France, on May 11-13, 2017.

Macrophage phagocytosis cracking the defect code in COPD.

In the normal non-diseased lung, various macrophage populations maintain homeostasis and sterility by ingesting and clearing inhaled particulates, pathogens and apoptotic cells from the local environment. This process of phagocytosis leads to the degradation of the internalized material, coordinated induction of gene expression, antigen presentation and cytokine production, implicating phagocytosis as a central regulator of innate immunity. Phagocytosis is extremely efficient and any perturbation of this function is deleterious. In inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD), despite their increased numbers, macrophages demonstrate significantly reduced phagocytic capacity of bacteria and apoptotic cells. This defect could play a role in dysbiosis of the lung microbiome contributing to disease pathophysiology. In this review, we will discuss lung macrophages, describe phagocytosis and its related downstream processes and the reported phagocytosis defects in COPD. Finally, we will briefly examine current strategies that focus on restoring the phagocytic capabilities of lung macrophages that may have utility in COPD.