Tunneling nanotubes and related structures: molecular mechanisms of formation and function.

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention.

Evaluation of Poly(I:C) and combination of CpG ODN plus Montanide ISA adjuvants to enhance the efficacy of outer membrane vesicles as an acellular vaccine against Brucella melitensis infection in mice.

Brucellosis is the most common zoonotic disease worldwide and still there is no vaccine for human use. The commercial animal vaccines also have major problems that limit their use. Therefore, there is a need for an effective Brucella vaccine which is multivalent and produces a good protective immunity with minimal disadvantages. Due to their heterogeneous composition and diverse functions, OMVs are promising acellular vaccine candidates against brucellosis. In the present study, the potential of Poly(I:C) or CpG ODN 1826+ Montanide ISA 70 VG adjuvant formulations were evaluated to enhance the immunity and protection levels conferred by OMVs against Brucella challenge in mice. The results indicated that both vaccine regimens were able to induce strong Th1-biased responses and confer protective levels significantly higher than REV.1 live vaccine. With regard to the results, it is concluded that OMVs in either adjuvant can be introduced as a new vaccine candidate against B. melitensis infection.

Surfactant protein D regulates caspase-8-mediated cascade of the intrinsic pathway of apoptosis while promoting bleb formation.

Surfactant-associated protein D (SP-D) is a soluble innate immune collectin present on many mucosal surfaces. We recently showed that SP-D suppresses the extrinsic pathway of apoptosis by downregulating caspase-8 activation. However, the effects of SP-D on the intrinsic pathway of apoptosis are not clearly understood. In the intrinsic pathway, cytochrome c is released by mitochondria into the cytoplasm. Oxidation of cytochrome c by cytochrome c oxidase activates the apoptosome and caspase-9 cascade. Both caspase-8- and caspase-9-mediated branches are activated in the intrinsic pathway of apoptosis; however, little is known about the relevance of the caspase-8 pathway in this context. Here we studied the effects of SP-D on different branches of the intrinsic pathway of apoptosis using UV-irradiated Jurkat T-cells. We found that SP-D does not inhibit the caspase-9 branch of apoptosis and the relevance of the caspase-8-related branch became apparent when the caspase-9 pathway was inhibited by blocking cytochrome c oxidase. Under these conditions, SP-D reduces the activation of caspase-8, executioner caspase-3 and exposure of phosphatidylserine (PS) on the membranes of dying cells. By contrast, SP-D increases the formation of nuclear and membrane blebs. Inhibition of caspase-8 confirms the effect of SP-D is unique to the caspase-8 pathway. Overall, SP-D suppresses certain aspects of the intrinsic pathway of apoptosis via reduction of caspase-8 activation and PS flipping while at the same time increasing membrane and nuclear bleb formation. This novel regulatory aspect of SP-D could help to regulate intrinsic pathway of apoptosis to promote effective blebbing and breakdown of dying cells.

Analyzing the Dynamics of Signaling Microclusters.

T cell antigen receptor (TCR) stimulation induces recruitment and accumulation of various types of signaling molecules and forms signaling microclusters. The dynamics of the microclusters are important for regulating the quality and quantity of T cell activation. We describe here our protocols for analysis of signaling microclusters by using supported planar bilayers.

The Mast Cell Antibody-Dependent Degranulatory Synapse.

Mast cells are key effector cells in inflammation that can be activated by specific antigens via IgE or IgG binding on their FcR. Aggregation of mast cell Fc receptors by cell-bound antigens induces mast cell polarized degranulation toward the stimulatory cell, a process named antibody-dependent degranulatory synapse (ADDS). This polarized degranulation allows mast cells to expose bioactive material embedded in the granule matrix toward the antibody-targeted cell and is accompanied by the formation of a signaling area at the cell-cell contact site. In this chapter, we describe (1) how to stimulate mast cells with cell-bound antigens and (2) how to monitor ADDS formation and to investigate ADDS characteristics by confocal microscopy.

[USE OF VIBRIO CHOLERAE SURFACE STRUCTURES FOR SPECIFIC PROPHY- LAXIS AND DIAGNOSTICS OF CHOLERA].

The need for efficient and cost-effective cholera vaccine hasn't lost its actuality in view of the emergence of new strains leading to severe clinical forms of cholera and capable to replace strains of the seventh.cholera pandemic, and in connection with the threat of cholera spreading beyond the borders of endemic countries. In this review data from literature sources are presented about the use of outer membrane proteins, vesicles, cell ghosts of the cholera causative agent in specific prophylaxis and diagnostics of the disease.

Common Cell Shape Evolution of Two Nasopharyngeal Pathogens.

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.

Outer membrane vesicles from pathogenic bacteria initiate an inflammatory response in human endothelial cells.

INTRODUCTION: Gram-negative bacteria release outer membrane vesicles (OMVs) during growth that contain various membrane components involved in eliciting an inflammatory response, including lipopolysaccharide and virulence factors. However, little is known about the role of OMVs in sepsis. The objective of this study was to determine how OMVs, derived from Escherichia (E.) coli, elicit the cellular responses involved in activating the inflammatory cascade, and to determine whether additional virulence factors in pathogenic OMVs augment the inflammatory response. METHODS: Human umbilical endothelial cells were inoculated with OMVs from non-pathogenic E. coli (npOMV) or pathogenic E. coli (pOMV) and analyzed for adhesion protein synthesis, cytokine production, and necrosis factor (NF)-κB translocation. RESULTS: Flow cytometry demonstrated that human umbilical vein endothelial cells exposed to npOMV or pOMV significantly increased expression of E-selectin and intercellular adhesion molecule, with a large population of cells demonstrating increased expression of both proteins. Interleukin-6 levels were significantly elevated by 4 h after exposure to npOMV and pOMVs. NF-κB translocation to the nucleus was shown to be induced by npOMV and pOMVs. However, the role of additional virulence factors associated with pOMVs remains undefined. CONCLUSIONS: Both npOMVs and pOMVs are capable of initiating the inflammatory cascade in endothelial cells. OMVs trigger NF-κB translocation to the nucleus, resulting in up-regulation of adhesion molecules and cytokines, presumably for the recruitment of leukocytes. By eliciting an inflammatory response, OMVs could facilitate the transition from a localized infection to a systemic response, and ultimately sepsis.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction.

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell-cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II(+) cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx(3)cr1(GFP) and CD11c(eYFP) transgenic mice revealed that MNTs form de novo at a rate of 15.5 µm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell-cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

The molecular basis of induction and formation of tunneling nanotubes.

Tunneling nanotubes (TNTs) and associated structures are recently recognized structures for intercellular communication. They are F-actin-containing thin protrusions of the plasma membrane of a cell and allow a direct physical connection to the plasma membranes of remote cells. TNTs and associated structures serve as mediators for intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, several pathogens have been shown to exploit these structures to spread among cells. Because of their contribution to normal cellular functions and importance in pathological conditions, studies on TNTs and related structures have accelerated over the past few years. These studies have revealed key molecules for their induction and/or formation; HIV Nef and M-Sec can induce the formation of TNTs in coordination with the remodeling of the actin cytoskeleton and vesicle trafficking.

Intranasal immunization with nontypeable Haemophilus influenzae outer membrane vesicles induces cross-protective immunity in mice.

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

Outer membrane vesicles as a candidate vaccine against edwardsiellosis.

Infection with Edwardsiella tarda, a gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1ß, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis.

Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins.

Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

Membrane ruffles capture C3bi-opsonized particles in activated macrophages.

A widespread belief in phagocyte biology is that FcgammaR-mediated phagocytosis utilizes membrane pseudopods, whereas Mac-1-mediated phagocytosis does not involve elaborate plasma membrane extensions. Here we report that dynamic membrane ruffles in activated macrophages promote binding of C3bi-opsonized particles. We identify these ruffles as components of the macropinocytosis machinery in both PMA- and LPS-stimulated macrophages. C3bi-particle capture is facilitated by enrichment of high-affinity Mac-1 and the integrin-regulating protein talin in membrane ruffles. Membrane ruffle formation and C3bi-particle binding are cytoskeleton dependent events, having a strong requirement for F-actin and microtubules (MTs). MT disruption blunts ruffle formation and PMA- and LPS-induced up-regulation of surface Mac-1 expression. Furthermore, the MT motor, kinesin participates in ruffle formation implicating a requirement for intracellular membrane delivery to active membrane regions during Mac-1-mediated phagocytosis. We observed colocalization of Rab11-positive vesicles with CLIP-170, a MT plus-end binding protein, at sites of particle adherence using TIRF imaging. Rab11 has been implicated in recycling endosome dynamics and mutant Rab11 expression inhibits both membrane ruffle formation and C3bi-sRBC adherence to macrophages. Collectively these findings represent a novel membrane ruffle "capture" mechanism for C3bi-particle binding during Mac-1-mediated phagocytosis. Importantly, this work also demonstrates a strong functional link between integrin activation, macropinocytosis and phagocytosis in macrophages.

Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria.

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

Differential mechanisms of microparticle transfer toB cells and monocytes: anti-inflammatory propertiesof microparticles.

Microparticles are small vesicles released from the plasma membrane of various cell types independently of apoptosis or cell death, are transferred between cells, and carry membrane proteins from one cell to another. We have studied the mechanism of uptake of microparticles by monocytes and B cells. The transfer of microparticles to B cells was almost completely dependent on complement. Incubation of microparticles with serum resulted in opsonization of microparticles with the complement cleavage product iC3b. The subsequent transfer to B cells was mediated by the complement receptor CR2. The interaction between iC3b-opsonized microparticles and B cells reduced the activation of B cells as measured by expression of MHC class II, CD86 and CD25. In contrast, transfer of microparticles to monocytes was only partially complement dependent, but involved calcium and annexin V, and was found to change the cytokine profile of monocytes towards a reduced release of the pro-inflammatory cytokines GM-CSF and TNF-alpha and an increased release of the anti-inflammatory cytokine IL-10. These data show that microparticles are taken up by B cells and monocytes by different mechanisms and modulate the activation of monocytes and B cells towards an anti-inflammatory phenotype. Microparticles might be involved in counterbalancing pro-inflammatory signals arising from tissue injury or inflammation.

Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD82 in T lymphocytes.

Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation molecules through cell-to-cell contacts. The tetraspanin CD82 has previously been shown to act as a cytoskeleton-dependent costimulation molecule. We show here that CD82 engagement leads to the tyrosine phosphorylation and association of both the Rho GTPases guanosine exchange factor Vav1 and adapter protein SLP76, suggesting that Rho GTPases participate in CD82 signaling. Indeed, broad inactivation of all Rho GTPases, or a specific blockade of RhoA, Rac1 or Cdc42, inhibited the morphological changes linked to CD82 engagement but failed to modulate the inducible association of CD82 with the actin network. Rho GTPase inactivation, as well as actin depolymerization, reduced the ability of CD82 to phosphorylate Vav and SLP76 and to potentiate the phosphorylation of two early TcR signaling intermediates: the tyrosine kinases ZAP70 and membrane adapter LAT. Taken together, this suggests that an amplification loop, via early Vav and SLP76 phosphorylations and Rho-GTPases activation, is initiated by CD82 association with the cytoskeleton, which permits cytoskeletal rearrangements and costimulatory activity. Moreover, the involvement of CD82 in the formation of the immunological synapse is strongly suggested by its accumulation at the site of TcR engagement. This novel link between a tetraspanin and the Rho GTPase cascade could explain why tetraspanins, which are known to form heterocomplexes, are involved in cell activation, adhesion, growth and metastasis.