Search

Many innate immune receptors function collaboratively to detect and elicit immune responses to pathogens, but the physical mechanisms that govern the interaction and signaling crosstalk between the receptors are unclear. In this study, we report that the signaling crosstalk between Fc gamma receptor (FcγR) and Toll-like receptor (TLR)2/1 can be overall synergistic or inhibitory depending on the spatial proximity between the receptor pair on phagosome membranes. Using a geometric manipulation strategy, we physically altered the spatial distribution of FcγR and TLR2 on single phagosomes. We demonstrate that the signaling synergy between FcγR and TLR2/1 depends on the proximity of the receptors and decreases as spatial separation between them increases. However, the inhibitory effect from FcγRIIb on TLR2-dependent signaling is always present and independent of receptor proximity. The overall cell responses are an integration from these two mechanisms. This study presents quantitative evidence that the nanoscale proximity between FcγR and TLR2 functions as a key regulatory mechanism in their signaling crosstalk.

Key Message: Short review focussing on the role and targeting of vacuolar substructure in plant immunity and pathogenesis. Plants lack specialized immune cells, therefore each plant cell must defend itself against invading pathogens. A typical plant defense strategy is the hypersensitive response that results in host cell death at the site of infection, a process largely regulated by the vacuole. In plant cells, the vacuole is a vital organelle that plays a central role in numerous fundamental processes, such as development, reproduction, and cellular responses to biotic and abiotic stimuli. It shows divergent membranous structures that are continuously transforming. Recent technical advances in visualization and live-cell imaging have significantly altered our view of the vacuolar structures and their dynamics. Understanding the active nature of the vacuolar structures and the mechanisms of vacuole-mediated defense responses is of great importance in understanding plant-pathogen interactions. In this review, we present an overview of the current knowledge about the vacuole and its internal structures, as well as their role in plant-microbe interactions. There is so far limited information on the modulation of the vacuolar structures by pathogens, but recent research has identified the vacuole as a possible target of microbial interference.

Ion channels facilitate the movement of ions across the plasma and organellar membranes. A recent symposium brought together scientists who study ion channels and transporters in immune cells, which highlighted advances in this emerging field and served to chart new avenues for investigating the roles of ion channels in immunity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Autophagy acts as an intracellular host defense system against invading pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we revealed Rab GTPase network that regulate autophagosome formation against GAS. A unique set of Rab GTPases coordinates autophagy to enable to form huge autophagosomes surrounding GAS by linking recycling endosomes and trans Golgi-network. We also found that NLRP4, one of intracellular pathogen recognition receptor, directs Rho signaling to facilitate autophagosome formation. In this article, we would like to show our findings on how host autophagy regulators coordinate autophagy during GAS infection.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40 phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40 phox and p67 phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67 phox , the key cytosolic subunit of the oxidase, and p40 phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67 phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40 phox and p67 phox at the phagosomal membrane., (© Society for Leukocyte Biology.)

The mitochondrial antiviral signaling (MAVS) protein is a central adaptor protein required for antiviral innate immune signaling. To facilitate its roles in innate immunity, MAVS localizes to multiple intracellular membranous compartments, including the mitochondria, the mitochondrial-associated ER membrane (MAM), and peroxisomes. Studies of MAVS function therefore often require an analysis of MAVS localization. To detect MAVS protein on intracellular membranes, biochemical fractionation to isolate MAMs, mitochondria, or peroxisomes can be used. Further, immunofluorescence with antibodies against specific membrane markers can be used to visualize MAVS distribution throughout the cell. Here, we describe the biochemical fractionation and immunofluorescence protocols used to detect MAVS subcellular localization.

Cover legend: Macrophages phagocytosing RFP-labeled E.coli. GFP-APPL2 labels the phagosomal membrane. Image produced by N. Condon. See Yeo et al. Traffic 2016; 17(9):1014-1026. Read the full article on doi:10.1111/tra.12415., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils., (© Society for Leukocyte Biology.)

Cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494 bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-β expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-β expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-β activation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The dengue virus (DENV) circulates between humans and mosquitoes and requires no other mammals or birds for its maintenance in nature. The virus is well-adapted to humans, as reflected by high-level viraemia in patients. To investigate its high adaptability, the DENV induction of host type-I interferon (IFN) was assessed in vitro in human-derived HeLa cells and compared with that induced by the Japanese encephalitis virus (JEV), a closely related arbovirus that generally exhibits low viraemia in humans. A sustained viral spread with a poor IFN induction was observed in the DENV-infected cells, whereas the JEV infection resulted in a self-limiting and abortive infection with a high IFN induction. There was no difference between DENV and JEV double-stranded RNA (dsRNA) as IFN inducers. Instead, the dsRNA was poorly exposed in the cytosol as late as 48 h post-infection (p.i.), despite the high level of DENV replication in the infected cells. In contrast, the JEV-derived dsRNA appeared in the cytosol as early as 24 h p.i. Our results provided evidence for the first time in DENV, that concealing dsRNA in the intracellular membrane diminishes the effect of the host defence mechanism, a strategy that differs from an active suppression of IFN activity.

The membrane landscape of a cell often changes drastically upon infection by a virus. In the case of the well-studied positive strand RNA virus poliovirus, the short infection cycle induces vesicles and tubular structures early in infection, and double-membraned vesicles late in infection. In this review, the current understanding of membrane changes in a PV-infected cell, the host and viral factors that facilitate these changes, and how these changes may promote virus replication will be discussed. Host factors involved in membrane rearrangement during infection include components of the COPI and COPII secretory pathways, lipid kinases, and the autophagy pathway. The roles of cellular membranes include acting as a scaffold for the RNA replication complex and roles in exit of mature virus. Finally, recent studies suggesting that not all picornaviruses are truly 'non-enveloped' are discussed in the context of the field, raising the possibility that cell-derived membranes play a role in delivering poliovirus particles to the extracellular space., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Microbial nucleic acids induce potent innate immune responses by stimulating the expression of type I interferons. Cyclic GMP-AMP synthase (cGAS) is a cytosolic dsDNA sensor mediating the innate immunity to microbial DNA. cGAS is activated by dsDNA and catalyze the synthesis of a cyclic dinucleotide cGAMP with 2',5' and 3',5'phosphodiester linkages. cGAMP binds to the adaptor STING located on the endoplasmic reticulum membrane and mediates the recruitment and activation of the protein kinase TBK1 and transcription factor IRF3. Phosphorylated IRF3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The crystal structures of cGAS and its complex with dsDNA, STING and its complex with various cyclic dinucleotides have been determined recently. Here we summarize the results from these structural studies and provide an overview about the mechanism of cGAS activation by dsDNA, the catalytic mechanism of cGAS, and the structural basis of STING activation by cGAMP., (Published by Elsevier Ltd.)

The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

Antibodies directed to intracellular neural antigens have been mainly described in paraneoplastic peripheral neuropathies and mostly includes anti-Hu and anti-CV2/CRMP5 antibodies. These antibodies occur with different patterns of neuropathy. With anti-Hu antibody, the most frequent manifestation is sensory neuronopathy with frequent autonomic involvement. With anti-CV2/CRMP5 the neuropathy is more frequently sensory and motor with an axonal or mixed demyelinating and axonal electrophysiological pattern. The clinical pattern of these neuropathies is in keeping with the cellular distribution of HuD and CRMP5 in the peripheral nervous system. Although present in high titer, these antibodies are probably not directly responsible for the neuropathy. Pathological and experimental studies indicate that cytotoxic T-cells are probably the main effectors of the immune response. These disorders contrast with those in which antibodies recognize a cell surface antigen and are probably responsible for the disease. The neuronal cell death and axonal degeneration which result from T-cell mediated immunity explains why treating these disorders remains challenging., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Interferon inducible transmembrane (IFITM) proteins are a recently discovered family of cellular anti-viral proteins that restrict the replication of a number of enveloped and non-enveloped viruses. IFITM proteins are located in the plasma membrane and endosomal membranes, the main portals of entry for many viruses. Biochemical and membrane fusion studies suggest IFITM proteins have the ability to inhibit viral entry, possibly by modulating the fluidity of cellular membranes. Here we discuss the IFITM proteins, recent work on their mode of action, and future directions for research., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Noncanonical autophagy is utilized by phagocytes to kill and digest extracellular pathogens. This process is initiated at the cell surface by receptors that recruit elements of the autophagy machinery, like LC3, to the phagosome. Also known as LC3-associated phagocytosis, the intersection of autophagy and phagocytosis was initially described as a pathway that limits the proliferation of engulfed pathogens by expediting phagosome maturation. Emerging evidences suggest that this pathway confers previously unsuspected versatility to the immune response as it regulates functions like the interferon pathway, dead cell clearance, and antigen presentation. Here we review recent advances in understanding the functional consequences of linking the autophagy machinery to phagocytosis in innate immunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

Phagocytes such as dendritic cells (DC) and macrophages employ phagocytosis to take up pathogenic bacteria into phagosomes, digest the bacteria and present the bacteria-derived peptide antigens to the adaptive immunity. Hence, efficient antigen presentation depends greatly on a well-regulated phagocytosis process. Lipids, particularly phosphoinositides, are critical components of the phagosomes. Phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3 ] is formed at the phagocytic cup, and as the phagosome seals off from the plasma membrane, rapid disappearance of PI(3,4,5)P3 is accompanied by high levels of phosphatidylinositol-3-phosphate (PI3P) formation. The sorting nexin (SNX) family consists of a diverse group of Phox-homology (PX) domain-containing cytoplasmic and membrane-associated proteins that are potential effectors of phosphoinositides. We hypothesized that SNX3, a small sorting nexin that contains a single PI3P lipid-binding PX domain as its only protein domain, localizes to phagosomes and regulates phagocytosis in DC. Our results show that SNX3 recruits to nascent phagosomes and silencing of SNX3 enhances phagocytic uptake of bacteria by DC. Furthermore, SNX3 competes with PI3P lipid-binding protein, early endosome antigen-1 (EEA1) recruiting to membranes. Our results indicate that SNX3 negatively regulates phagocytosis in DC possibly by modulating recruitment of essential PI3P lipid-binding proteins of the phagocytic pathways, such as EEA1, to phagosomal membranes., (© 2012 Blackwell Publishing Ltd.)

Membranes of endolysosomal compartments in macrophages are often damaged by physical or chemical effects of particles ingested through phagocytosis or by toxins secreted by intracellular pathogens. This study identified a novel inducible activity in macrophages that increases resistance of phagosomes, late endosomes, and lysosomes to membrane damage. Pretreatment of murine macrophages with LPS, peptidoglycan, TNF-α, or IFN-γ conferred protection against subsequent damage to intracellular membranes caused by photooxidative chemistries or by phagocytosis of ground silica or silica microspheres. Phagolysosome damage was partially dependent on reactive oxygen species but was independent of the phagocyte oxidase. IFN-γ-stimulated macrophages from mice lacking the phagocyte oxidase inhibited escape from vacuoles by the intracellular pathogen Listeria monocytogenes, which suggested a role for this inducible renitence (resistance to pressure) in macrophage resistance to infection by pathogens that damage intracellular membranes. Renitence and inhibition of L. monocytogenes escape were partially attributable to heat shock protein-70. Thus, renitence is a novel, inducible activity of macrophages that maintains or restores the integrity of endolysosomal membranes.

Neutrophils are pivotal to host defence during infectious diseases. However, activated neutrophils may also cause undesired tissue damage. Ample examples include small-vessel inflammatory diseases (vasculitis) that are associated with anti-neutrophil cytoplasmic autoantibodies (ANCA) residing in the patients' plasma. In addition to being an important diagnostic tool, convincing evidence shows that ANCA are pathogenic. ANCA-neutrophil interactions induce important cellular responses that result in highly inflammatory necrotizing vascular damage. The interaction begins with ANCA binding to their target antigens on primed neutrophils, proceeds by recruiting transmembrane molecules to initiate intracellular signal transduction and culminates in activation of effector functions that ultimately mediate the tissue damage., (© 2012 The Author. Clinical and Experimental Immunology © 2012 British Society for Immunology.)

Dengue virus causes ∼50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that complex assembly and activity occur in parallel. However, information about the oxidase assembly in individual phagosomes in live cells is scarce. We studied the dynamic behavior of the crucial cytosolic NADPH oxidase component p67(phox) during phagocytosis by videomicroscopy. p67(phox) is involved in the regulation of electron flow from NADPH to oxygen, leading to superoxide radical formation inside the phagosome. p67(phox)-citrine, expressed in myeloid PLB-985 cells, accumulated at the phagosomal membrane during phagocytosis of yeast particles. Using photobleaching techniques (FRAP, FLIP), we demonstrated that p67(phox)-citrine diffused freely in this phagosomal membrane, but the phagosomal pool of p67(phox)-citrine did not exchange with the cytosolic pool. This result suggests that once assembled in the NADPH oxidase complex, p67(phox) is stable in this complex. Furthermore, the time of the presence of p67(phox)-citrine at the phagosome increased substantially in the presence of complement in the opsonizing serum compared with decomplemented serum. PI(3)P also accumulated around phagosomes for twice as long in the presence of complement. The presence of p67(phox)-citrine was correlated with the duration of phagosomal ROS production in different opsonization conditions. These data support the critical role of p67(phox) for ROS production on the level of individual phagosomes.

Parasites like malaria and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of plants. The plastid (known as the apicoplast) is non-photosynthetic but retains many hallmarks of its ancestry including a circular genome that it synthesises proteins from and a suite of biosynthetic pathways of cyanobacterial origin. In this review, the discovery of the apicoplast and its integration, function and purpose are explored. New insights into the apicoplast fatty acid biosynthesis pathway and some novel roles of the apicoplast in vaccine development are reviewed.

Plants have evolved various means for controlled and organized cell destruction, known as programmed cell death (PCD). In plant immune responses against microbial infection, hypersensitive cell death as a form of PCD is a crucial event to prevent the spread of biotrophic pathogens. Recent live cell imaging techniques have revealed dynamic features and significant roles of cytoskeletons and the vacuole during defense responses and the PCD. Actin microfilaments (MFs) focus on the infection sites and function as tracks for the polar transport of antimicrobial materials. To accomplish hypersensitive cell death, further dynamic changes in cytoskeletons are induced. MFs play a role in the structural and functional regulation of the vacuole, leading to execution of the PCD. We here overview spatiotemporal dynamic changes in the cytoskeletons and the vacuoles triggered by signals from pathogens, and propose a hypothetical model for MF-regulated vacuole-mediated PCD in plant immunity.

FcγR-mediated phagocytosis is a cellular event that is evolutionary conserved to digest IgG-opsonized pathogens. Pseudopod formation during phagocytosis is a limiting step in managing the uptake of particles, and in this paper, we show that the conventional kinesin is involved in both receptor and membrane delivery to the phagocytic cup. Expression of a mutant kinesin isoform (GFP dominant negative mutant of kinesin H chain [EGFP-Kif5B-DN]) in RAW264.7 cells significantly reduced binding of IgG-sheep RBCs when macrophages were faced with multiple encounters with opsonized particles. Scanning electron microscopy analysis of EGFP-Kif5B-DN-expressing cells challenged with two rounds of IgG-sheep RBCs showed sparse, extremely thin pseudopods. We saw disrupted Rab11 trafficking to the phagocytic cup in EGFP-Kif5B-DN-transfected cells. Our particle overload assays also implicated phagosome membrane recycling in pseudopod formation. We observed reduced phagosome fission and trafficking in mutant kinesin-expressing cells, as well as reduced cell surface expression of FcγRs and Mac-1 receptors. In conclusion, anterograde trafficking via kinesin is essential for both receptor recycling from the phagosome and delivery of Rab11-containing membrane stores to effect broad and functional pseudopods during FcγR-mediated phagocytosis.

TLR9 recognizes unmethylated CpG DNA and induces innate immune responses. TLR9 activation is a multistep process requiring proteolytic cleavage and trafficking to endolysosomal compartments for ligand-induced signaling. However, the rules that govern the dynamic subcellular trafficking for TLR9 after pathogen uptake have not been established. In this study, we demonstrate that uptake of Aspergillus fumigatus conidia induced drastic spatial redistribution of TLR9 to the phagosomal membrane of A. fumigatus-containing phagosomes but not to bead-containing phagosomes in murine macrophages. Specific TLR9 recruitment to the fungal phagosome was consistent using A. fumigatus spores at different germination stages and selected mutants affecting the display of Ags on the fungal cell surface. Spatiotemporal regulation of TLR9 compartmentalization to the A. fumigatus phagosome was independent of TLR2, TLR4, and downstream TLR signaling. Our data demonstrate that the TLR9 N-terminal proteolytic cleavage domain was critical for successful intracellular trafficking and accumulation of TLR9 in CpG-containing compartments and A. fumigatus phagosomal membranes. Our study provides evidence for a model in which A. fumigatus spore phagocytosis by macrophages specifically induces TLR9 recruitment to A. fumigatus phagosomes and may thereby mediate TLR9-induced antifungal innate immune responses.

The role of astrocytes as structural and metabolic support for neurons is known since the beginning of the last century. Because of their strategic localization between neurons and capillaries they can monitor and control the level of synaptic activity by providing energetic metabolites to neurons and remove excess of neurotransmitters. During the last two decades number of papers further established that the astrocytic plasma-membrane G-protein coupled receptors (GPCR) can sense external inputs (such as the spillover of neurotransmitters) and transduce them as intracellular calcium elevations and release of chemical transmitters such as glutamate. The chemokine CXCR4 receptor is a GPCR widely expressed on glial cells (especially astrocytes and microglia). Activation of the astrocytic CXCR4 by its natural ligand CXCL12 (or SDF1 alpha) results in a long chain of intracellular and extracellular events (including the release of the pro-inflammatory cytokine TNFalpha and prostanglandins) leading to glutamate release. The emerging role of CXCR4-CXCL12 signalling axis in brain physiology came from the recent observation that glutamate in astrocytes is released via a regulated exocytosis process and occurs with a relatively fast time-scale, in the order of few hundred milliseconds. Taking into account that astrocytes are electrically non-excitable and thus exocytosis rely only on a signalling pathway that involves the release Ca(2+) from the internal stores, these results suggested a close relationship between sites of Ca(2+) release and those of fusion events. Indeed, a recent observation describes structural sub-membrane microdomains where fast ER-dependent calcium elevations occur in spatial and temporal correlation with fusion events., ((c) 2010 Elsevier B.V. All rights reserved.)

The adenovirus E1A oncogene induces innate immune rejection of tumors by sensitizing tumor cells to apoptosis in response to injuries, such as those inflicted by macrophage-produced TNF alpha and NO. E1A sensitizes cells to TNF by repressing its activation of NF-kappaB-dependent, antiapoptotic defenses. This suggested the hypothesis that E1A blockade of the NF-kappaB activation response might be the central mechanism of E1A induced cellular sensitivity to other proapoptotic injuries, such as macrophage-produced NO. However, creation of E1A-positive NIH-3T3 mouse cell variants with high-level, NF-kappaB-dependent resistance to TNF did not coselect for resistance to apoptosis induced by either macrophage-NO or chemical-NO, as the hypothesis would predict. E1A expression did block cellular recovery from NO-induced mitochondrial injury and converted the reversible, NO-induced cytostasis response of cells to an apoptotic response. This viral oncogene-induced phenotypic conversion of the cellular injury response of mouse and human cells was mediated by an E1A-related increase in NO-induced activation of caspase-2, an apical initiator of intrinsic apoptosis. Blocking caspase-2 activation or expression eliminated the NO-induced apoptotic response of E1A-positive cells. These results define an NF-kappaB-independent pathway through which the E1A gene of human adenovirus sensitizes mouse and human cells to apoptosis by enhancement of caspase-2-mediated mitochondrial injury.

Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.

The airway epithelium represents a primary site for the introduction and deposition of potentially pathogenic microorganisms into the body, through inspired air. The epithelial mucosa is an important component of the innate immune system that recognizes conserved structures in microorganisms and initiates appropriate signaling to recruit and activate phagocytic cells to the airways. This review focuses on how airway epithelial cells sense and respond to the presence of bacterial pathogens. The major signaling cascades initiated by epithelial receptors that lead to phagocyte recruitment to the airways as well as the ability of the epithelium to regulate inflammation are discussed., (Copyright (c) 2007 Wiley-Liss, Inc.)

Proteomics provides a powerful tool to characterize the protein content of an organelle. However, identifications obtained through mass spectrometry and database searching only make sense if the organelle sample is not heavily cross-contaminated. Besides the proteomic analysis, which gives an overview of possible cross-contamination, biochemical methods can be used to assess sample purity. These methods use specific markers that are detected and measured. Here, we describe the use of immunological, enzymatic, lipid, and pigment markers that allow the purity of chloroplast envelope fractions to be estimated.

Pemphigoid gestationis (PG) is an autoimmune sub-epidermal bullous dermatosis of pregnancy associated with circulating autoantibodies targeting the extracellular non-collagenous (NC) 16A domain of bullous pemphigoid (BP) 180 antigen. In order to determine whether BP180 regions other than NC16A are recognized by PG autoantibodies, we have analyzed the reactivity of 15 PG patient sera against several BP180 antigenic sites by sensitive methods such as immunological screening and ELISA. Most PG sera tested (13 of 15) reacted with an epitope (amino acid 508-541) mapped in the NC16A domain. Of note, nine of 15 PG patient sera reacted with at least one additional antigenic site other than NC16A. Specifically, two epitopes in the BP180 extracellular domain and five epitopes in the intracellular one were recognized by three and seven PG sera, respectively. In addition, a representative intracellular epitope was recognized by PG autoantibodies as a portion of BP180 antigen both in denaturating and native conditions. Finally, reactivity against epitopes additional to NC16A was also detected at an early stage of the disease. The identification and characterization of hitherto unrecognized epitopes targeted by PG patient autoantibodies provide novel insights into the pathophysiology of humoral immune response to BP180 in PG.

Responses of circulating hemocytes were studied in Lymnaea stagnalis exposed to 10, 30, 90, and 270 microg/L fomesafen for 24 and 504 h. Flow cytometry was used to quantify fomesafen-induced production of reactive oxygen species (ROS), phagocytic activity on Escherichia coli, and oxidative burst when hemocytes were challenged by E. coli or phorbol 12-myristate-13-acetate (PMA). Lysosomal membrane damage was assessed, using the neutral-red retention time (NRRT) assay. Exposure to fomesafen for 24 h resulted in increase in ROS levels and decreases in phagocytosis and the oxidative burst in PMA-stimulated hemocytes. After 504 h, intracellular levels of ROS returned to normal, but phagocytosis of E. coli was still inhibited and the associated oxidative burst significantly reduced. After both durations of exposure, decreases of NRRT indicated that lysosome membrane fragility increased with fomesafen concentration. Potential implications for the health and survival of the snails and consequences on populations are discussed.

T cells play an important role in the initiation and the progression of rheumatoid arthritis (RA) and depletion of potentially pathogenic T cells was suggested as an important therapeutic protocol. We determined if rosmarinic acid (RosA), known as a secondary metabolite from herbal plants, had apoptotic activity toward T cells from RA patients and further verified target T-cell subsets. CD3(+)CD25(+) activated T-cell subsets from most of the RA patients displayed significantly higher apoptosis rates than did the PBMCs and total CD3(+) T cells. Furthermore, activated and effector CD4(+) T cells, including CD4(+)CD25(+) and CD4(+)CD45RO(+) T cells, had a tendency of being more susceptible to RosA-induced apoptosis than that of resting and naïve T-cell subsets. RosA induced the release of cytochrome c from mitochondria and the blockage of mitochondrial depolarization inhibited apoptosis. Taken together, these results suggest that RosA induces apoptosis of activated T-cell subsets from RA patients via a mitochondrial pathway.

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.

Exosomes are MHC-bearing vesicles secreted by a wide array of cells. We have previously shown that donor-haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC-mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short-term LF 15-0195 (LF) treatment, which blocks the maturation of DCs, so that donor-MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF-treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor-specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor-specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor-MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor-specific allograft tolerance.

Signals from the precursor BCR (preBCR) cause proliferation and differentiation of progenitor (pro-) B cells into pre-B cells. Given the very low amounts of surface preBCRs and the demonstrated cell autonomy of preBCR signaling, we examined the possible occurrence of preBCR signal propagation from intracellular membranes such as the endoplasmic reticulum (ER) and the trans-Golgi network (TGN) in transformed and primary pro-B cells. PreBCRs composed of normal Ig mu or truncated Dmu heavy chains (HCs) were redirected to intracellular sites via localization sequences appended to the HC cytoplasmic tail. PreBCR complexes retained in the TGN or shunted from the TGN to lysosomes were as or 50% as active as the corresponding wild-type preBCRs in directing preBCR-dependent events, including CD2 and CD22 expression and proliferation in primary pro-B cells. This occurred despite their low to undetectable surface expression in transformed cells, which otherwise allowed significant surface accumulation of wild-type preBCRs. In contrast, ER-retained preBCRs were inactive. These results suggest that preBCR signaling is remarkably tolerant of dramatic changes in its subcellular distribution within post-ER compartments and support the possibility that the preBCR can activate signaling pathways in the TGN as well as the plasma membrane.

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over-expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C-terminally truncated form of N2. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full-length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co-expression of the C-terminal fragment of YFP fused to APP and the N-terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.

Conclusion: Intracellular cysteine cathepsins are pro-apoptotic factors involved in activation of caspases in two oral squamous cell carcinoma (SCC) cell lines., Objective: To study the possible involvement of lysosomal cathepsins in oral SCC cell apoptosis., Material and Methods: Apoptosis was induced in the two human oral SCC cell lines UT-SCC-20A and UT-SCC-24A using naphthazarin or anti-Fas antibodies, and was studied by analysis of caspase activity and nuclear morphology. Involvement of lysosomal cathepsins was investigated using the cysteine cathepsin inhibitor z-FA-FMK and the cathepsin D inhibitor pepstatin A. The amounts of cellular and soluble Fas death receptor were determined by ELISA., Results: Release of cathepsins from the lysosomes to the cytosol was observed early in apoptosis. Cysteine cathepsins were found to be involved in activation of caspases in response to treatment with naphthazarin or anti-Fas antibodies, but inhibition of cysteine cathepsin activity was not sufficient to prevent cell death. Moreover, inhibition of cysteine cathepsin activity resulted in increased expression of the Fas death receptor, suggesting involvement of extracellular cysteine cathepsins in death receptor shedding.

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.

Elimination of tumor cells by cytotoxic T lymphocytes (CTL) is mediated by two major pathways: the granule exocytosis and the death receptor pathway, transduced by Fas, TNF and TRAIL. The usage of these distinct pathways in the alloreactive setting across major and minor HLA barriers still remains controversial. We generated CTLs against allogeneic Epstein-Barr virus (EBV)-transformed cell lines (LCL) from HLA-unmatched healthy donors and assessed their cytotoxicity by flow cytometrically measuring mitochondrial membrane permeability (MMP) of target cells. Mitochondrial apoptosis induced by CTL was abrogated by selectively inhibiting the granule exocytosis-mediated pathway with Concanamycin A (CMA). Conversely, apoptosis was not decreased in the presence of the caspase 8 inhibitor IETD, which is specific for all death receptor pathways. In general, caspases were not involved in MMP as shown using the pan-caspase inhibitor zVAD. This effector mechanism was preserved when using purified CD4 + and CD8 + T-lymphocyte subsets to generate CTL. We further showed, that death receptor signalling was not used as a salvage mechanism when granule exocytosis was inhibited even at longer incubation times sufficient for slow kinetic death receptor caspase signalling. Our results clearly demonstrate that mitochondrial apoptosis induced by human alloreactive CTLs is mainly mediated by granule exocytosis but not by death receptor caspase dependent pathways. Furthermore, the granule exocytosis pathway does not require caspases to induce MMP.

Two membrane compartments of Escherichia coli ghosts, representing empty bacterial cell envelopes, were investigated as carriers of foreign antigens. By subcutaneous immunisation of mice the immunogenicity of bacterial ghosts carrying the Hepatitis B virus core 149 protein (HBcAg-149) as model antigen anchored either in the inner or the outer membrane of E. coli was compared. Both systems induced significant immune responses against the foreign target antigen, the HBcAg-149, in mice. Results indicate that bacterial ghosts provide an excellent carrier system for antigen delivery.

Apoptosis contributes to the pathology of gastric epithelial cell damage that characterizes Helicobacter pylori infection. The secreted peptidyl prolyl cis, trans-isomerase of H. pylori, HP0175 executed apoptosis of the gastric epithelial cell line AGS in a dose- and time-dependent manner. The effect of HP0175 was confirmed by generating an isogenic mutant of H. pylori disrupted in the HP0175 gene. The apoptosis-inducing ability of this mutant was impaired compared with that of the wild type. The effect of HP0175 was mediated through TLR4. Preincubation of the gastric epithelial cell line AGS with anti-TLR4 mAb inhibited apoptosis induced by HP0175. Downstream of TLR4, apoptosis signal-regulating kinase 1 activated MAPK p38, leading to the caspase 8-dependent cleavage of Bid, its translocation to the mitochondria, mitochondrial pore formation, cytochrome c release, and activation of caspases 9 and 3. We show for the first time that a secreted bacterial Ag with peptidyl prolyl cis,trans-isomerase activity signals through TLR4, and that this Ag executes gastric epithelial cell apoptosis through a signaling pathway in which TLR4 and apoptosis signal-regulating kinase 1 are central players.

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system.

Despite many studies on the topic, plasma cells found in human periapical chronic inflammatory lesions (granulomas) continue to present unresolved issues. In this study, we tried to assess quantitatively and qualitatively the nature of plasma cells of 4 human periapical granulomas. Samples were analyzed for relative amounts of IgG-, IgM-, IgA-, and IgE-positive plasma cells by immunohistochemistry, and for morphological changes by transmission electron microscopy (TEM). By immunohistochemistry, many plasma cells stained positively with anti-IgG and anti-IgM antibodies; fewer cells reacted with anti-IgE and anti-IgA. Russell Bodies, controversial aspects of plasma cell maturation, showed positive reactivity of the superficial layer only to antibodies against IgG and IgM. By TEM analysis, phenotypes of normal and dysfunctional plasma cells (Mott cells) were evident. Russell Bodies appeared as intra- or extracellular round vesicles, with an homogeneous internal core, and an external membrane, resembling rough endoplasmatic reticulum (RER). We can conclude that mucosal immune response is not the predominant type in the periapical lesions examined. Positive immunoreaction for IgG and IgM of Russell Bodies may be due to the residual RER membrane, whereas components of yet unidentified nature may occupy the internal core.

During phagocytosis and phagosome maturation, complex membrane traffic events must be coordinated. We have observed, using fluorescent fluid-phase and membrane markers, that in the human neutrophil, internalization of nonopsonized, Gram-positive bacteria, but not of latex beads, is accompanied by a rapid and localized formation of pinosomal structures. This pinocytic response is calcium-dependent but insensitive to actin cytoskeleton disruption and wortmannin treatment. Contrary to what we observe, endosomal structures usually are considered to participate in phagosome formation by providing necessary membrane to forming phagosomes. Instead, our results show a coupling between neutrophil secretory and membrane-retrieval processes during phagosome maturation, and we suggest that the observed, localized pinocytic response is linked to the secretion of azurophilic granules toward nascent phagosomes. Accordingly, M and M-like protein-expressing Streptococcus pyogenes bacteria, which are able to survive inside neutrophil phagosomes, inhibit both the secretion of azurophilic granules to phagosomes and pinosome formation.

Since dendritic cells (DC) participate in both innate and adaptive immunity, their survival and expansion is tightly controlled. Little is known about the mechanisms of DC apoptosis. PGE(2), an arachidonic acid metabolite, plays an essential role in DC migration. We propose a novel function for PGE(2) as a DC survival factor. Our studies demonstrate that PGE(2) protects DC in vitro against apoptosis induced by withdrawal of growth factors or ceramide. DC matured in conditions that inhibit endogenous PGE(2) release are highly susceptible to apoptosis and exogenous PGE(2) re-establishes the more resistant phenotype. The antiapoptotic effect is mediated through EP-2/EP-4 receptors and involves the PI3K --> Akt pathway. PGE(2) leads to increased phosphorylation of Akt, protection against mitochondrial membrane compromise, and decreased caspase 3 activity. Macroarray data indicate that PGE(2) leads to the down-regulation of a number of proapoptotic molecules, i.e., BAD, several caspases, and granzyme B. In vivo, higher numbers of immature and Ag-loaded CFSE-labeled DC are present in the draining lymph nodes of mice inoculated with PGE(2) receptor agonists, compared with animals treated with ibuprofen or controls injected with PBS. This suggests that PGE(2) acts as an endogenous antiapoptotic factor for DC and raises the possibility of using PGE(2) agonists to increase the survival of Ag-loaded DC following in vivo administration.

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.