Dissection of the Dislocation Pathway for Type I Membrane Proteins with a New Small Molecule Inhibitor, Eeyarestatin

Molecular Biology of the Cell. Apr, 2004  |  Pubmed ID: 14767067

The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor alpha. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.

CX3CR1-mediated Dendritic Cell Access to the Intestinal Lumen and Bacterial Clearance

Science (New York, N.Y.). Jan, 2005  |  Pubmed ID: 15653504

Dendritic cells (DCs) and macrophages are critical to innate and adaptive immunity to the intestinal bacterial microbiota. Here, we identify a myeloid-derived mucosal DC in mice, which populates the entire lamina propria of the small intestine. Lamina propria DCs were found to depend on the chemokine receptor CX3CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens. CX3CR1 was also found to control the clearance of entero-invasive pathogens by DCs. Thus, CX3CR1-dependent processes, which control host interactions of specialized DCs with commensal and pathogenic bacteria, may regulate immunological tolerance and inflammation.

In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

Infection and Immunity. Jul, 2006  |  Pubmed ID: 16790753

Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

Recruitment of CD63 to Cryptococcus Neoformans Phagosomes Requires Acidification

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2006  |  Pubmed ID: 17043215

The subcellular localization of the cluster of differentiation 63 (CD63) tetraspanin and its interaction with the class II MHC antigen presentation pathway were examined in the context of phagocytosis by live cell imaging, by using monomeric red fluorescent protein-tagged mouse CD63 expressed in primary bone marrow-derived cell cultures. Upon phagocytosis of Cryptococcus neoformans and polystyrene beads, CD63 was recruited selectively to C. neoformans-containing phagosomes in a MyD88-independent acidification-dependent manner. Bead-containing phagosomes, within a C. neoformans-containing cell, acidified to a lesser extent and failed to recruit CD63 to a level detectable by microscopy. CD63 recruitment to yeast phagosomes occurred independently of class II MHC and LAMP-1. These observations indicate that the composition of distinct phagosomal compartments within the same cell is determined by phagosomal cargo and may affect the outcome of antigen processing and presentation.

Immunoglobulin G Signaling Activates Lysosome/phagosome Docking

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2006  |  Pubmed ID: 17110435

An important role of IgG antibodies in the defense against microbial infections is to promote the ingestion and killing of microbes by phagocytes. Here, we developed in vivo and in vitro approaches to ask whether opsonization of particles with IgG enhances intracellular targeting of lysosomes to phagosomes. To eliminate the effect of IgG on the ingestion process, cells were exposed to latex beads at 15-20 degrees C, which allows engulfment of both IgG-coated and uncoated beads but prevents the fusion of lysosomes with phagosomes. Upon shifting the temperature to 37 degrees C, phagosomes containing IgG beads matured significantly faster into phagolysosomes as judged by colocalization with lysosomal markers. The IgG effect was independent of other particle-associated antigens or serum factors. Lysosome/phagosome attachment was also quantified biochemically with a cytosol-dependent scintillation proximity assay. Interactions were enhanced significantly in reactions containing cytosol from mouse macrophages that had been exposed to IgG-coated beads, indicating that IgG signaling modulates the cytosolic-targeting machinery. Similar results were obtained with cytosol from primary human monocytes, human U-937 histiocytic lymphoma cells and from Chinese hamster ovary (CHO) cells transfected with a human IgG (Fcgamma) receptor. IgG-induced activation is shown to affect the actin-dependent tethering/docking stage of the targeting process and to proceed through a pathway involving protein kinase C. These results provide a rare example of an extracellular signal controlling membrane targeting on the level of tethering and docking. We propose that this pathway contributes to the role of antibodies in the protection against microbial infections.

Abdominal Abscesses Due to Actinomycosis After Laparoscopic Cholecystectomy: Case Reports and Review

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Jan, 2007  |  Pubmed ID: 17173208

We describe 2 patients who presented to a health care facility with abdominal abscesses years after undergoing laparoscopic cholecystectomy that was complicated by gallstone spillage. In both patients, sample cultures yielded Actinomyces species and enteric organisms. In 1 patient, crystallographic analysis of abscess debris confirmed the presence of gallstones. Actinomyces species is a rare cause of abdominal abscesses that should be considered in this patient population.

Tubulation of Class II MHC Compartments is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2007  |  Pubmed ID: 17513769

Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

Treatment of Refractory Babesia Microti Infection with Atovaquone-proguanil in an HIV-infected Patient: Case Report

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Dec, 2007  |  Pubmed ID: 18190320

A patient with acquired immune deficiency syndrome presented with babesiosis 6 months after presumed tick exposure. Despite initial treatment with azithromycin and atovaquone, followed by quinine and clindamycin, he experienced an increasing parasite load. Finally, red blood cell exchange transfusion, anti-Babesia therapy, and the addition of atovaquone-proguanil to the treatment regimen led to symptomatic improvement and elimination of parasitemia. Low-level parasitemia recurred 20 weeks later and was eradicated by administration of atovaquone-proguanil monotherapy. Atovaquone-proguanil appears to have activity against babesiosis and should be studied as a potential therapy for patients with refractory babesiosis.

The Known Unknowns of Antigen Processing and Presentation

Nature Reviews. Immunology. Aug, 2008  |  Pubmed ID: 18641646

The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention.

Hodgkin's Lymphoma Masquerading As Vertebral Osteomyelitis in a Man with Diabetes: a Case Report

Journal of Medical Case Reports. 2010  |  Pubmed ID: 20370895

Infection and malignancy often have common characteristics which render the differential diagnosis for a prolonged fever difficult. Imaging and tissue biopsy are crucial in making a correct diagnosis, though differentiating between chronic osteomyelitis and malignancy is not always straightforward as they possess many overlapping features.

TLR9 is Actively Recruited to Aspergillus Fumigatus Phagosomes and Requires the N-terminal Proteolytic Cleavage Domain for Proper Intracellular Trafficking

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2010  |  Pubmed ID: 21059889

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.

Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions

PloS One. 2010  |  Pubmed ID: 21217821

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

The Duality of Aspergillus Terreus: Differential Immune Responses to Distinct Conidia

Virulence. May-Jun, 2011  |  Pubmed ID: 21636969

Toll-like Receptor 9 Modulates Macrophage Antifungal Effector Function During Innate Recognition of Candida Albicans and Saccharomyces Cerevisiae

Infection and Immunity. Dec, 2011  |  Pubmed ID: 21947771

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens. Macrophages sample the phagosomal content and orchestrate the innate immune response. Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA and is activated by fungal DNA. Here we demonstrate that specific triggering of TLR9 recruitment to the macrophage phagosomal membrane is a conserved feature of fungi of distinct phylogenetic origins, including Candida albicans, Saccharomyces cerevisiae, Malassezia furfur, and Cryptococcus neoformans. The capacity to trigger phagosomal TLR9 recruitment was not affected by a loss of fungal viability or cell wall integrity. TLR9 deficiency has been linked to increased resistance to murine candidiasis and to restriction of fungal growth in vivo. Macrophages lacking TLR9 demonstrate a comparable capacity for phagocytosis and normal phagosomal maturation compared to wild-type macrophages. We now show that TLR9 deficiency increases macrophage tumor necrosis factor alpha (TNF-α) production in response to C. albicans and S. cerevisiae, independent of yeast viability. The increase in TNF-α production was reversible by functional complementation of the TLR9 gene, confirming that TLR9 was responsible for negative modulation of the cytokine response. Consistently, TLR9 deficiency enhanced the macrophage effector response by increasing macrophage nitric oxide production. Moreover, microbicidal activity against C. albicans and S. cerevisiae was more efficient in TLR9 knockout (TLR9KO) macrophages than in wild-type macrophages. In conclusion, our data demonstrate that TLR9 is compartmentalized selectively to fungal phagosomes and negatively modulates macrophage antifungal effector functions. Our data support a model in which orchestration of antifungal innate immunity involves a complex interplay of fungal ligand combinations, host cell machinery rearrangements, and TLR cooperation and antagonism.

Dynamic Virulence: Real-time Assessment of Intracellular Pathogenesis Links Cryptococcus Neoformans Phenotype with Clinical Outcome

MBio. 2011  |  Pubmed ID: 21954307

While a myriad of studies have examined host factors that predispose persons to infection with the opportunistic fungal pathogen Cryptococcus neoformans, comparatively little has been done to examine how virulence factor differences among cryptococcal isolates may impact outcome. In the recent report by Alanio et al. (A. Alanio, M. Desnos-Ollivier, and F. Dromer, mBio 2:e00158-11, 2011), novel flow cytometry-based techniques were employed to demonstrate an association between the phenotype of C. neoformans-macrophage interactions, as measured by phagocytosis and intracellular replication, and patient outcomes, as determined by positive cultures on therapy and survival. These experiments establish that the prognosis of patients with cryptococcosis is influenced by the phenotypic properties of the infecting fungal isolate.

Fusarium Pathogenesis Investigated Using Galleria Mellonella As a Heterologous Host

Fungal Biology. Dec, 2011  |  Pubmed ID: 22115447

Members of the fungal genus Fusarium are capable of manifesting in a multitude of clinical infections, most commonly in immunocompromised patients. In order to better understand the interaction between the fungus and host, we have developed the larvae of the greater wax moth, Galleria mellonella, as a heterologous host for fusaria. When conidia are injected into the haemocoel of this Lepidopteran system, both clinical and environmental isolates of the fungus are able to kill the larvae at 37 °C, although killing occurs more rapidly when incubated at 30 °C. This killing was dependent on several other factors besides temperature, including the Fusarium strain, the number of conidia injected, and the conidia morphology, where macroconidia are more virulent than their microconidia counterpart. There was a correlation in the killing rate of Fusarium spp. when evaluated in G. mellonella and a murine model. In vivo studies indicated G. mellonella haemocytes were capable of initially phagocytosing both conidial morphologies. The G. mellonella system was also used to evaluate antifungal agents, and amphotericin B was able to confer a significant increase in survival to Fusarium-infected larvae. The G. mellonella-Fusarium pathogenicity system revealed that virulence of Fusarium spp. is similar, regardless of the origin of the isolate, and that mammalian endothermy is a major deterrent for Fusarium infection and therefore provides a suitable alternative to mammalian models to investigate the interaction between the host and this increasingly important fungal pathogen.

The Tetraspanin CD82 is Specifically Recruited to Fungal and Bacterial Phagosomes Prior to Acidification

Infection and Immunity. Mar, 2011  |  Pubmed ID: 21149584

CD82 is a member of the tetraspanin superfamily, whose physiological role is best described in the context of cancer metastasis. However, CD82 also associates with components of the class II major histocompatibility complex (MHC) antigen presentation pathway, including class II MHC molecules and the peptide-loading machinery, as well as CD63, another tetraspanin, suggesting a role for CD82 in antigen presentation. Here, we observe the dynamic rearrangement of CD82 after pathogen uptake by imaging CD82-mRFP1 expressed in primary living dendritic cells. CD82 showed rapid and specific recruitment to Cryptococcus neoformans-containing phagosomes compared to polystyrene-containing phagosomes, similar to CD63. CD82 was also actively recruited to phagosomes containing other pathogenic fungi, including Candida albicans and Aspergillus fumigatus. Recruitment of CD82 to fungal phagosomes occurred independently of Toll-like receptor (TLR) signaling. Recruitment was not limited to fungi, as bacterial organisms, including Escherichia coli and Staphylococcus aureus, also induced CD82 recruitment to the phagosome. CD82 intersected the endocytic pathway used by lipopolysaccharide (LPS), implicating CD82 in trafficking of small, pathogen-associated molecules. Despite its partial overlap with lysosomal compartments, CD82 recruitment to C. neoformans-containing phagosomes occurred independently of phagosome acidification. Kinetic analysis of fluorescence imaging revealed that CD82 and class II MHC simultaneously appear in the phagosome, indicating that the two proteins may be associated. Together, these data show that the CD82 tetraspanin is specifically recruited to pathogen-containing phagosomes prior to fusion with lysosomes.

Dragon (repulsive Guidance Molecule B) Inhibits IL-6 Expression in Macrophages

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2011  |  Pubmed ID: 21187450

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.

Use of Fungal Derived Polysaccharide-conjugated Particles to Probe Dectin-1 Responses in Innate Immunity

Integrative Biology : Quantitative Biosciences from Nano to Macro. Feb, 2012  |  Pubmed ID: 22200052

The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of β-1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of "fungal like particles" was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified β-1,3-glucan derived from Saccharomyces cerevisiae. The morphology of the β-1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the β-1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating β-1,3-glucan beads with laminarinase, a specific β-1,3-gluconase, the reactivity of the anti-β-1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly β-1,3-glucan. TNF-α was also measured by stimulating bone-marrow derived macrophages with the β-1,3-glucan beads, and showed a dose dependent response compared to soluble β-glucan, insoluble β-1,3-glucan, uncoated beads, and soluble β-1,3-glucan mixed with uncoated beads. Finally, β-1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. β-1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. These data describe a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.