CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Exposure to siRNA-GalNAc Conjugates in Systems of the Standard Test Battery for Genotoxicity.

Registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-O-methyl ribose modifications of multiple nucleotides in the ICH battery of assays, all of which have uniformly yielded negative results. To verify these negative genotoxicity results, in this study we confirm test article exposure using toolkit small interfering RNAs (siRNAs) representative of those in the clinic. In the Ames test, the highest uptake of the siRNA-GalNAc conjugates occurred at 1 h postdose in all bacterial strains independent of siRNA sequence or chemistry (up to ∼14,000 siRNA molecules per cell), followed by metabolic degradation of the parent siRNA at 6, 24, and 48 h postdose. siRNA-GalNAc conjugates were internalized by bacteria as assessed by protection from the addition of nucleases to the culture media following uptake and by the requirement of cell lysis for detection of the siRNA. In the in vitro chromosome aberration assay, uptake was observed in Chinese hamster ovary cells (up to ∼5,500 siRNA molecules per cell at 21 h postdose) and in CD3+ human peripheral blood lymphocytes (up to ∼500 siRNA molecules per cell at 21 h postdose). In the in vivo micronucleus assay in rat bone marrow, exposure to parent siRNA was 100-350 µg of antisense strand per gram of protein at 24 and 48 h postlimit dose of 2 g/kg. Loss of terminal nucleotides was detected in bone marrow by mass spectrometry, indicating exposure to monomer metabolites as well. Negative genotoxicity results were also confirmed in an in vitro double-strand DNA break assay in HeLa and HepG2 cells where exposure was maximized using transfection reagents. Thus negative genotoxicity assay results for siRNA-GalNAc conjugates were valid and not the result of poor or no intracellular exposure.

Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing ß-1,3 Glucan.

Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate ß-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that ß-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing ß-1,3 glucan beads. Dectin-1-mediated spleen tyrosine kinase activation is required for TLR9 trafficking to ß-1,3 glucan-, A. fumigatus-, and C. albicans-containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of ß-1,3 glucan by Dectin-1 triggers TLR9 trafficking to ß-1,3 glucan-containing phagosomes, which may be critical in coordinating innate antifungal defense.

Shielding of Lipid Nanoparticles for siRNA Delivery: Impact on Physicochemical Properties, Cytokine Induction, and Efficacy.

Formulation of short interfering RNA (siRNA) into multicomponent lipid nanoparticles (LNP) is an effective strategy for hepatic delivery and therapeutic gene silencing. This study systematically evaluated the effect of polyethylene glycol (PEG) density on LNP physicochemical properties, innate immune response stimulation, and in vivo efficacy. Increased PEG density not only shielded LNP surface charge but also reduced hemolytic activity, suggesting the formation of a steric barrier. In addition, increasing the PEG density reduced LNP immunostimulatory potential as reflected in cytokine induction both in vivo and in vitro. Higher PEG density also hindered in vivo efficacy, presumably due to reduced association with apolipoprotein E (ApoE), a protein which serves as an endogenous targeting ligand to hepatocytes. This effect could be overcome by incorporating an exogenous targeting ligand into the highly shielded LNPs, thereby circumventing the requirement for ApoE association. Therefore, these studies provide useful information for the rational design of LNP-based siRNA delivery systems with an optimal safety and efficacy profile.

Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host.

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.

Toll-like receptor 9 modulates macrophage antifungal effector function during innate recognition of Candida albicans and Saccharomyces cerevisiae.

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.

The tetraspanin CD82 is specifically recruited to fungal and bacterial phagosomes prior to acidification.

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.

TLR9 is actively recruited to Aspergillus fumigatus phagosomes and requires the N-terminal proteolytic cleavage domain for proper intracellular trafficking.

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.

Fibroblast-like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: evidence of a link between an increased myofibroblast-like phenotype and high-inflammation synovitis.

OBJECTIVE: Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues. METHODS: Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues. RESULTS: Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor beta/activin A-inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue. CONCLUSION: Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.

Genomics in the immune system.

The analysis of gene expression in tissues, cells, and biologic systems has evolved in the last decade from the analysis of a selected set of genes to an efficient high throughput whole-genome screening approach of potentially all genes expressed in a tissue or cell sample. Development of sophisticated methodologies such as microarray technology allows an open-ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and define the samples' unique biology. This discovery-based research provides the opportunity to characterize either new genes with unknown function or genes not previously known to be involved in a biologic process. The latter category may hold surprises that sometimes urge us to redirect our thinking. Here, we review the impact of large-scale gene expression profiling by DNA-microarray technology on basic and clinical aspects of immunology.

Rheumatoid arthritis is a heterogeneous disease: evidence for differences in the activation of the STAT-1 pathway between rheumatoid tissues.

OBJECTIVE: To generate a molecular description of synovial tissue from rheumatoid arthritis (RA) patients that would allow us to unravel novel aspects of pathogenesis and to identify different forms of disease. METHODS: We applied complementary DNA microarray analysis to profile gene expression, with a focus on immune-related genes, in affected joint tissues from RA patients and in tissues from osteoarthritis (OA) patients as a control. To validate microarray data, real-time polymerase chain reaction was performed on genes of interest. RESULTS: The gene expression signatures of synovial tissues from RA patients showed considerable variability, resulting in the identification of at least two molecularly distinct forms of RA tissues. One class of tissues revealed abundant expression of clusters of genes indicative of an involvement of the adaptive immune response. Detailed analysis of the expression profile provided evidence for a prominent role of an activated signal transducer and activator of transcription 1 pathway in these tissues. The expression profiles of another group of RA tissues revealed an increased tissue remodeling activity and a low inflammatory gene expression signature. The gene expression pattern in the latter tissues was reminiscent of that observed in the majority of OA tissues. CONCLUSION: The differences in the gene expression profiles provide a unique perspective for distinguishing different pathogenetic RA subsets based on molecular criteria. These data reflect important aspects of molecular variation that are relevant for understanding the biologic dysregulation underlying these subsets of RA. This approach may also help to define homogeneous groups for clinical studies and evaluation of targeted therapies.