Alzheimer's disease protease-containing plasma extracellular vesicles transfer to the hippocampus via the choroid plexus.

BACKGROUND: Plasma extracellular vesicles (pEV) can harbor a diverse array of factors including active proteases and the amyloid-precursor-protein (APP) cleavage product Aß, involved in plaque formation in Alzheimer`s diseases (AD). A potential role of such vesicles in AD pathology is unexplored. METHODS: In a case-control study of randomly selected patients with AD and other neurological diseases (n = 14), and healthy controls (n = 7), we systematically analyzed the content of pEV, using different assay systems. In addition, we determined their entry path into brain tissue, employing animal (mice) injection experiments with ex vivo generated EV that were similar to AD-pEV, followed by multi antigen analysis (MAA) of brain tissue (n = 4 per condition). The results were compared with an IHC staining of human brain tissue in a small cohort of AD patients (n = 3) and controls with no neurodegenerative diseases (n = 3). FINDINGS: We show that pEV levels are considerably upregulated in AD patients. Besides numerous inflammatory effectors, AD-pEV contained α-, ß- and γ-secretases, able to cleave APP in in target cells. In vitro generated EV with similar characteristics as AD-pEV accumulated in the choroid plexus (CP) of injected animals and reached primarily hippocampal neurons. Corroborating findings were made in human brain samples. An inhibitor of hyaluronic-acid-synthetase (HAS) blocked uploading of proteases and Hyaluronan onto EV in vitro and abolished CP targeting in animal injection experiments. INTERPRETATION: We conclude that protease-containing pEV could be part of a communication axis between the periphery and the brain that could be become detrimental depending on pEV concentration and duration of target cell impact. FUNDING: See the Acknowledgements section.

Plasma-derived extracellular vesicles discriminate type-1 allergy subjects from non-allergic controls.

BACKGROUND: Allergies are on the rise globally, with an enormous impact on affected individuals' quality of life as well as health care resources. They cause a wide range of symptoms, from slightly inconvenient to potentially fatal immune reactions. While allergies have been described and classified phenomenologically, there is an unmet need for easily accessible biomarkers to stratify the severity of clinical symptoms. Furthermore, biomarkers marking the success of specific immunotherapy are urgently needed. OBJECTIVES: Plasma extracellular vesicles (pEV) play a role in coordinating the immune response and may be useful future biomarkers. A pilot study on differences in pEV content was carried out between patients with type I allergy, suffering from rhinoconjunctivitis with or without asthma, and voluntary non-allergic donors. METHODS: We examined pEV from 38 individuals (22 patients with allergies and 16 controls) for 38 chemokines, cytokines, and soluble factors using high-throughput data mining approaches. RESULTS: Patients with allergies had a distinct biomarker pattern, with 7 upregulated (TNF-alpha, IL-4, IL-5, IL-6, IL-17F, CCL2, and CCL17) and 3 downregulated immune mediators (IL-11, IL-27, and CCL20) in pEV compared to controls. This reduced set of 10 factors was able to discriminate controls and allergic patients better than the total array. CONCLUSIONS: The content of pEV showed potential as a target for biomarker research in allergies. Plasma EV, which are readily measurable via blood test, may come to play an important role in allergy diagnosis. In this proof-of-principle study, it could be shown that pEV's discriminate patients with allergies from controls. Further studies investigating whether the content of pEVs may predict the severity of allergic symptoms or even the induction of tolerance to allergens are needed.

Evidence for liver and peripheral immune cells secreting tumor-suppressive extracellular vesicles in melanoma patients.

BACKGROUND: Before and after surgery melanoma patients harbor elevated levels of extracellular vesicles in plasma (pEV), suppressing tumor cell activity. However, due to technical reasons and lack of cell-specific biomarkers, their cellular origin remains obscure. METHODS: We mimicked the interaction of tumor cells with liver cells and PBMC in vitro, and compared newly secreted EV-associated miRNAs and protein factors with those detected in melanoma patient`s pEV. FINDINGS: Our results suggest that pEV from melanoma patients are secreted in part by residual or relapsing tumor cells, but also by liver and peripheral blood mononuclear cells (PBMC). Our approach identified factors that were seemingly associated either with tumor cell activity, or the counteracting immune system, including liver cells. Notably, the presence/absence of these factors correlated with the clinical stage and tumor relapse. INTERPRETATION: Our study may provide new insights into the innate immune defense against tumor cells and implies that residual tumor cells could be more active than previously thought. In addition we provide some preliminary evidence that pEV marker patterns could be used to predict cancer relapse.

Innate extracellular vesicles from melanoma patients suppress ß-catenin in tumor cells by miRNA-34a.

Upon tumor development, new extracellular vesicles appear in circulation. Our knowledge of their relative abundance, function, and overall impact on cancer development is still preliminary. Here, we demonstrate that plasma extracellular vesicles (pEVs) of non-tumor origin are persistently increased in untreated and post-excision melanoma patients, exhibiting strong suppressive effects on the proliferation of tumor cells. Plasma vesicle numbers, miRNAs, and protein levels were elevated two- to tenfold and detected many years after tumor resection. The vesicles revealed individual and clinical stage-specific miRNA profiles as well as active ADAM10. However, whereas pEV from patients preventing tumor relapse down-regulated ß-catenin and blocked tumor cell proliferation in an miR-34a-dependent manner, pEV from metastatic patients lost this ability and stimulated ß-catenin-mediated transcription. Cancer-induced pEV may constitute an innate immune mechanism suppressing tumor cell activity including that of residual cancer cells present after primary surgery.

HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection.

Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvß3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c(+) CD14(+) cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity.

CD83 and GRASP55 interact in human dendritic cells.

CD83 is one of the best known surface markers for mature human dendritic cells (DCs). The full-length 45 kDa type-I membrane-bound form (mbCD83) is strongly glycosylated upon DCs maturation. As co-stimulatory properties of CD83 are attributed to mbCD83 surface expression is required for efficient T-cell stimulation by mature DCs. By yeast two-hybrid screening, we were able to identify GRASP55 as interaction partner of CD83. DCs maturation induces endogenous CD83 protein expression with simultaneous regulation of CD83 glycosylation, interaction and co-localization with GRASP55 and CD83 surface exposure. GRASP55 is especially known for its role in maintaining Golgi architecture, but also plays a role in Golgi transport of specific cargo proteins bearing a C-terminal valine residue. Here we additionally demonstrate that binding of CD83 and GRASP55 rely on the C-terminal TELV-motif of CD83. Mutation of this TELV-motif not only disrupted binding to GRASP55, but also altered the glycosylation pattern of CD83 and reduced its membrane expression. Here we show for the first time that GRASP55 interacts with CD83 shortly after induction of DC maturation and that this interaction plays a role in CD83 glycosylation as well as in surface expression of CD83 on DCs.

Induction of HIV transcription by Nef involves Lck activation and protein kinase C theta raft recruitment leading to activation of ERK1/2 but not NF kappa B.

The Nef protein of HIV-1 is a key promoter of disease progression, owing to its dramatic yet ill-defined impact on viral replication. Previously, we have shown that Nef enhances Tat-mediated transcription in a manner depending on Lck and the cytoplasmic sequestration of the transcriptional repressor embryonic ectodermal development [corrected]. In this study, we report that Lck is activated by Nef and targets protein kinase Ctheta downstream, leading to the translocation of the kinase into membrane microdomains. Although microdomain-localized protein kinase Ctheta is thought to induce the transcription factor NFkappaB, we unexpectedly failed to correlate Nef-induced signaling events with enhanced NFkappaB activity. Instead, we observed an increase in ERK MAPK activity. We conclude that Nef-mediated signaling cooperates with Nef-induced derepression and supports HIV transcription through an ERK MAPK-dependent, but NFkappaB-independent, pathway.

HIV Nef enhances Tat-mediated viral transcription through a hnRNP-K-nucleated signaling complex.

Although dispensable in vitro, HIV Nef enables high-level viral replication in infected hosts by an as yet unexplained mechanism. Previously, we proposed that Nef functionally cooperates with the viral transactivator Tat by derepressing the viral promoter via a Nef-associated kinase complex (NAKC). Here, we demonstrate that hnRNP-K, a host factor thought to facilitate crosstalk between kinases and gene expression, interacts with Nef and, as part of NAKC, nucleates Nef-interacting kinases, including Lck, PKCdelta, and PI-3 kinase, leading to Lck and Erk1/2 activation. This strongly increased HIV transcription, which depended on Tat and the NF-kB motif in the viral promoter, but not on NF-kB activation. Depletion of hnRNP-K in a Jurkat model of HIV latency increased Erk1/2 activity and greatly augmented HIV reactivating stimuli. We conclude that hnRNP-K coordinates membrane signaling with transcriptional derepression through Erk1/2 and is targeted by HIV to enable Tat-mediated transcription.

Novel (n)PKC kinases phosphorylate Nef for increased HIV transcription, replication and perinuclear targeting.

The N-terminus of the human immunodeficiency virus (HIV) pathogenicity factor Nef associates with a protein complex (NAKC for Nef-associated kinase complex) that contains at least two kinases: the tyrosine kinase Lck and a serine kinase activity which was found to phosphorylate Lck and the Nef N-terminus. Here we show that this serine kinase activity is mediated by members of the novel Protein Kinase C (nPKC) subfamily, PKCdelta and theta. Association with the Nef N-terminus was sufficient to activate PKC leading to phosphorylation of Nef in vitro on a conserved serine residue at position 6. Mutation of serine 6 or coexpression of a transdominant negative PKC mutant significantly reduced Nef-stimulated HIV transcription and replication in resting PBMC. When analyzing the molecular mechanisms, we found that mutating serine 6 moderately affected myristoylation of Nef and its association with Pak2 activity, whereas CD4 downmodulation was not inhibited. More interestingly, this mutation abolished the typical perinuclear localization of Nef in T cells. We conclude that the activation of nPKCs by Nef is required to increase viral replication/infectivity and direct the subcellular localization of Nef.

HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane.

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.