[Abstract unavailable

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1β is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1β and HMGB1 respond at different times to different stimuli: IL-1β secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.

Interleukin 1␤ (IL-1␤), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a "leaderless" secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1␤ out of the cell. Indeed, although most of the IL-1␤ precursor (proIL-1␤) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1␤ and the endolysosomal hydrolase cathepsin D or for both IL-1␤ and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1␤ is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1␤ secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1␤ from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1␤ but deplete the vesicular proIL-1␤ content, indicating that exocytosis of proIL-1␤-containing vesicles is regulated by ATP and osmotic conditions. INTRODUCTIONInterleukin 1 (IL-1) 1 is a multifunctional cytokine and a major soluble mediator of inflammation (Dinarello, 1991). Although its biological activity is extracellular, this protein lacks a secretory signal sequence (Rubartelli and Sitia, 1997), raising the question of how it can be transported out of the cell. Two forms of IL-1 exist, ␣ and ␤; however, studies on IL-1 secretion mostly focused on IL-1␤, which is the major extracellular form in humans (Dinarello, 1991). IL-1␤ is synthesized by monocytes upon activation as a 35-kDa precursor, which accumulates in the cytosol (Singer et al., 1988) and is proteolytically processed to the mature form of 17 kDa by the caspase IL-1␤-converting enzyme (ICE) (Cerretti et al., 1992;Thornberry et al., 1992). ICE is present in the cell cytosol (Ayala et al., 1994;Singer et al., 1995) as a p45 inactive polypeptide. Where and how maturation of ICE to the active (p10/p20) form and the processing of IL-1␤ take place are unclear; however, the mature form of IL-1␤ seems to be either ¶ Corresponding author. E-mail address: annarub@hp380.ist. et al., 1990). Although the levels of basal release of IL-1␤ are quite low (Rubartelli et al., 1990), secretion is dramatically induced by extracellular ATP (Hogquist et al., 1991;Rubartelli et al., 1993;Perregaux and Gabel, 1994), which can be autocrinally produced by activated monocytes (Ferrari et al., 1997) and interact with P2Z purinoreceptors expressed on their membrane (Hickman et al., 1994;Di Virgilio, 1995). The cellular pathway underlying this ATP-driven secretion is, however, unknown. We have previously shown that IL-1␤ is ...

Blocking the activity of IL-1␤ has entered the clinical arena of treating autoimmune diseases. However, a successful outcome of this approach requires a clear definition of the mechanisms controlling IL-1␤ release. These are still unclear as IL-1␤, lacking a secretory signal peptide, follows a nonclassical pathway of secretion. Here, we analyze the molecular mechanism(s) undergoing IL-1␤ processing and release in human monocytes and provide a unifying model for the regulated secretion of the cytokine. Our data show that in a first step, pro-caspase-1 and endotoxininduced pro-IL-1␤ are targeted in part to specialized secretory lysosomes, where they colocalize with other lysosomal proteins.

It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1alpha (HIF-1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein-Barr Virus (EBV), Latent Membrane Protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF-1α is detectable in exosomes, and that LMP1 significantly increases levels of HIF-1α in exosomes. HIF-1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We show also that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with Epithelial Mesenchymal Transition (EMT). In addition we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, since exosome-mediated delivery of active and inactive forms of HIF-1α results in reciprocal changes in expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

The presentation of exogenous protein antigens in a major histocompatibility complex class I–restricted fashion to CD8+ T cells is called cross-presentation. We demonstrate that cross-presentation of soluble viral antigens (derived from hepatitis C virus [HCV], hepatitis B virus [HBV], or human immunodeficiency virus) to specific CD8+ T cell clones is dramatically improved when antigen-presenting dendritic cells (DCs) are pulsed with the antigen in the presence of chloroquine or ammonium chloride, which reduce acidification of the endocytic system. The export of soluble antigen into the cytosol is considerably higher in chloroquine-treated than in untreated DCs, as detected by confocal microscopy of cultured cells and Western blot analysis comparing endocytic and cytosolic fractions. To pursue our findings in an in vivo setting, we boosted groups of HBV vaccine responder individuals with a further dose of hepatitis B envelope protein vaccine with or without a single dose of chloroquine. Although all individuals showed a boost in antibody titers to HBV, six of nine individuals who were administered chloroquine showed a substantial CD8+ T cell response to HBV antigen, whereas zero of eight without chloroquine lacked a CD8 response. Our results suggest that chloroquine treatment improves CD8 immunity during vaccination.

For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.

Key Points• FAO is a crucial metabolic pathway for leukemic cell proliferation and apoptosis.• FAO inhibitors represent a novel targeted approach for leukemia treatment.Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose-and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes. (Blood. 2015;126(16):1925-1929