Judith M. Silverman

An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37°C ± pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-α. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Significance Amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease, is associated with mutation and misfolding of the Cu/Zn superoxide dismutase (SOD1) protein. Prior studies found that mutant misfolded SOD1 can convert wild-type (WT) SOD1 to a misfolded form inside living cells in a prion-like fashion. We now report that misfolded WT SOD1 can be transmitted from cell to cell, and that propagated protein misfolding can be perpetuated. Misfolded SOD1 transmission between cells can be mediated through release and uptake of protein aggregates or via small membrane-bounded transport vesicles called exosomes. These mechanisms may help explain why sporadic ALS, without a known genetic cause, can spread systematically from region to region in a progressive manner. Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

Leishmania Exosomes Modulate Innate and Adaptive Immune Responses through Effects on Monocytes and Dendritic Cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ–producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100−/− L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100−/− leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100−/− exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

Exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes.

The release of exosomes and other microvesicles by diverse prokaryotic and eukaryotic cells and organisms was first appreciated early in the 20th century. The functional properties of these organelles, however, have only recently been the focus of rigorous investigation. In this review, we discuss the release of microvesicles of varying complexity by diverse microbial pathogens. This includes vesicle secretion by Gram‐negative bacteria, eukaryotic parasites of the kinetoplast lineage and opportunistic fungal pathogens of both the ascomycetes and basidiomycetes lineages. We also discuss vesicle release from mammalian cells brought about as a result of infection with bacteria, viruses and prions. In addition, we review the evidence showing that in their specific microenvironments, release of these organelles from diverse pathogens contributes to pathogenesis. Germane to this and based upon recent findings with Leishmania, we propose a model whereby exosome release by an intracellular pathogen serves as a general mechanism for effector molecule delivery from eukaryotic pathogen to host cell cytosol. These new findings linking exosomes and other microvesicles to infection biology have important implications for understanding the immune response to infection and for the design of research strategies aimed at the development of novel therapeutics and vaccines.

Leishmania Exosomes Deliver Preemptive Strikes to Create an Environment Permissive for Early Infection

Herein, we review evidence supporting a role for Leishmania exosomes during early infection. We suggest a model in which Leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for Leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Leishmania exosomes influence the phenotype of treated immune cells. For example, wild-type (WT) exosomes attenuate interferon-γ-induced pro-inflammatory cytokine production (TNF-α) by Leishmania-infected monocytes while conversely enhancing production of the anti-inflammatory cytokine IL-10. The Leishmania proteins GP63 and elongation factor-1α (EF-1α) are found in secreted vesicles and are likely important effectors responsible for these changes in phenotype. GP63 and EF-1α access host cell cytosol and activate multiple host protein-tyrosine phosphatases (PTPs). Activation of these PTPs negatively regulates interferon-γ signaling and this prevents effective expression of the macrophage microbicidal arsenal, including TNF-α and nitric oxide. In addition to changing macrophage phenotype, WT vesicles dampen the immune response of monocyte-derived dendritic cells and CD4+ T lymphocytes. This capacity is lost when the protein cargo of the vesicles is modified, specifically when the amount of GP63 and EF-1α in the vesicles is reduced. It appears that exosome delivery of effector proteins results in activation of host PTPs and the negative regulatory effects of the latter creates a pro-parasitic environment. The data suggest that Leishmania exosomes secreted upon initial infection are capable of delivering effector cargo to naïve target cells wherein the cargo primes host cells for infection by interfering with host cell signaling pathways.

Myeloid Cell IL-10 Production in Response to Leishmania Involves Inactivation of Glycogen Synthase Kinase-3β Downstream of Phosphatidylinositol-3 Kinase

Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3β is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3β, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3β markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3β negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3β promotes disease pathogenesis.

Secreted virulence factors and immune evasion in visceral leishmaniasis

Evasion or subversion of host immune responses is a well‐established paradigm in infection with visceralizing leishmania. In this review, we summarize current findings supporting a model in which leishmania target host regulatory molecules and pathways, such as the PTP SHP‐1 and the PI3K/Akt signaling cascade, to prevent effective macrophage activation. Furthermore, we describe how virulence factors, secreted by leishmania, interfere with macrophage intracellular signaling. Finally, we discuss mechanisms of secretion and provide evidence that leishmania use a remarkably adept, exosome‐based secretion mechanism to export and deliver effector molecules to host cells. In addition to representing a novel mechanism for trafficking of virulence factors across membranes, recent findings indicate that leishmania exosomes may have potential as vaccine candidates.

Exosome-dependent and independent mechanisms are involved in prion-like transmission of propagated Cu/Zn superoxide dismutase misfolding

Amyotrophic lateral sclerosis (ALS), a fatal adult-onset degenerative neuromuscular disorder with a poorly defined etiology, progresses in an orderly spatiotemporal manner from one or more foci within the nervous system, reminiscent of prion disease pathology. We have previously shown that misfolded mutant Cu/Zn superoxide dismutase (SOD1), mutation of which is associated with a subset of ALS cases, can induce endogenous wild-type SOD1 misfolding in the intracellular environment in a templating fashion similar to that of misfolded prion protein. Our recent observations further extend the prion paradigm of pathological SOD1 to help explain the intercellular transmission of disease along the neuroaxis. It has been shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or released to the extracellular environment on the surface of exosomes secreted from living cells. Furthermore, once propagation of misfolded wild-type SOD1 has been initiated in human cell culture, it continues over multiple passages of transfer and cell growth. Propagation and transmission of misfolded wild-type SOD1 is therefore a potential mechanism in the systematic progression of ALS pathology.

HUMANIZED PMN310 SHOWS ENHANCED THERAPEUTIC POTENTIAL BY BINDING TOXIC LOW MOLECULAR WEIGHT Aβ OLIGOMERS WHILE AVOIDING ARIA-RELATED BINDING TO Aβ DEPOSITS IN AD PATIENT BRAINS

P2-048 HUMANIZED PMN310 SHOWS ENHANCED THERAPEUTIC POTENTIAL BY BINDING TOXIC LOW MOLECULARWEIGHTAb OLIGOMERS WHILE AVOIDING ARIARELATED BINDING TO Ab DEPOSITS IN AD PATIENT BRAINS Johanne Kaplan, Ebrima Gibbs, Judith M. Silverman, Jing Wang, Xubiao Peng, Steven S. Plotkin, Neil R. Cashman, ProMIS Neurosciences, Toronto, ON, Canada; University of British Columbia, Vancouver, BC, Canada. Contact e-mail: johanne.kaplan@ promisneurosciences.com