Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported ...that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.
Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH ...domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.
Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting ...activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.
Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans ...and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that β-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that β-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, β-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble β-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating β-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.
Natural killer (NK) cells are a subset of immune effectors that directly bind and kill fungi via a perforin-dependent mechanism. The receptor mediating this activity and its potential role in disease ...remain unknown. Using an unbiased approach, we determined that NKp30 is responsible for recognition and killing of the fungal pathogens Cryptococcus and Candida. NKp30 was required for NK cell-fungal conjugate formation, phosphatidylinositol 3-kinase (PI3K) signaling, and perforin release. Because fungal infections are a leading cause of death in AIDS patients, we examined NKp30 expression in HIV-infected patients. NK cells from these patients had diminished NKp30 expression, defective perforin release, and blunted microbicidal activity. Surprisingly, interleukin-12 (IL-12) restored NKp30 expression and fungal killing. Thus, the NKp30 receptor plays a critical role in NK cell antifungal cytotoxicity, and diminished expression of NKp30 is responsible for defective antifungal activity of NK cells from HIV-infected patients, which can be corrected with IL-12.
•An unbiased approach identifies NKp30 as a NK cell fungicidal receptor•NKp30 mediates direct binding, signaling, and perforin release in response to fungi•NK cells from HIV patients had diminished NKp30 expression and blunted fungal killing•IL-12 restored NKp30 expression and fungal killing in NK cells from HIV patients
Neutrophils encounter and 'prioritize' many chemoattractants in their pursuit of bacteria. Here we tested the possibility that the phosphatase PTEN is responsible for the prioritization of ...chemoattractants. Neutrophils induced chemotaxis by two separate pathways, the phosphatidylinositol-3-OH kinase (PI(3)K) phosphatase and tensin homolog (PTEN) pathway, and the p38 mitogen-activated protein kinase pathway, with the p38 pathway dominating over the PI(3)K pathway. Pten(-/-) neutrophils could not prioritize chemoattractants and were 'distracted' by chemokines when moving toward bacterial chemoattractants. In opposing gradients, PTEN became distributed throughout the cell circumference, which inhibited all PI(3)K activity, thus permitting 'preferential' migration toward bacterial products via phospholipase A(2) and p38. Such prioritization was defective in Pten(-/-) neutrophils, which resulted in defective bacterial clearance in vivo. Our data identify a PTEN-dependent mechanism in neutrophils to prioritize, 'triage' and integrate responses to multiple chemotactic cues.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The fidelity of synaptic transmission depends on the integrity of the protein machinery at the synapse. Unfolded synaptic proteins undergo refolding or degradation in order to maintain synaptic ...proteostasis and preserve synaptic function, and buildup of unfolded/toxic proteins leads to neuronal dysfunction. Many molecular chaperones contribute to proteostasis, but one in particular, cysteine string protein (CSPα), is critical for proteostasis at the synapse. In this study we report that exported vesicles from neurons contain CSPα. Extracellular vesicles (EV's) have been implicated in a wide range of functions. However, the functional significance of neural EV's remains to be established. Here we demonstrate that co-expression of CSPα with the disease-associated proteins, polyglutamine expanded protein 72Q huntingtin
°
or superoxide dismutase-1 (SOD-1
leads to the cellular export of both 72Q huntingtin
°
and SOD-1
via EV's. In contrast, the inactive CSPα
mutant does not facilitate elimination of misfolded proteins. Furthermore, CSPα-mediated export of 72Q huntingtin
°
is reduced by the polyphenol, resveratrol. Our results indicate that by assisting local lysosome/proteasome processes, CSPα-mediated removal of toxic proteins via EVs plays a central role in synaptic proteostasis and CSPα thus represents a potential therapeutic target for neurodegenerative diseases.
Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic ...expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.
Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in ...investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.
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