Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells.

Extracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells. Abbreviations: NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin.

Natural Killer-Derived Exosomal miR-186 Inhibits Neuroblastoma Growth and Immune Escape Mechanisms.

In neuroblastoma, the interplay between immune cells of the tumor microenvironment and cancer cells contributes to immune escape mechanisms and drug resistance. In this study, we show that natural killer (NK) cell-derived exosomes carrying the tumor suppressor microRNA (miR)-186 exhibit cytotoxicity against MYCN-amplified neuroblastoma cell lines. The cytotoxic potential of these exosomes was partly dependent upon expression of miR-186. miR-186 was downregulated in high-risk neuroblastoma patients, and its low expression represented a poor prognostic factor that directly correlated with NK activation markers (i.e., NKG2D and DNAM-1). Expression of MYCN, AURKA, TGFBR1, and TGFBR2 was directly inhibited by miR-186. Targeted delivery of miR-186 to MYCN-amplified neuroblastoma or NK cells resulted in inhibition of neuroblastoma tumorigenic potential and prevented the TGFß1-dependent inhibition of NK cells. Altogether, these data support the investigation of a miR-186-containing nanoparticle formulation to prevent tumor growth and TGFß1-dependent immune escape in high-risk neuroblastoma patients as well as the inclusion of ex vivo-derived NK exosomes as a potential therapeutic option alongside NK cell-based immunotherapy.Significance: These findings highlight the therapeutic potential of NK cell-derived exosomes containing the tumor suppressor miR-186 that inhibits growth, spreading, and TGFß-dependent immune escape mechanisms in neuroblastoma.

Biological roles and potential applications of immune cell-derived extracellular vesicles.

Extracellular vesicles (EVs) deliver bioactive macromolecules (i.e. proteins, lipids and nucleic acids) for intercellular communication in multicellular organisms. EVs are secreted by all cell types including immune cells. Immune cell-derived EVs modulate diverse aspects of the immune system to either enhance or suppress immune activities. The extensive effects of immune cell-derived EVs have become the focus of great interest for various nano-biomedical applications, ranging from the medical use of nanoplatform-based diagnostic agents to the development of therapeutic interventions as well as vaccine applications, and thus may be ideal for 'immune-theranostic'. Here, we review the latest advances concerning the biological roles of immune cell-derived EVs in innate and acquired immunity. The intercellular communication amongst immune cells through their EVs is highlighted, showing that all immune cell-derived EVs have their unique function(s) in immunity through intricate interaction(s). Natural-killer (NK) cell-derived EVs, for example, contain potent cytotoxic proteins and induce apoptosis to targeted cancer cells. On the other hand, cancer cell-derived EVs bearing NK ligands may evade immune surveillance and responses. Finally, we discuss possible medical uses for the immune cell-derived EVs as a tool for immune-theranostic: as diagnostic biomarkers, for use in therapeutic interventions and for vaccination.

Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells.

Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission electron microscope; PBMC: peripheral blood mononuclear cells; FBS: foetal bovine serum.

Alpha7 nicotinic acetylcholine receptor is required for amyloid pathology in brain endothelial cells induced by Glycoprotein 120, methamphetamine and nicotine.

One of the most challenging issues in HIV-associated neurocognitive disorders (HAND) caused by HIV-1 virotoxins and drug abuse is the lack of understanding the underlying mechanisms that are commonly associated with disorders of the blood-brain barrier (BBB), which mainly consists of brain microvascular endothelial cells (BMEC). Here, we hypothesized that Glycoprotein 120 (gp120), methamphetamine (METH) and nicotine (NT) can enhance amyloid-beta (Aß) accumulation in BMEC through Alpha7 nicotinic acetylcholine receptor (α7 nAChR). Both in vitro (human BMEC) (HBMEC) and in vivo (mice) models of BBB were used to dissect the role of α7 nAChR in up-regulation of Aß induced by gp120, METH and NT. Aß release from and transport across HBMEC were significantly increased by these factors. Methyllycaconitine (MLA), an antagonist of α7 nAChR, could efficiently block these pathogenic effects. Furthermore, our animal data showed that these factors could significantly increase the levels of Aß, Tau and Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in mouse cerebrospinal fluid (CSF) and Aß in the mouse brains. These pathogenicities were significantly reduced by MLA, suggesting that α7 nAChR may play an important role in neuropathology caused by gp120, METH and NT, which are the major pathogenic factors contributing to the pathogenesis of HAND.

Coexpression Network Analysis of Benign and Malignant Phenotypes of SIV-Infected Sooty Mangabey and Rhesus Macaque.

To explore the differences between the extreme SIV infection phenotypes, nonprogression (BEN: benign) to AIDS in sooty mangabeys (SMs) and progression to AIDS (MAL: malignant) in rhesus macaques (RMs), we performed an integrated dual positive-negative connectivity (DPNC) analysis of gene coexpression networks (GCN) based on publicly available big data sets in the GEO database of NCBI. The microarray-based gene expression data sets were generated, respectively, from the peripheral blood of SMs and RMs at several time points of SIV infection. Significant differences of GCN changes in DPNC values were observed in SIV-infected SMs and RMs. There are three groups of enriched genes or pathways (EGPs) that are associated with three SIV infection phenotypes (BEN+, MAL+ and mixed BEN+/MAL+). The MAL+ phenotype in SIV-infected RMs is specifically associated with eight EGPs, including the protein ubiquitin proteasome system, p53, granzyme A, gramzyme B, polo-like kinase, Glucocorticoid receptor, oxidative phosyphorylation and mitochondrial signaling. Mitochondrial (endosymbiotic) dysfunction is solely present in RMs. Specific BEN+ pattern changes in four EGPs are identified in SIV-infected SMs, including the pathways contributing to interferon signaling, BRCA1/DNA damage response, PKR/INF induction and LGALS8. There are three enriched pathways (PRR-activated IRF signaling, RIG1-like receptor and PRR pathway) contributing to the mixed (BEN+/MAL+) phenotypes of SIV infections in RMs and SMs, suggesting that these pathways play a dual role in the host defense against viral infections. Further analysis of Hub genes in these GCNs revealed that the genes LGALS8 and IL-17RA, which positively regulate the barrier function of the gut mucosa and the immune homeostasis with the gut microbiota (exosymbiosis), were significantly differentially expressed in RMs and SMs. Our data suggest that there exists an exo- (dysbiosis of the gut microbiota) and endo- (mitochondrial dysfunction) symbiotic imbalance (EESI) in HIV/SIV infections. Dissecting the mechanisms of the exo-endo symbiotic balance (EESB) that maintains immune homeostasis and the EESI problems in HIV/SIV infections may lead to a better understanding of the pathogenesis of AIDS and the development of novel interventions for the rational control of this disease.

Pathogenic Triad in Bacterial Meningitis: Pathogen Invasion, NF-κB Activation, and Leukocyte Transmigration that Occur at the Blood-Brain Barrier.

Bacterial meningitis remains the leading cause of disabilities worldwide. This life-threatening disease has a high mortality rate despite the availability of antibiotics and improved critical care. The interactions between bacterial surface components and host defense systems that initiate bacterial meningitis have been studied in molecular and cellular detail over the past several decades. Bacterial meningitis commonly exhibits triad hallmark features (THFs): pathogen penetration, nuclear factor-kappaB (NF-κB) activation in coordination with type 1 interferon (IFN) signaling and leukocyte transmigration that occur at the blood-brain barrier (BBB), which consists mainly of brain microvascular endothelial cells (BMEC). This review outlines the progression of these early inter-correlated events contributing to the central nervous system (CNS) inflammation and injury during the pathogenesis of bacterial meningitis. A better understanding of these issues is not only imperative to elucidating the pathogenic mechanism of bacterial meningitis, but may also provide the in-depth insight into the development of novel therapeutic interventions against this disease.

The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence.

Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus.

How Cryptococcus interacts with the blood-brain barrier.

Cryptococcus demonstrates predilection for invasion of the brain, but the mechanism by which Cryptococcus crosses the blood-brain barrier (BBB) to cause brain invasion is largely unknown. In order for Cryptococcus to cross the BBB, there must be a way to either cross human brain microvascular endothelial cells, which are the main constitute of the BBB, or go in between tight junctions. Recent evidence of human brain microvascular endothelial cell responses to transcellular brain invasions includes membrane rearrangements, intracellular signaling pathways and cytoskeletal activations. Several Cryptococcal genes related to the traversal of BBB have been identified, including CPS1, ITR1a, ITR3c, PLB1, MPR1, FNX1 and RUB1. In addition, Cryptococcus neoformans-derived microvesicles may contribute to cryptococcal brain invasion. Paracellularly, Cryptococcus may traverse across BBB using either routes utilizing plasmin, ammonia or macrophages in a Trojan horse mechanism.

Alpha7 nicotinic acetylcholine receptor is required for blood-brain barrier injury-related CNS disorders caused by Cryptococcus neoformans and HIV-1 associated comorbidity factors.

BACKGROUND: Cryptococcal meningitis is the most common fungal infection of the central nervous system (CNS) in HIV/AIDS. HIV-1 virotoxins (e.g., gp41) are able to induce disorders of the blood-brain barrier (BBB), which mainly consists of BMEC. Our recent study suggests that α7 nAChR is an essential regulator of inflammation, which contributes to regulation of NF-κB signaling, neuroinflammation and BBB disorders caused by microbial (e.g., HIV-1 gp120) and non-microbial [e.g., methamphetamine (METH)] factors. However, the underlying mechanisms for multiple comorbidities are unclear. METHODS: In this report, an aggravating role of α7 nAChR in host defense against CNS disorders caused by these comorbidities was demonstrated by chemical [inhibitor: methyllycaconitine (MLA)] and genetic (α7(-/-) mice) blockages of α7 nAChR. RESULTS: As shown in our in vivo studies, BBB injury was significantly reduced in α7(-/-) mice infected with C. neoformans. Stimulation by the gp41 ectodomain peptide (gp41-I90) and METH was abolished in the α7(-/-) animals. C. neoformans and gp41-I90 could activate NF-κB. Gp41-I90- and METH-induced monocyte transmigration and senescence were significantly inhibited by MLA and CAPE (caffeic acid phenethyl ester, an NF-κB inhibitor). CONCLUSIONS: Collectively, our data suggest that α7 nAChR plays a detrimental role in the host defense against C. neoformans- and HIV-1 associated comorbidity factors-induced BBB injury and CNS disorders.

Exosome-mediated transfer of microRNAs within the tumor microenvironment and neuroblastoma resistance to chemotherapy.

BACKGROUND: How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL). METHODS: Coculture experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR-155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Student's t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. RESULTS: miR-21 mean fold change (f.c.) was 12.08±0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51±0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P <.001). Mean tumor volumes in Dotap-miR-155 compared with Dotap-scrambled were 322.80±120mm(3) and 76.00±39.3mm(3), P = .002 at day 24, respectively. Patients with high CD163 infiltrating NBLs had statistically significantly higher intratumoral levels of miR-155 (P = .04) and lower levels of TERF1 mRNA (P = .02). CONCLUSIONS: These data indicate a unique role of exosomic miR-21 and miR-155 in the cross-talk between NBL cells and human monocytes in the resistance to chemotherapy, through a novel exosomic miR-21/TLR8-NF-кB/exosomic miR-155/TERF1 signaling pathway.

Circulating brain microvascular endothelial cells (cBMECs) as potential biomarkers of the blood-brain barrier disorders caused by microbial and non-microbial factors.

Despite aggressive research, central nervous system (CNS) disorders, including blood-brain barrier (BBB) injury caused by microbial infection, stroke, abused drugs [e.g., methamphetamine (METH) and nicotine], and other pathogenic insults, remain the world's leading cause of disabilities. In our previous work, we found that dysfunction of brain microvascular endothelial cells (BMECs), which are a major component of the BBB, could be caused by nicotine, meningitic pathogens and microbial factors, including HIV-1 virulence factors gp41 and gp120. One of the most challenging issues in this area is that there are no available cell-based biomarkers in peripheral blood for BBB disorders caused by microbial and non-microbial insults. To identify such cellular biomarkers for BBB injuries, our studies have shown that mice treated with nicotine, METH and gp120 resulted in increased blood levels of CD146+(endothelial marker)/S100B+ (brain marker) circulating BMECs (cBMECs) and CD133+[progenitor cell (PC) marker]/CD146+ endothelial PCs (EPCs), along with enhanced Evans blue and albumin extravasation into the brain. Nicotine and gp120 were able to significantly increase the serum levels of ubiquitin C-terminal hydrolase 1 (UCHL1) (a new BBB marker) as well as S100B in mice, which are correlated with the changes in cBMECs and EPCs. Nicotine- and meningitic E. coli K1-induced enhancement of cBMEC levels, leukocyte migration across the BBB and albumin extravasation into the brain were significantly reduced in alpha7 nAChR knockout mice, suggesting that this inflammatory regulator plays an important role in CNS inflammation and BBB disorders caused by microbial and non-microbial factors. These results demonstrated that cBMECs as well as EPCs may be used as potential cell-based biomarkers for indexing of BBB injury.

Factors required for activation of urease as a virulence determinant in Cryptococcus neoformans.

UNLABELLED: Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni(2+) into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria. IMPORTANCE: Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans.

Cryptococcus neoformans-derived microvesicles enhance the pathogenesis of fungal brain infection.

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.

Identification of genes from the fungal pathogen Cryptococcus neoformans related to transmigration into the central nervous system.

BACKGROUND: A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis. METHODOLOGY/PRINCIPAL FINDINGS: Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the "Trojan horse" model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA. CONCLUSIONS/SIGNIFICANCE: This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry.

Vimentin and PSF act in concert to regulate IbeA+ E. coli K1 induced activation and nuclear translocation of NF-κB in human brain endothelial cells.

BACKGROUND: IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities. METHODOLOGY/PRINCIPAL FINDINGS: IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/ß phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/ß phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus. CONCLUSION/SIGNIFICANCE: These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis.

The autophagy regulator Rubicon is a feedback inhibitor of CARD9-mediated host innate immunity.

Assembly of a scaffold consisting of CARD9, BCL10, and MALT1 (CBM complex) is critical for effective signaling by multiple pattern recognition receptors (PRRs) including Dectin and RIG-I. The RUN domain Beclin-1-interacting cysteine-rich-containing Rubicon protein associates constitutively with the Beclin-UVRAG-Vps34 complex under normal conditions to regulate autophagy. Rubicon also interacts with the phagocytic NADPH-oxidase complex upon TLR stimulation to induce potent antimicrobial responses. Here, we show Rubicon is a physiological feedback inhibitor of CBM-mediated PRR signaling, preventing unbalanced proinflammatory responses. Upon Dectin-1- or RIG-I-mediated activation, Rubicon dynamically exchanges binding partners from 14-3-3ß to CARD9 in a stimulation-specific and phosphorylation-dependent manner, disassembling the CBM signaling complex and ultimately terminating PRR-induced cytokine production. Remarkably, Rubicon's actions in the autophagy complex, phagocytosis complex, and CBM complex are functionally and genetically separable. Rubicon thus differentially targets signaling complexes, depending on environmental stimuli, and may function to coordinate various immune responses against microbial infection.

Hyaluronic acid receptor CD44 deficiency is associated with decreased Cryptococcus neoformans brain infection.

Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44(-/-)) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain.

Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells.

BACKGROUND: Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown. METHODS: In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated. RESULTS: We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network. CONCLUSION: These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.

Retinoid-regulated FGF8f secretion by osteoblasts bypasses retinoid stimuli to mediate granulocytic differentiation of myeloid leukemia cells.

Signaling from the human hematopoietic stem cell (HSC) niche formed by osteoblastic cells regulates hematopoiesis. We previously found that retinoic acid receptor alpha (RARα), a transcription factor activated by retinoic acid (RA), mediates both granulocytic and osteoblastic differentiation. This effect depends on decreased phosphorylation of serine 77 of RARα (RARαS77) by the cyclin-dependent kinase-activating kinase (CAK) complex, a key cell-cycle regulator. In this article, we report that, by suppressing CAK phosphorylation of RARα, RA induces FGF8f to mediate osteosarcoma U2OS cell differentiation in an autocrine manner. By contrast, paracrine FGF8f secreted into osteoblast-conditioned medium by U2OS cells transduced with FGF8f or a phosphorylation-defective RARαS77 mutant, RARαS77A, bypasses RA stimuli to cross-mediate granulocytic differentiation of different types of human leukemic myeloblasts and normal primitive hematopoietic CD34(+) cells, possibly through modulating mitogen-activated protein kinase (MAPK) pathways. Further experiments using recombinant human FGF8f (rFGF8f) stimuli, antibody neutralization, and peptide blocking showed that paracrine FGF8f is required for mediating terminal leukemic myeloblast differentiation. These studies indicate a novel regulatory mechanism of granulocytic differentiation instigated by RA from the HSC niche, which links loss of CAK phosphorylation of RARα with paracrine FGF8f-mediated MAPK signaling to mediate leukemic myeloblast differentiation in the absence of RA. Therefore, these findings provide a compelling molecular rationale for further investigation of paracrine FGF8f regulation, with the intent of devising HSC niche-based FGF8f therapeutics for myeloid leukemia, with or without RA-resistance.