Dynamic bidirectional regulation of NLRC3 and gammaherpesviruses during viral latency in B lymphocytes.

While most NOD-like receptors (NLRs) are predominately expressed by innate immune cells, NLRC3, an inhibitory NLR of immune signaling, exhibits the highest expression in lymphocytes. The role of NLRC3 or any NLRs in B lymphocytes is completely unknown. Gammaherpesviruses, including human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68), establish latent infection in B lymphocytes, which requires elevated NF-κB. This study shows that during latent EBV infection of human B cells, viral-encoded latent membrane protein 1 (LMP1) decreases NLRC3 transcript. LMP1-induced-NF-κB activation suppresses the promoter activity of NLRC3 via p65 binding to the promoter. Conversely, NLRC3 inhibits NF-κB activation by promoting the degradation of LMP1 in a proteasome-dependent manner. In vivo, MHV-68 infection reduces Nlrc3 transcripts in splenocytes, and Nlrc3-deficient mice show greater viral latency than controls. These results reveal a bidirectional regulatory circuit in B lymphocytes, where viral latent protein LMP1 reduces NLRC3 expression, while NLRC3 disrupts gammaherpesvirus latency, which is an important step for tumorigenesis.

Reconstitution of Injectable Poly-d,l-lactic Acid: Efficacy of Different Diluents and a New Accelerating Method.

Injectable poly-d,l-lactic acid (PDLLA) is a new collagen-stimulating filler containing PDLLA microspheres and carboxymethyl cellulose. It is available as a lyophilized powder that must be reconstituted with a diluent before administration. The aims of this study were to investigate the efficacy of different diluents and a new accelerating "back-and-forth" method. METHODS: Six different diluents, sodium bicarbonate, sterile water for injection (SWFI), normal saline, lidocaine, lidocaine with epinephrine (lidocaine + E), and mannitol, were tested. The recommended "vortex" method for preparation of thin suspensions and a new back-and-forth method suitable for both thin and thick suspensions were compared. Gross and microscopic views of the prepared suspensions were examined. RESULTS: Using the vortex method, only mannitol and SWFI are found to be effective reconstitution diluents for injectable PDLLA. Using the back-and-forth method, all six diluents can be used for reconstitution of injectable PDLLA. Moreover, the time needed for reconstitution of injectable PDLLA by this back-and-forth method is very short, regardless of the thickness of the suspension. CONCLUSIONS: Clinically, only SWFI can be used for reconstitution of injectable PDLLA by "hand-shaking" or vortex method. To accelerate the reconstitution time especially when using small amount of SWFI, back-and-forth is the method of choice. Besides, when SWFI is not available, other diluents such as normal saline, lidocaine, or lidocaine + E can be used by this novel back-and-forth reconstitution method.

Vaccine adjuvant activity of a TLR4-activating synthetic glycolipid by promoting autophagy.

Toll-like receptors (TLRs) play crucial roles in host immune defenses. Recently, TLR-mediated autophagy is reported to promote immune responses via increasing antigen processing and presentation in antigen presenting cells. The present study examined whether the synthetic TLR4 activator (CCL-34) could induce autophagy to promote innate and adaptive immunity. In addition, the potential of CCL-34 as an immune adjuvant in vivo was also investigated. Our data using RAW264.7 cells and bone marrow-derived macrophages showed that CCL-34 induced autophagy through a TLR4-NF-κB pathway. The autophagy-related molecules (Nrf2, p62 and Beclin 1) were activated in RAW264.7 cells and bone marrow-derived macrophages under CCL-34 treatment. CCL-34-stimulated macrophages exhibited significant antigen-processing activity and induced the proliferation of antigen-specific CD4+T cells as well as the production of activated T cell-related cytokines, IL-2 and IFN-γ. Furthermore, CCL-34 immunization in mice induced infiltration of monocytes in the peritoneal cavity and elevation of antigen-specific IgG in the serum. CCL-34 treatment in vivo did not cause toxicity based on serum biochemical profiles. Notably, the antigen-specific responses induced by CCL-34 were attenuated by the autophagy inhibitor, 3-methyladenine. In summary, we demonstrated CCL-34 can induce autophagy to promote antigen-specific immune responses and act as an efficient adjuvant.

HIV-1 Tat Interacts with a Kaposi's Sarcoma-Associated Herpesvirus Reactivation-Upregulated Antiangiogenic Long Noncoding RNA, LINC00313, and Antagonizes Its Function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining cancer with abnormal angiogenesis. The high incidence of KS in human immunodeficiency virus (HIV)-infected AIDS patients has been ascribed to an interaction between HIV type 1 (HIV-1) and KSHV, focusing on secretory proteins. The HIV-1 secreted protein HIV Tat has been found to synergize with KSHV lytic proteins to induce angiogenesis. However, the impact and underlying mechanisms of HIV Tat in KSHV-infected endothelial cells undergoing viral lytic reactivation remain unclear. Here, we identified LINC00313 as a novel KSHV reactivation-activated long noncoding RNA (lncRNA) that interacts with HIV Tat. We found that LINC00313 overexpression inhibits cell migration, invasion, and tube formation, and this suppressive effect was relieved by HIV Tat. In addition, LINC00313 bound to polycomb repressive complex 2 (PRC2) complex components, and this interaction was disrupted by HIV Tat, suggesting that LINC00313 may mediate transcription repression through recruitment of PRC2 and that HIV Tat alleviates repression through disruption of this association. This notion was further supported by bioinformatics analysis of transcriptome profiles in LINC00313 overexpression combined with HIV Tat treatment. Ingenuity Pathway Analysis (IPA) showed that LINC00313 overexpression negatively regulates cell movement and migration pathways, and enrichment of these pathways was absent in the presence of HIV Tat. Collectively, our results illustrate that an angiogenic repressive lncRNA, LINC00313, which is upregulated during KSHV reactivation, interacts with HIV Tat to promote endothelial cell motility. These results demonstrate that an lncRNA serves as a novel connector in HIV-KSHV interactions.IMPORTANCE KS is a prevalent tumor associated with infections with two distinct viruses, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the virus-virus interaction associated with KS formation has focused on secretory factors. HIV Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migration-regulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection.

NLRP12 Regulates Anti-viral RIG-I Activation via Interaction with TRIM25.

Establishing the balance between positive and negative innate immune mechanisms is crucial for maintaining homeostasis. Here we uncover the regulatory crosstalk between two previously unlinked innate immune receptor families: RIG-I, an anti-viral cytosolic receptor activated type I interferon production, and NLR (nucleotide-binding domain, leucine repeat domain-containing protein). We show that NLRP12 dampens RIG-I-mediated immune signaling against RNA viruses by controlling RIG-I's association with its adaptor MAVS. The nucleotide-binding domain of NLRP12 interacts with the ubiquitin ligase TRIM25 to prevent TRIM25-mediated, Lys63-linked ubiquitination and activation of RIG-I. NLRP12 also enhances RNF125-mediated, Lys48-linked degradative ubiquitination of RIG-I. Vesicular stomatitis virus (VSV) infection downregulates NLRP12 expression to allow RIG-I activation. Myeloid-cell-specific Nlrp12-deficient mice display a heightened interferon and TNF response and are more resistant to VSV infection. These results indicate that NLRP12 functions as a checkpoint for anti-viral RIG-I activation.

CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo.

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE: Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.

Central Nervous System Melioidosis Mimics Malignancy: A Case Report and Literature Review.

BACKGROUND: Central nervous system (CNS) melioidosis is notorious because of the difficulty in bacteria eradication and the destruction of brain structures. Early manifestation of CNS melioidosis mimics malignancy or stroke. We present a case of CNS melioidosis that initially manifested as malignancy. CASE DESCRIPTION: A 30-year-old man presented with sudden onset of left limb weakness and seizure. Computed tomography of the brain showed a low-density lesion over the right parietal lobe, and magnetic resonance imaging showed a well-enhanced lobulated lesion. Neuronavigation-guided open surgery was performed but failed to find a malignancy. The patient presented 3 days later with sudden loss of consciousness, pupil dilation, and high fever. Emergent craniectomy was performed for severe right hemisphere swelling with midline shift. After craniectomy, pus was found in the previous operative field. Burkholderia pseudomallei was cultured from pus and blood samples 1 week after collection. The brain lesion developed into an organized abscess and led to mass effect and ventriculitis. Extraventricular drainage and débridement was performed repeatedly accompanied by systemic and intraventricular antibiotic administration. After 4 months of treatment, the patient achieved a complete consciousness recover while left hemiparesis. CONCLUSIONS: CNS melioidosis requires accurate pathogen identification and appropriate long-term antibiotic treatment for eradication of bacteria and prevention of relapse. Débridement and adequate drainage provide better infection control and outcome.

DcR3 suppresses influenza virus-induced macrophage activation and attenuates pulmonary inflammation and lethality.

UNLABELLED: Influenza A virus (IAV) infects macrophages and stimulates innate immunity receptors and sensors to produce proinflammatory cytokines and chemokines, which are responsible for IAV-induced pulmonary inflammation and injury. Decoy receptor 3 (DcR3) is a soluble protein belonging to the tumor necrosis factor receptor superfamily (TNFRSF), and is able to skew macrophage differentiation into an M2 phenotype. We demonstrated that DcR3 attenuated IAV-induced secretion of proinflammatory cytokines and chemokine from macrophages, and mitigated pulmonary infiltration and reduce lethality. Proteome-wide phosphoproteomic mapping revealed that DcR3 not only activated STK10, a negative regulator of cell migration, but also inactivated PKC-α, which are crucial for the activation of ERK and JNK in human macrophages. Furthermore, less pulmonary infiltration with lower levels of proinflammatory cytokines and chemokine in bronchoalveolar lavage fluid (BALF) were observed in DcR3-transgenic mice. Moreover, recombinant DcR3.Fc and heparan sulfate proteoglycan binding domain of DcR3.Fc (HBD.Fc) fusion proteins attenuated weight loss and protected mice from IAV-induced lethality. Thus, DcR3-mediated protection is not only via suppression of proinflammatory cytokine and chemokine release, but also via activation of STK10 to inhibit cell infiltration. DcR3 fusion proteins may become therapeutic agents to protect host from IAV-induced lethality in the future. KEY MESSAGE: • DcR3 suppresses IAV-induced cytokine secretion.• DcR3 inhibits IAV-induced JNK and ERK activation in human macrophages.• DcR3 downregulates TLR3 and 7 expressions in human macrophages.• DcR3 protects mice from IAV-induced lethality.

Distinct regulation of dengue virus-induced inflammasome activation in human macrophage subsets.

Macrophages (Mϕ) are the major source of inflammatory cytokines and are target cells for dengue virus (DV) replication. However, Mϕ are heterogeneous and their phenotypic and functional diversities are influenced by cytokines that regulate their differentiation, tissue distribution, and defense against invading pathogens. In vitro, human primary macrophages are derived from peripheral blood CD14+ monocytes in the presence of macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF). These are essential for developing tissue/resting macrophages (M-Mϕ) and inflammatory macrophages (GM-Mϕ), respectively. While IFN production is similar between M-Mϕ and GM-Mϕ, M-Mϕ cannot produce IL-1ß after DV infection. In contrast, GM-Mϕ is more susceptible to DV infection and DV triggers CLEC5A in GM-Mϕ to activate NLRP3 inflammasomes, which in turn release IL-18 and IL-1ß that are critical for Th17 activation and contribute to disease severity. Thus, GM-Mϕ is more representative than M-Mϕ for investigating inflammasome activation in dengue infection, and is invaluable for revealing the molecular mechanism of pathogen-induced inflammatory reaction. Distinct phenotypes of macrophage subsets under the influence of M-CSF and GM-CSF raise the question of optimal conditions for culturing primary macrophages to study host-pathogen interaction.

CLEC5A is critical for dengue virus-induced inflammasome activation in human macrophages.

Persistent high fever is one of the most typical clinical symptoms in dengue virus (DV)-infected patients. However, the source of endogenous pyrogen (eg, IL-1ß) and the signaling cascade leading to the activation of inflammasome and caspase-1, which are essential for IL-1ß and IL-18 secretion, during dengue infection have not been elucidated yet. Macrophages can be polarized into distinct phenotypes under the influence of GM-CSF or M-CSF, denoted as GM-Mϕ and M-Mϕ, respectively. We found that DV induced high levels of IL-1ß and IL-18 from GM-Mϕ (inflammatory macrophage) and caused cell death (pyroptosis), whereas M-Mϕ (resting macrophage) did not produce IL-1ß and IL-18 on DV infection even with lipopolysaccharide priming. This observation demonstrates the distinct responses of GM-Mϕ and M-Mϕ to DV infection. Moreover, up-regulation of pro-IL-1ß, pro-IL-18, and NLRP3 associated with caspase-1 activation was observed in DV-infected GM-Mϕ, whereas blockade of CLEC5A/MDL-1, a C-type lectin critical for dengue hemorrhagic fever and Japanese encephalitis virus infection, inhibits NLRP3 inflammasome activation and pyrotopsis in GM-Mϕ. Thus, DV can activate NLRP3 inflammasome via CLEC5A, and GM-Mϕ plays a more important role than M-Mϕ in the pathogenesis of DV infection.

Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell.

Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE-based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys-regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF-κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism-based chemopreventive strategy for prostate cancer.

CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality.

CLEC5A/MDL-1, a member of the myeloid C-type lectin family expressed on macrophages and neutrophils, is critical for dengue virus (DV)-induced hemorrhagic fever and shock syndrome in Stat1⁻/⁻ mice and ConA-treated wild type mice. However, whether CLEC5A is involved in the pathogenesis of viral encephalitis has not yet been investigated. To investigate the role of CLEC5A to regulate JEV-induced neuroinflammation, antagonistic anti-CLEC5A mAb and CLEC5A-deficient mice were generated. We find that Japanese encephalitis virus (JEV) directly interacts with CLEC5A and induces DAP12 phosphorylation in macrophages. In addition, JEV activates macrophages to secrete proinflammatory cytokines and chemokines, which are dramatically reduced in JEV-infected Clec5a⁻/⁻ macrophages. Although blockade of CLEC5A cannot inhibit JEV infection of neurons and astrocytes, anti-CLEC5A mAb inhibits JEV-induced proinflammatory cytokine release from microglia and prevents bystander damage to neuronal cells. Moreover, JEV causes blood-brain barrier (BBB) disintegrity and lethality in STAT1-deficient (Stat1⁻/⁻) mice, whereas peripheral administration of anti-CLEC5A mAb reduces infiltration of virus-harboring leukocytes into the central nervous system (CNS), restores BBB integrity, attenuates neuroinflammation, and protects mice from JEV-induced lethality. Moreover, all surviving mice develop protective humoral and cellular immunity against JEV infection. These observations demonstrate the critical role of CLEC5A in the pathogenesis of Japanese encephalitis, and identify CLEC5A as a target for the development of new treatments to reduce virus-induced brain damage.

Cytotoxic effects of celecoxib on Raji lymphoma cells correlate with aggravated endoplasmic reticulum stress but not with inhibition of cyclooxygenase-2.

Inhibition of cyclooxygenase 2 (COX-2) by the selective COX-2 inhibitor celecoxib has been suggested as potentially useful for B-cell lymphoma therapy. However, additional pharmacological activities of celecoxib have been discovered and have challenged the notion that its antitumor effects are mediated primarily via the inhibition of COX-2. To shed light on this issue, we have investigated the effects of different pharmacological agents with greatly varying COX-2 inhibitory potency in Raji lymphoma cells in vitro. We found that cytotoxic potency of these compounds did not at all correlate with their COX-2 inhibitory activity; in fact, the most potent COX-2 inhibitors lacked the ability to kill Raji cells. Instead, the cytotoxic outcome was closely aligned with these agents' ability to trigger endoplasmic reticulum (ER) stress, which could be further enhanced by bortezomib, an agent with known ER stress-inducing potency. Together, these results indicate that celecoxib's cytotoxic effects on Raji lymphoma cells do not involve the inhibition of COX-2.

CLEC5A is critical for dengue-virus-induced lethal disease.

Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A-DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A-DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-alpha, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases.