Involvement of NOX2-derived ROS in human hepatoma HepG2 cell death induced by Entamoeba histolytica.

Entamoeba histolytica is an enteric tissue-invasive protozoan parasite causing amoebic colitis and liver abscesses in humans. Amoebic contact with host cells activates intracellular signaling pathways that lead to host cell death via generation of caspase-3, calpain, Ca2+ elevation, and reactive oxygen species (ROS). We previously reported that various NADPH oxidases (NOXs) are responsible for ROS-dependent death of various host cells induced by amoeba. In the present study, we investigated the specific NOX isoform involved in ROS-dependent death of hepatocytes induced by amoebas. Co-incubation of hepatoma HepG2 cells with live amoebic trophozoites resulted in remarkably increased DNA fragmentation compared to cells incubated with medium alone. HepG2 cells that adhered to amoebic trophozoites showed strong dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, suggesting intracellular ROS accumulation within host cells stimulated by amoebic trophozoites. Pretreatment of HepG2 cells with the general NOX inhibitor DPI or NOX2-specific inhibitor GSK 2795039 reduced Entamoeba-induced ROS generation. Similarly, Entamoeba-induced LDH release from HepG2 cells was effectively inhibited by pretreatment with DPI or GSK 2795039. In NOX2-silenced HepG2 cells, Entamoeba-induced LDH release was also significantly inhibited compared with controls. Taken together, the results support an important role of NOX2-derived ROS in hepatocyte death induced by E. histolytica.

Signaling Role of NADPH Oxidases in ROS-Dependent Host Cell Death Induced by Pathogenic Entamoeba histolytica.

All living organisms are destined to die. Cells, the core of those living creatures, move toward the irresistible direction of death. The question of how to die is critical and is very interesting. There are various types of death in life, including natural death, accidental death, questionable death, suicide, and homicide. The mechanisms and molecules involved in cell death also differ depending on the type of death. The dysenteric amoeba, E. histolytica, designated by the German zoologist Fritz Schaudinn in 1903, has the meaning of tissue lysis; i.e., tissue destroying, in its name. It was initially thought that the amoebae lyse tissue very quickly leading to cell death called necrosis. However, advances in measuring cell death have allowed us to more clearly investigate the various forms of cell death induced by amoeba. Increasing evidence has shown that E. histolytica can cause host cell death through induction of various intracellular signaling pathways. Understanding of the mechanisms and signaling molecules involved in host cell death induced by amoeba can provide new insights on the tissue pathology and parasitism in human amoebiasis. In this review, we emphasized on the signaling role of NADPH oxidases in reactive oxygen species (ROS)-dependent cell death by pathogenic E. histolytica.

Naegleria fowleri Induces Jurkat T Cell Death via O-deGlcNAcylation.

The pathogenic free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis, a fatal infection, by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves. N. fowleri can induce host cell death via lytic necrosis. Similar to phosphorylation, O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), respectively. However, the detailed mechanism of host cell death induced by N. fowleri is unknown. In this study, we investigated whether N. fowleri can induce the modulation of O-GlcNAcylated proteins during cell death in Jurkat T cells. Co-incubation with live N. fowleri trophozoites increased DNA fragmentation. In addition, incubation with N. fowleri induced a dramatic reduction in O-GlcNAcylated protein levels in 30 min. Moreover, pretreatment of Jurkat T cells with the OGA inhibitor PUGNAc prevented N. fowleri-induced O-deGlcNAcylation and DNA fragmentation. These results suggest that O-deGlcNAcylation is an important signaling process that occurs during Jurkat T cell death induced by N. fowleri.

Trichomonas vaginalis-secreted cysteinyl leukotrienes promote migration, degranulation and MCP-1 production in mast cells.

Trichomonas vaginalis, a flagellated extracellular protozoan parasite that infects the human genitourinary tract, is usually transmitted by sexual contact. Our previous study showed that the leukotriene B4 (LTB4 ), a T vaginalis-secreted lipid mediator, induces interleukin (IL)-8 production and promotes mast cell degranulation and migration via BLT1 in human. In this study, we investigated whether T vaginalis produces another leukotrienes and whether it causes increased MCP-1 production, mast cell migration and degranulation by activating mast cells. We found that cysteinyl leukotrienes (CysLTs) were contained in T vaginalis-derived secretory product (TvSP) by ELISA. The TvSP-stimulated human mast cell line (HMC-1) exhibited significantly increased monocyte chemoattractant protein-1 (MCP-1) secretion compared to the unstimulated cells. Inhibition of NOX2 activation of cells by treatment of NOX inhibitor or NOX2 siRNA reduced TvSP-stimulated MCP-1 production in HMC-1 cells. It was also confirmed that the receptor for CysLTs is expressed in mast cells. The CysLT receptor (CysLTR) antagonist inhibited TvSP-stimulated MCP-1 production of mast cells, as well as ROS production, migration and degranulation of mast cells, and reduced phospho-NF-kB expression. These results suggest that T vaginalis-secreted CysLTs promote migration, degranulation and MCP-1 production in human mast cells through CysLT receptor-mediated NOX2 activation.

Phylogenetic Characteristics of Echinococcus granulosus Sensu Lato in Uzbekistan.

Echinococcosis occurs mainly in areas with heavy livestock farming, such as Central Asia, America, and Australia. Echinococcus granulosus sensu lato (s.l.) infection causes echinococcosis in intermediate hosts, such as sheep, cattle, goats, camels, and horses. Numerous cases of echinococcosis occur in Uzbekistan as stock farming is a primary industry. Epidemiological and genetic studies of E. granulosus s.l. are very important for mitigating its impact on public health and the economy; however, there are no such studies on E. granulosus s.l. in Uzbekistan. In the present study, to determine which genotypes exist and are transmitted, we isolated Echinococcus sp. from definitive hosts (one isolate each from jackal and dog) and intermediate hosts (52 isolates from humans and 6 isolates from sheep) in Uzbekistan and analyzed the isolates by sequencing 2 mitochondrial DNA components (cox1 and nad1). The results showed that all of isolates except one belonged to the E. granulosus sensu stricto (s.s.) G1 and G3 genotypes. Phylogenetic analysis based on cox1 sequences showed that 42 isolates from humans, 6 isolates from sheep, and one isolate from jackal were the G1 genotype, whereas the remaining 8 isolates from human and the one isolate from dog were the G3 genotype. These results suggest that the G1 and G3 genotypes of E. granulosus s.s. are predominant in Uzbekistan, and both wild animals and domestic animals are important for maintaining their life cycle. Only one isolate from human sample was confirmed to be E. eqiinus (G4 genotype), which is known to be for the first time.

Prevalence of Intestinal Helminth Infections in Dogs and Two Species of Wild Animals from Samarkand Region of Uzbekistan.

This study aimed to determine the prevalence of intestinal helminth parasitic infections and associated risk factors for the human infection among the people of Samarkand, Uzbekistan. Infection status of helminths including Echinococcus granulosus was surveyed in domestic and wild animals from 4 sites in the Samarkand region, Uzbekistan during 2015-2018. Fecal samples of each animal were examined with the formalin-ether sedimentation technique and the recovery of intestinal helminths was performed with naked eyes and a stereomicroscope in total 1,761 animals (1,755 dogs, 1 golden jackal, and 5 Corsac foxes). Total 658 adult worms of E. granulosus were detected in 28 (1.6%) dogs and 1 (100%) golden jackal. More than 6 species of helminths, i.e., Taenia hydatigena, Dipylidium caninum, Diplopylidium nolleri, Mesocestoides lineatus, Toxocara canis, and Trichuris vulpis, were found from 18 (1.0%) dogs. Six (T. hydatigena, Toxascaris leonina, Alaria alata, Uncinaria stenocephala, D. caninum, and M. lineatus) and 2 (D. nolleri and M. lineatus) species of helminths were also detected from 5 Corsac foxes and 1 golden jackal, respectively. Taeniid eggs were found in 2 (20%) out of 10 soil samples. In the present study, it was confirmed that the prevalences of helminths including E. granulosus are not so high in domestic and wild animals. Nevertheless, the awareness on the zoonotic helminth infections should be continuously maintained in Uzbekistan for the prevention of human infection.

NOX4 activation is involved in ROS-dependent Jurkat T-cell death induced by Entamoeba histolytica.

AIMS: Entamoeba histolytica can induce host cell death through induction of various intracellular signalling pathways. The responses triggered by E. histolytica are closely associated with tissue pathogenesis and immune evasion. Although E. histolytica can induce reactive oxygen species (ROS) in host cells, which NADPH oxidase (NOX) isoform contributes to amoeba-triggered Jurkat T-cell death is unclear. In this study, we investigated the signalling role of NOX4-derived ROS in E. histolytica-induced Jurkat T-cell death process. METHODS AND RESULTS: In resting-state Jurkat T cells, NOX4 is strongly expressed. When Jurkat T cells were incubated with live E. histolytica trophozoites, intracellular ROS was significantly increased compared to cells incubated with medium alone. E. histolytica-induced ROS production was inhibited by pretreating Jurkat T cells with a NOX inhibitor. In addition, pretreating Jurkat T cells with a NOX inhibitor (Diphenyleneiodonium chloride) effectively blocked E. histolytica-induced phosphatidylserine (PS) exposure and DNA fragmentation of host cells. Moreover, siRNA-mediated knockdown of NOX4 protein expression in Jurkat T cells prevented E. histolytica-induced ROS generation and DNA fragmentation. CONCLUSION: These results suggest that NOX4 has a critical role in ROS-dependent cell death process in Jurkat T cells induced by E. histolytica.

Therapeutic effects of Echinococcus granulosus cystic fluid on allergic airway inflammation.

Previous studies showed that Echinococcus granulosus infection reduces allergic airway inflammation in experimentally infected hosts and the cystic fluid of E. granulosus is known to activate regulatory T (CD4+CD25+Foxp3+T, Treg) cells. To evaluate the effects of cystic fluid of E. granulosus on allergic airway inflammation, we investigated the regulation of the inflammatory reaction by cystic fluid using an allergic airway inflammation animal model. Cystic fluid was administered to C57BL/6 mice seven times every other day, after which allergic airway inflammation was induced using ovalbumin and aluminum. The airway resistance, number of eosinophils and other immune cells in the bronchoalveolar lavage fluid, and levels of Th2 and Th17-related cytokines were significantly reduced by cystic fluid pre-treatment in allergic airway inflammation-induced mice. The number IL-4+CD4+ T cells decreased, the number of Treg cells increased in the lung-draining lymph nodes and spleen of cystic fluid pre-treated mice. In conclusion, E. granulosus-derived cystic fluid may alleviate the Th2 allergic airway inflammatory response via Treg cells. Further studies of the immune regulation of cystic fluid may lead to the development of therapeutic agents for immune disorders.

O-deGlcNAcylation is required for Entamoeba histolytica-induced HepG2 cell death.

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. E. histolytica can induce host-cell apoptosis by initiating various intracellular signaling mechanisms closely associated with tissue pathogenesis and parasitic immune evasion. O-GlcNAcylation, similar to phosphorylation, is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, whether O-GlcNAc alterations in host cells affect E. histolytica-induced cell death and which signal molecules participate in E. histolytica-induced deglycosylation remain unknown. In this study, co-incubation of HepG2 cells with E. histolytica increased DNA fragmentation and LDH release as compared with control cells. Additionally, Gal-lectin-mediated amoebic adherence of live trophozoites to HepG2 cells decreased O-GlcNAcylated protein levels within 5 min. We also observed a rapid decrease in cellular OGT protein level, but not OGA, in HepG2 cells in a contact-dependent manner. Furthermore, HepG2 pretreatment with OGA inhibitors or OGA siRNA prevented E. histolytica-induced O-deGlcNAcylation, DNA fragmentation, and LDH release. Our results suggested that E. histolytica-induced O-deGlcNAcylation in HepG2 cells was an important process required for hepatocyte cell death induced by E. histolytica adherence.

BLT1-mediated O-GlcNAcylation is required for NOX2-dependent migration, exocytotic degranulation and IL-8 release of human mast cell induced by Trichomonas vaginalis-secreted LTB4.

Trichomonas vaginalis is a sexually-transmitted protozoan parasite that causes vaginitis and cervicitis. Although mast cell activation is important for provoking tissue inflammation during infection with parasites, information regarding the signaling mechanisms in mast cell activation and T. vaginalis infection is limited. O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues that functions as a critical regulator of intracellular signaling, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We investigated if O-GlcNAcylation was associated with mast cell activation induced by T. vaginalis-derived secretory products (TvSP). Modified TvSP collected from live trichomonads treated with the 5-lipooxygenase inhibitor AA861 inhibited migration of mast cells. This result suggested that mast cell migration was caused by stimulation of T. vaginalis-secreted leukotrienes. Using the BLT1 antagonist U75302 or BLT1 siRNA, we found that migration of mast cells was evoked via LTB4 receptor (BLT1). Furthermore, TvSP induced protein O-GlcNAcylation and OGT expression in HMC-1 cells, which was prevented by transfection with BLT1 siRNA. TvSP-induced migration, ROS generation, CD63 expression and IL-8 release were significantly suppressed by pretreatment with OGT inhibitor ST045849 or OGT siRNA. These results suggested that BLT1-mediated OGlcNAcylation was important for mast cell activation during trichomoniasis.

Comparison of Secretome Profile of Pathogenic and Non-Pathogenic Entamoeba histolytica.

The obligatory intracellular protozoan parasite Entamoeba histolytica causes amebic dysentery and liver abscess. E. histolytica adheres to the host tissues in a contact-dependent manner. E. histolytica excretory-secretory products (ESP) might play critical roles during invasion. We comparatively analyzed the secretome profile of E. histolytica pathogenic HM-1:IMSS and non-pathogenic Rahman strains. The two ESP revealed similar but distinct spotting patterns. In both ESP, alcohol dehydrogenase, enolase 1, and transketolase, which control classical carbohydrate metabolism and other moonlighting effects, constituted the most abundant fractions. We recognized differently secreted molecules. Secretion of cytoskeletal organization proteins (actin, actin binding protein, and EHI_068510), protein remodeling amino peptidase, and multifunctional elongation factor 1-α were increased in Rahman. Conversely, carbohydrate metabolizing enolase 1, alcohol dehydrogenase, transketolase, calponin, phosphoglucose mutase, malic enzyme and EHI_156420, xenobiotic scavenging superoxide dismutase and EHI_140740, and pyruvate:ferredoxin oxidoreductase and coronin (carbohydrate metabolism/detoxification) showed reduced secretion. Transcription levels of some genes involved in these processes also decreased. Changes of secretory behavior, especially decreased secretion of multifunctional carbohydrate metabolizing enzymes and detoxifying proteins that importantly participated in amoeba pathogenesis might reflect avirulent nature of Rahman strain in the host.

SNAP23-Dependent Surface Translocation of Leukotriene B4 (LTB4) Receptor 1 Is Essential for NOX2-Mediated Exocytotic Degranulation in Human Mast Cells Induced by Trichomonas vaginalis-Secreted LTB4.

Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women and itself secretes lipid mediator leukotriene B4 (LTB4). Mast cells are important effector cells of tissue inflammation during infection with parasites. Membrane-bridging SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes are critical for fusion during exocytosis. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce exocytosis in mast cells, information regarding the signaling mechanisms between mast cell activation and TvSP is limited. In this study, we found that SNAP23-dependent surface trafficking of LTB4 receptor 1 (BLT1) is required for nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-mediated exocytotic degranulation of mast cells induced by TvSP. First, stimulation with TvSP induced exocytotic degranulation and reactive oxygen species (ROS) generation in HMC-1 cells. Next, TvSP-induced ROS generation and exocytosis were strongly inhibited by transfection of BLT1 small interfering RNA (siRNA). TvSP induced trafficking of BLT1 from the cytosol to the plasma membrane. We also found that knockdown of SNAP23 abrogated TvSP-induced ROS generation, exocytosis, and surface trafficking of BLT1 in HMC-1 cells. By coimmunoprecipitation, there was a physical interaction between BLT1 and SNAP23 in TvSP-stimulated HMC-1 cells. Taken together, our results suggest that SNAP23-dependent surface trafficking of BLT1 is essential for exocytosis in human mast cells induced by T. vaginalis-secreted LTB4 Our data collectively demonstrate a novel regulatory mechanism for SNAP23-dependent mast cell activation of T. vaginalis-secreted LTB4 involving surface trafficking of BLT1. These results can help to explain how the cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses.

Practical Algorisms for PCR-RFLP-Based Genotyping of Echinococcus granulosus Sensu Lato.

Echinococcus granulosus sensu lato (s.l.) is a causative agent of cystic echinococcosis or cystic hydatid disease in humans and domestic and wild animals. The disease is a serious health problem in countries associated with poverty and poor hygiene practices, particularly in livestock raising. We introduced a practical algorism for genotyping the parasite, which may be useful to many developing countries. To evaluate the efficiency of the algorism, we genotyped 3 unknown strains isolated from human patients. We found that unknowns 1 and 3 were included in G1, G2, and G3 genotypes group and unknown 2 was included in G4 genotype (Echinococcus equinus) according to the algorisms. We confirmed these results by sequencing the 3 unknown isolates cox1 and nad1 PCR products. In conclusion, these new algorisms are very fast genotype identification tools that are suitable for evaluating E. granulosus s.l. isolated from livestock or livestock holders, particularly in developing countries.

Thymic stromal lymphopoietin regulates eosinophil migration via phosphorylation of l-plastin in atopic dermatitis.

Infiltration of eosinophils in atopic dermatitis (AD), which contains inflammatory molecules and cytokines, recruits more inflammatory cells and causes further skin damage. Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine that induces the proinflammatory Th2 immune response and plays an important role in allergic disease. In this study, we aimed to identify a novel protein that regulates TSLP in eosinophils to further understand the role of eosinophils in atopic dermatitis. Using a proteomics approach, we identified the TSLP-inducible protein l-plastin and confirmed upregulation of l-plastin and p-l-plastin in TSLP-treated human eosinophilic leukaemic (EoL-1) cells and in eosinophils from AD patients. Migration assays showed that migration of eosinophils increased when cells were treated with TSLP and when cells were treated with TSLP and an additional cytokine such as interleukin (IL)-3, IL-4, IL-5 or IL-13, when compared to migration of untreated eosinophils. We also confirmed a positive correlation between phosphorylation of l-plastin and an increase in migration of TSLP and cytokine-treated eosinophils. In addition, phosphorylation of l-plastin was sensitive to PKCßII inhibition. Our results suggest that TSLP-induced phosphorylation of l-plastin affects eosinophil migration, which may be mediated by the protein kinase C signalling pathway in atopic dermatitis, thus suggesting p-l-plastin as a potential drug target for eosinophil-targeted allergy therapy.

Activation of MAPK Is Required for ROS Generation and Exocytosis in HMC-1 Cells Induced by Trichomonas vaginalis-Derived Secretory Products.

Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47(phox) in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling.

Modulation of dendritic cell function by Trichomonas vaginalis-derived secretory products.

Trichomoniasis caused by the parasitic protozoan Trichomonas vaginalis is the most common sexually transmitted disease in the world. Dendritic cells are antigen presenting cells that initiate immune responses by directing the activation and differentiation of naïve T cells. In this study, we analyzed the effect of Trichomonas vaginalis-derived Secretory Products on the differentiation and function of dendritic cells. Differentiation of bone marrow-derived dendritic cells in the presence of T. vaginalis-derived Secretory Products resulted in inhibition of lipopolysaccharide-induced maturation of dendritic cells, down-regulation of IL-12, and up-regulation of IL-10. The protein components of T. vaginalis-derived Secretory Products were shown to be responsible for altered function of bone marrow- derived dendritic cells. Chromatin immunoprecipitation assay demonstrated that IL-12 expression was regulated at the chromatin level in T. vaginalis-derived Secretory Productstreated dendritic cells. Our results demonstrated that T. vaginalis- derived Secretory Products modulate the maturation and cytokine production of dendritic cells leading to immune tolerance.

Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

Degradation of the transcription factors NF-κB, STAT3, and STAT5 is involved in Entamoeba histolytica-induced cell death in Caco-2 colonic epithelial cells.

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

NOX2-derived ROS-mediated surface translocation of BLT1 is essential for exocytosis in human eosinophils induced by LTB4.

BACKGROUND: Leukotriene B4 (LTB4) is a proinflammatory lipid mediator that elicits eosinophil exocytosis, leading to allergic inflammation. However, the detailed intracellular signaling mechanisms of eosinophil exocytosis induced by LTB4 are poorly understood. Herein, we report that NADPH oxidase (NOX)2-derived reactive oxygen species (ROS)-mediated BLT1 migration to the cell surface is required for exocytosis in human eosinophils induced by LTB4. METHODS: Peripheral blood eosinophils were purified and stimulated for up to 60 min with LTB4. The signaling role of NOX2-derived ROS in BLT1-dependent exocytosis in LTB4-stimulated eosinophils was investigated. RESULTS: Stimulating eosinophils with LTB4 induced intracellular ROS production and surface upregulation of the exocytosis marker protein CD63 via BLT1-mediated signaling. LTB4 induced p47(phox) phosphorylation and 91(phox) expression required for NOX2 activation in a BLT1-dependent manner. Pretreatment with NOX2 inhibitors, but not mitochondria inhibitor, prevented LTB4-induced ROS generation and exocytosis. At 30 min after stimulation with LTB4, BLT1 expression at the cell surface was upregulated. LTB4-triggered surface upregulation of BLT1 was also blocked by inhibition of ROS generation with NOX2 inhibitors. Moreover, stimulation for 30 min with LTB4 resulted in the interaction of BLT1 with NOX2 by immunoprecipitation. LTB4-induced ROS generation, surface upregulation of BLT1 and exocytosis was also inhibited by pretreatment with a lipid raft disruptor, protein kinase C inhibitor, or Src kinase inhibitor. CONCLUSION: These results suggest that NOX2-derived ROS-mediated BLT1 trafficking to the cell surface plays a key role in the exocytosis of human eosinophils induced by LTB4.

Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.