Young Ah Lee

Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.

Eosinophil-Mediated Tissue Inflammatory Responses in Helminth Infection

Eosinophilic leukocytes function in host protection against parasitic worms. In turn, helminthic parasites harbor specific molecules to evade or paralyze eosinophil-associated host immune responses; these molecules facilitate the migration and survival of parasitic helminths in vivo. This competition between eosinophil and worm leads to stable equilibria between them. An understanding of such dynamic host-eosinophil interactions will help us to uncover mechanisms of cross talk between host and parasite in helminth infection. In this review, we examine recent findings regarding the innate immune responses of eosinophils to helminthic parasites, and discuss the implications of these findings in terms of eosinophil-mediated tissue inflammation in helminth infection.

Entamoeba histolytica induces cell death of HT29 colonic epithelial cells via NOX1-derived ROS.

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

NOX1 participates in ROS-dependent cell death of colon epithelial Caco2 cells induced by Entamoeba histolytica

Entamoeba histolytica, which causes amebic colitis and occasional liver abscesses in humans, can induce host cell death through apoptosis and necrosis. Recently, we have demonstrated that E. histolytica can induce cell death in neutrophils via diphenyleneiodonium-sensitive NADPH oxidase (NOX)-derived reactive oxygen species (ROS). Although there are enzyme systems similar to the phagocyte NADPH oxidase system in many non-phagocytic cell types, the signaling role of NOX-derived ROS in cell death of human colon epithelial cells induced by E. histolytica remains obscure. Incubation of colon epithelial Caco2 tumor cell lines with amebic trophozoites resulted in intracellular ROS generation and cell death in a caspase-independent manner. Pretreatment with DPI, an inhibitor of NOX, strongly decreased E. histolytica-induced cell death in Caco2 cells. As identified by RT-PCR, NOX1 transcripts were highly expressed in Caco2 cells. siRNA-mediated suppression of NOX1 protein significantly inhibited E. histolytica-induced cell death and ROS response in Caco2 cells. These results suggest that NOX1 participates in the ROS-dependent cell death of colon epithelial cells induced by amebic adhesion during the early phase of intestinal amebiasis.

Leukotriene B4 receptor BLT-mediated phosphorylation of NF-κB and CREB is involved in IL-8 production in human mast cells induced by Trichomonas vaginalis-derived secretory products

Trichomonas vaginalis is a protozoan parasite that causes acute tissue inflammation in vaginal trichomoniasis. In this study, we investigated the signaling mechanisms through which T. vaginalis-derived secretory products (TvSP) induce chemokine IL-8 production in human mast cells. Stimulation with TvSP induced up-regulation of IL-8 protein secretion in HMC-1 cells. In addition, TvSP induced phosphorylation of transcription factors NF-κB and CREB in HMC-1 cells. Pretreatment of TvSP with lipase, but not heat or proteinase K strongly abolished the stimulatory effect on IL-8 production. Moreover, TvSP-induced IL-8 production and phosphorylation of NF-κB or CREB were inhibited when HMC-1 cells were stimulated with modified TvSP collected from 5-lipooxygenase inhibitor-treated trichomonads. Indeed, T. vaginalis-secreted lipid mediator LTB(4) (700pg/ml) from 1×10(7) trichomonads. Furthermore, pretreatment of HMC-1 cells with antagonists for LTB(4) receptors BLT1 or BLT2 abolished the stimulatory effects of TvSP. Finally, TvSP-induced IL-8 production was inhibited by pretreatment with IκB or CREB inhibitors. These results suggest that T. vaginalis-derived secretory lipid mediator LTB(4) induces IL-8 production in mast cells via BLT-dependent activation of NF-κB and CREB.

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 p47phox phosphorylation and 91phox 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.

NF-κB and CREB Are Involved in IL-8 Production of Human Neutrophils Induced by Trichomonas vaginalis- Derived Secretory Products

Trichomonas vaginalis is a flagellated lumen-dwelling extracellular protozoan parasite that causes human trichomoniasis via sexual intercourse. Human neutrophils play a crucial role in acute tissue inflammatory responses in T. vaginalis infection. In this study, we investigated the signaling mechanism of neutrophil responses when stimulated with T. vaginalis-derived secretory products (TvSP), which were collected from 1×107 live trichomonads. Incubation of human neutrophils isolated from peripheral blood with TvSP induced up-regulation of IL-8 protein secretion. In addition, stimulation with TvSP induced phosphorylation of NF-κB and CREB in neutrophils. Moreover, TvSP-induced IL-8 production was also significantly inhibited by pretreatment of neutrophils with iκB inhibitor or CREB inhibitor. These results suggest that transcription factors NF-κB and CREB are involved in IL-8 production in human neutrophils induced by stimulation with T. vaginalis infection.

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 p47phox 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.

Calpains are Involved in Entamoeba histolytica-Induced Death of HT-29 Colonic Epithelial Cells

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and µ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and µ-calpain may be involved in colon epithelial cell death induced by E. histolytica.

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.