Nguyen Thi Xuan

Ion Channels Modulating Mouse Dendritic Cell Functions

Ca2+-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca2+]i upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca2+]i, which was due to Ca2+ release from intracellular stores and influx of extracellular Ca2+ across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca2+ release-activated Ca2+ channels (CRAC). These currents were highly selective for Ca2+, exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca2+-dependent inactivation. In addition, the LPS-induced increase of [Ca2+]i was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K+ (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-α and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the ICRAC inhibitor SKF-96365. Taken together, our results demonstrate that Ca2+ influx in LPS-stimulated DCs occurs via Ca2+ release-activated Ca2+ channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.

Stimulation of Suicidal Erythrocyte Death by α-Lipoic Acid

Α-lipoic acid, a nutrient with both, antioxidant and oxidant activity induces apoptosis in a variety of cells. Owing to its proapoptotic potency Α-lipoic acid has been suggested for the therapy of cancer. Α-Lipoic acid stimulates apoptosis by induction of oxidative stress and subsequent activation of caspases. Oxidative stress could similarly trigger caspase activation and suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling and cell shrinkage. Eryptosis is triggered by increase of cytosolic Ca2+ concentration and/or ceramide formation. The present study explored whether Α -lipoic acid influences eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in FACS analysis, cytosolic Ca2+ concentration from Fluo3 fluorescence, caspase activation and ceramide formation utilizing respective antibodies, cytosolic ATP concentration from a luciferase-assay. Within 48 hours, exposure to Α-lipoic acid (10 - 75 mM) significantly decreased forward scatter, increased cytosolic Ca2+ concentration, decreased ATP concentration, activated caspase 3, stimulated formation of ceramide and triggered annexin V-binding. Glucose depletion (48 h) was followed by decrease of forward scatter and increase of annexin V-binding, effects significantly augmented in the presence of Α-lipoic acid (20 mM). Oxidative stress (30 min 0.3 mM tert-butylhydroperoxide) similarly triggered annexin binding, an effect slightly but significantly blunted by Α-lipoic acid. In conclusion, Α-lipoic acid triggers eryptosis but by the same token counteracts eryptosis during oxidative stress. Α-lipoic acid sensitive eryptosis may lead to anemia and derangements of microcirculation.

Ca2+-dependent functions in peptidoglycan-stimulated mouse dendritic cells.

Peptidoglycans (PGN) from bacterial cell walls may modify the course of an infection with bacterial pathogens. The present study explored the effect of PGN on cytosolic Ca²⁺ activity, cytokine production and phagocytosis of mouse dendritic cells (DCs), essential cells in the initiation and direction of antigen-specific T cell responses. Exposure of DCs to PGN was followed by a rapid increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i), which was due to Ca²⁺ release from intracellular stores and influx of extracellular Ca²⁺ across the cell membrane. In DCs isolated from Toll-like receptor 2 (TLR2) deficient mice the effect of PGN on [Ca²⁺]_i was dramatically impaired. The PGN-induced increase of [Ca²⁺]_i was dependent on voltage-gated K⁺ (Kv) channel activity. PGN-induced increase of [Ca²⁺]_i was significantly blunted by margatoxin (MgTx) and perhexiline maleate (PM), inhibitors of Kv1.3 and Kv1.5, respectively. PGN further stimulated the release of tumour necrosis factor α (TNFα), interleukin-12 (IL-12) and interleukin-10 (IL-10), an effect significantly blunted by PM and the specific blocker of store-operated Ca²⁺ channels SKF-96365. Moreover, phagocytic capacity was dramatically increased in PGN-stimulated DCs in the presence of either Kv channel inhibitors or SKF-96365. The observations disclose Ca²⁺ and Kv channel-dependent cytokine production and phagocytosis in PGN-stimulated DCs.

Effect of thymoquinone on mouse dendritic cells.

Thymoquinone, a component of Nigella sativa is known to confer protection against tumour growth due to stimulation of tumour cell apoptosis. Moreover, thymoquinone has remarkable anti-inflammatory potency. Surprisingly, despite its powerful influence on inflammation and its immunomodulatory effects, little is known about its effect on dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC maturation and cytokine release is triggered by bacterial components such as lipopolysaccharides (LPS). The present study explored whether thymoquinone modifies LPS-induced DC maturation, survival and cytokine release. To this end, mouse bone marrow derived DCs were treated with LPS and different concentrations of thymoquinone and the surface expression of CD11c, CD86, MHCII, CD54 and CD40 was determined by FACS analysis, the formation of the interleukins 10 (IL-10) and 12 (IL-12p70) as well as TNF-α by ELISA, caspase activation by FITC-labelled antibodies (FACS), cell membrane scrambling by annexin V binding (FACS) and Akt and ERK1/2 phosphorylation by Western blotting. LPS increased the percentage of CD11c+CD86+, CD11c+MHCII+, CD11c+CD40+ and CD11c+CD54+ cells and stimulated the release of IL-10, IL-12p70 and TNF-α. These effects were blunted by thymoquinone in a concentration dependent manner (1-20 µM). Moreover, LPS decreased and thymoquinone increased caspase 3 and caspase 8 activation and annexin V binding. Moreover, LPS-induced phosphorylation of prosurvival kinases Akt and ERK1/2 was abrogated by thymoquinone. In conclusion, thymoquinone compromises the maturation, cytokine release and survival of DCs.

Regulation of calcium signaling in dendritic cells by 1,25-dihydroxyvitamin D3

Dendritic cells (DCs) are antigen‐presenting cells that provide a link between innate and adaptive immunity. Ca2+‐dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], a secosteroid hormone, that, in addition to its wellestablished action on Ca2+ homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca2+ activity. The present study explored whether 1,25(OH)2D3 modifies the intracellular Ca2+ concentration ([Ca2+]i) in DCs. Here we show that mouse DCs expressed K+‐independent (NCX1–3) and K+‐dependent (NCKX1, 3, 4, and 5) Na+/Ca2+ exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca2+]i, an effect significantly blunted by prior incubation with 1,25(OH)2D3. 1,25(OH)2D3 increased the membrane abundance of the NCKX1 protein, up‐regulated the K+‐ and Na+‐dependent Ca2+ entry and enhanced the K+‐dependent Na+/Ca2+ exchanger currents. The NCKX blocker 3′,4′‐dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)2D3 on the LPS‐induced increase of [Ca2+]i. Expression of the costimulatory molecule CD86 was down‐regulated by 1,25(OH)2D3, an effect reversed by DBZ. In summary, 1,25(OH)2D3 blunts the LPS‐induced increase in [Ca2+]i by stimulation of Na+/Ca2+ exchangerdependent Ca2+ extrusion, an effect that contributes to 1,25(OH)2D3‐mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function.—Shumilina, E., Xuan, N. T., Matzner, N., Bhandaru, M., Zemtsova, I. M., Lang, F. Regulation of calcium signaling in dendritic cells by 1,25‐dihydroxyvitamin D3. FASEB J. 24, 1989–1996 (2010). www.fasebj.org

Effect of bacterial lipopolysaccharide on Na(+)/H(+) exchanger activity in dendritic cells.

The function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, is stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS). In macrophages, ROS formation is paralleled by activation of the Na+/H+ exchanger, a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether LPS influence Na+/H+ exchanger activity in DCs. The DCs were isolated from murine bone marrow, cell volume was estimated from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, apoptosis from annexin V binding, cytosolic pH (pHi) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Na+/H+ exchanger activity from the Na+ dependent realkalinization following an ammonium pulse. Exposure of DCs to LPS (1 µg/ml) led to a transient increase of Na+/H+ exchanger activity. Moreover, LPS increased forward scatter and ROS formation and decreased apoptosis. The NHE1 inhibitor cariporide (10 µM) virtually abrogated Na+/H+ exchanger activity, inhibited LPS-induced cell swelling, blunted LPS-induced ROS formation and reversed the antiapoptotic effect of LPS. Na+/H+ exchanger activity was stimulated by oxidative stress and LPS induced stimulation of NHE activity was abolished in presence of ROS chelators (Tempol, Tiron and Vitamin C). In conclusion, LPS treatment transiently upregulates the Na+/H+ exchanger in DCs, an effect required for the effects of LPS on DC survival, cell volume and ROS formation.

Triggering of dendritic cell apoptosis by xanthohumol

Xanthohumol, a flavonoid from beer with anticancer activity is known to trigger apoptosis in a variety of tumor cells. Xanthohumol further has anti-inflammatory activity. However, little is known about the effect of xanthohumol on survival and function of immune cells. The present study thus addressed the effect of xanthohumol on dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. To this end, mouse bone marrow-derived DCs were treated with xanthohumol with subsequent assessment of enzymatic activity of acid sphingomyelinase (Asm), ceramide formation determined with anti-ceramide antibodies in FACS and immunohistochemical analysis, caspase activity utilizing FITC conjugated anti-active caspase 8 or caspase 3 antibodies in FACS and by Western blotting, DNA fragmentation by determining the percentage of cells in the sub-G1 phase and cell membrane scrambling by annexin V binding in FACS analysis. As a result, xanthohumol stimulated Asm, enhanced ceramide formation, activated caspases 8 and 3, triggered DNA fragmentation and led to cell membrane scrambling, all effects virtually absent in DCs from gene targeted mice lacking functional Asm or in wild-type cells treated with sphingomyelinase inhibitor amitriptyline. In conclusion, xanthohumol stimulated Asm leading to caspase activation and apoptosis of bone marrow-derived DCs.

Phosphoinositide 3-Kinase Dependent Regulation of Kv Channels in Dendritic Cells

The phosphoinositide 3 (PI3) kinase plays a pivotal role in the regulation of dendritic cells (DCs), antigen-presenting cells that are able to initiate primary immune responses and to establish immunological memory. PI3 kinase is an endogenous suppressor of interleukin 12 (IL-12) production in DCs that is triggered by Toll-like receptor signaling. Inhibition of IL-12 production limits T helper 1 (Th1) polarization. On the other hand, PI3 kinase is an important regulator of various ion channels. The present study aimed to explore whether ion channels in DCs are regulated by PI3 kinase and whether they are important for DC function. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by patch clamp. As a result, DCs express voltage-gated K+ channels (Kv), which are blocked by Stichodactyla helianthus toxin (ShK, 2.5 nM). A significant upregulation of Kv currents was observed upon maturation of DCs as induced by stimulation of the cells with lipopolysaccharide (LPS, 0.1 µg/ml, 48 h). A dramatic increase of Kv current amplitude was observed following preincubation of the cells with LY294002 (100 nM), a specific inhibitor of PI3 kinase. PI3 kinase inhibitor wortmannin (100 nM) similarly increased Kv current. LY294002 treatment was further followed by a significant increase of IL-12 production. ShK (100 nM) significantly blunted the stimulation of IL-12 release by LPS but not when the cells were first pretreated with LY294002. The observations point to Kv channel sensitive and Kv channel insensitive regulation of DC function.

Stimulation of Mouse Dendritic Cells by Gum Arabic

Gum Arabic (GA), a nonabsorbable nutrient manufactured from the exudate of Acacia senegal, is composed of a complex polysaccharide. GA is used by the pharmaceutical and food industry as an emulsifier but may, at an appropriate dosage, modify intestinal transport. Dendritic cells (DCs) can protrude between epithelial cells and sense the composition of the lumen. As DCs are stimulated by bacterial polysaccharides, we hypothesized that GA may similarly stimulate DCs. To test that hypothesis, mouse DCs were treated with either LPS or GA and expression of maturation markers, phagocytotic activity, cytokine production and ability to stimulate CD4+ T cells in allogenic mixed leukocyte reaction (allo-MLR) was analyzed. As a result both LPS and GA increased the percentage of CD11c+CD86+, CD11c+MHCII+, CD11c+CD40+, CD54-expressing DCs and decreased their phagocytic activity. Both LPS and GA stimulated the production of IL-6, IL-10, IL12p70 and TNFΑ in a p38- and/or ERK-dependent manner. GA treatment led to an enhanced IL-10 secretion, whereas LPS was more effective on IL-6 and IL-12p70 production. Both LPS- and GA-stimulated DCs enhanced CD4+ T cell proliferation but the profile of cytokines produced in allo-MLR was different. High levels of IL-10 and IL-6 were observed in the presence of GA-treated DCs, whereas IFN-Γ and IL-12p70 production was similar with LPS- or GA-treated DCs. LPS upregulated p38 and transiently ERK1/2, while GA led to more sustained activation of ERK1/2, only. In conclusion, the observations reveal a powerful immunomodulatory effect of GA.

Association of two variants of the interferon-alpha receptor-1 gene with the presentation of hepatitis B virus infection

Interferon-alpha (IFNalpha) is a critical mediator of immunity to hepatitis B virus (HBV) infection. Although IFN has been used in the treatment of viral hepatitis for more than a decade, the role of IFN-alpha-receptor in HBV infection has not been intensively studied. We have evaluated the impact of two variants of the IFNAR1 gene on the outcome of HBV infection. Four hundred and fifty eight HBV-infected Vietnamese patients, with well-characterised clinical profiles including all forms of hepatic disease, and 160 non-infected, healthy Vietnamese individuals were enrolled in the study. Of these patients, 54 had acute hepatitis B, 88 had chronic hepatitis B, 118 had liver cirrhosis, 146 had a hepatocellular carcinoma and 52 were asymptomatic carriers of HBV. We analysed two SNPs for unequal distribution between these groups. The first SNP, rs1012335 is situated in intron 3 of the interferon alpha receptor 1 (IFNAR1). A C at position 17470 in the IFNAR1 on both chromosomes was detected more frequently in HBV-infected patients compared to healthy controls (OR: 2.6; 95% CI: 1.46-4.72, p < 0.001). The same homozygosity is also associated with higher concentrations of AST and ALT (aspartate and alanine amino-transferase) in the plasma of the patients. The second SNP (rs2257167) is situated in exon 4, causing a change of amino acids from Val (GTT) to Leu (CTT). Subjects having GTT on both chromosomes were more frequent in the healthy control group (OR: 0.54, 95% CI: 0.35-0.84, p = 0.004) and had lower plasma ALT concentrations. The findings indicate that two variants of the IFNAR1 gene are associated with the clinical presentation of HBV infection.