Pilar García-Peñarrubia

Implication of reactive oxygen species in the antibacterial activity against Salmonella Typhimurium of hepatocyte cell lines

We recently described the antibacterial activity of a murine hepatocyte cell line stimulated with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and lipopolysaccharide (LPS) against intracellular Salmonella organisms. Here we show for the first time the existence of basal antibacterial activity in cultured hepatocyte cell lines. Thus treatment of resting and stimulated hepatocytes with catalase or superoxide dismutase increased bacterial number recovered per monolayer, which suggests that the mechanism involved with antibacterial activity of hepatocytes is mediated by reactive oxygen species (ROS). Also, the capacity of these cell lines to generate intracellular peroxides under resting and stimulated conditions was investigated. This revealed that IL-1 and LPS did not induce any increase in the amount of intracellular peroxides by themselves, but they primed IFN-gamma for maximal induction of peroxides. The intracellular amount of peroxides was highly increased on stimulation with IFN-gamma, IL-1, and LPS, and it was strongly inhibited by catalase. This explains that the mechanism whereby this enzyme inhibits antibacterial activity takes place by decreasing the intracellular pool of peroxides. In turn, experiments performed in the presence of several inhibitors of metabolic pathways involved in ROS generation suggested that cyclo-oxygenase are a source of these species in hepatocyte cell lines. These results attribute a prominent role to the generation of peroxides as effector molecules of antibacterial activity in hepatocyte cell lines. Thus these cells displayed a moderate basal level, which increased on stimulation with proinflammatory cytokines such as IFN-gamma, IL-1, and bacterial products such as LPS. Finally, it has been also shown for the first time that IFN-gamma stimulation induces production of peroxides in human and murine hepatocyte cell lines.

Role of trehalose-6P phosphatase (TPS2) in stress tolerance and resistance to macrophage killing in Candida albicans☆

Disruption of the TPS2 gene encoding the only trehalose-6P phosphatase activity in Candida albicans caused a pleiotropic defective phenotype, maintaining the cell wall integrity and the ability to form chlamydospores. A homozygous tps2Delta/tps2Delta showed reduced growth at high temperatures and a marked sensitivity to heat shock (42 degrees C) and severe oxidative exposure (50mM H(2)O(2)). Reintroduction of the TPS2 gene reversed these alterations. A more detailed study of the antioxidant response showed that exponential tps2Delta null cells displayed an adaptive response to oxidative stress as well as cross-tolerance between temperature and oxidative stress. Differential measurement of trehalose and trehalose-6P, using reliable new HPLC methodology, revealed a significant accumulation of trehalose-6P in tps2Delta cells, which was enhanced after oxidative exposure. In contrast, the level of trehalose-6P in parental cells was virtually undetectable, and oxidative treatment only induced the synthesis of free trehalose. A transitory increase in the expression of TPS2 and TPS1 genes was promoted in wild-type cells in response to acute (50mM) but not gentle (5mM) oxidative exposure. TPS1 and TPS2 oxidative-induced transcriptions were completely absent from the tps2Delta mutant. Exponential blastoconidia from both parental and tps2Delta/tps2Delta strains were completely phagocytosed by murine and human macrophages, triggering a subsequent proinflammatory response manifested by the release of TNF-alpha. Reflecting the lower resistance to oxidative stress displayed by the tps2Delta mutant, intracellular survival in resting and IFN-gamma and LPS-stimulated macrophages was also diminished. Taken together, our results confirm the mainly protective role played by the trehalose biosynthetic pathway in the cellular response to oxidative stress and subsequently in the resistance to phagocytosis in C. albicans, a defensive mechanism in which TPS2 would be involved.

Peritoneal macrophage priming in cirrhosis is related to ERK phosphorylation and IL-6 secretion

Eur J Clin Invest 2010; 41 (1): 8–15

The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation

BackgroundThe development of ascites in cirrhotic patients generally heralds a deterioration in their clinical status. A differential gene expression profile between alcohol- and hepatitis C virus (HCV)-related cirrhosis has been described from liver biopsies, especially those associated with innate immune responses. The aim of this work was to identify functional differences in the inflammatory profile of monocyte-derived macrophages from ascites in cirrhotic patients of different etiologies in an attempt to extrapolate studies from liver biopsies to immune cells in ascites. To this end 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (nu2009=u200915) or alcohol (nu2009=u200930) were studied. Cytokines and the cell content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation in peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined.ResultsA different pattern of leukocyte migration to the peritoneal cavity and differences in the primed status of macrophages in cirrhosis were observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral blood cell subpopulations could be observed, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in the HCV than the alcohol etiology. HCV-related cirrhosis etiology was associated with a decreased inflammatory profile in ascites compared with the alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were observed in ascitic fluid from the HCV group. Isolated peritoneal monocyte-derived macrophages maintained their primed status in vitro throughout the 24 h culture period. The level of ERK1/2 phosphorylation was higher in ALC peritoneal macrophages at baseline than in HCV patients, although the addition of LPS induced a greater increase in ERK1/2 phosphorylation in HCV than in ALC patients.ConclusionsThe macrophage inflammatory status is higher in ascites of alcohol-related cirrhotic patients than in HCV-related patients, which could be related with differences in bacterial translocation episodes or regulatory T cell populations. These findings should contribute to identifying potential prognostic and/or therapeutic targets for chronic liver diseases of different etiology.

Role of MAP Kinases and PI3K‐Akt on the cytokine inflammatory profile of peritoneal macrophages from the ascites of cirrhotic patients

Several new approaches targeting inflammation associated with different diseases are in clinical development.

Glycoconjugate expression on the cell wall of tps1/tps1 trehalose-deficient Candida albicans strain and implications for its interaction with macrophages

The yeast Candida albicans has developed a variety of strategies to resist macrophage killing. In yeasts, accumulation of trehalose is one of the principal defense mechanisms under stress conditions. The gene-encoding trehalose-6-phosphate synthase (TPS1), which is responsible for trehalose synthesis, is induced in response to oxidative stress, as in phagolysosomes. Mutants unable to synthesize trehalose are sensitive to oxidative stress in vitro. In mice, the TPS1-deficient strain, tps1/tps1, displays a lower infection rate than its parental strain (CAI4). We have previously demonstrated the reduced binding capacity of tps1/tps1 and its lower resistance to macrophages. At the same time, its outer cell wall layer was seen to be altered. In this study, we show that depending on the culture conditions, the tps1/tps1 strain regulates the carbohydrate metabolism in a different way to CAI4, as reflected by the enhanced β-mannosylation of cell wall components, especially at the level of the 120 kDa glycoprotein species, accessible at the cell surface of tps1/tps1 when cultured in liquid medium, but not on solid medium. This leads to changes in its surface properties, as revealed by decreased hydrophobicity, and the lower levels of ERK1/2 phosphorylation and tumor necrosis factor-α (TNF-α) production in macrophages, thus increasing the resistance to these cells. In contrast, in solid medium, in which over-glycosylation was less evident, tps1/tps1 showed similar macrophage interaction properties to CAI4, but was less resistant to killing, confirming the protective role of trehalose. Thus, the lack of trehalose is compensated by an over-glycosylation of the cell wall components in the tps1/tps1 mutant, which reduces susceptibility to killing.

Collateral sensitivity to cold stress and differential BCL-2 family expression in new daunomycin-resistant lymphoblastoid cell lines.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells is one of the main causes of chemotherapy failure in cancer, and, usually, is due to the increased expression of P-glycoprotein (MDR-1, P-gp, ABCB1), a pump that expels chemotherapeutics from the cell and/or regulates apoptosis. Thus, it is fundamental to find drugs or stress stimuli with a capacity to induce apoptosis in such cells and to identify the mechanisms involved. We address this matter in human cells and establish new daunomycin (DNM)-resistant cell lines (IM-9R) by exposing the parental lymphoblastic cells (IM-9) to increasing doses of the anti-neoplastic drug, daunomycin. The resistance level of IM-9R cell lines, MDR-1 expression and functionality, collateral sensitivity and Bcl-2 and caspases protein expression are analyzed. As a result, we show for the first time that, unlike the parental cells, human lymphoblastic resistant cells exhibit collateral sensitivity to cold stress, confirming that this phenomenon is not exclusive to murine leukemic cells, but a broader one associated with the acquisition of drug resistance. Furthermore, the new resistant cell lines undergo a significant increase in active caspase-3 and -9 levels and drastic changes in Bcl-2 family protein expression during the process of MDR phenotype acquisition.

Mathematical modelling and computational study of two-dimensional and three-dimensional dynamics of receptor–ligand interactions in signalling response mechanisms

Cell signalling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signalling pathways. But the binding process still presents many enigmas and, by analogy with surface catalytic reactions, two different mechanisms can be conceived: the first mechanism is related to the Eley–Rideal (ER) mechanism, i.e. the bulk-dissolved ligand interacts directly by pure three-dimensional (3D) diffusion with the specific surface receptor; the second mechanism is similar to the Langmuir–Hinshelwood (LH) process, i.e. 3D diffusion of the ligand to the cell surface followed by reversible ligand adsorption and subsequent two-dimensional (2D) surface diffusion to the receptor. A situation where both mechanisms simultaneously contribute to the signalling process could also occur. The aim of this paper is to perform a computational study of the behavior of the signalling response when these different mechanisms for ligand-receptor interactions are integrated into a model for signal transduction and ligand transport. To this end, partial differential equations have been used to develop spatio-temporal models that show trafficking dynamics of ligands, cell surface components, and intracellular signalling molecules through the different domains of the system. The mathematical modeling developed for these mechanisms has been applied to the study of two situations frequently found in cell systems: (a) dependence of the signal response on cell density; and (b) enhancement of the signalling response in a synaptic environment.

Regulatory role of PI3K-protein kinase B on the release of interleukin-1β in peritoneal macrophages from the ascites of cirrhotic patients.

Great effort has been paid to identify novel targets for pharmaceutical intervention to control inflammation associated with different diseases. We have studied the effect of signalling inhibitors in the secretion of the proinflammatory and profibrogenic cytokine interleukin (IL)‐1β in monocyte‐derived macrophages (M‐DM) obtained from the ascites of cirrhotic patients and compared with those obtained from the blood of healthy donors. Peritoneal M‐DM were isolated from non‐infected ascites of cirrhotic patients and stimulated in vitro with lipopolysaccharide (LPS) and heat‐killed Candida albicans in the presence or absence of inhibitors for c‐Jun N‐terminal kinase (JNK), mitogen‐activated protein kinase kinase 1 (MEK1), p38 mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K). The IL1B and CASP1 gene expression were evaluated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). The expression of IL‐1β and caspase‐1 were determined by Western blot. IL‐1β was also assayed by enzyme‐linked immunosorbent assay (ELISA) in cell culture supernatants. Results revealed that MEK1 and JNK inhibition significantly reduced the basal and stimulated IL‐1β secretion, while the p38 MAPK inhibitor had no effect on IL‐1β levels. On the contrary, inhibition of PI3K increased the secretion of IL‐1β from stimulated M‐DM. The activating effect of PI3K inhibitor on IL‐1β release was mediated mainly by the enhancement of the intracellular IL‐1β and caspase‐1 content release to the extracellular medium and not by increasing the corresponding mRNA and protein expression levels. These data point towards the role of MEK1 and JNK inhibitors, in contrast to the PI3K‐protein kinase B inhibitors, as potential therapeutic tools for pharmaceutical intervention to diminish hepatic damage by reducing the inflammatory response mediated by IL‐1β associated with liver failure.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug‐resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti‐neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P‐glycoprotein (MDR1, P‐gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P‐gp over‐expression, but not their sensitive counterparts, are hypersensitive to cold‐induced cell death when exposed to temperatures below 4°C. The transfection of parental cells with a P‐gp‐expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P‐gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase‐3 at physiological temperature (37°C) in MDR cells compared with their parental cell line. Treatment with a caspase‐3 inhibitor partially rescues MDR leukemic cells from cold‐induced apoptosis, which suggests that the cell death mechanism may require caspase‐3 activity. Taken together, these findings demonstrate that P‐gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer. J. Cell. Biochem. 113: 1416–1425, 2012.