Patterns of Heterochromatin Transitions Linked to Changes in the Expression of Plasmodium falciparum Clonally Variant Genes.

The survival of malaria parasites in the changing human blood environment largely depends on their ability to alter gene expression by epigenetic mechanisms. The active state of Plasmodium falciparum clonally variant genes (CVGs) is associated with euchromatin characterized by the histone mark H3K9ac, whereas the silenced state is characterized by H3K9me3-based heterochromatin. Expression switches are linked to euchromatin-heterochromatin transitions, but these transitions have not been characterized for the majority of CVGs. To define the heterochromatin distribution patterns associated with the alternative transcriptional states of CVGs, we compared H3K9me3 occupancy at a genome-wide level among several parasite subclones of the same genetic background that differed in the transcriptional state of many CVGs. We found that de novo heterochromatin formation or the complete disruption of a heterochromatin domain is a relatively rare event, and for the majority of CVGs, expression switches can be explained by the expansion or retraction of heterochromatin domains. We identified different modalities of heterochromatin changes linked to transcriptional differences, but despite this complexity, heterochromatin distribution patterns generally enable the prediction of the transcriptional state of specific CVGs. We also found that in some subclones, several var genes were simultaneously in an active state. Furthermore, the heterochromatin levels in the putative regulatory region of the gdv1 antisense noncoding RNA, a regulator of sexual commitment, varied between parasite lines with different sexual conversion rates. IMPORTANCE The malaria parasite P. falciparum is responsible for more than half a million deaths every year. P. falciparum clonally variant genes (CVGs) mediate fundamental host-parasite interactions and play a key role in parasite adaptation to fluctuations in the conditions of the human host. The expression of CVGs is regulated at the epigenetic level by changes in the distribution of a type of chromatin called heterochromatin. Here, we describe at a genome-wide level the changes in the heterochromatin distribution associated with the different transcriptional states of CVGs. Our results also reveal a likely role for heterochromatin at a particular locus in determining the parasite investment in transmission to mosquitoes. Additionally, this data set will enable the prediction of the transcriptional state of CVGs from epigenomic data, which is important for the study of parasite adaptation to the conditions of the host in natural malaria infections.

Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum.

Human to vector transmission of malaria requires that some blood-stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here, we study this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually committed parasite stages that precede the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route in which PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon re-invasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Reanalysis of published single-cell RNA-sequencing (RNA-seq) data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle.

Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum.

Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt within hosts Plasmodium falciparum undergoes drastic switches in gene expression. To identify regulatory regions in the parasite genome, we performed genome-wide profiling of chromatin accessibility in two culture-adapted isogenic subclones at four developmental stages during the intraerythrocytic cycle by using the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs) localize preferentially at transcriptional start sites (TSSs). Chromatin accessibility by ATAC-seq is predictive of active transcription and of the levels of histone marks H3K9ac and H3K4me3. Our assay allows the identification of novel regulatory regions including TSS and enhancer-like elements. We show that the dynamics in the accessible chromatin profile matches temporal transcription during development. Motif analysis of stage-specific ATAC-seq sites predicts the in vivo binding sites and function of multiple ApiAP2 transcription factors. At last, the alternative expression states of some clonally variant genes (CVGs), including eba, phist, var and clag genes, associate with a differential ATAC-seq signal at their promoters. Altogether, this study identifies genome-wide regulatory regions likely to play an essential function in the developmental transitions and in CVG expression in P. falciparum.

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes.

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

A transcriptional switch underlies commitment to sexual development in malaria parasites.

The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci prone to spontaneous activation. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

A molecular switch in the efficiency of translation reinitiation controls expression of var2csa, a gene implicated in pregnancy-associated malaria.

Plasmodium falciparum malaria parasites export the protein PfEMP1 to the surface of infected erythrocytes, enabling them to adhere to receptors in the microvasculature and thereby avoid clearance by the spleen. The gene var2csa encodes the form of PfEMP1 that binds specifically within the placenta, causing pregnancy-associated malaria, and appears to not be expressed in the absence of a placenta. We previously described an upstream open reading frame (uORF) that is responsible for repression of translation of the downstream ORF (dORF) that encodes VAR2CSA, thus keeping the gene silent when parasites infect non-pregnant individuals. To elucidate the molecular mechanism by which this repression is overcome during pregnancy, we stably transformed parasites with reporter gene constructs designed to detect switches in the efficiency of dORF translation. We found that proper regulation of switching relies on two separate components, (i) active translation of the uORF and (ii) sequence-specific characteristics of the surrounding transcript, which together control the ability of the ribosome complex to reinitiate a second round of translation and thus express VAR2CSA. These results provide the first details of a molecular switch that allows parasites take advantage of the unique niche provided by the placenta.

CD14 and TLR4 mediate cytokine release promoted by electronegative LDL in monocytes.

AIMS: Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes. METHODS AND RESULTS: Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells. CONCLUSIONS: CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations.

Effect of statin and fibrate treatment on inflammation in type 2 diabetes. A randomized, cross-over study.

A randomized, crossover study compared the effects of atorvastatin, gemfibrozil and their combination on inflammatory markers in type 2 diabetes. C-reactive protein (CRP), lipoprotein-associated phospholipase A2 (Lp-PLA2), secretory phospholipase A2 (sPLA2), interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP1) and tumor necrosis factor α (TNFα) were measured. Both lipid-lowering drugs had positive, complementary and additive effects on inflammatory markers, which were closely related to baseline inflammatory status.

Immunochemical analysis of the electronegative LDL subfraction shows that abnormal N-terminal apolipoprotein B conformation is involved in increased binding to proteoglycans.

Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).

Proteomic analysis of electronegative low-density lipoprotein.

Low density lipoprotein is a heterogeneous group of lipoproteins that differs in lipid and protein composition. One copy of apolipoprotein (apo)B accounts for over 95% of the LDL protein, but the presence of minor proteins could disturb its biological behavior. Our aim was to study the content of minor proteins in LDL subfractions separated by anion exchange chromatography. Electropositive LDL [LDL(+)] is the native form, whereas electronegative LDL [LDL⁻] is a minor atherogenic fraction present in blood. LC-ESI MS/MS analysis of both LDL fractions identified up to 28 different proteins. Of these, 13 proteins, including apoB, were detected in all the analyzed samples. LDL⁻ showed a higher content of most minor proteins. Statistical analysis of proteomic data indicated that the content of apoE, apoA-I, apoC-III, apoA-II, apoD, apoF, and apoJ was higher in LDL⁻ than in LDL(+). Immunoturbidimetry, ELISA, or Western blot analysis confirmed these differences. ApoJ and apoF presented the highest difference between LDL(+) and LDL⁻ (>15-fold). In summary, the increased content of several apolipoproteins, and specifically of apoF and apoJ, could be related to the physicochemical characteristics of LDL⁻, such as apoB misfolding, aggregation, and abnormal lipid composition.

Receptores implicados en la inducción de citoquinas promovida por la LDL electronegativa en monocitos / Receptors involved in cytokine induction promoted by electronegative LDL in monocytes

Introducción La LDL electronegativa (LDL[−]) es una fracción minoritaria de la LDL total que se encuentra en circulación y presenta diferentes propiedades inflamatorias, siendo una de las más relevantes la inducción de citoquinas en células endoteliales y mononucleares. Sin embargo, no se conoce el mecanismo por el cual la LDL(−) ejerce su acción (..) (AU)

Introduction Elecronegative LDL (LDL(−)) is a small fraction of the total circulating LDL and has different inflammatory properties, one of the most important being the induction of cytokines in endotelial cells and monocytes. However, the mechanism by which LDL (−) exercises its action at cellular level is not known. The objective of this study was to evaluate the receptors involved in LDL (−) binding in monocytes, and how the liberation of cytokines (..) (AU)

Aggregated electronegative low density lipoprotein in human plasma shows a high tendency toward phospholipolysis and particle fusion.

Aggregation and fusion of lipoproteins trigger subendothelial retention of cholesterol, promoting atherosclerosis. The tendency of a lipoprotein to form fused particles is considered to be related to its atherogenic potential. We aimed to isolate and characterize aggregated and nonaggregated subfractions of LDL from human plasma, paying special attention to particle fusion mechanisms. Aggregated LDL was almost exclusively found in electronegative LDL (LDL(-)), a minor modified LDL subfraction, but not in native LDL (LDL(+)). The main difference between aggregated (agLDL(-)) and nonaggregated LDL(-) (nagLDL(-)) was a 6-fold increased phospholipase C-like activity in agLDL(-). agLDL(-) promoted the aggregation of LDL(+) and nagLDL(-). Lipoprotein fusion induced by α-chymotrypsin proteolysis was monitored by NMR and visualized by transmission electron microscopy. Particle fusion kinetics was much faster in agLDL(-) than in nagLDL(-) or LDL(+). NMR and chromatographic analysis revealed a rapid and massive phospholipid degradation in agLDL(-) but not in nagLDL(-) or LDL(+). Choline-containing phospholipids were extensively degraded, and ceramide, diacylglycerol, monoacylglycerol, and phosphorylcholine were the main products generated, suggesting the involvement of phospholipase C-like activity. The properties of agLDL(-) suggest that this subfraction plays a major role in atherogenesis by triggering lipoprotein fusion and cholesterol accumulation in the arterial wall.

HDL and electronegative LDL exchange anti- and pro-inflammatory properties.

Electronegative LDL [LDL(-)] is a minor modified LDL subfraction present in blood with inflammatory effects. One of the antiatherogenic properties of HDL is the inhibition of the deleterious effects of in vitro modified LDL. However, the effect of HDL on the inflammatory activity of LDL(-) isolated from plasma is unknown. We aimed to assess the putative protective role of HDL against the cytokine released induced in monocytes by LDL(-). Our results showed that LDL(-) cytokine release was inhibited when LDL(-) was coincubated with HDL and human monocytes and also when LDL(-) was preincubated with HDL and reisolated prior to cell incubation. The addition of apoliprotein (apo)AI instead of HDL reproduced the protective behavior of HDL. HDL preincubated with LDL(-) promoted greater cytokine release than native HDL. Incubation of LDL(-) with HDL decreased the electronegative charge, phospholipase C-like activity, susceptibility to aggregation and nonesterified fatty acid (NEFA) content of LDL(-), whereas these properties increased in HDL. NEFA content in LDL appeared to be related to cytokine production because NEFA-enriched LDL induced cytokine release. HDL, at least in part through apoAI, inhibits phospholipase-C activity and cytokine release in monocytes, thereby counteracting the inflammatory effect of LDL(-). In turn, HDL acquires these properties and becomes inflammatory.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles.

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Electronegative LDL induces Fas and modifies gene expression in mononuclear cells.

Electronegative LDL (LDL(-)) is a minor modified LDL subfraction that promotes cytokine release by human mononuclear cells. The aim of the current study was to evaluate changes in gene expression induced by LDL(-) versus native LDL in lymphocytes and monocytes. Therefore, mononuclear cells were incubated with these LDL subfractions and their effects on expression in human whole genome were analyzed by gene array. Differential expression of the genes was quantified by real-time RT-PCR. LDL(-) altered the gene expression pattern, particularly of inflammatory genes. LDL(-) down-regulated CD36 and colony-stimulating factor 1 receptor (CSF1R) genes and up-regulated Fas expression and Fas protein on cellular membrane. LDL(-) seemed to promote the alterations in these genes by activation of NF-kB and inhibition of AP1 and PPARG. In conclusion, LDL(-) induced changes in gene expression in monocytes and lymphocytes. Fas up-regulation suggests a proinflammatory action; however, CSF1R and CD36 down-regulation could decrease monocyte differentiation and activation. Therefore, LDL(-) promoted not only inflammatory effects but also counteracting actions in circulating mononuclear cells.

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level.

Electronegative LDL [LDL(-)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(-) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(-) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(-) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(-) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(-) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(-) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression.

Novel phospholipolytic activities associated with electronegative low-density lipoprotein are involved in increased self-aggregation.

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.

Pro-inflammatory action of LDL(-) on mononuclear cells is counteracted by increased IL10 production.

OBJECTIVE: LDL(-) is a minor LDL subfraction that induces inflammatory factor release by endothelial cells. Since LDL(-) is present in plasma, its interaction with leucocytes, a cell type involved in atherosclerosis phenomena, is feasible; therefore, the aim of the current study was to evaluate LDL(-) effect on lymphocytes and monocytes isolated from human plasma. METHODS AND RESULTS: Mononuclear cells were incubated with LDL(+) and LDL(-) and expression and release of several inflammatory mediators were analyzed by protein membrane assay, ELISA and real-time RT-PCR. LDL(-) induced a significantly increased production versus LDL(+) in MCP1, GRObeta, GROgamma, IL6, IL8 and IL10 in monocytes as well as in lymphocytes. These induced molecules are inflammatory, except for IL10 which is considered an anti-inflammatory cytokine. Therefore, the role of IL10 was evaluated in experiments where exogenous IL10 or antibodies anti-IL10 or anti-IL10 receptor were added. IL10 addition diminished the release of the other factors induced by LDL(-) near to basal production both at protein and RNA level. In contrast, the antibody anti-IL10 increased inflammatory cytokine release around two-fold, whereas the antibody anti-IL10 receptor produced a lower effect. CONCLUSIONS: LDL(-) promoted inflammatory cytokine production in leucocytes; however, it also induced IL10 that minimized this effect. Therefore, IL10 developed a significant role in counteracting the LDL(-) inflammatory action.

Dietary phytosterols modulate T-helper immune response but do not induce apparent anti-inflammatory effects in a mouse model of acute, aseptic inflammation.

Although most studies have focused on the cholesterol-lowering activity of phytosterols, other biological actions have been ascribed to these plant sterol compounds, one of which is a potential immune modulatory effect. To gain insight into this issue, we used a mouse model of acute, aseptic inflammation induced by a single subcutaneous turpentine injection. Hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)) mice, fed with or without a 2% phytosterol supplement, were treated with turpentine or saline and euthanized 48 h later. No differences were observed in spleen lymphocyte subsets between phytosterol- and control-fed apoE(-/-) mice. However, cultured spleen lymphocytes of apoE(-/-) mice fed with phytosterols and treated with turpentine showed increased IL-2 and IFN-gamma secretion (T-helper type1, Th1 lymphocyte cytokines) compared with turpentine-treated, control-fed animals. In contrast, there was no change in Th2 cytokines IL-4 and IL-10. Phytosterols also inhibit intestinal cholesterol absorption in wild-type C57BL/6J mice but, in this case, without decreasing plasma cholesterol. Spleen lymphocytes of turpentine-treated C57BL/6J mice fed with phytosterols also showed increased IL-2 production, but IFN-gamma, IL-4 and IL-10 production was unchanged. The Th1/Th2 ratio was significantly increased both in phytosterol-fed apoE(-/-) and C57BL/6J mice. We conclude that phytosterols modulate the T-helper immune response in vivo, in part independently of their hypocholesterolemic effect in a setting of acute, aseptic inflammation. Further study of phytosterol effects on immune-based diseases characterized by an exacerbated Th2 response is thus of interest.

Inducción de citocinas por efecto de la LDL electronegativa en monocitos y linfocitos / Cytokine induction by the effect of electronegative LDL on monocytes and lymphocytes

Introducción y objetivo. La lipoproteína de baja densidad (LDL) electronegativa (LDL[­]) es una fracción minoritaria modificada de la LDL presente en circulación plasmática con propiedades aterogénicas e inflamatorias. Se ha descrito que la LDL(­) induce, en cultivos de células endoteliales, la producción de diversos mediadores de la inflamación, así como apoptosis y/o citotoxicidad. Sin embargo, no se ha evaluado previamente su efecto sobre otros tipos celulares, como células en circulación, sobre las que es más posible su interacción durante la circulación plasmática. Por ello, el objetivo fue analizar las citocinas, los factores de crecimiento y otras moléculas proinflamatorias implicadas en el proceso arteriosclerótico que son inducidos por la LDL(­) en monocitos y linfocitos aislados de sangre periférica. Material y métodos. La LDL total fue aislada mediante ultracentrifugación y se separaron las fracciones electropositiva (LDL[+] o LDL nativa) y electronegativa por cromatografía de intercambio aniónico. Se incubaron monocitos y linfocitos aislados de voluntarios normolipémicos con las LDL durante 20 h; se utilizó lipopolisacárido (LPS) como control positivo. Se valoró en los sobrenadantes celulares la producción de 42 mediadores inflamatorios relacionados con la arteriosclerosis mediante un array de proteínas. Se cuantificó por ELISA las proteínas inducidas y mediante PCR a tiempo real se evaluó si su inducción era transcripcional. Resultados y conclusión. La LDL(­) indujo una mayor liberación y expresión, comparada con la LDL(+), de interleucina (IL) 6, IL-8, IL-10, MCP-1, growth-related oncogene (GRO) (GROß y GRO*, tanto en monocitos como en linfocitos. Así pues, la LDL(­) es capaz de inducir en células mononucleares la producción de factores implicados en el proceso inflamatorio que actúan en diferentes estadios de la lesión arteriosclerótica (AU)

Introduction and objective. Electronegative low-density lipoprotein (LDL(­)) is a minor modified LDL fraction present in plasma with atherogenic and inflammatory properties. In cultured endothelial cells, LDL(­) has been reported to induce production of several mediators of inflammation, as well as apoptosis and/or cytotoxicity. However, the effect of LDL(­) on other cell types, such as white blood cells ­with which its interaction is more feasible during plasma circulation­ has not previously been evaluated. Therefore, the objective of this study was to analyze the cytokines, growth factors and other proinflammatory molecules involved in the atherosclerotic process that are induced by LDL(­) in monocytes and lymphocytes isolated from peripheral blood. Material and methods. Total LDL was isolated by ultracentrifugation and electropositive (LDL(+) or native LDL) and electronegative fractions were separated by anion-exchange chromatography. Monocytes and lymphocytes isolated from normolipemic volunteers were incubated with LDLs for 20 h; lipopolysaccharide was used as positive control. The production of 42 inflammatory mediators related to atherosclerosis was determined in cell supernatants by protein array. Induced proteins were quantified by ELISA assays. The question of whether induction was transcriptional was determined by real time-polymerase chain reaction. Results and conclusion. LDL(­) induced greater release and expression of interleukin (IL)-6, IL-8, IL-10, MCP-1, GROß and GRO* than did LDL(+) in monocytes and lymphocytes. Therefore, in mononuclear cells, LDL(­) is able to induce production of several factors involved in the inflammatory process that act on different stages of the atherosclerotic lesion (AU)