Sepsis is a major challenge in medicine. It is a common and frequently fatal condition, despite the use of specific antibiotics, aggressive operative intervention, nutritional support, and life support therapies. The incidence of sepsis is still increasing, with mortality rates between 30% and 70% (1). There is now consensus that sepsis is accompanied by the inability to properly regulate the inflammatory response. Therefore, more efficient strategies to modulate the inflammation in sepsis are needed (2-4).
Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction between the host immune system and the bacteria or bacterial wall components. One of the most known and studied of these pathogen-derived molecules is lipopolysaccharide (LPS), the main glycolipid component of the outer membrane of gram-negative bacteria. Recognition of LPS by host cells initiates an inflammatory reaction that can reproduce many features of the gram-negative infection and, for this reason, LPS is widely used as an inducer of immune and inflammatory responses (5).
The inflammatory and immune responses may be modified by dietary lipids (6,7). An epidemiological study developed with Greenland Eskimos revealed a low incidence of inflammatory and autoimmune diseases associated with diets containing fish oil, rich in n-3 fatty acids (8). Similarly, a low incidence of inflammatory and coronary heart diseases has been observed among people using the Mediterranean diet in which the primary lipid source is olive oil rich in n-9 fatty acids (9). Moreover, attempts to improve the clinical course of critically ill patients through the modulation of dietary fatty acids showed encouraging results (7).
Fatty acids are important as sources of energy, structural components of membranes, signaling molecules, and precursors of eicosanoids. The mediators derived from polyunsaturated fatty acids released from membrane phospholipids seem to modulate immune responses and regulate the production of cytokines during inflammatory processes (6,7,10-12). Of interest, dietary fatty acids have been demonstrated to modify the composition of cellular lipid domains (6). Accumulating evidence points to an important role of lipid domains, including lipid rafts and caveolae from plasma membrane and from intracellular lipid-rich domains, such as lipid bodies (LB), in cellular signaling and innate immunity (6,13). Leukocyte LB function as an intracellular store of localization of arachidonic acid and of enzymes related to eicosanoid synthesis, including mitogen-activated protein kinase, phospholipase A2, cyclooxygenase, and lipooxygenase (14-18). Recently, we demonstrated that LB formed in leukocytes after LPS stimulation are intracellular sites for paracrine eicosanoid synthesis, increasing in leukocytes from patients with sepsis in comparison with healthy subjects (19). LB are rapidly inducible and can be modulated by exogenous fatty acid administration. Unsaturated fatty acids such as arachidonic acid (20:4n-6) stimulate LB formation in a dose-dependent manner, whereas LB are not induced in the presence of saturated fatty acids, indicating that LB formation depends on the number of double bonds in the fatty acid (14-16).
In the present study, we examined whether diets of different fatty acid composition could interfere with the inflammatory process observed during LPS-induced shock.
MATERIALS AND METHODS
LPS from Escherichia coli (serotype 0111:B4) was obtained from Sigma (St. Louis, MO). Osmium tetroxide (OsO4) was obtained from Electron Microscopy Science (Fort Washington, PA).
Animals and diets
Female C57Bl/6J mice weighing 20 g were obtained from FIOCRUZ (Rio de Janeiro, Brazil). Mice were housed in a room at 25°C, with an alternating light/dark cycle of 12 h and free access to pelleted diet and water. All manipulations with animals were in accordance to guidelines of the Council for International Organization of Medical Sciences and the Brazilian College for Animal Experimentation. The experiments described in this work received prior approval from the Oswaldo Cruz Foundation Animal Welfare Committee. The diets used included a commercial chow (CC) for laboratory rodents (Purina, São Paulo, Brazil) and four experimental diets containing 7% (w/w) of commercial canola oil (CO), sesame oil (SeO), soybean oil (SO), or virgin olive oil (OO). These four experimental diets were prepared according to the American Institute of Nutrition (AIN-93) for laboratory rodent diets, containing vitamin E and other antioxidant compounds (20). These oils were chosen according to the content of oleic, linoleic, and linolenic acids. All diets were stored at 4°C before use and were provided fresh to the animals every 2 days. Mice were killed between 8:30 and 9:30 a.m. under CO2 atmosphere.
Fatty acid composition of the oils and diets used
The oils used in chow preparation were analyzed by capillary gas chromatography for detection of fatty acids. Oleic acid (18:1n-9) is the predominant fatty acid in CO and OO. SeO and SO are rich in linoleic acid (18:2n-6). These data are similar to those described in the literature for all oils used (21), showing no loss of fatty acid content during diet preparation (70°C for 12 h) and that the 18:2n-6/18:3n-3 ratio was not altered. Fatty acid composition of each diet was analyzed by capillary gas chromatography. Briefly, all samples were directly transesterified (22) and the resulting fatty acid methyl esters in hexane were injected into a gas chromatograph (GC-14B; Shimadzu, Columbia, MD) with split injection. Separation was achieved with an Omegawax-320 capillary column (Supelco, Bellefonte, PA) using temperature gradient elution, and fatty acids were identified after comparison of retention times of analytes with those of certified standards (Sigma). Fatty acid contents are expressed as weight percentages (wt %) of total fatty acids. Table 1 summarizes the data obtained.
Endotoxic shock and peritoneal lavage
Mice were fed with different diets for 6 weeks. After this period, endotoxic shock was induced in mice with an intraperitoneal injection of LPS (400 μg/cavity) to a final volume of 200 μL. Control animals received sterile saline. Ninety minutes or 6 h after injection, mice were killed and the peritoneal cavity was opened and rinsed with 5 mL of cold saline. Total leukocyte counts were performed in a Neubauer chamber, and differential leukocyte counts were performed on a Cytospin 3 (Shandon, Pittsburgh, PA) spots stained with Diff-Qwick (Shandon). Survival of mice fed with CC or test diets and injected with saline or LPS was determined daily for 7 days in separate groups of 10 animals for each diet condition.
LB staining and enumeration
LB were evaluated in leukocytes (106 cells/mL) collected in peritoneal fluid 90 min or 6 h after saline or LPS injection. Leukocytes on cytospin spots (while still moist) were fixed in 3.7% formaldehyde prepared in Ca2+/Mg2+-free Hanks' balanced salt solution (HBSS, pH 7.4), rinsed in 0.1 M cacodylate buffer (pH 7.4), stained with 1.5% OsO4 for 30 min, rinsed in distilled water, immersed in 1.0% thiocarbohydrazide for 5 min, rinsed in 0.1 M cacodylate buffer, restained in 1.5% OsO4 for 5 min, rinsed in distilled water, and then dried and mounted. The morphology of fixed cells was observed, and LB were enumerated by light microscopy with a 100× objective lens in 50 consecutively scanned leukocytes.
Leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) assays
Lipid mediators were measured directly in the supernatant from cell-free peritoneal lavage obtained 90 min after saline or LPS injection or 6 h after preincubation of peritoneal leukocytes (106 cells/mL) in HBSS containing Ca2+ and Mg2+ and stimulation with 0.5 μM A23187 for 15 min. Reactions were stopped on ice, and samples were centrifuged at 500g for 10 min at 4°C. LTB4 and PGE2 were assayed in the cell-free supernatant by enzyme-linked immunoassay (EIA) according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI).
TNF-α from plasma and IL-6, IL-10, and MCP-1 from peritoneal fluid collected 90 min after saline or LPS injection were measured by enzyme-linked immunoabsorbant assay (ELISA) according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).
Results are expressed as means ± SEM and were analyzed by means of one-way analysis of variance (ANOVA) followed by the Bonferroni student's t test (Sigma Stat, v 1.0) with the level of significance set at P ≤ 0.05. Survival curves were generated with Prism computer software (GraphPad, San Diego, CA), and comparisons between curves were made by the Mantel-Cox log-rank test.
Body weight and food intake in mice fed with different diets
The intake of CC or the four tested diets led to identical gains in body weight (Fig. 1A). The food intake was similar for each of the four test diets (Fig. 1B), and was lower than in mice fed with CC. Energy consumption was also lower for the test diets. This may be related to the composition of CC that stimulates mice to increase food intake.
Survival rate after LPS-induced endotoxic shock
After 6 weeks on CC or a test diet, animals received an i.p. injection of saline or 400 μg of LPS in the peritoneal cavity. Six hours after LPS, mice clearly displayed the symptoms of endotoxic shock, such as decreased motor activities, ruffled fur, diarrhea, and ocular exudates, as described in Reference 23. Figure 2 shows that 24 h after LPS, 80% of mice fed with SO died. In 48 h, 70% to 80% of mice fed with CC, SeO, or CO died. However, mice fed with a diet enriched with OO were resistant to endotoxic shock, with a 60% survival rate at 168 h. Saline-injected mice had 100% survival rate for the time evaluated (data not shown).
Cytokine levels in LPS-induced shock
To investigate the effect of diets on the cytokine profile, IL-6, IL-10, and MCP-1 levels in peritoneal lavage and TNF-α levels in plasma were evaluated 90 min after i.p. injection of saline or LPS (400 μg). As shown in Figure 3A, LPS induced an increase in IL-6 levels with all diets used. Although IL-6 levels appeared to be lower in CO- and OO-fed mice, the differences were not significant. LPS also increased IL-10 concentrations in relation to saline-injected mice, regardless of diet used (Fig. 3B). In contrast, Figure 3C shows that the increment in MCP-1 concentrations observed after the endotoxic shock for CC, CO, SeO, and SO diets almost disappeared in OO-fed mice. In plasma, LPS induced significantly higher concentrations of TNF-α in mice fed with CC, SeO, or SO than in mice fed with CO or OO (Fig. 3D).
Lipid mediators 90 min after LPS-induced shock
The effects of the different dietary fatty acids on LB formation and on lipid mediators were analyzed 90 min after LPS-induced shock. As shown in Figure 4A, there is an increase in the number of LB present in neutrophils after LPS injection in mice fed with CC, CO, SeO, and SO in relation to saline-injected mice. The increase in LB was not observed in mice fed with an OO-enriched diet. Figure 4 shows that PGE2 (Fig. 4B) and LTB4 (Fig. 4C) concentrations increased after LPS injection, but were significantly lower in mice fed with OO than in mice fed with the other diets.
Neutrophil influx in LPS-induced shock
To investigate whether diet could modify neutrophil influx, a hallmark of acute inflammatory processes, cells were counted in peritoneal lavage 90 min after endotoxic shock. As shown in Figure 5, LPS induced an important influx of neutrophils into the peritoneal cavity, and this was significantly lower in mice fed with OO than for the other diets. Similar results were obtained when neutrophil numbers were evaluated 6 h after endotoxic shock (data not shown). Mononuclear leukocytes in peritoneal lavage were also counted, and no difference was found among dietary groups (data not shown).
Lipid mediators 6 h after LPS-induced shock
The production of lipid mediators by cells recovered from the peritoneal cavity 6 h after LPS-induced shock was also investigated. As shown in Figure 6, and confirming our previous results (17), in peritoneal leukocytes primed by a previous contact with LPS, there was a significant increase in the number of LB. Interestingly, the increase in LB numbers was significantly reduced in leukocytes obtained from animals fed with OO. Stimulation of peritoneal cells in vitro with 0.5 μM of the calcium ionophore, A23187, induced a marked production of PGE2 and LTB4 in CC-, CO-, SeO-, and SO-fed mice in relation to saline-injected mice. In contrast, in mice fed with OO, there was a significant reduction of PGE2 and LTB4 production.
This study shows for the first time that mice fed with OO have improved survival to endotoxic shock and that the protective mechanism may involve modulation of LB formation and decreased production of inflammatory mediators.
The severity of sepsis can be influenced by the nutritional status of humans and animals. The beneficial effect of consuming OO on endotoxic shock observed in the present study was also detected in mice fed with diets containing SeO, followed by cecal ligation and puncture (CLP), and in guinea pigs fed with fish oil, followed by endotoxin injection (24,25). Such an increase in survival may be explained by the ability of dietary lipids to modulate the production of cytokines and lipid mediators in response to bacterial endotoxin (7,11). Consumption of OO over 6 weeks led to lower levels of IL-6 and TNF-α, similar to what was observed with CO, suggesting that diets containing lipids rich in oleic acid (n-9) may modulate synthesis of proinflammatory cytokines induced by LPS. However, only in mice fed with OO was MCP-1 production almost completely blocked, suggesting that other components of this oil may also exert an important role in modulating cytokines involved in the inflammatory response. Similarly, LB formation was reduced in mice fed with OO, and this was accompanied by a reduction in the synthesis of PGE2 and total blockage of LTB4 production.
Extra virgin and virgin OO are unique among dietary lipids in that they are obtained from seeds without any extraction by solvents; thus, the chemical composition of all natural components in the seeds is retained (26). It has been demonstrated that minor components of OO, such as oleuropein, tyrosol, and other phenolic compounds, have anti-eicosanoid and antioxidant effects in leukocytes (26,27). Administration of OO to healthy volunteers over 3 weeks provided effective protection against low-density lipoprotein oxidation, decreasing oxidative stress markers, and increasing high-density lipoprotein cholesterol levels in the peripheral blood (28).
Curiously, the synthesis of LTB4 and MCP-1 triggered by the endotoxic shock was almost completely abolished with the consumption of OO. This was accompanied by a significant decrease in neutrophil influx into the peritoneal cavity and a decrease in the formation of LB in leukocytes. LB are nonmembrane-bound lipid-rich cytoplasmic inclusions that characteristically increase in number during inflammatory conditions, including adult respiratory distress syndrome and sepsis (19,29). Previously, it has been demonstrated that LB in leukocytes are compartmentalization sites for arachidonic acid, phospholipase A2, and eicosanoid-forming enzymes (14-18), and they are involved in the enhanced production of prostaglandins and leukotrienes (15-19,30). These findings are consistent with our present results showing that an increase in LB numbers correlates with increased LTB4 and PGE2 release by these cells after activation with submaximal concentrations of A23187. Conversely, agents that inhibited LB formation in vitro inhibited the priming response for enhanced eicosanoid release (15,16), which may explain the results obtained with the consumption of dietary virgin oil. LTB4 is a potent chemotactic agent for neutrophils and it has been shown to play an important role in the accumulation of neutrophils in experimental models of sepsis (31,32). MCP-1 stimulates the production of LTB4 from peritoneal macrophages in vitro and, in turn, a specific LTB4 receptor antagonist inhibited CLP-induced neutrophil and macrophage influx that was accompanied by a reduced level of MCP-1 in peritoneum (32). Moreover, the lack of MCP-1 in knockout mice abolished leukocyte accumulation and reduced the level of LTB4 (33). Elevated levels of MCP-1 have been detected in plasma of patients with peritonitis (34), as well as after administration of endotoxin in animals or human volunteers (35,36). Elevated MCP-1 levels observed in mice consuming diets containing CO, SeO, or SO may serve as an indirect mediator to attract neutrophils via the production of LTB4 after LPS, suggesting a cross-talk between these mediators during LPS-endotoxic shock, as demonstrated before for septic peritonitis (31) and pleurisy (33).
Analysis of these data leads us to speculate that the beneficial effect of dietary virgin OO against endotoxic shock may be associated with inhibition of LB formation and MCP-1 production with an impact on the generation of LTB4 and on recruitment of activated leukocytes. There is also a possibility that after 6 weeks, consumption of virgin OO led to a modification of cellular lipid domains such as in the composition of lipid rafts, or promoted the production of 5-series of leukotrienes, which are 10- to 100-fold less potent than those from 4-series in developing the inflammatory process (7,37). These and other possibilities to explain the beneficial effect of virgin OO on septic shock are now under investigation.
The authors thank Dr. Martha M. Sorenson for her careful revision of this manuscript.
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