Eicosapentaenoic (EPA) and Docosahexaenoic (DHA) Acid Differentially Modulate Rat Neutrophil Function In Vitro
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- Paschoal, V.A., Vinolo, M.A.R., Crisma, A.R. et al. Lipids (2013) 48: 93. doi:10.1007/s11745-012-3726-6
Fish oils are used as therapeutic agents in chronic inflammatory diseases. The omega-3 fatty acids (FA) found in these oils are mainly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The anti-inflammatory properties of fish oils are attributed to both omega-3 fatty acids. However, it is unknown whether such effects are due to either EPA or DHA. In this study, the effects of EPA and DHA on rat neutrophil function in vitro were compared. Both EPA and DHA increased the production of H2O2 when cells were stimulated or not with lipopolysaccharides (LPS). However, EPA was more potent than DHA in triggering an increase in superoxide release by cells in the basal condition or when stimulated with phorbol myristate acetate (PMA) or zymosan. Only DHA increased the phagocytic capacity and fungicidal activity of neutrophils. Both FA increased the release of tumor necrosis factor-α (TNF-α) in nonstimulated cells, but only EPA increased the production of cytokine-inducing neutrophil chemoattractant-2 (CINC-2) in the absence or presence of LPS, whereas production of interleukin-1 beta (IL-1β) was only increased by DHA in the presence of LPS. In addition, there was no alteration in the production of nitric oxide. In conclusion, we show herein that EPA and DHA can differently modulate aspects of the neutrophil response, which may be relevant for the development of therapies rich in one or other FA depending on the effect required.
Keywordsn-3 Fatty acidsReactive oxygen speciesCytokinesCINC-2TNF-αIL-1βPhagocytosisFungicidal activity
Analysis of variance
Cytokine-inducing neutrophil chemoattractant-2
Enzyme-linked immunosorbent assay
Fetal bovine serum
Reduced nicotinamide adenine dinucleotide phosphate
Nucleotide-binding protein oligomerization domain
Phorbol myristate acetate
Polyunsaturated fatty acid
Reactive nitrogen species
Reactive oxygen species
Roswell park memorial institute
Tumor necrosis factor-α
Neutrophils play a key role in inflammatory and immune responses. These cells are the first to reach the inflammatory site, where they internalize, kill, and digest pathogens. In this regard, several mechanisms including the release of the content of their granules and the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and oxidized halides are activated and act together to eliminate invading microorganisms . These cells also present receptors including toll-like receptors (TLRs) and nucleotide-binding protein oligomerization domains (NLRs) in their cell membrane and cytoplasm for detection of pathogen-associated molecular patterns. Once activated, these receptors activate several intracellular pathways that lead to the production of proinflammatory cytokines, such as TNF-α and IL-1β, chemokines, including CXCL3/CINC-2, and lipid mediators [2–6].
Fish oil, rich in omega-3 PUFA, modulates immune and inflammatory responses. Beneficial anti-inflammatory effects of these oils have been demonstrated in several pathological conditions including type II diabetes mellitus, hypertriglyceridemia , and cardiovascular disease [8, 9]. It has been shown that intake of oils rich in omega-3 FA modifies several aspects of leukocyte function including lymphocyte proliferation [10, 11], cytokine synthesis, antibody production , and expression of membrane surface proteins . Regarding neutrophils, previous studies have shown that pretreatment with omega-3 FA reduces the adherence and tissue infiltration of these cells  and the intake of oil rich in EPA causes a dose-dependent decrease in neutrophil respiratory burst . The proposed mechanisms for these effects include changes in FA composition of cell membranes. Incorporation of omega-3 FA affects membrane fluidity, composition of lipid rafts, distribution and function of receptors , and production of second messengers , such as phosphatidylinositol triphosphate (IP3) and diacylglycerol , and direct effects through surface and intracellular “fatty acid receptors” .
As mentioned above, fish oil has immunomodulatory effects; however, it is unknown whether these effects are due to either EPA or DHA or a combination of both omega-3 fatty acids. Therefore, the aim of this study is to compare the effects of EPA and DHA on the function of rat neutrophils in vitro.
Materials and Methods
Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, phorbol myristate acetate (PMA), sodium bicarbonate, l-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), lucigenin, horseradish peroxidase (HRP) type II, penicillin, streptomycin, lipopolysaccharide (LPS—Escherichia coli strain 0111:B4), and oyster glycogen were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI) 1640 culture medium, fetal bovine serum (FBS), and Amplex Ultrared® were purchased from Invitrogen (Carlsbad, CA, USA). Stock solutions of the fatty acids were prepared in ethanol as described in a previous study from the group .
Male Wistar rats weighing 200 ± 20 g were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo. Rats were maintained at 23 °C under a light:dark cycle of 12:12 h. Animals received standard diet and water ad libitum.
The procedures used in this study were approved by the Ethics Committee on Animal Experimentation of the Institute of Biomedical Sciences, University of São Paulo (protocol no. 103).
Isolation of Neutrophils
Rat neutrophils were obtained by peritoneal lavage with 50 mL phosphate-buffered saline (PBS), 3 h after i.p. injection of 10 mL freshly prepared 1 % (w/v) oyster glycogen solution in sterile PBS. By this procedure, which has been previously published by our group [21–23], more than 90 % of the cells obtained were neutrophils.
Fatty Acid Cytotoxicity as Determined by Plasma Membrane Integrity and DNA Fragmentation
Before performing the functional assays with neutrophils, membrane integrity and deoxyribonucleic acid (DNA) fragmentation of cells incubated with different concentrations of FA for 4 or 18 h were evaluated by flow cytometry using propidium iodide , as described in previous studies of our group .
Determination of Reactive Oxygen Species (ROS)
ROS production by neutrophils was analyzed using two methods: the fluorimetric method using Amplex Ultrared®, and lucigen-amplified chemiluminescence assay. For the assay with Amplex Ultrared®, 96-well plates were prepared containing 5 × 105 cells in 250 mL PBS supplemented with 1 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose and 10 % FBS, Amplex Ultrared® (50 mM), and HRP type II (0.1 U/mL) in the absence or presence of EPA or DHA (12.5, 25, 50, 100, and 150 μM) and PMA (10 nM), resulting in 200 μL final volume. The assays were performed in duplicate. The plate was incubated in the dark at 37 °C in a humid atmosphere for 1 h, followed by reading by fluorometer (Synergy HT multimode microplate reader) at 530 nm excitation/590 nm emission . Results are expressed as fluorescence over 1 h by subtracting the blank reading (same reagents, but without cells).
For the lucigen-amplified chemiluminescence assay, 96-well white plates were prepared containing 5 × 105 cells in 250 mL PBS with 1 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose and 10 % FBS. Production of O2·− was monitored after addition of lucigenin (1 mM) by luminometer (EG&G Berthold microplate luminometer; Berthold Technologies, Germany) for 60 min [21, 27]. Negative controls containing all components, except the cells, were used to assess whether there was any interference in the determinations. In these experiments, positive control containing PMA or opsonized zymosan (zymosan A from Saccharomyces cerevisiae; Sigma) was also included. Analysis of the results was performed using the integral of the curve areas obtained for each sample.
Evaluation of Phagocytosis and Fungicidal Activity
Candida albicans (ATCC Y-537) was kindly provided by Prof. Dr. Sandro Rogerio de Almeida (Mycology Laboratory, Department of Clinical Analyses and Toxicology, Faculty of Pharmaceutical Sciences, University of São Paulo) in Sabouraud agar (Difco® BD, Le Pont de Claix, France, Europe). A suspension of C. albicans was obtained with PBS, and the concentration was adjusted to 2.5 × 106/mL cells. This suspension was opsonized by incubation in the presence of rat serum for 30 min at 37 °C.
Opsonized C. albicans (1 yeast/5 cells) was added to cells in sterile PBS supplemented with 1 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose and 10 % FBS, and the samples were incubated in the presence of vehicle, EPA or DHA at 37 °C with gentle shaking for 40 min. An aliquot of this suspension was adhered to glass cover slips using a cytocentrifuge. Cover slips were stained with May-Grünwald and Giemsa solutions (Sigma Chemical Co., St. Louis, MO, USA) . For analysis of phagocytosis, at least 100 cells were counted. Cells that had at least one Candida albicans internalized were regarded as having performed phagocytosis [29–31]. Fungicidal activity was evaluated by counting at least 100 cells that phagocytized C. albicans, and the result was expressed by a score according to criteria established by Corazzini , as previously described in another study of our group .
Determination of Nitric Oxide (NO) and Cytokines
Neutrophils (2.5 × 106 cells/mL) were incubated in RPMI 1640 culture medium containing 10 % FBS in the absence or presence of LPS (5 μg/mL E. coli 0111:B4; Sigma) and various concentrations of EPA or DHA for 18 h, as previously published by our group . Cell supernatant was collected at the end of the incubation period and stored at −80 °C until measurement of nitrite and cytokines. Nitrite was quantified using Griess reagent . Determination of TNF-α, IL-1β, and CINC-2/CXCL3 produced in vitro by neutrophils was performed by enzyme-linked immunosorbent assay (ELISA) using DuoSet kits from R&D Systems (Minneapolis, MN, USA) according to the procedures provided by the manufacturer.
The results are expressed as mean ± standard error of mean (SEM). The program Prism 5.02 (Graph Pad Software, Inc., San Diego, CA, USA) was used to perform the statistical analysis. Comparisons between groups were performed by one-way analysis of variance (ANOVA) and Dunnett post-test. For some comparisons, two-way ANOVA with Bonferroni post-test was used. Differences were considered significant when P < 0.05.
Cytotoxicity of DHA and EPA on Neutrophils
Before performing the functional assays with neutrophils, membrane integrity and DNA fragmentation of cells incubated with different concentrations of FA for 4 or 18 h were evaluated by flow cytometry using propidium iodide. After 4 h, loss of plasma membrane integrity was observed with 250 μM DHA and 300 μM EPA, and DNA fragmentation at 150 μM DHA and 200 μM EPA (Supplementary Fig. 1). After 18 h of incubation, 150 μM EPA and 75 μM DHA induced significant loss of membrane integrity. DNA fragmentation was observed at EPA 100 μM and DHA 75 μM (Supplementary Fig. 2). Based on these results, we established the nontoxic concentrations of FA to be used in the neutrophil function assays. For the evaluation of ROS and phagocytosis/killing of C. albicans (<4 h cell incubation), the maximal concentrations of EPA and DHA used were 150 and 100 μM, respectively. Incubation of neutrophils for longer periods (evaluation of cytokines and NO) was performed using a maximum concentration of 50 μM.
Production of ROS
The effect of EPA and DHA on ROS production by neutrophils was investigated using two methods: the fluorimetric method using Amplex Ultrared®, which detects mostly intra- and extracellular hydrogen peroxide , and lucigen-amplified chemiluminescence assay, a method that measures mainly extracellular superoxide (O2·−) [27, 35].
Production of Hydrogen Peroxide (H2O2)
Production of Superoxide Onion (O2·−)
Phagocytosis and Fungicidal Activity
Production of Cytokines and NO by Neutrophils
Neutrophils were incubated with different concentrations of EPA and DHA for 18 h in the presence or absence of LPS. The concentration of proinflammatory cytokines TNF-α and IL-1β, the chemokine CINC-2/CXCL3, and NO was then measured in the cell supernatant.
Summary of the effects observed in this study
Cytotoxicity (18 h)
Membrane integrity without stimulus
DNA fragmentation without stimulus
Membrane integrity with LPS
DNA fragmentation with LPS
Phagocytosed (40 min)
Fungicidal activity (40 min)
↑ 25 and ↓ 50
Previous studies have shown that ingestion of omega-3 FA-rich oils affects the function of cells, including production of inflammatory mediators (cytokines, RNS, ROS, eicosanoids), neutrophil recruitment (expression of adhesion molecules and chemokines), and effector function (phagocytosis and microorganism killing). However, it is unknown whether the effects of omega-3 FA on neutrophils are due to either EPA or DHA. Herein, we show that DHA and EPA presented similar effects regarding production of ROS by neutrophils. Nevertheless, for other functional parameters including cytokine production, phagocytosis, and killing, these fatty acids presented different effects.
Phagocytosis, a process by which neutrophils internalize particles/cells, was shown to be significantly increased by DHA but not by EPA. This results is in contrast with the findings of previous studies performed with goat neutrophils  and monocytes  and murine macrophages , in which both FA increased phagocytosis. Differences in the experimental protocol in aspects such as cell type and state of activation, particle/cell used for evaluation of phagocytosis, and concentration/period of incubation may, at least in part, account for this discrepancy. Despite the fact that the precise underlying mechanism involved in the differential effect of EPA and DHA in neutrophil phagocytosis remains to be elucidated, we speculate that the difference in the degree of unsaturation between these two FA may be involved. Cells treated with fatty acids incorporate them into membrane phospholipids, affecting cell membrane fluidity , thus playing an important role in phagocytic activity . Moreover, the effects of fatty acid incorporation on phagocytic activity are correlated with the degree of unsaturation; i.e., cells treated with fatty acids with more double bonds present higher phagocytic capacity , possibly explaining the higher capacity of DHA to stimulate phagocytosis compared with EPA.
In addition to their microbicidal effect, RNS also play an important role by modulating inflammatory and immune responses . Activated neutrophils produce large amounts of NO via inducible nitric oxide synthase (iNOS) . Previous studies have shown that EPA and DHA can affect the production of this mediator in macrophages in different ways depending on the concentration of the fatty acid and the period of incubation; For example, in J774 macrophages incubated for 48 h with EPA or DHA at 10 and 25 μM, respectively, an increase in NO production has been shown, whereas at 50 and 100 μM the production of this mediator was inhibited . In addition, Komatsu et al.  demonstrated that NO production and iNOS expression in RAW264 cells and peritoneal macrophages are inhibited when cells are treated with 60 μM DHA but are not altered when they are treated with 15 μM for 24 h in the presence of LPS (1 μg/mL). In contrast to the results obtained in macrophages, we have shown that EPA and DHA have no effect on neutrophil production of NO, demonstrating that these cells present a different response to omega-3 fatty acids compared with macrophages.
Production of O2·− in activated neutrophils occurs predominantly via the NADPH oxidase system [1, 44] after being stimulated, for example, by opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine (fMLP), or PMA . Subsequently, O2·− can be converted to H2O2 and more reactive oxidants such as hypochlorous and hypobromous acids . Both EPA and DHA increased the production of H2O2 and O2·− in the absence or presence of PMA, although at different concentrations. In the presence of zymosan particles, the production of O2·− was only increased by the treatment with EPA. Similar results were found in human neutrophils , however goat neutrophils treated with EPA or DHA presented no alteration or a reduction in the production of ROS in the absence or presence of PMA . Such discrepancy may occur due to different methods of quantification of ROS or the activation state of the cells. Given that treatment with the NADPH oxidase inhibitor DPI  completely blocked ROS production induced by FA and opsonized zymosan, it is likely that EPA and DHA increase the production of ROS via NADPH oxidase.
During the inflammatory response, several cytokines are secreted by neutrophils and macrophages due to the activation of pattern recognition receptors (PRRs) present in their membranes by components and/or products of microorganisms or necrotic cells . The production of cytokines was differently affected by the different concentrations of EPA or DHA and the activation state of the cell. For instance, the production of IL-1β was not altered by treatment with EPA and DHA. However, in the presence of LPS, DHA increased such production whereas EPA did not cause any change. EPA or DHA at 25 μM increased TNF-α production. However, at 50 μM, DHA reduced its production whereas EPA maintained a higher production. The production of CINC-2 was increased by neutrophils treated with EPA, with and without LPS, whereas treatment with DHA had no effect. It should be noted that, in the absence of LPS, DHA only increased the production of TNF-α, whereas EPA increased TNF-α and CINC-2 production.
Despite the recognized anti-inflammatory effects of omega-3 fatty acids both in vitro [50–53] and in vivo [54–57], the proinflammatory effects of EPA and DHA observed in the present study are in accordance with several other studies which demonstrate that omega-3 PUFA increases TNF-α release in vivo [58, 59] and in vitro by peritoneal macrophages [60–66]. The proinflammatory effect of the omega-3 FA on neutrophils may accelerate the beginning of the immune response, thus leading to a faster resolution, preventing a long-lasting inflammation. Moreover, the in vivo anti-inflammatory effects caused by the omega-3 FA are a result of their actions on various cells involved in the inflammatory response, including macrophages and endothelial cells. Also, omega-3 FA may present proinflammatory effects on neutrophils when they are not under the influence of other cells, as is observed during an inflammatory response .
In conclusion, EPA and DHA modulate various rat neutrophil functions. These FA showed distinct actions on cytokine production, phagocytosis, and fungicidal activity of neutrophils. EPA and DHA had similar effects on the production of ROS, although at different concentrations. These differences may be explained by the fact that EPA and DHA differently alter the fatty acid composition of membrane phospholipids, changing the ligand–receptor binding affinity. These results contribute to the elucidation of how EPA and DHA influence neutrophil function and the inflammatory response.
This study is supported by FAPESP, CNPq, and CAPES. The authors acknowledge the technical support of José Roberto Mendonça.