Dietary fish oil increases the proportion of a specific neutrophil subpopulation in blood
and total neutrophils in peritoneum of mice following endotoxin–induced inflammation
Hildur H. Arnardottir*+ Jona Freysdottir§+ and Ingibjorg Hardardottir*
*Department of Biochemistry and Molecular Biology and §Department of Immunology,
Faculty of Medicine, Biomedical Center, University of Iceland, 101 Reykjavik, Iceland,
+Center of Rheumatology Research and Department of Immunology, Landspitali – The
University Hospital of Iceland, Reykjavik, Iceland
Corresponding author: Ingibjorg Hardardottir, Professor, Department of Biochemistry and
Molecular Biology, Faculty of Medicine, Biomedical Center, University of Iceland,
Vatnsmyrarvegur 16, IS-101 Reykjavik, Iceland, Tel. 354 525 4885, Fax 354 525 4886,
Running title: Effects of dietary fish oil on neutrophils in mice
This work was supported by grants from the Icelandic Research Fund (www.rannis.is) and the
University of Iceland Research Fund (www.hi.is/en/research_degrees).
Key words: Fish oil, LPS, endotoxin-induced inflammation, chemokines, neutrophils, CCL3
Abbreviations used: AA, arachidonic acid; DHA, docosahexaenoic acid; DMEM, Dulbecco's
Modified Eagle Medium; EPA, eicosapentaenoic acid; G-CSF, granulocyte colony-
stimulating factor; HLA, human leukocyte antigen; IL, interleukin; LPS, lipopolysaccharide;
mAb, monoclonal antibodies; MHC, major histocompatibility complex; NK, natural killer;
PBS, phosphate buffered saline; PG, prostaglandin; PUFA, polyunsaturated fatty acids; TNF,
tumor necrosis factor.
Omega-3 polyunsaturated fatty acids may have beneficial effects in inflammation where neutrophil
migration and activation is of importance. The effects of dietary fish oil on neutrophil numbers and
subpopulations in healthy mice and mice with endotoxin-induced inflammation were determined.
Mice were fed a control diet with or without 2.8% fish oil and half of them injected
intraperitoneally with endotoxin. Blood, peritoneal lavage, bone marrow and spleen were
collected. Expression of cell surface molecules was analyzed by flow cytometry and
chemokine concentrations determined by ELISA. Dietary fish oil did not alter the proportion
of total neutrophils in blood but increased the proportion of a specific subpopulation of
neutrophils 48 h following endotoxin administration. This subpopulation of neutrophils expressed
higher levels of CD11b, Ly6G and MHC-II, suggesting a different role for these neutrophils in the
inflammatory response. Dietary fish oil did not affect neutrophil numbers in the peritoneum of
healthy mice but 12 h after endotoxin administration there were fewer neutrophils in the
peritoneum of mice fed the fish oil diet than in mice fed the control diet. However, 48 h after
endotoxin administration mice fed the fish oil diet had more neutrophils in peritoneum than
mice fed the control diet. These results indicate that although dietary fish oil may delay
recruitment of neutrophils from blood to the peritoneum early in inflammation it has potential
to increase the number of peritoneal neutrophils later, which may be of benefit as impaired
neutrophil migration and activation has been associated with immunosuppression late in
Dietary fish oil, or omega-3 polyunsaturated fatty acids (PUFA), affect inflammatory responses and
are thought to have anti-inflammatory effects. Although inflammation is beneficial, uncontrolled or
prolonged inflammation can cause harm and needs to be tightly regulated. Inflammation is
characterized by a complex sequence of events involving alterations in the inflammatory
mediator network as well as rearrangement of innate immune cell populations and changes in
their activation status . Neutrophils have an essential role in host defense, but are also
thought to be able to promote persistent inflammatory responses and tissue injury, which can
be detrimental to the host [2, 3]. They are recruited out of the bloodstream in response to
chemokines and after activation, sequentially discharge granules, which contain a large panel
of antimicrobial agents . They also recruit other innate cells to the site of inflammation
through the release of chemokines and antimicrobial peptides with chemotactic properties .
The pro-inflammatory role of neutrophils has been elucidated in studies using a monoclonal
antibody (RB6-8C5) that binds to Ly6G, a specific granulocyte surface marker, but also to
Ly6C, an antigen broadly expressed on immune cells, including monocytes, dendritic cells, T
cells, natural killer (NK) cells, NKT cells and eosinophils, in addition to neutrophils. Use of
the Ly6G-specific antibodies 1A8 and NIMP-R14 has greatly improved identification of
mouse neutrophils and several studies have now shown that neutrophils, in addition to having
pro-inflammatory effects, also exhibit anti-inflammatory properties, secreting high amounts
of anti-inflammatory cytokines and that neutrophil depletion during a chronic infection
promotes inflammation [6, 7, 8]. Furthermore, recent studies have shown that subpopulations
of neutrophils have the ability to present antigen and provide help for T cells  as well as for
splenic B cells .
In severe inflammation, or sepsis, the initial hyper-inflammatory phase is followed by an
anti-inflammatory or immunosuppressive phase [11, 12]. In the immunosuppressive phase the
neutrophils may become dysregulated and despite having increased levels of activation markers,
such as CD11b, ICAM-1, myeloperoxidase and CD66b, their adherence and transmigration is
impaired . Decreased neutrophil recruitment is also, in part, due to down-regulation of the
chemokine receptor CXCR2 in response to both lipopolysaccharide (LPS) and high levels of
the chemokine CXCL8 (or CXCL1 and CXCL2 in mice) . Failure of neutrophil migration
results in impaired bacterial clearance, weakened host defense and increased risk of secondary
infections [13, 15]. As omega-3 PUFA are considered to have beneficial effects in severe
inflammation, it is surprising that in several in vitro studies, omega-3 PUFA have been shown to
hinder neutrophil adhesion and migration. In a recent study by Yates et al.  DHA,
although not EPA, inhibited adhesion of neutrophils to the endothelium by reducing surface
expression of E-selectin on endothelial cells following cytokine stimulation . In another
study from the same laboratory, EPA reduced migration of neutrophils across vascular
endothelium by decreasing formation of prostaglandin (PG)D2, instead generating PGD3, that
antagonizes the action of PGD2, but PGD2 provides a necessary signal for neutrophils to
traverse the endothelium . In addition, pretreatment of endothelial cells with DHA down-
regulated tumor necrosis factor (TNF)-α-induced endothelial cell surface expression of P-
selectin and decreased TNF-α-induced neutrophil adhesion . Furthermore, it has been
demonstrated that the omega-3-derived resolvin D2 inhibits adherence of neutrophils to
activated endothelial cells in a mouse cremaster model  and that resolvin E1 reduces
infiltration of neutrophils in murine peritonitis .
The effects of dietary fish oil, or omega-3 PUFA, on neutrophil populations or neutrophil
migration in vivo have not been examined. In the present study we show that dietary fish oil
may delay neutrophil migration to the peritoneum of mice in vivo, but increase their numbers
in the peritoneum late in the inflammation, as well as increasing the proportion of a specific
subpopulation of neutrophils in the circulation that may have a different role in the
inflammatory process than other neutrophils.
2.1. Mice and diets
Female C57BL/6 mice weighing between 18 and 20 g (Taconic Europe, Ejby, Denmark)
were housed five or eight per cage in a humidity (45-55%) and temperature (23-25°C)
controlled environment with a 12 h light and dark cycle. All procedures with animals
complied with NRC´s Guide for the Care and Use of Laboratory Animals and were approved
by the Experimental Animal Committee, Ministry for the Environment in Iceland. Mice were
acclimated for one week prior to initiation of the experiment. They were randomly assigned to
either a group fed control diet (D07121302; Research Diets Inc., New Brunswick, NJ) or a
group fed fish oil diet (D07121303; Research Diets Inc.) for 6 weeks. The composition of the
diets was based on a typical American diet, i.e. the "US17" diet formulated by Monsanto (St.
Louis, MO) and Research Diets Inc. with minor modification by the authors (Table 1). Energy
distribution of the diets was as follows: carbohydrate, 44%; fat, 35%; and protein, 21%. The
fish oil diet contained 28 g/kg menhaden fish oil (Omega Protein, Reedville, VA), which was
added at the expense of safflower oil (Welch, Holme & Clark Co. Inc., Newark, NJ). To
adjust for the arachidonic acid (AA) content in the fish oil diet, AA ethyl ester (Nu-Check-
Prep, Elysian, MN) (0.5 g/kg) was added to the control diet. The fatty acid composition of the
diets is shown in Table 2. In brief, the fish oil diet contained 10.6 g/kg omega-3 PUFA (4.0
g/kg EPA and 2.5 g/kg DHA) and the control diet 3.4 g/kg omega-3 PUFA (undetectable
levels of EPA and DHA). To prevent oxidation the diets were aliquoted into daily portions
and were stored under an atmosphere of nitrogen at -20°C. All mice were provided fresh food
daily and had free access to food and water.
2.2. Body mass and food intake
Weight of the mice was monitored weekly throughout the experiments. Food was provided
daily and any remaining food from the previous day discarded. Food intake was determined
as the difference between the food supplied and the amount of food left.
2.3. Induction of inflammation.
Mice were injected intraperitoneally with LPS (0.5 mg/kg; E. coli, serotype 055:B5, Sigma
Aldrich) in phosphate buffered saline (PBS) (Invitrogen, Paisley, UK). At indicated time
points they were anesthetized with a (1:1) mixture of hypnorm (VetaPharma Ltd, Leeds, UK)
and dormicum (Roche, Basel, Switzerland). Blood was collected via axillary bleeding and the
mice killed by cervical dislocation.
2.4. Collection of blood and peritoneal lavage
Blood was collected into EDTA-K coated tubes (Sarstedt, Nümbrect, Germany) for flow
cytometry analysis or Eppendorf tubes for whole blood collection. Serum was collected and
stored at -70°C until analyzed. For collection of peritoneal lavage, cold PBS without calcium
or magnesium was injected into the peritoneum, it massaged gently and peritoneal lavage
collected. Cells were spun down and the supernatant collected and stored at -70°C. Peritoneal
cells were washed with PBS and resuspended in Dulbecco's Modified Eagle Medium (DMEM)
with Gluta-MAX-I (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Invitrogen).
2.5. Collection of bone marrow and spleen cells
Right femurs and tibiae were cut at the diaphyses and bone marrow cells were flushed out by
repeated injections of DMEM supplemented with 10% fetal bovine serum (Invitrogen).
Spleens were passed through a cell strainer (BD Bioscience, San Jose, CA) to acquire a single
cell suspension. Red blood cells were lysed with ACK lysing buffer (0.15 M NH4Cl, 1 mM
KHCO3, 0.1 mM Na2EDTA) and the cells washed and resuspended in 5 ml DMEM.
2.6. Cell count and phenotypic characterization of leukocytes by flow cytometry
For total blood cell numbers and number of neutrophils, 50 µL of blood were measured
precisely into Trucount tubes (BD Biosciences) and stained with monoclonal antibodies (mAb)
against Ly6G. Peritoneal, spleen and bone marrow cells were counted using Trypan blue and
Countess automated cell counter (Invitrogen).
For phenotypic analysis of leukocytes, 100 µl of blood or 0.3 x 106 bone marrow, spleen
or peritoneal cells were pre-incubated with a (1:1) mixture of normal rat:normal mouse serum
(2%) (AbD Serotec, Kidlington, UK). The cells were stained with fluorochrome-labeled mAb
against CD11b (Mac-1), CD14, CD62L (L-selectin), MHC-II (eBioscience, San Diego, CA),
Ly6G (clone 1A8 that only binds to Ly6G but not Ly6C, BD Bioscience) and CXCR2 (R&D
Systems, Abington, UK). Bone marrow, spleen and peritoneal cells were additionally stained
with mAb against B220, CD90.2 and NK1.1 (eBioscience) for exclusion of lymphocytes. Red
blood cells in blood samples were lysed with FACS Lysing Solution (BD Bioscience). All
samples were washed and resuspended in FACS staining buffer (PBS with 0.5% BSA, 2 mM
EDTA, 0.1% sodium azide). Appropriate isotypic controls were used to set the quadrants and
evaluate background staining. Samples were collected on FACScalibur (BD Biosciences) and
data analyzed using Cell Quest (BD Biosciences) and FlowJo (Tree Star Inc., Ashland, OR).
Blood, bone marrow, spleen and peritoneal neutrophils were identified as cells expressing the
granulocyte marker Ly6G (clone 1A8) and the chemokine receptor CXCR2 but lacking
expression of the lymphocyte markers B220, CD90.2 and NK1.1. The neutrophils also
expressed CD11b, CD14, CD62L and MHC-II.
2.7. Activation of resident peritoneal cells ex vivo and intracellular cytokine staining by flow
Peritoneal cells in DMEM (2.5 x 105 cells/250 µl/well) were cultured in a 48-well flat bottom
plate at 37°C with 5% CO2. Non-adherent cells were discarded and the adherent cells washed
twice with PBS and cultured further in 250 µl DMEM supplemented with 5% autologous
serum for 24 h in the absence (negative control) or presence of 1 µg/ml LPS (E. coli 055:195;
Difco Laboratories, Detroit, MI). For cytokine measurements, the cells were spun down after
24 h culture and the supernatant collected and stored at -70°C until analyzed with ELISA. For
intracellular staining, brefeldin A (3 µg/ml; eBioscience) was added to the cell culture for the
last 6 h of the 24 h LPS stimulation. The supernatant was removed and the cells scraped from
the plate after being treated with cold PBS containing 20 mM EDTA for 15 minutes. The cells
were stained with mAb against CD11b and against B220, NK1.1 and CD90.2 (for exclusion
of contaminating lymphocytes) and then, after fixation in 4% formaldehyde, permeabilization
with 0.1% saponin in FACS staining buffer and blocking with a 1:1 normal mouse:normal rat
serum (20%), with mAb against CCL3 (eBioscience). Cells were analyzed on FACScalibur as
2.8. Chemokine and cytokine analysis
Concentrations of chemokines and cytokines in serum and peritoneal fluid were measured
with Duo Set ELISA kits (R&D Systems).
2.9. Data analysis
Data are expressed as mean and standard error of the mean and were analyzed using two-way
ANOVA and Tukey’s post hoc test except in Figure 3 in which unpaired student’s t-test was
used to determine whether differences between the two dietary groups were statistically
significant at a single time point. Statistical analysis was performed using SPSS software,
version 17 (SPSS Inc., Chicago, IL). A P value of <0.05 was considered statistically
significant. All experiments were performed at least three times.
3.1. Mouse growth and dietary intake
There was no difference in the mean daily food intake, weight gain of mice not receiving LPS
or weight loss of mice receiving LPS between mice in the two dietary groups (Supplemental
3.2. Effects of dietary fish oil on blood neutrophils in healthy mice and mice with endotoxin-
Dietary fish oil did not affect the number of total neutrophils in healthy mice or mice with
endotoxin-induced inflammation, although at 12 and 24 h there was a trend towards more
neutrophils in blood from mice fed the fish oil diet compared with that in blood from mice fed
the control diet (Fig. 1A). Dietary fish oil did not alter the proportion of total neutrophils (of
total blood cells) prior to or following LPS administration (data not shown). Neutrophils from
healthy mice were homogenous in size and granularity, but 8, 12 and 48 h after administration
of LPS two distinct populations of neutrophils were present in blood, one resembling the
population present in blood prior to LPS administration (N1) and another population consisting of
less granular and slightly larger neutrophils (N2) (Figs. 1B and C) that had higher expression of
CD11b, Ly6G and MHC-II than the N1 neutrophils, but similar levels of CXCR2 and CD62L (data
not shown). At 8 and 12 h after administration of LPS there was no difference in the proportion of
the two neutrophil populations in mice fed the different diets (Fig. 1D). However, at 48 h following
LPS administration, the N2 subpopulation was larger in proportion in mice fed the fish oil diet (Figs.
1C and D) than in mice fed the control diet (Figs. 1B and D). N2 neutrophils from mice fed the fish
oil diet had higher expression levels of Ly6G 12 h after administration of LPS (2342 ± 205) than N2
neutrophils from mice fed the control diet (1640 ± 108, P<0.05). There was no difference in
expression levels of Ly6G on N2 neutrophils from mice fed the different diets at 8 or 48 h after
induction of inflammation. Dietary fish oil did not affect Ly6G expression levels on N1 neutrophils
in healthy mice or mice with endotoxin-induced inflammation, nor expression of other surface
markers on N1 or N2 neutrophils, at any of the time points examined (data not shown).
3.3. Effects of dietary fish oil on bone marrow and spleen neutrophils in healthy mice and
mice with endotoxin-induced inflammation
Dietary fish oil did not affect the number or the proportion of neutrophils, nor expression
levels of surface markers on neutrophils, in either bone marrow or spleen in healthy mice or
in mice with endotoxin-induced inflammation (data not shown).
3.4. Effects of dietary fish oil on serum concentrations of neutrophil chemoattractants in mice
with endotoxin-induced inflammation
Murine CXCL1 and CXCL2 are potent neutrophil chemoattractants that bind to the
chemokine receptor CXCR2 and recruit neutrophils to the site of infection or inflammation
and CCL3 has also been implicated to play a role in the recruitment of neutrophils [21, 22].
Dietary fish oil did not affect serum levels of CXCL1 in healthy mice or mice killed 3 h or 48
h after endotoxin-induced inflammation (Table 3). CXCL2 and CCL3 were not detected in
serum from healthy mice or in serum from mice that were killed 48 h after endotoxin-induced
inflammation (Table 3). However, fish oil fed mice that were killed 3 h after endotoxin-
induced inflammation had lower serum concentrations of CXCL2 but higher serum
concentrations of CCL3 than mice fed the control diet (Table 3). The effects of dietary fish oil
on serum concentrations of the cytokines granulocyte colony-stimulating factor (G-CSF) and
interleukin (IL)-33 were examined as G-CSF stimulates the release of neutrophils from the
bone marrow and IL-33 prevents down-regulation of CXCR2 on neutrophils during acute
inflammation and thus enhances neutrophil migration to the site of infection . G-CSF was
not detected in serum from healthy mice and there was no difference in G-CSF levels in
serum from mice in the two dietary groups killed 3 and 48 h after induction of inflammation
(Table 3). IL-33 was not detected in serum from healthy mice or mice with endotoxin-induced
inflammation (data not shown).
3.5. Effects of dietary fish oil on peritoneal neutrophils in healthy mice and mice with
The number of total neutrophils was similar in the peritoneum of healthy mice fed the fish oil
diet and healthy mice fed the control diet (Fig. 2A). Following administration of LPS there
was a delay in the increase in the number of neutrophils in the peritoneum of mice fed the fish
oil diet compared with that of mice fed the control diet where at 12 h after LPS administration
fewer neutrophils were observed in the peritoneum of mice fed the fish oil diet than in mice
fed the control diet (Fig. 2A). However, at 48 h there were more neutrophils present in the
peritoneum of mice fed the fish oil diet compared with that of mice fed the control diet (Fig.
2B) and the proportion of neutrophils of total peritoneal cells was significantly greater in mice
fed the fish oil diet (Figs. 2D and E) than in mice fed the control diet (Figs. 2C and E).
3.6. Effects of dietary fish oil on peritoneal concentrations and peritoneal cell production of
Dietary fish oil did not affect peritoneal concentrations of CXCL1 or CXCL2 prior to or 3 or
48 h after induction of inflammation (Table 3). CCL3 was not detected in peritoneal fluid of
healthy mice or mice killed 48 h after endotoxin-induced inflammation (Table 3). However,
dietary fish oil increased the concentration of CCL3 in peritoneal fluid from mice killed 3 h
after endotoxin-induced inflammation (Table 3; Fig. 3A). When peritoneal cells from mice
fed control and fish oil diet were collected and stimulated with LPS ex vivo, peritoneal cells
from mice fed the fish oil diet secreted more CCL3 than peritoneal cells from mice fed the
control diet (Fig. 3B) and the amount of CCL3 produced by each cell was higher among
peritoneal cells from mice fed the fish oil diet than mice fed the control diet (Fig. 3C). In
contrast, dietary fish oil decreased the number of CD11b+ resident peritoneal macrophages
secreting CCL3 in the peritoneum (Fig. 3D).
Although omega-3 PUFA may be of benefit in inflammation, their effects on migration and
dysregulation of neutrophils, which is thought to be of importance in defense failure during the
immunosuppressive phase observed late in the inflammatory process, have not been reported.
The results from the current study show that dietary fish oil affects the proportion of a
subpopulation of neutrophils in blood and the number and proportion of total neutrophils in
peritoneum of mice with endotoxin-induced inflammation. The most striking finding is that
late in the inflammation, or 48 h after endotoxin administration, there were around 40% more
neutrophils in the peritoneum of mice fed the fish oil diet than in mice fed the control diet and
the proportion of neutrophils of total cells in the peritoneum was around 80% higher in mice
fed the fish oil diet than in mice fed the control diet. At this time point there was no difference
in expression levels of any of the surface markers examined, indicating that the activation
state of the neutrophils was not affected by the fish oil diet. These results indicate that dietary
fish oil has the potential to enhance migration of neutrophils to the site of inflammation late in
the inflammatory phase, without affecting their activation status, which may be of benefit
during the immunosuppressive phase of severe inflammation.
The higher number of neutrophils in the peritoneum of mice fed the fish oil diet 48 h
following induction of inflammation was preceded by fewer neutrophils in the peritoneum at
12 (and perhaps also 24) h, indicating that infiltration of neutrophils into the peritoneum was
delayed in mice fed the fish oil diet compared with that in mice fed the control diet. The
tendency towards more neutrophils being present in blood at 12 and 24 h after induction of
inflammation in mice fed the fish oil diet compared with that in mice fed the control diet
supports the notion that there may have been a delay in the recruitment of neutrophils from
the blood to the peritoneum in mice fed the fish oil diet and that they accumulated in the
circulation at that time. The delayed recruitment of neutrophils from blood to the peritoneum
may be explained by the omega-3 PUFA inhibiting adhesion or transmigration of the
neutrophils as results from several in vitro and in vivo studies indicate that omega-3 PUFA, or
lipid mediators derived from them, can affect endothelial adhesion and/or migration of
neutrophils [16, 20]. These studies all demonstrate that omega-3 PUFA, or mediators derived
from them, affect neutrophil endothelial adhesion or transendothelial migration although the
mechanism by which they do so differ. Therefore, in the present study, the effects of fish oil
to decrease neutrophil migration to the peritoneum at early time points in the inflammatory
phase may be explained by any of these mechanisms. That the reduction in neutrophil
migration in mice fed the fish oil diet at 12 and 24 h after induction of inflammation was
overcome at 48 h indicates that other mechanisms may mediate neutrophil migration later in
The only other study that has examined the effects of dietary fish oil on neutrophil
recruitment into the peritoneum in vivo did so following infection with L. monocytogenes and
showed no effect of fish oil on neutrophil numbers in peritoneum 24 h following infection
. It is not surprising that the results from that study differ from the results from the present
study as the mechanism of neutrophil recruitment to the peritoneum in response to L.
monocytogenes differs from the mechanism of neutrophil recruitment in response to E. coli
(or LPS), the former being CD11b/CD18-independent but the latter CD11b/CD18-dependent
The effects of the fish oil diet on peritoneal concentrations of the chemokines involved in
neutrophil recruitment did not shed light on the mechanism by which fish oil affected
neutrophil numbers in the peritoneum. Peritoneal concentrations of the neutrophil
chemoattractants CXCL1, CXCL2 and CCL3 peaked within 4 h and were back to almost
undetectable levels by 8 h (data not shown). The increase in peritoneal concentration of CCL3
in mice fed the fish oil diet was evident at 3 h, when there were few neutrophils in the
peritoneum and no difference in their numbers between mice fed the different diets. When the
number of neutrophils started to increase, there were fewer neutrophils in the peritoneum of
mice fed the fish oil diet, which is in contrast to the higher concentration of CCL3 in the
peritoneum few hours earlier. Neither did expression levels of CXCR2 on the neutrophils
explain the delay in neutrophil recruitment to the peritoneum, as there was no difference in
the expression levels of CXCR2 on neutrophils from mice fed the different diets.
Although there was no difference in neutrophil numbers in bone marrow from mice with
endotoxin-induced inflammation fed the different diets, there was a trend towards higher
serum levels of G-CSF in mice fed the fish oil diet compared with that in mice fed the control
diet 12 h after induction of inflammation. G-CSF stimulates the release of neutrophils from
the bone marrow into the circulation  and the trend towards an increase in G-CSF levels
in blood is consistent with the trend towards increased numbers of neutrophils in blood from
mice fed the fish oil diet at 12 and 24 h after induction of inflammation. Serum levels of
CCL3 were also higher in mice fed the fish oil diet compared with that in mice fed the control
diet, but serum levels of CCL3 peaked around 2 h after administration of LPS, long before the
trend towards higher neutrophil numbers appeared in blood from mice fed the fish oil diet.
In the present study, mice fed the fish oil diet had a higher proportion of N2 neutrophils
in the circulation than mice fed the control diet. The N2 neutrophils expressed higher levels of
CD11b, Ly6G and MHC-II than the N1 population. The higher expression levels of CD11b
on the N2 neutrophils suggests that they may be in a more activated state than the N1
neutrophils [27, 28] or have immunosuppressive properties similar to human neturophils with
high expression levels of CD11b . Their expression of MHC-II also indicates that they
can be involved in antigen presentation to T cells as has been shown for mouse neutrophils
expressing MHC-II . The presence of two neutrophil populations in the circulation upon
induction of inflammation was intriguing as several recent studies have emerged showing that
neutrophils are far from being a homogeneous population with only the traditional phagocytic
and bactericidal function. These studies have shown neutrophils to be able to secrete high
levels of IL-10, suppress T cell responses, possess antigen-presenting function and providing
help for T cells and a population of neutrophils located in the marginal zone of spleen have
been shown to induce immunoglobulin class switching, somatic hypermutation and antibody
production by activating marginal zone B cells [6, 7, 8, 9, 10].
The results from the present study show that dietary fish oil increases the proportion of a
specific subpopulation of circulating neutrophils that may have a different role in the
inflammatory process than other neutrophils. They also show that although dietary fish oil
may delay the recruitment of neutrophils from blood to the peritoneum at early time points
after onset of inflammation it increases the number of peritoneal neutrophils at a later time
point. The increased number of neutrophils at the site of inflammation at a late time point in
the inflammatory process may shed light on the mechanism by which omega-3 PUFA can
have beneficial effects in severe inflammation, in which impaired neutrophil migration and
activation has been associated with immunosuppression and defense failure. In addition,
although neutrophils are the main effector cells during inflammation they can also control
excessive inflammatory responses by secreting anti-inflammatory cytokines and may have a
previously unsuspected regulatory role during inflammation [6, 7]. Whether these late
arriving neutrophils observed in the mice fed dietary fish oil are of this anti-inflammatory
type remains to be investigated.
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Fig. 1. Effects of dietary fish oil on the number of total neutrophils (A), size and granularity
of the neutrophils (B-C) and proportion of N2 neutrophils of total neutrophils (D) prior to
and/or following LPS administration. Mice were fed a control diet (dashed line, open bar) or a
diet supplemented with 2.8% fish oil (solid line, black bar) for 6 weeks. They were injected
with LPS (0.5 mg/kg) or not and sacrificed at indicated time points (A,D) or at 48 h (B,C).
Blood was collected and cells stained with monoclonal antibodies and counted using
TruCount (A) and analyzed by flow cytometry (A-D). Values are means ± SEM, n=3-5 (A),
n=8 (D). Representative forward and side scatter dot plots of blood cells from mice fed the
control (B) and the fish oil (C) diets 48 h after administration of LPS, grey dots, neutrophils
(N1 and N2); black dots, other cells. *Different from control, P<0.05.
Fig. 2. Effects of dietary fish oil on the number of total peritoneal neutrophils (A, B), size and
granularity of the neutrophils (C-D) and proportion of neutrophils of total peritoneal cells (E)
prior to and/or following administration of LPS. Mice were fed a control diet (dashed line,
open bar) or a diet supplemented with 2.8% fish oil (solid line, black bar) for 6 weeks. They
were injected with LPS (0.5 mg/kg) or not and sacrificed at indicated time points (A) or at 48
h (B-E). Peritoneal cells were collected and stained with monoclonal antibodies and analyzed
with flow cytometry. Values are means ± SEM, n= 3-5 (A), n=11 (B, E). Representative
forward and side scatter dot plots of peritoneal cells from mice fed control (C) or fish oil (D)
diets 48 h after administration of LPS, grey dots, neutrophils (N); black dots, other cells. *
Different from control, P<0.05.
Fig. 3. Effects of dietary fish oil on peritoneal concentrations of CCL3 (A), LPS-induced
CCL3 secretion by resident peritoneal cells ex vivo (B), expression levels of CCL3 on
peritoneal cells (C) and the proportion of CCL3 secreting cells of total CD11b+ peritoneal
cells (D). Mice were fed a control diet (open bar) or a diet supplemented with 2.8% fish oil
(black bar) for 6 weeks. Mice were injected with LPS (0.5 mg/kg) and peritoneal fluid
collected 3 h later (A). Peritoneal cells (1x106 cells/ml) were collected from healthy mice and
stimulated with LPS (1 µg/ml) for 24 h and supernatant collected (B) and chemokine
concentrations measured with ELISA (A, B). Brefeldin A was added to peritoneal cells in
culture for the last 6 h of the stimulation and expression levels of CCL3 on CD11b+ peritoneal
cells (C) and the percentage of CD11b+ peritoneal cells secreting CCL3 (D) measured with
flow cytometry. Values are means ± SEM, n=14-17 (A), n=17 (B), n=13-16 (C-D). *
Different from control, P<0.05.
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