Dietary Omega-3 Fatty Acids Increase Survival and Decrease BacterialLoad in Mice Subjected to Staphylococcus aureus-Induced Sepsis

Sara L. Svahn,a Marcus A. Ulleryd,a Louise Grahnemo,a,b Marcus Ståhlman,c Jan Borén,c Staffan Nilsson,d John-Olov Jansson,a

Maria E. Johanssona

Department of Physiology, Institute of Neuroscience and Physiology,a and Department of Rheumatology and Inflammation Researchb and Department of Molecular andClinical Medicine,c Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; Department of Mathematical Statistics, Chalmers University of Technology,Gothenburg, Swedend

Sepsis caused by Staphylococcus aureus is increasing in incidence. With the alarming use of antibiotics, S. aureus is prone to be-come methicillin resistant. Antibiotics are the only widely used pharmacological treatment for sepsis. Interestingly, mice fedhigh-fat diet (HFD) rich in polyunsaturated fatty acids have better survival of S. aureus-induced sepsis than mice fed HFD richin saturated fatty acids (HFD-S). To investigate what component of polyunsaturated fatty acids, i.e., omega-3 or omega-6 fattyacids, exerts beneficial effects on the survival of S. aureus-induced sepsis, mice were fed HFD rich in omega-3 or omega-6 fattyacids for 8 weeks prior to inoculation with S. aureus. Further, mice fed HFD-S were treated with omega-3 fatty acid metabolitesknown as resolvins. Mice fed HFD rich in omega-3 fatty acids had increased survival and decreased bacterial loads compared tothose for mice fed HFD-S after S. aureus-induced sepsis. Furthermore, the bacterial load was decreased in resolvin-treated micefed HFD-S after S. aureus-induced sepsis compared with that in mice treated with vehicle. Dietary omega-3 fatty acids increasethe survival of S. aureus-induced sepsis by reversing the deleterious effect of HFD-S on mouse survival.

Sepsis is a deadly disease with an increasing incidence world-wide (1). It is the leading cause of death in intensive care units,

with a mortality rate of 30 to 70% (2). Today, antibiotics are theonly widely used pharmacological treatment for sepsis (3). Staph-ylococcus aureus and other Gram-positive bacteria account for themajor part of its increased incidence (1, 4). Resistance to antibi-otics is a growing problem worldwide. S. aureus bacteria are athreat to human health, given that they have obtained a gene thatallows them to become methicillin resistant (5). In intensive careunits in Europe, more than 50% of the isolates of S. aureus aremethicillin resistant (6).

Sepsis is considered a two-phase disease with an initial hyper-inflammatory state, followed by a later hypoimmune state, andmost deaths occur in the hypoimmune state (2, 7). A large portionof the previous efforts to develop new treatments have been fo-cused on dampening the hyperinflammatory state (8). However,these strategies have led to very minor results. A better under-standing of factors that can influence the mortality rates duringthe different stages of sepsis might lead to new treatments, in ad-dition to antibiotics.

In previous studies by our research group, we found that theimmune system was negatively affected by high-fat diet (HFD)rich in saturated fatty acids (HFD-S), resulting in decreased sur-vival of septic S. aureus-induced infection, compared with a low-fat diet (LFD) (9). Interestingly, in a later study, we found thatmice fed HFD rich in polyunsaturated fatty acids (HFD-P) hadincreased survival of septic S. aureus-induced infection and de-creased bacterial loads in the kidneys compared with those formice fed HFD-S (10).

Polyunsaturated fatty acids, such as those found in HFD-P, canbe divided mainly into omega-3 and omega-6 fatty acids on thebasis of the location of the first double valence bond in relation tothe end of the carbon chain (omega carbon). They can further bedivided into cis and trans fatty acids on the basis of the configura-tion of the two hydrogen atoms on either side of the double bond.

If the two hydrogen atoms are on the same side of the chain, thefatty acid is said to have a cis configuration, whereas if the twohydrogen atoms are on opposite sides of the chain, the fatty acid issaid to have a trans configuration. Omega-6 fatty acids, especiallyarachidonic acid, are considered to have essentially proinflamma-tory properties, whereas omega-3 fatty acids are considered tohave anti-inflammatory properties (11). For example, patientswith inflammatory diseases such as rheumatoid arthritis and ath-erosclerosis have been reported to benefit from dietary omega-3fatty acids (12, 13).

The innate immune response, in particular, the activation ofneutrophils, is the first line of defense against bacterial infectiousdiseases (14). We have previously shown that mice fed HFD-Phave a higher frequency of neutrophils in their bone marrow thanmice fed HFD-S. The omega-3 fatty acid metabolites known asresolvins are a group of biologically active metabolites that havebeen found to enhance neutrophils’ capacity to phagocytose Esch-erichia coli (15). It has also been shown that the survival of micewas increased by treatment with resolvin D2 (RvD2) after cecalligation and puncture, an experimental model of multibacterialsepsis (15). Similar results with increased survival by treatmentwith RvD1 have been shown after inoculation with E. coli (16).

In the present study, we investigated if HFD rich in omega-3

Received 14 November 2015 Returned for modification 23 December 2015Accepted 2 February 2016

Accepted manuscript posted online 8 February 2016

Citation Svahn SL, Ulleryd MA, Grahnemo L, Ståhlman M, Borén J, Nilsson S,Jansson J-O, Johansson ME. 2016. Dietary omega-3 fatty acids increase survivaland decrease bacterial load in mice subjected to Staphylococcus aureus-inducedsepsis. Infect Immun 84:1205–1213. doi:10.1128/IAI.01391-15.

Editor: A. Camilli

Address correspondence to Maria E. Johansson, maria.e.johansson@neuro.gu.se.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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fatty acids (HFD-�3) or omega-6 fatty acids (HFD-�6) is mostefficient at increasing survival and decreasing bacterial loads inmice with septic S. aureus-induced infection. We found that micefed HFD-�3 survived better than mice fed HFD-�6 or HFD-S andhad decreased bacterial loads compared to those for mice fedHFD-S. Also, mice fed HFD-S had a lower frequency of phagocy-tosing neutrophils in their circulation. Moreover, we found thatmice fed HFD-S had decreased bacterial loads when treated withresolvins after inoculation with S. aureus.

MATERIALS AND METHODSExperimental protocol. The experimental design used in this study isshown in Fig. 1. In summary, C57BL/6 mice were fed LFD, HFD-S, HFD-�3, or HFD-�6 for 8 weeks. Following 8 weeks on the different diets, micewere either intravenously (i.v.) inoculated with S. aureus or terminated formeasurements of immune functions by flow cytometry. Bacterial loadswere analyzed in infected animals 6 days after inoculation, and survivalwas monitored for 17 days after inoculation. The animals remained ontheir respective diets after inoculation. The measurements of immuneparameters included determination of the frequencies of different im-mune cells in bone marrow and the frequency of neutrophils from thecirculation that had phagocytosed pHrodo Green S. aureus BioParticles(pHrodo particles; Life Technologies, CA, USA). To investigate if mice fedHFD-S would benefit from treatment with resolvins, mice were onceagain fed for 8 weeks, but this time only HFD-S. Following 8 weeks onHFD-S, mice were i.v. inoculated with S. aureus. On days 1 to 4, mice weretreated i.v. with the vehicle, RvD1 (Cayman Chemical, Ann Arbor, MI,USA), or RvD2 (Cayman Chemical). Bacterial loads and survival wereonce again analyzed in infected animals 6 days after inoculation and mon-itored for 17 days after inoculation, respectively. All experiments wereapproved in advance by the local ethics committee for animal care at theUniversity of Gothenburg.

Diets. The compositions of the diets used are shown in Table 1. Micewere randomized to receive LFD (D12450B; 3.9 kcal/g, 10 kcal% fat, 20kcal% protein, 70 kcal% carbohydrate; Research Diets, New Brunswick,NJ, USA), HFD-S (D12492; 5.2 kcal/g, 60 kcal% fat, 20 kcal% protein, 20kcal% carbohydrate; Research Diets), HFD-�3 (D090120501; same fat,protein, and carbohydrate composition as HFD-S but 31% of lard ex-

changed for ROPUFA 75EE; Research Diets), or HFD-�6 (D10031504;same fat, protein, and carbohydrate composition as HFD-S but 69% oflard exchanged for safflower oil; Research Diets). The HFDs were matchedto have similar carbohydrate and protein contents and to differ only in fatcomposition and concentration. The concentration of omega-3 fatty acidswas almost 21-fold higher in HFD-�3 (24.7%) than in HFD-�6 (1.2%).In contrast, the concentration of omega-6 fatty acids was almost 3-foldhigher in HFD-�6 (62.9%) than in HFD-�3 (23.4%). The amounts oftrans fatty acids were similar in HFD-S and HFD-�3 (0.7 and 0.5%, re-spectively) and lowest in HFD-�6 (0.2%).

Bacterial inoculation, survival, and bacterial loads. Mice were inoc-ulated i.v. through the tail vein with 0.2 ml of S. aureus LS-1 solutioncontaining 3.0 � 107 to 5.4 � 107 CFU. For bacterial load determination,mice were terminated 6 days after inoculation. Both kidneys were har-vested aseptically and prepared as previously described (10). During thetime the mice were infected, they were continuously examined for signs ofsevere sickness: motionlessness, isolation, piloerection, dehydration, andhypothermia. If a mouse showed three or more of these five signs, it waseuthanized and considered dead by sepsis.

Body weight and composition. Body weight was determined at thestart of the experiment and every second week until inoculation. Fat andlean body masses were analyzed 3 days before inoculation by dual-energyX-ray absorptiometry (DXA; PIXImus2; Lunar GE Medical Systems,Madison, WI, USA). During the measurements, the mice were placedunder inhalation anesthesia with isoflurane (Forene; Abbot Scandinavia,Solna, Sweden).

Flow cytometry. (i) Collection and staining of bone barrow. Micewere anesthetized and then perfused with physiological saline at a pres-sure of 100 mm Hg for 10 min. Bone marrow cells were harvested from thefemur and tibia and stored in phosphate-buffered saline (PBS) with 1%fetal calf serum (FCS) on ice until preparation. Bone marrow cell suspen-sions were centrifuged, incubated with Pharm Lyse (BD Biosciences,Franklin Lakes, NJ, USA) for 6 min on ice to lyse red blood cells, washed,and resuspended in PBS with 1% FCS. Cell concentrations were deter-mined with an automated cell counter (Bio-Rad, Hercules, CA, USA), and3 � 105 cells from each sample were used for staining. To avoid unspecificbinding via Fc-receptor interactions, cells were incubated with Fc block

FIG 1 Experimental design. Seven-week-old mice were randomized into dif-ferent diet groups. After 8 weeks on the diets, mice were either terminated forcollection of bone marrow and blood for flow cytometry analysis or inoculatedwith S. aureus for bacterial load or survival analysis. Mice inoculated with S.aureus were either terminated after 6 days (6d) for bacterial load analysis ormonitored continuously for up to 17 days (17d) for survival analysis. Anotherset of mice were fed only HFD-S. After 8 weeks on the diet, mice were inocu-lated with S. aureus and randomized to receive i.v. treatment with the vehicle,RvD1, or RvD2. Bacterial load and survival analyses were conducted in thesame way as described above. Experimental groups: LFD, HFD-S, HFD-�3,HFD-�6, HFD-S and treatment with the vehicle (vehicle), HFD-S and treat-ment with RvD1, and HFD-S and treatment with RvD2. *, LFD was not in-cluded in the first survival study.

TABLE 1 Energy densities and compositions of the experimental dietsused in this study

Parameter LFD HFD-S HFD-�3 HFD-�6

Energy density (kcal/g) 3.9 5.2 5.2 5.2

Macronutrients (% kcal)Protein 20 20 20 20Carbohydrate 70 20 20 20Fat 10 60 60 60

Fat sources (% of total fat)Soybean oil 55.6 9.3 9.3 9.3Lard 44.4 90.7 62.2 27.8ROPUFA 75EE 28.5Safflower oil 63.0

Fatty acids (% by wt of total fatty acids)� SFAa 22.7 32.0 23.6 16.0� MUFAb 29.8 36.0 28.3 19.9� PUFAc 47.5 32.0 48.1 64.1� n-3 total fat 5.2 2.1 24.7 1.2� n-6 total fat 42.4 29.9 23.4 62.9n-6/n-3 8.2 14.1 0.9 51.9

a SFA, saturated fatty acids.b MUFA, monounsaturated fatty acids.c PUFA, polyunsaturated fatty acids.

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(2.4G2; BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at roomtemperature. The cells were labeled with the following antibodies: anti-mouse Ly6G-fluorescein isothiocyanate (FITC; clone 1A8; BD Biosci-ences), anti-mouse CD115-allophycocyanin (APC; clone AFS98; BioLeg-end, San Diego, CA, USA), anti-mouse CD117-phycoerythrin (PE; clone2B8; BioLegend), and 7-aminoactinomycin D (7AAD; BD Biosciences)for detection of live/dead cells. Cells were examined with a FACSCantoAflow cytometer (BD Biosciences). Instrument setting was performed withComp-Beads (BD Biosciences), and data analyses were performed withthe FACSDiva software (version 6.1.3; BD Biosciences). Flow cytometryanalysis was started with a leukocyte gate and thereafter performed with alive gate with 7AAD. Neutrophils were identified as Ly6G� CD115� cells(17), hematopoietic precursor cells were identified as CD117� cells (18),and monocytes were identified as CD115� cells (19). Flow cytometryresults are presented as the frequency of living leukocytes. For the gatingstrategy used for bone marrow, see Fig. 4A.

(ii) Collection and preparation of the blood for phagocytosis mea-surement. Mice were anesthetized, and their blood was collected tran-scardially into heparin tubes and stored at room temperature until furtherpreparation. pHrodo Green S. aureus BioParticles (pHrodo particles; LifeTechnologies, CA, USA) were prepared according to the manufacturer’sinstructions and incubated with an equal volume of mouse serum at 37°Cfor 10 min. From each animal, 100 �l of whole blood was used for thesample, as well as for the negative control. Negative-control samples wereincubated with 10 �l of 100 �g/ml cytochalasin D (Sigma-Aldrich, MO,USA) dissolved in dimethyl sulfoxide at 37°C for 10 min. The negativecontrol with cytochalasin D was used to determine the background me-dian fluorescence intensity (MFI) when the phagocytosis process hadbeen stopped. A 20-�l volume of the pHrodo-serum suspension wasadded to the samples, as well as to the negative control, and they wereincubated at 37°C for 45 min. All tubes were then transferred to ice to stopthe phagocytosis. The red blood cells were then lysed, and the sampleswere washed twice and resuspended in PBS with 1% FCS. To avoid un-specific binding via Fc-receptor interactions, cells were incubated withFc block (BD Biosciences) for 15 min at room temperature. The cellswere labeled with anti-mouse CD45-peridinin chlorophyll protein(PerCp; clone 30-F11; BD Biosciences) and anti-mouse Ly6G-APC(clone 1A8; BD Biosciences) antibodies. The cells were examined witha FACSCantoA flow cytometer (BD Biosciences). Instrument setting wasperformed with Comp-Beads (BD Biosciences) for the antibodies andcells for pHrodo particles. Data analyses were performed with the FACS-Diva software (version 6.1.3; BD Biosciences). Flow cytometry analysis ofphagocytosis capacity was started with a singlet gate, followed by forwardand side scatter, gating leukocytes. Phagocytosing neutrophils were iden-tified as CD45� Ly6G� pHrodo�. For the gating strategy used for blood,see Fig. 5A. The MFI represents the median amount of pHrodo particlesengulfed by neutrophils after subtraction of the MFI of the negative con-trol (MFI sample – MFI negative control).

Blood hematology analysis. Blood samples were collected transcardi-ally and stored in EDTA tubes on ice until blood analysis was conducted.Blood was analyzed with a VetH5 blood analyzer (Abaxis, Union City, CA,USA). This device is capable of discriminating between granulocytes andother leukocytes but not among neutrophils, eosinophils, and basophils.

Cytokine analysis of serum. Serum was collected and stored at �80°Cuntil further preparation. The cytokines included were interleukin-1�(IL-1�), IL-6, IL-10, IL-17A, gamma interferon (IFN-�), monocyte che-motactic protein 1 (MCP-1), and tumor necrosis factor alpha (TNF-).The concentrations of the cytokines were determined with a Luminex kitprepared according to the manufacture’s specifications (Bio-Rad, Hercu-les, CA, USA).

Free fatty acid analysis. Plasma (25 to 50 �l) was spiked with 43.5nmol of the internal standard (C23:0 fatty acid) and extracted by the auto-mated BUME method (20). After evaporation of the total extracts, the freefatty acids were derivatized to methyl esters with trimethylsilyldiazometh-ane (2 M hexane solution; Sigma-Aldrich, Sweden). After 2 h of de-

rivatization at room temperature, the samples were evaporated and re-constituted in 200 �l of heptane. The fatty acid methyl esters wereanalyzed with a 6890N gas chromatograph (Agilent, Santa Clara, CA,USA) coupled to a flame ionization detector. For analysis, 1 �l of theheptane phase was injected (1:10 split) and separated on a DB23 column(60 m by 0.25 mm [inner diameter], 0.15 �m; J&W 122-2361) with alinear gradient of 160 to 230°C at 4°C/min. Quantification of the fatty acidmethyl esters was done by relating the endogenous signals to the signalfrom the internal standard.

Survival and bacterial loads in resolvin experiments. To investigateif mice fed HFD-S would benefit from treatment with resolvins, mice werefed for 8 weeks, but this time only HFD-S. At 15 weeks of age, micewere i.v. inoculated with S. aureus. For more detailed information aboutthe inoculation and diet, see the previous sections. On days 1 to 4, micewere treated i.v. with the vehicle (0.5% ethanol in saline), RvD1 (1 or 10ng/dose), or RvD2 (1 or 10 ng/dose). Bacterial loads and survival wereonce again analyzed in infected animals 6 days after inoculation and mon-itored for 17 days after inoculation, respectively.

Statistical analysis. Analysis of the survival of mice fed LFD, HFD-S,HFD-�3, or HFD-�6 was done with the log rank test, and the results areillustrated as Kaplan-Meier curves. To investigate the correlation betweenbody weight and survival, Cox regression analysis was performed withbody weight as a covariate. Statistical analysis of the bacterial load andflow cytometry analysis consisted of a two-way analysis of variance(ANOVA) with the experiments and experimental days as factors. Eachfactor was considered a nuisance factor. The bacterial load and flow cy-tometry data are expressed as the estimated marginal mean plus standarderror of the mean (SEM). Body weight and composition were analyzed byANOVA, followed by Tukey’s post hoc test. When Levene’s test revealedunequal variance, Dunnett’s T3 post hoc test was used for comparisons ofnumerical data among the four experimental groups. In this case, the dataare expressed as the mean plus SEM. Analysis of the survival of mice fedHFD-S and treated with the vehicle, RvD1, or RvD2 was done with the logrank test, and the results are illustrated as Kaplan-Meier curves. Statisticalanalysis of bacterial loads after resolvin treatment consisted of a two-wayANOVA with contrast and the experiments as a factor. The experimentwas considered a nuisance factor. Differences in sample numbers weredue to laboratory errors or lack of sample material. All tests were twosided, and P 0.05 was considered significant. All of the statistical anal-yses were carried out with the SPSS software (version 18.0.2 for Windows;IBM Corporation, Armonk, NY, USA).

RESULTSMice fed HFD-�3 have better survival and decreased bacterialloads after S. aureus infection compared with mice fed HFD-S.Survival at day 17 after i.v. inoculation with S. aureus was 4.7-foldgreater among mice fed HFD-�3 than among mice fed HFD-S and2.7-fold greater than among mice fed HFD-�6 (P 0.001 and P �0.04, respectively) (Fig. 2A). However, there was no difference insurvival between mice fed HFD-S and those fed HFD-�6 (Fig.2A). As shown in Fig. 2B, bacterial loads 6 days after i.v. inocula-tion mirrored the survival data. Mice fed HFD-S had a 3.5-fold-greater bacterial load than mice fed LFD or HFD-�3 (P 0.001and P 0.001, respectively) and a 1.5-fold-greater bacterial loadthan mice fed HFD-�6 (P � 0.05). There was no differenceamong the bacterial loads of mice fed LFD, HFD-�3, orHFD-�6 (Fig. 2B).

S. aureus may use exogenous fatty acids for incorporation in itscell membrane (21). This energy-saving mechanism can poten-tially result in enhanced growth of S. aureus. We therefore inves-tigated serum saturated fatty acid content. The saturated fatty acidcontents of all of the groups were similar, except for mice fedHFD-�3, where the saturated fatty acid content was increased(Table 2).

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Uninfected mice fed HFD-�3 are less obese than mice fedHFD-S but obese compared with mice fed LFD. Obesity per semay increase susceptibility to infection (22). To investigate possi-ble effects of the different experimental diets on body weight andcomposition, we performed a DXA analysis. After 8 weeks on thedifferent experimental diets, mice fed LFD had 36% lower bodyweight (P 0.001) and 59% lower fat mass (P 0.001) than mice fedHFD-S, whereas mice fed HFD-�3 had only 16% lower body weight(Fig. 3A; P � 0.002) and 27% lower fat mass (Fig. 3B; P 0.001) thanmice fed HFD-S. Mice fed HFD-�6 had a body weight and fat masssimilar to those of mice fed HFD-S. At the same time, the lean massdid not differ between any of the experimental groups (Fig. 3A andB). Further, the increased body weight of mice fed HFD-S did notinfluence survival (P � 0.56).

Uninfected mice fed HFD-�3 have a higher frequency ofneutrophils in their bone marrow than mice fed HFD-S. To in-

vestigate the dietary effect on neutrophils, the frequency of neu-trophils in the bone marrow of uninfected mice was analyzed. Forthe gating strategy used, see Fig. 4A. Mice fed HFD-�3 had 9%higher frequency of Ly6G� neutrophils in bone marrow (P 0.001) than mice fed HFD-S, 10% higher (P 0.001) than micefed LFD, and 8% higher (P 0.01) than mice fed HFD-�6 (Fig.4B). However, there was no difference among the frequenciesof neutrophils in the bone marrow of mice fed LFD, HFD-S, orHFD-�6.

To investigate if dietary fats can affect the frequency of progen-itor cells in uninfected mice, bone marrow cells were stained withthe general hematopoietic precursor marker CD117. Mice fedHFD-�3 had a 10% higher frequency of CD117� precursor cellsin their bone marrow (P 0.05) than mice fed HFD-S and a 12%higher frequency (P 0.05) than mice fed HFD-�6. At the sametime, mice fed LFD had a 10% higher frequency of CD117� pre-

FIG 2 Survival and bacterial loads. (A) Survival at 0 to 17 days after S. aureus inoculation after 8 weeks on HFD-S, HFD-�3, or HFD-�6. The log rank test wasused to determine statistically significant differences (n � 20 mice per group). (B) Bacterial loads in the kidneys at day 6 after S. aureus inoculation after 8 weekson LFD, HFD-S, HFD-�3, or HFD-�6. Two-way ANOVA with experiment as a nuisance factor was used to determine statistically significant differences (n �18, 16, 18, and 10 mice per group, respectively). The data shown are the estimated marginal mean plus SEM. *, P 0.05; ***, P 0.001. Experimental groups:LFD, HFD-S, HFD-�3, and HFD-�6.

TABLE 2 Hematology analysis, serum cytokine levels, and free fatty acid contents of serum

Parameter LFD HFD-S HFD-�3 HFD-�6

WBCa count 2.1 � 0.3h 3.1 � 0.3 3.5 � 0.2e 3.3 � 0.4e

LYMb count 1.6 � 0.3 2.4 � 0.2 2.4 � 0.2 2.3 � 0.3GRANc count 0.4 � 0.1 0.6 � 0.1 0.9 � 0.1e 0.8 � 0.1

Concn of:IL-1� 928 � 107 951 � 131 997 � 59 801 � 94IL-6 30 � 6 34 � 7 41 � 4 33 � 4IL-10 164 � 25 250 � 45 239 � 23 191 � 30IL-17 983 � 154 1,262 � 134 1,146 � 136 1,098 � 172IFN-� 281 � 38 335 � 61 316 � 27 259 � 41MCP-1 1,050 � 150 1,024 � 152 1,063 � 65 890 � 118TNF- 24,646 � 3,944 28,270 � 5,428 27,118 � 2,970 25,051 � 4,395

SFAd content (mol%) 44.6 � 1.7 45.9 � 1.0 54.5 � 0.9e,f,g 45.9 � 1.8a WBC, white blood cells.b LYM, lymphocytes.c GRAN, granulocytes.d SFA, saturated fatty acids.e P 0.05 versus LFD.f P 0.05 versus HFD-S.g P 0.05 versus HFD-�6.h Values are means � standard errors of the means (SEM).

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cursor cells (P 0.05) than mice fed HFD-�6. There was nodifference in the frequency of hematopoietic progenitor cells in bonemarrow between mice fed HFD-�3 or LFD, between mice fed LFD orHFD-S, or between mice fed HFD-S or HFD-�6 (Fig. 4C).

The frequency of CD115� monocytes in bone marrow wasinvestigated since they also have the capacity to phagocytose bac-teria. Mice fed LFD had 7% (P 0.05), 10% (P � 0.01), and 13%(P � 0.001) higher frequencies of CD115� monocytes than micefed HFD-S, HFD-�3, or HFD-�6, respectively. There was no dif-ference in the frequency of monocytes in bone marrow amongmice fed HFD-S, HFD-�3, or HFD-�6 (Fig. 4D).

Mice fed HFD-S have a decreased frequency of neutrophilsphagocytosing pHrodo particles compared with that for micefed LFD or HFD-�3. Not only is the frequency of neutrophilsimportant for the survival of sepsis, but neutrophil phagocytosingcapacity is crucial for survival during an infection. To investigate ifthe diet also affects the capacity of neutrophils to phagocytosebacteria, blood from the mice was challenged with pHrodo parti-cles and analyzed by flow cytometry. Circulating neutrophils frommice fed HFD-S had a lower capacity to phagocytose pHrodoparticles than those from mice fed LFD (2.7%; P � 0.01) orHFD-�3 (2.5%; P 0.05) (Fig. 5B). The neutrophils of micefed HFD-S had a slightly and nonsignificantly lower (2.0%; P �0.057) capacity to phagocytose pHrodo particles than those ofmice fed HFD-�6. There was no difference between any of thegroups in the MFI of the neutrophils that had phagocytosedpHrodo particles (Fig. 5C).

There was no difference in the white blood cell counts of micefed HFD-S, HFD-�3, or HFD-�6. To investigate the dietary ef-fect on the immune cells in circulation, the white blood cell, gran-ulocyte, and lymphocyte concentrations in blood were analyzed.Mice fed HFD-�3 or HFD-�6 had higher white blood cell con-centrations in their circulation than mice fed LFD (Table 2). Therewas no difference between mice fed LFD and mice fed HFD-S oramong mice fed HFD-S, HFD-�3, or HFD-�6. Further, mice fedHFD-�3 had a greater granulocyte concentration in their circula-tion than mice fed LFD, but there were no differences among micefed LFD, HFD-S, or HFD-�6. The diet did not affect the lympho-cyte concentration in the circulation in any of the groups.

There was no systemic inflammation in any of the mice fedthe different diets. Since inflammation can affect the concentra-tion of neutrophils both in bone marrow and in circulation, thelevels of several inflammatory cytokines (IL-1�, IL-6, IL-10, IL-

17A, IFN-�, MCP-1, and TNF-) in serum were investigated.There were no differences between the groups in any of thesecytokines (Table 2).

Mice fed HFD-S might benefit from treatment with resolvinsafter inoculation with S. aureus. Resolvins are metabolites ofomega-3 fatty acids. Given the beneficial effects of dietary ome-ga-3 fatty acids described above, we hypothesized that the negativeeffects of HFD-S could be reduced by treating mice with i.v. re-solvins after inoculation with S. aureus. As shown in Fig. 6, feedingmice HFD-S and treating them with resolvins on days 1 to 4 afterinoculation did not influence survival at day 17. However, moreresolvin-treated mice than vehicle-treated mice survived to day 8after inoculation (85 and 60% survival, respectively) (Fig. 6A).Further, treatment with resolvins decreased bacterial loads in thekidneys 6 days after S. aureus inoculation (P 0.05) (Fig. 6B).

DISCUSSIONDietary omega-3 fatty acids increase the survival of mice after S.aureus-induced sepsis. In this study, we investigated the effects ofdietary omega-3 and omega-6 fatty acids on the immune systemand survival after S. aureus-induced sepsis. Our results show thataddition of dietary omega-3 fatty acids to dietary HFD led togreater mouse survival of S. aureus-induced sepsis than a diet richin saturated fatty acids or omega-6 fatty acids. Additionally, theresults show that mice fed HFD-�3, HFD-�6, or LFD had de-creased bacterial loads in their kidneys compared to those for micefed HFD-S, indicating that feeding mice HFD-�3 can reverse thedeleterious effects of saturated fatty acids on survival of S. aureus-induced sepsis. Mice fed HFD-�6 also had decreased bacterialloads in their kidneys compared to those for mice fed HFD-S, butthe difference was not great enough to affect their survival. S.aureus could benefit from using exogenous fatty acids for incor-poration in the cell membrane (21). This energy-saving mecha-nism can potentially result in enhanced growth of S. aureus andcould potentially explain the increased bacterial loads and the sub-sequent mortality rate of mice fed HFD-S. Still, this is not likelysince the saturated fatty acids contents of the different diets arequite similar. Further, the saturated fatty acid content in serum ofmice fed HFD-S was not increased after 8 weeks. Our findingsagree with our previous studies showing that mice fed HFD-S havean impaired immune response compared to that of mice fed LFD(9). The findings of this study are also in line with our recentfinding that mice fed HFD-P have greater survival and a higher

FIG 3 Body weight and composition. (A) Body weights of uninfected mice after 8 weeks on a diet. One-way ANOVA with Dunnett’s T3 post hoc test was used todetermine statistically significant differences (n � 10 mice per group). (B) Lean mass and fat mass of uninfected mice after 8 weeks on a diet, measured by DXA. One-wayANOVA with Dunnett’s post hoc test for lean mass and Tukey’s test for fat mass were used to determine statistically significant differences. The data shown are the meanplus SEM (n � 10 mice per group). **, P 0.01; ***, P 0.001. Experimental groups: LFD, HFD-S, HFD-�3, and HFD-�6.

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frequency of neutrophils in their bone marrow than mice fedHFD-S (10). Thus, our results show that omega-3 fatty acids arethe most probable fatty acids needed to exert the beneficial prop-erties of a polyunsaturated fatty acid diet.

The effects of dietary fatty acids on immune function are notcorrelated with body fat mass. The type of dietary fat seems to bemore important for the immune system than the total amount ofdietary fat consumed and the resulting obesity. In line with ourprevious findings, there was no correlation between body weightand survival; hence, the increased body weight of mice fed HFD-Sdid not influence their survival.

Dietary omega-3 fatty acids increase survival of sepsis andstimulate the immune system. In the literature, it is debatedwhether patients die from sepsis because of underactivity of theimmune system coupled with enhanced bacterial growth or be-cause of an overactivity of the immune system that impairs phys-iological functions and decreases the survival of the host (2, 7, 23).The present study shows that the bacterial load 6 days after inoc-ulation was decreased in mice fed HFD-�3 compared to that inmice fed HFD-S, indicating that the immune systems of mice fedHFD-�3 were more effective. The 3.5-fold difference in the bac-terial loads of mice fed HFD-�3 and those fed HFD-S may not

FIG 4 Leukocyte frequency in bone marrow. (A) Gating strategy for neutrophils, monocytes, and hematopoietic precursor cells in bone marrow. SSC, sidescatter; FSC, forward scatter. (B) Frequency of Ly6G� neutrophils in bone marrow (n � 10 mice per group). (C) Frequency of CD117� precursor cells in bonemarrow (n � 10 mice per group). (D) Frequency of CD115� monocytes in bone marrow (n � 10 mice per group). The data in panels B to D were compared bytwo-way ANOVA with experiment as a nuisance factor and are the estimated marginal mean plus SEM. *, P 0.05; **, P 0.01; ***, P 0.001. The results arepresented as the frequency of living leukocytes. Experimental groups: LFD, HFD-S, HFD-�3, and HFD-�6.

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seem a dramatic one; however, since an infection is a dynamicrelationship between the host’s immune system and the bacte-rial growth rate, it is plausible that more dramatic differencescould be present at earlier time points. Further, as the differ-ence between the mortality rates of mice fed HFD-�3 and micefed HFD-�6 occurred from about 1 week after inoculation, itseems likely that the decreased bacterial loads of mice fedHFD-�3 are associated with their greater survival.

Still, we cannot exclude the possibility that other factors, suchas bacterial virulence and/or direct antimicrobial effects of ome-

ga-3 fatty acids on S. aureus, could contribute to the improvedsurvival of mice fed HFD-�3. To address whether sera from micefed HFD-�3 contained any inhibitory substances influencing bac-terial growth, we used the disk diffusion method; however, wefound no evidence that the sera of mice fed HFD-�3 containedany inhibitory substances (data not shown). Hence, we concludethat mice fed HFD-�3 survive S. aureus-induced sepsis because ofa greater capacity to clear bacteria.

Dietary omega-3 fatty acids stimulate neutrophil frequencyand function. The immune response to bacterial infections, espe-

FIG 5 Capacity of circulating neutrophils to phagocytose pHrodo particles. (A) Gating strategy for pHrodo-phagocytosing neutrophils in circulation for bothfrequency and MFI. SSC, side scatter. (B) Frequencies of circulating CD45� Ly6G� pHrodo� neutrophils (n � 10 mice per group). (C) MFI of circulating CD45�

Ly6G� pHrodo� neutrophils (n � 10, 8, 9, and 8 mice per group, respectively). The data in panels A and B were compared by two-way ANOVA with theexperimental day as a nuisance factor and are the estimated marginal mean plus the SEM. *, P 0.05; **, P 0.01. Experimental groups: LFD, HFD-S, HFD-�3,and HFD-�6.

FIG 6 Survival and bacterial loads after treatment with resolvins. (A) Survival on days 0 to 17 after S. aureus inoculation after 8 weeks on HFD-S. On days 1 to4, mice received the vehicle (0.5% ethanol in saline), RvD1, or RvD2. The log rank test was used to determine statistically significant differences (n � 20 mice pergroup). (B) Bacterial loads in the kidneys on day 6 after S. aureus inoculation after 8 weeks on HFD-S. Two-way ANOVA with the experiment as a nuisance factorwas used to determine statistically significant differences (n � 23, 28, and 22 mice per group, respectively). The data shown are the estimated marginal mean plusSEM. *, P 0.05. Experimental groups: vehicle, RvD1, and RvD2.

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cially for defense against S. aureus, is greatly dependent on neu-trophils (24, 25). Therefore, the frequency of neutrophils in thebone marrow of uninfected mice after 8 weeks on different dietswas analyzed. We have previously shown that mice fed a diet richin polyunsaturated fatty acids (a mixture of both omega-3 andomega-6 fatty acids) also had a higher frequency of neutrophils intheir bone marrow (10). This was confirmed in the present studyin mice fed HFD-�3. In contrast, monocytes in bone marrow werenot affected by the type of fat in the diet, indicating that the effectof omega-3 fatty acids on neutrophils may be specific to neutro-phils only and not to all phagocytosing innate immune cells. Itshould be noted that the increased frequency of neutrophils inbone marrow in mice fed HFD-�3 does not automatically result inan increased frequency of neutrophils in blood; rather, it seemsthat neutrophils are retained within the bone marrow, ready to bereleased upon stimulation (26).

The frequency of circulating neutrophils that phagocytosedpHrodo particles was higher in mice fed HFD-�3 than in mice fedHFD-S and similar to that in mice fed LFD. Thus, it is most likelythat the increased survival is attributable not to a single mecha-nism but rather to a combination of an increased frequency ofneutrophils in the bone marrow and improved phagocytosing ca-pacity contributing to the increased survival of mice fed HFD-�3.

Further, a previous study by our group also indicated that di-etary polyunsaturated fatty acids increase the migration capacityof neutrophils (10). Taken together, these findings indicate thatpolyunsaturated fatty acids, especially omega-3 fatty acids, affectboth the frequency of neutrophils in bone marrow and their func-tion in circulation.

Dietary omega-3 fatty acids may both stimulate neutrophilfunction and inhibit inflammation. Clinically, a high frequencyof neutrophils, especially in blood, is regarded as a sign of anongoing infection with concomitant inflammation. However, wedid not see any differences in circulating granulocytes in the pres-ent study. Further, we have previously shown that mice fed a dietrich in polyunsaturated fatty acids (a mixture of omega-3 andomega-6 fatty acids) also had an increased frequency of neutro-phils in their bone marrow. Their increased frequency of neutro-phils did not seem to be a symptom of inflammation since they didnot have greater levels of inflammatory markers in their serumthan mice fed HFD-S in the uninfected state (10). This is furtherconfirmed in the present study, where none of the diets affectedthe levels of the inflammatory cytokines IL-1�, IL-6, IL-10, IL-17A, IFN-�, MCP-1, and TNF- in serum. Thus, the increasedneutrophil frequency found in bone marrow is not due to ongoingsystemic inflammation. Actually, dietary omega-3 fatty acids havebeen suggested to suppress inflammation (11). Hence, these find-ings suggest that it is possible to stimulate the capacity to fightinfections (e.g., with HFD-�3) without causing the deleteriouseffects associated with chronic inflammation, i.e., a type ofchronic immune activation in the absence of infection.

Dietary omega-3 fatty acids stimulate hematopoietic precur-sor cells. We wanted to investigate if diet affects the frequency ofCD117� hematopoietic precursor cells in bone marrow. Mice fedHFD-�3 had a higher frequency of hematopoietic precursor cellsthan mice fed HFD-S or HFD-�6. Therefore, it might be specu-lated that mice fed HFD-�3 are more prepared to quickly mobi-lize a leukocyte defense in general to fight off bacteria.

Potentially beneficial effects of resolvins on the bacterialload in sepsis. In vivo, omega-3 fatty acids can be metabolized into

a novel substance group named resolvins (15). It has previouslybeen suggested that treatment with RvD1 and RvD2 can increasethe survival of sepsis induced by cecal ligation and puncture; how-ever, the mechanism responsible for this remains unclear (15, 27).It has been suggested that resolvins affect the migration capacity ofneutrophils (28). Since our previous findings indicate that poly-unsaturated fatty acids also affect the migration capacity of neu-trophils and our present study shows that omega-3 fatty acidsincrease the capacity of neutrophils to phagocytose, we decided toinvestigate if mice fed HFD-S could be saved by treatment withresolvins after S. aureus-induced sepsis. The overall survival ofresolvin- and vehicle-treated mice on day 17 after inoculation didnot differ. However, more resolvin-treated mice than vehicle-treated mice survived to day 8 after inoculation (85 and 60% sur-vival, respectively). Further, there was a trend toward increasedsurvival of mice treated with resolvins on days 7 and 9 after inoc-ulation. Moreover, on day 6 after inoculation, mice treated withresolvins had decreased bacterial loads in their kidneys comparedto those for mice treated with the vehicle. Taken together, theseresults suggest that mice fed HFD-S can benefit from treatmentwith resolvins after S. aureus-induced sepsis; however, treatmentwith the doses used in the present study and only treatmentduring days 1 to 4 after S. aureus-induced sepsis were not suffi-cient to affect the overall mortality rate 17 days after inocula-tion. Further studies are warranted, as resolvins are an inter-esting potential treatment for S. aureus-induced sepsis. Thatthey can be administered after the infection is manifested bythe results of both this study and a previous one (15, 16), andthey do not require pretreatment, as diet does.

The differences between the effects of omega-3 and omega-6fatty acids. Our finding that a diet containing omega-3 fatty acids,but not omega-6 fatty acids, can stimulate the immune system andproduce a mortality rate during infection lower than that of micefed HFD-S is of interest in relation to earlier studies on the healtheffects of omega-3 and omega-6 fatty acids. Arachidonic acid,which is an omega-6 fatty acid, is a precursor of both prostaglan-dins and leukotrienes. Prostaglandins and leukotrienes are be-lieved to be mostly proinflammatory, while omega-3 fatty acidmetabolites are mostly anti-inflammatory (11). For example, pa-tients with the inflammatory disease rheumatoid arthritis benefitfrom dietary omega-3 fatty acids (12). Dietary omega-3 fatty ac-ids are also believed to decrease the risk of cardiovascular dis-ease, possibly thanks to suppressed inflammation, but the ben-eficial effect of omega-3 fatty acids in this context may be rathersmall (13). The exact relationship between the various effects ofdietary omega-3 and omega-6 fatty acids remains to be eluci-dated.

For technical reasons, the omega-3 fatty acid diet in the presentstudy contained some omega-6 fatty acids (about as much as theomega-3 fatty acids). In contrast, the omega-6 fatty acid diet onlycontained 1/50 omega-3 fatty acids and should reflect a pure ome-ga-6 fatty acid effect. Therefore, what distinguishes the omega-3fatty acid diet from the omega-6 fatty acid diet is the presence ofomega-3 fatty acids rather than the absence of omega-6 fatty acids.Moreover, the contents of saturated fatty acids and monounsatu-rated fatty acids were only moderately lower in the omega-3 fattyacid diet than in the HFD-S diet. Importantly, this means thatomega-3 fatty acids themselves are likely to be the reason for thepositive effects of an omega-3 fatty acid diet. As omega-3 fatty

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acids are the precursors of resolvins, this is in line with the bene-ficial effects of resolvins on infections.

It may be speculated that the protective effect of omega-3 fattyacids is effective only in slightly immunosuppressed states, such asthat of mice fed HFD-S. Interestingly, a previous sepsis studydemonstrated the effect of resolvins on mice fed standard chow(27). Since resolvins are metabolites of omega-3 fatty acids, it ispossible that omega-3 fatty acids can act independently of otherdietary components; however, this needs to be investigated fur-ther.

Conclusions. In conclusion, our findings indicate that feedingmice omega-3 fatty acids results in better survival of sepsis thanthat of mice fed a diet rich in saturated fatty acids. The mechanismresponsible for this is most likely a combination of an increasedfrequency of neutrophils and precursor cells in bone marrow anda restored phagocytosis capacity. These factors, prior to infection,contribute to decreased bacterial growth. From a clinical perspec-tive, these results indicate that people eating a diet rich in satu-rated fatty acids, i.e., most of the people in the western world,could benefit from an increased intake of omega-3 fatty acids.

ACKNOWLEDGMENTS

We are grateful for the excellent technical assistance of Sansan Hua, PeterMicallef, and Karl Wendt. We thank Stefan Lange for expertise regardingbacterial growth, the disk diffusion analysis, and helpful comments on themanuscript.

We confirm that there are no conflicts of interests.

FUNDING INFORMATIONThis work was supported by grants from the Swedish Research Council(K2013-54X-09894-19-3 [J.-O.J.] and 2667 [M.E.J.]), the Swedish Societyfor Medical Research (M.E.J.), and the Sahlgrenska Center for Cardiovas-cular Metabolic Research (CMR, no. A305: 188, J.-O.J.), which is sup-ported by the Swedish Strategic Foundation, EC FP7 funding(Full4Health FP7-KBBE-2010 – 4-266408, J.-O.J.), the Magnus BergvallFoundation (M.E.J.), Längmanska Kulturfonden (M.E.J.), StiftelsenGamla trotjänarinnor (M.E.J.), OE och Edla Johanssons vetenskapligaStiftelse (M.E.J.), the Lars Hiertas Foundation (M.E.J.), the Åke WibergFoundation (M.E.J.), and Stiftelsen Tornspiran (S.L.S.).

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