Accepted Manuscript

Oral Omega-3 Fatty Acids Promote Resolution in Chemical Peritonitis

Alexander C. Chacon, BSc, Brett E. Phillips, PhD, Miranda A. Chacon, BSc, Deborah
Brunke-Reese, BSc, Shannon L. Kelleher, PhD, David I. Soybel, MD

PII: S0022-4804(16)30157-3
DOI: 10.1016/j.jss.2016.06.036
Reference: YJSRE 13834

To appear in: Journal of Surgical Research

Received Date: 5 February 2016

Revised Date: 1 June 2016
Accepted Date: 10 June 2016

Please cite this article as: Chacon AC, Phillips BE, Chacon MA, Brunke-Reese D, Kelleher SL, Soybel
DI, Oral Omega-3 Fatty Acids Promote Resolution in Chemical Peritonitis, Journal of Surgical Research
(2016), doi: 10.1016/j.jss.2016.06.036.

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 ACCEPTED MANUSCRIPT
Revised 05-25-2016

Oral Omega-3 Fatty Acids Promote Resolution in Chemical Peritonitis

Short title: Oral Omega-3s in Chemical Peritonitis

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Alexander C. Chacon, BScd, Brett E. Phillipsa, PhD, Miranda A. Chacon, BScd,

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Deborah Brunke-Reese, BSca , Shannon L. Kellehera,b,c, PhD, and David I. Soybel, MDa,b

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Departments of Surgerya, Cellular and Molecular Physiologyb, and Pharmacologyc, Pennsylvania State

University College of Medicined, Hershey, Pennsylvania, USA.

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*Corresponding Author:
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David I. Soybel MD
Department of Surgery
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Penn State College of Medicine

500 University Drive
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PO Box 850, Mail Code H149

Hershey, PA 17033
Tel 717-531-5272
FAX 717-531-0884
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Email dsoybel@hmc.psu.edu
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Authors’ contributions: A.C.C., B.E.P., S.L.K., and D.I.S. designed the study. A.C.C., B.E.P., M.A.C., and
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D.B-R. executed the experiments. A.C.C., B.E.P., and D.I.S. interpreted the data and wrote the

manuscript. All authors discussed, revised the manuscript for intellectual content, and gave approval of

the final version.

Disclosures: A.C.C., B.E.P., M.A.C., D.B-R., S.L.K., and D.I.S. reported no proprietary or commercial

interest in any product mentioned or concept discussed in the article.

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Abstract

Introduction

Recent studies suggest that purified omega-3 fatty acids may attenuate acute inflammation and hasten

the transition to healing. In this study, we tested the hypothesis that pre-treatment with omega-3 rich

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fish oil (FO) would promote resolution of peritoneal inflammation through production of specific lipid

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mediators.

Methods

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C57/BL6 mice were given a daily 200 μL oral gavage of saline (CTL) or FO (1.0-1.5 g/kg/d DHA and 1.3-2.0

g/kg/d EPA) for 7 days before chemical peritonitis was induced with thioglycollate. Peritoneal lavage

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fluid was collected before induction and at days 2 and 4 post peritonitis onset. Prostaglandin E2 (PGE2),
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Leukotriene B4 (LTB4), Resolvin D1 (RvD1), and the composition of immune cell populations were

examined in peritoneal lavage exudates. Cells harvested from the peritoneum were assessed for
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macrophage differentiation markers, phagocytosis, and LPS-induced cytokine secretion profiles (IL-6, IL-
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10, IL-1β, TNFα).

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Results

The ratio of RvD1 to pro-inflammatory PGE2 and LTB4 was increased in the peritoneal cavity of FO
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supplemented animals. FO induced a decrease in the number of monocytes in the lavage fluid, with no

change in the number of macrophages, neutrophils, or lymphocytes. Macrophage phagocytosis and

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M1/M2 mRNA markers were unchanged by FO with the exception of decreased PPARγ expression. FO
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increased ex vivo TNFα secretion after stimulation with LPS.

Conclusions

Our findings provide evidence that nutraceutically-relevant doses of FO supplements given before and

during chemical peritonitis shift the balance of lipid mediators towards a pro-resolution, anti-
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inflammatory state without drastically altering the number or phenotype of local innate immune cell

populations.

Keywords: Fish Oil, Inflammation, Resolution, Peritonitis, Resolvin D1, Leukotriene B4

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Introduction

Resolution of local and systemic inflammation is critical in the process of recovery from

peritonitis. Once thought to be a passive process involving depletion of pro-inflammatory mediators,

resolution is now known to be an active, cell-mediated progression involving the actions of specific lipids

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and proteins as drivers of the resolving response. Novel discoveries 1-3 have illuminated the second half

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of this bimodal response to immune challenge, which is dictated in part by bioactive lipid mediators with

varied functions. As reviewed elsewhere4, pro-resolution lipid mediators - resolvins, maresins and

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protectins – appear during the first phase of inflammation and are capable of inducing the resolution

program, which includes the eventual deceleration of recruitment of neutrophils and acceleration of

recruitment of macrophages to inflamed spaces3,5.

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macrophage population is switched from M1 (pro-inflammation) to M2 (pro-resolution)6, which includes

decreased production and/or down-regulation of pro-inflammatory cytokines7,8 and alterations in

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immune cell shape and morphology consistent with the scavenging of non-viable cells and material, a

process known as efferocytosis9. It has been postulated that altered ratios of pro-resolution to pro-
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inflammation lipids (prostaglandins, leukotrienes) may effectively dictate the character of an

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inflammatory response by shifting the balance of pro- and anti-inflammatory cytokines and
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chemokines10.

Endogenous production of pro-resolving lipid mediators depends on availability of specific

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precursors in the omega-3 (n-3) fatty acid class, including DHA (docosahexaenoic acid) and EPA
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(eicosapentaenoic acid). Thus, it has been hypothesized that resolution of certain states of chronic

inflammation might be interrupted by treatment with these compounds, either in purified form or in the

form of fish oil (FO) supplements. As the FO industry has grown to become a nearly $2 billion market

and recent surveys have shown that 1/3 of Americans taking nutritional supplements may be using a FO

supplement11, the need exists to carefully examine how omega-3 administration may affect medical
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care, especially in relation to invasive procedures. Indeed, the benefits of purified and dietary

supplement forms have been observed in some settings associated with the surgical patient. Recent

meta-analyses show that enteral immunonutrition regimes, which include omega-3 supplements, may

optimize recovery of patients who undergo elective surgery for GI cancer, especially when delivered in

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the perioperative period12,13, while some parenteral models of omega-3 supplementation have also

shown beneficial effects14,15. In murine models a similar mixed picture exists, but in congruence with

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human models a majority of studies providing FO supplementation over periods of 5 days to 12 weeks

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display downstream resolution-favoring changes in mRNA expression of classical M1/M2 macrophage

markers of maturation16, inflammatory cytokine and lipid profile17-19, efferocytosis20, and immune cell

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oxidative potency6, with additional reports indicating that oral FO takes 24-72 hours to induce beneficial
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metabolic effects7.

It remains unclear what sorts of benefits would be observed in settings of more acute
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inflammation such as recovery from surgery 21,22. Critical concerns in design of such studies include the
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demonstration that FO supplementation actually results in increased production of pro-resolution lipid

mediators and that they are available in the inflamed space. Also important is the need to characterize
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changes in profiles of pro- and anti-inflammation cytokines resulting from such supplementation23-25.
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Lastly, it remains unclear whether dietary supplements can elicit the phenotypic switch to resolution

with efficacy similar to that of purified forms of DHA/EPA but with less toxicity.
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In this study, we utilized a mouse model of sterile chemical peritonitis to characterize the local
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transition from acute inflammation to resolution in response to dietary FO supplements that may be

obtained commercially. Thioglycollate is a relatively mild chemical irritant which provides a

reproducible and consistent approach to study cascades of inflammation and resolution in the mouse

and rat analogous to those in chemical and mechanical peritonitis conditions such as gastric acid leak,

abdominal trauma, and surgical bowel irritation, without resulting in the presence of confounding
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polymicrobial infection26. Peritoneal injection of thioglycollate has been utilized as a low mortality, low

systemic sequelae method to recruit and subsequently harvest inflammation-driving resident

macrophages for the purposes of in vitro and ex vivo study, and captures cells transitioning between

acute inflammation and active resolution (3-4 days post-injection) with peak inflammatory response in

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the first 1-2 days and complete clearance by day 7-827. Using this model, we find that relevant doses of

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oral FO favor production and local appearance of resolution-phase lipid mediators but may yet be

unable to elicit the dramatic changes in cell number and cytokine production that would be required to

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effect a clear benefit in accelerating recovery from an acute sterile inflammatory insult.

Materials/Methods

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Animals and Experimental Design

All animal procedures were approved and conducted in accordance with the Institutional Animal
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Care and Use Committee of Penn State Milton S. Hershey College of Medicine. Male C57BL/6 mice were
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obtained commercially (Taconic, Hudson, NY) and individually housed in polycarbonate cages with a 12

h light/dark cycle. Solely male mice were utilized, as recent reports have displayed differences in
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leukotriene biosynthesis between sexes following chemical peritonitis28. Animals were fed ad libitum
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with the commercial AIN-93M (MP Biomedicals, Santa Ana, CA) diet containing 4% soybean oil for 28

days starting at 10-12 weeks of age. On days 21 through 28, mice were given 200 µL daily oral gavages
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of PBS as control (CTL) or Marinol C-38 (FO) (Stepan, Maywood NJ), a proprietary fish oil blend
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containing 17% DHA and 24% EPA (approximately 1.0-1.5 g/kg/d DHA and 1.3-2.0 g/kg/d EPA). At day

28, mice from each group were either euthanized to establish baseline infiltrative and secretory

characteristics of immune cell populations within the peritoneum (Day 0) or 1mL 3% thioglycollate was

injected into the peritoneal cavity to induce a chemical peritonitis, as established in prior studies27.

Mice given thioglycollate were euthanized at day 2 or 4 post-injection. Mouse diets and oral gavages
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were continued throughout the additional duration of chemical peritonitis. Once euthanized, mouse

blood was collected via cardiac puncture and plasma was isolated via centrifugation at 1000 rcf and

immediately flash-frozen in liquid nitrogen. Additionally, the peritoneal cavity was rinsed with 5mL cold

1X PBS, the peritoneal lavage exudate was collected, and the cellular and liquid exudate fractions were

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isolated via centrifugation at 3000 rcf. The supernatant was flash-frozen.

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Ex vivo cytokine release and bioparticle phagocytosis

Peritoneal exudate cells from 6-8 mice per group were isolated and counted via

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hemocytometry. 2.5X105 cells from mice 2 and 4 days post-injection were suspended in 200 µL media,

plated on 96 well plates and incubated for 16 h with and without LPS (100 ng/mL). Cells from day 0 mice

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at baseline were not utilized as cell numbers were too low for all assays except for cytologic analysis.
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Following stimulation, culture media was removed and assessed for cytokine content (IL-6, IL-10, IL-1β,

and TNFα) by ELISA according to the manufacturer’s protocols (Duoset, R&D Systems, Minneapolis, MN).
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Immune cell phagocytosis was assessed on the same cells via incubation with fluorescent E. coli particles
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(Vybrant Phagocytosis Assay Kit (Life Technologies, Frederick, MD)) for 2 h, after which intracellular

fluorescence was measured in a Synergy-2 fluorescent plate reader at 480 nm excitation and 520 nm
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emission settings.
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Cytology of infiltrated exudate cells

1X105 exudate cells from mice at baseline or at 2 and 4 days post-injection were independently
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plated on Superfrost microscope slides using a CytoPro centrifugation system, allowed to dry for 1 h,
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and fixed with 50 μL MeOH. Slides were stained with 50 μL Wright/Giemsa stain to differentiate

immune cell types. Briefly, on one slide per mouse and 4 field counts per slide, cell types were

determined according to cell morphology, nuclear characteristics, cytoplasmic contents, and color. Cells

were deemed vacuolar if more than 50% of the cytoplasm consisted of vacuoles. To ensure objective

determination of cell morphology and identification of cellular debris, two individuals independently
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assessed unidentified samples (results averaged) to allow for the novel characterization of macrophage

vacuole content.

Gene expression profiling by quantitative PCR

Cells harvested 4 days post-thioglycollate injection (90-95% macrophages) were assessed for

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macrophage differentiation markers via quantitative PCR (qPCR). RNA was isolated with the Totally RNA

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kit (Ambion, Naugatuck, CT) and 1 µg was used to generate cDNA with the ImProm-II Reverse

Transcription System (Promega, Madison, WI). qPCR was performed with the QuantStudio 12K Flex

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machine (ThermoFisher, Waltham, MA) using the FastStart Universal SYBR Green Master (ROX) mix

(Roche, Mannheim, Germany). All primers for Actin29, Arginase 130, CD3629, IL-630, iNOS30, PPARγ31,

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Ym230, and 12/15 LO32 were purchased from Integrated DNA Technologies (Coralville, IA), and consisted
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of sequences utilized in the literature.

Peritoneal exudate lipid assessment

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Frozen peritoneal exudate supernatant was assessed for Prostaglandin E2 (PGE2), Leukotriene
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B4 (LTB4) and Resolvin D1 (RvD1) content. Lipid fractions of the exudate supernatant were

concentrated using the Folch method33. Briefly, 2mL of sample was extracted using 8mL 2:1
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chloroform/methanol, vortexed thoroughly in polystyrene tubes, incubated 5 min, and centrifuged at

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4500 rpm for 20 min (room temperature). The organic (lower) phase was collected using sterile glass

pipettes and concentrated via vacuum centrifuge. Lipid content of isolated samples was assessed by
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ELISA according to company specifications (Cayman Chemical, Ann Arbor, MI). Total protein content in
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the exudate supernatant was assessed via Bradford Reagent (Sigma-Aldrich, St Louis, MO) and was

unchanged between treatment conditions (data not shown).

Statistical Analysis

Data are presented as means ± SEM. Data was assessed by Student t-test for comparison of

control and fish oil groups, or by Two-way ANOVA to examine treatment and time point interactions
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using GraphPad Prism, version 5.03. Bonferroni post-tests were run to examine significant effects of

time. A p-value of <0.05 was considered significant.

Results

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Supplement impact on functional parameters

No observable differences in alertness, grooming habits, posturing, or skin health of mice

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administered PBS or FO was noted. Basic assessments of mouse vitality, including baseline weight (the

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start of AIN 93 diet), weight change over the experimental period, and food intake were not different

between the groups (Table 1).

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Effect of Fish Oil on Immune Cell recruitment
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Recruitment of immune cells in response to chemical peritonitis in CTL and FO mice was determined

via Wright-Giemsa staining and differential classification. Thioglycollate induced significant recruitment
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of immune cells at days 2 and 4 (Figure 1). Monocyte number was significantly reduced with FO
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supplementation compared to control mice, p<0.05. Monocyte content was greater on day 2 than on

days 0 and 4. Cells derived from the monocyte lineage with mature characteristics (macrophages/
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[monocytes + macrophages]) were significantly increased with FO supplementation. FO did not

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significantly alter the total number of cells found in the peritoneal lavage before or after induction of

peritonitis with 3% thioglycollate. There were no significant differences in the numbers of macrophages,
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neutrophils, lymphocytes, plasma cells (Figure 1) or basophils, eosinophils, mast cells, or dead/dying
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cells in the peritoneal lavage (data not shown). It was observed that macrophages exhibited a more

vacuolar morphology following FO supplementation, though this effect was not significant, p=0.23.

Supplement impact on cytokine production, phagocytosis, and mRNA expression

Ex vivo cytokine secretion profiles in response to the danger signal LPS were examined in peritoneal cells

isolated on days 2 and 4. 16 h of LPS treatment induced a marked increase in secretion of IL-6, TNFα, IL-
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10, and IL-1β (Figure 2) from baseline, which was nearly undetectable without stimulation (data not

shown). Interestingly, TNFα secretion with LPS stimulation was greater in the FO group than the CTL

group, p<0.01. In both treatment conditions, secretion of IL-10 decreased and TNFα secretion increased

over time from day 2 to day 4, while IL-6 and IL-1β levels remained unchanged from day 2 to day 4

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regardless of treatment.

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To examine classical or alternative macrophage activation in response to chemical immune

challenge with or without FO supplementation, expression profiles of peritoneal cells at day 4 (90-95%

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macrophages) were determined via qPCR. PPARγ mRNA levels were significantly decreased with FO

supplementation, p<0.01 (Figure 2e). FO did not induce any differences in mRNA levels of Arginase 1,

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CD36, IL-6, iNOS, Ym2, or 12/15 LO in the recruited peritoneal immune cells.
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Ex vivo E. coli bioparticle phagocytosis was examined in peritoneal cells isolated on days 2 and 4

(Figure 2f). There were no significant differences in phagocytosis by recruited immune cells.
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Effect of fish oil on lipid mediator production

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Lipid mediators of inflammation were assessed in the peritoneal lavage. The ratio of anti-

inflammatory (RvD1) to pro-inflammatory (PGE2 or LTB4) lipid mediators was examined, as the balance
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between these two groups establishes the immune response. FO supplementation increased the
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RvD1/PGE2 ratio, RvD1/LTB4 ratio, and RvD1/ (PGE2+LTB4) ratio (Figure 3), p<0.05. Additionally, FO

reduced concentration of LTB4 in the peritoneal lavage (p<0.05), but elicited no significant change in
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RvD1 (p=0.16) or PGE2 (p=0.18). The concentration of LTB4 was elevated on day 2 as compared to days
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0 and 4. RvD1/LTB4 and RvD1/(PGE2 + LTB4) ratios were elevated on day 0 compared to days 2 and 4.

Discussion

The potential for specific natural and synthetic pro-resolving lipid compounds to alleviate

inflammation and hasten recovery from injury and illness has been well established in recent years34,35.
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Administration of highly processed nutraceutical agents may, however, be attended by unanticipated

toxicities or undesirable pharmacologic effects at high doses, with undetermined safety and utility of FO

supplementation pre and peri-operatively. Fish oil appears to be safe at doses up to 7 g/d, even in

patients on anticoagulant therapy, but may induce bleeding at doses of near 20 g/d in humans36, and the

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side effect profile of sub-acute or chronic supraphysiologic omega-3 fatty acids or targeted pro-resolving

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medicines is uncertain. Additionally, the translatability of studies incorporating pro-resolving lipids as

targeted agents, including the physiology dictating their effects on healing and convalescence in surgical

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illness, remains unclear. In this study of resolution from chemical peritonitis in mice, we demonstrated

that 7 days of oral fish oil gavage at a dose of approximately 6-9 g/kg/d (1.0-1.5 g/kg/d DHA and 1.3-2.0

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g/kg/d EPA) could increase the ratio of resolution phase lipid mediators to inflammatory lipid mediators
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while inducing only subtle alterations in immune cell type and number, cytokine production, and

functional status.
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As others have shown no change in Resolvin D1 content in serum37 and only modest changes in

D and E series Resolvins in the bone marrow following dietary omega-3s in comparable doses38, we
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sought to assess changes in inflammatory cell types and function in a specific, localized space to better
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understand transitions from inflammation to resolution in peritonitis. In response to oral omega-3

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supplementation, our data display a decrease in the number of monocytes present in the mouse

peritoneum following thioglycollate-induced chemical peritonitis, with no change in neutrophil or

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lymphocyte number. This stands in contrast to the results of prior chemical peritonitis studies in mice
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that have used intraperitoneal or intravenous administration of pro-resolution DHA and DHA- or EPA-

derived lipid mediator products, where moderate reductions in neutrophil influx39 as well as alterations

in efferocytotic capacity40-45 were noted. We also observed an increase in the ratio of macrophages/

(monocytes + macrophages) in the peritoneal space in mice supplemented with fish oil, with no change

in the number of macrophages present. This result indicates that cells derived from the monocytic cell
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line are present in the space following omega-3 supplementation; however, infiltrated or locally derived

early phase monocytic cells either do not migrate into the peritoneum in response to chemical challenge

or do not remain undifferentiated in the space for long before transitioning to macrophages. We did

note that cells from mice supplemented with fish oil were more vacuolar appearing, especially on day 2

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post injection; however, this result was not statistically significant and no change in bioparticle

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phagocytosis was observed in response to fish oil supplementation. Together, these results

demonstrate that oral FO supplementation does result in alterations in inflammatory cell recruitment

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and maturation in response to chemical peritonitis, but that this effect does not mimic the more potent

functional and phenotypic changes seen with administration of the downstream resolvins, maresins, and

protectins.

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In this study, we demonstrate that oral fish oil supplementation induced a significant increase in

TNFα secretion from isolated exudate cells challenged with LPS. This result is produced in a model of
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secondary macrophage activation, differing from models assessing systemic TNFα burden or models

assessing co-incubation of cells with resolvins and LPS42,46. Moreover, this effect is observed 2 and 4
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days downstream of in vivo chemical insult, perhaps indicative of retained ability of macrophages to
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elicit their cell recruitment and activation functions. Furthermore, in our day 4 exudate cellular
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population (90-95% macrophages), PPARγ mRNA expression was 27% lower in mice supplemented with

FO, with a limited response in mRNA message for classical M1/M2 phenotypic markers of macrophage
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differentiation. In vitro studies assessing the relationships of PPARγ lipid ligands, PPARγ binding, and
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downstream upregulation of macrophage differentiation markers such as CD36 (a surface receptor

implicated in efferocytosis) and Arginase 1 (a transaminase implicated in the classical M1/M2 switch)

have begun to uncover some as-of-yet unclear associations17,29,47-49. Akin to our results, one study

displayed a 26% decrease in PPARγ mRNA in mouse aortas following a 12 week fish oil diet; in another

study TNFα decreased PPARγ synthesis in macrophages50, perhaps providing a mechanism for the
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decreased PPARγ mRNA, unchanged particle phagocytosis and unchanged macrophage vacuolization in

this study. These results imply solely a mild change in M1/M2 macrophage phenotype occurs after

administration of short-term oral FO supplementation, in contrast to the potent effects of resolution-

phase lipid mediators6,42,46,51

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Our mouse model of FO supplementation displayed an increase in the ratio of pro-resolving

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RvD1 to pro-inflammatory LTB4 and PGE2, a novel finding which may indicate a relevant shift in

inflammatory balance favoring milder inflammation and improved resolution. This effect was not

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attributable to Resolvin D1 alone as it only displayed a trend of increasing concentration with FO

supplementation. Here, LTB4 decreased in response to FO. These findings suggest that the dietary

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omega-3 fatty acids DHA and EPA promote a pro-resolution lipid mediator milieu not only through the
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distinct resolution-phase actions of their downstream precursors, but also through suppression of

previously active inflammatory lipid cascades. LTB4 production is hindered by increasing pro-resolving
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metabolite concentrations51,52, and it has been shown that diminished LTB4 production results in

diminished neutrophil influx into the peritoneum in surgically induced bacterial peritonitis in mice53. We
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propose that therapies which increase the RvD1 to LTB4 ratio may hasten the recovery from
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inflammatory surgical illness.

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In conclusion, we have shown that relevant doses of FO in the mouse can induce a pro-resolving

lipid mediator milieu in the peritoneum without severely altering the recruitment of immune cells to the
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peritoneal space. This occurs during the transition phase from acute inflammation to active resolution.
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These results contrast those from studies utilizing purified lipid mediators, which have displayed great

potency in mouse and human models of inflammation. Further characterization of ratios between lipid

mediators in various disease states still remain to be identified, and approaches developed to intervene

are experimental. Many studies addressing the effects of resolvins, lipoxins, protectins, and maresins in

resolution from inflammatory conditions are underway, though basic dietary studies using whole fish oil
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and precursor lipid compounds should not be abandoned until more is known about lipid mediator

biology in surgical diseases.

In the setting where millions of Americans are already consuming dietary or supplemental fish

oil of their own accord, it is possible that under certain clinical circumstances oral preparations of lipid

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precursors such as DHA and EPA will result in mild to moderate accumulation of pro-resolving lipid

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mediators, with still unclear consequences on convalescence from surgery. This study provides a

foundation for understanding lipid and macrophage biology in acute inflammation in a controlled and

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non-infectious environment, helping to clarify the results of microbe-dependent pre-clinical models

studying omega-3 administration in bowel perforation, bacterial peritonitis, sepsis, and ileus. Additional

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studies of chemical peritonitis incorporating concomitant mono- or poly-microbial infection are
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warranted in order to fully understand the role that omega-3 supplementation may play in the

management of most clinical peritonitis scenarios. As others have shown, pre-, peri-, or post-
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operatively there may be opportunities to improve healing and convalescence using omega-3 therapies

without increasing the risk of post-operative surgical site complications and infection14, 54, 55, 56.
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Acknowledgements:
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E. coli bacterial endotoxin was a gift of Dr. James A. Lederer, (Brigham and Women’s Hospital, Boston,

MA). This study was funded by the Pennsylvania State University College of Medicine Department of
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Surgery.
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Figure Legends

Cover Legend. Representative Wright-Giemsa stain of cells isolated from mouse peritoneal lavages 2
days post-induction of chemical peritonitis, visualized via light microscopy. Slide displays monocytes,
macrophages, neutrophils, and lymphocytes from peritoneal lavages. Mice were treated with 7 days of
saline or fish oil oral gavage prior to induction of peritonitis with thioglycollate.
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Figure 1. Influence of FO supplementation on peritoneal immune cell populations recruited during

peritonitis. Wright-Giemsa stains of cells isolated from mice at day 0, 2, and 4 post-thioglycollate
injection were visualized via light microscopy for CTL mice (a, b, and c respectively) and FO mice (d, e, f).
Cells were classified by morphologic features and quantified. White bars indicate CTL, black bars
indicate FO. Results are presented as means ± SEM, with statistically significant treatment effects
denoted by * (2-way ANOVA, p < 0.05). Significant effects of time via 2-way ANOVA are not displayed
due to low cell content on day 0; effects due to time on days 2 and 4 are displayed based on Bonferroni

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post-tests signified by letters. The number of total live cells (g), monocytes (h), macrophages (i),
neutrophils (m), lymphocytes (n), and plasma cells (o) are presented as million cells infiltrated into the
peritoneum. Infiltrating macrophages were further characterized as avacuolar (less than 50% cytoplasm

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vacuolar, j) or vacuolar (>50%, k). Cells from the monocyte lineage deemed macrophages were
compared to total (Macrophages/ [monocytes+macrophages], l).

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Figure 2. Influence of FO supplementation on cytokine production, phagocytosis, and gene expression
of isolated peritoneal cells. White bars indicate CTL, black bars indicate FO. Results are presented as
means ± SEM, with statistically significant treatment effects denoted by * and significant time effects

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denoted by letters (2-way ANOVA, p < 0.05). 3% thioglycollate-elicited inflammatory cells (2 days and 4
days) were assessed for ex vivo LPS-stimulated (16 h) cytokine production, namely IL-6 (a), TNFα (b), IL-
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10 (c) and IL-1β (d). Recruited inflammatory cells (f) were assessed for ex vivo fluorescent particle
phagocytosis. qPCR was conducted on infiltrated peritoneal exudate cells on day 4 (90-95%
macrophages) to determine mRNA content of proteins involved the classical M1/M2 transition,
inflammatory cell efferocytosis, and fat metabolism (e). Results for mRNA are presented as means ±
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SEM and significance was determined via t-test with p < 0.05 (*). p-values less than 0.10 are shown.
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Figure 3. Influence of FO supplementation on content of lipid mediators in peritoneal exudates in

response to chemical peritonitis. White bars indicate CTL, black bars indicate FO. Results are expressed
as means ± SEM, with statistically significant treatment effects denoted by * and Bonferroni post-tests
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denoted by letters (2-way ANOVA, p < 0.05). Day 0, 2, and 4 post-thioglycollate injection peritoneal
exudate washings (5 mL) were assessed for concentration of Prostaglandin E2 (a), Leukotriene B4 (b),
and Resolvin D1 (c) via ELISA. Ratios between Resolvin D1 and Prostaglandin E2 content (R/P ratio, d),
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Resolvin D1 and Leukotriene B4 content (R/L ratio, e), and Resolvin D1 and the combination of
Prostaglandin E2 and Leukotriene B4 content (R/[P+L] ratio, f) were calculated.

Table 1. Significant findings are indicated by an arrow (p < 0.05). No change is indicated by a (-).
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Parameters displayed include baseline weight of animals (initiation of AIN 93 diet, in g), food intake (g)
animal weight change (at date of euthanization, in g), immune cell populations in the peritoneum at the
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time of sacrifice (million cells), ex vivo cytokine production in response to LPS (pg/mL), mRNA expression
of macrophage differentiation markers in cells harvested on day 4 (90-95% macrophages, relative),
bioparticle phagocytosis of harvested peritoneal cells (relative), and lipid mediators (Prostaglandin E2,
Leukotriene B4, and Resolvin D1) found in the exudate (pg/mL).

Table 1. Summary of effects of fish oil supplementation on biochemical parameters in response to

chemical peritonitis.

Treatment effect of FO
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Objective Animal Data

Animal Weight -
Food Intake -
Weight Change -

Peritoneal Cell Counts

PT
Total Live Cells -
Monocytes
Macrophages -

RI
Macrophages/(Monocytes+Macrophages)
Neutrophils -
Lymphocytes -

SC
Plasma Cells -

ex vivo cytokine production

U
IL-6 -
IL-10 -
AN
TNFα
IL-1β -
M

Macrophage gene expression

Arg1 -
CD36 -
D

IL-6 -
iNOS -
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PPARγ
Ym2 -
12/15 LO -
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ex vivo bioparticle phagocytosis -

C

Peritoneal exudate lipid mediators

PGE2 -
AC

LTB4
RvD1 -

RvD1/PGE2
RvD1/LTB4
RvD1/(PGE2+LTB4)
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a) b) c)

RI
U SC
d) e) f)

Live Cells
AN
Monocytes Macrophages
Cell Number

40
Cell Number

Cell Number
15 20
b
(1x10^6)

(1x10^6)

(1x10^6)
M
30 15
10
20 a 10
5 *
10 a* 5
*
0
D

0 0
0

4
g) Day h) Day i) Day
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Avacuolar Vacuolar Monocyte Lineage

Macrophages Macrophages Maturity
Macrophages)
Macrophages/
(Monocytes +
Cell Number

Cell Number

20 10 1.5
(1x10^6)

(1x10^6)
EP

15 8 *
6 1.0 *
10 *
4 0.5
5 2
0 0 0.0
C
0

4
0

j) Day k) Day l) Day

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Neutrophils Lymphocytes Plasma Cells

Cell Number
Cell Number

Cell Number

6 2.0 0.8
(1x10^6)
(1x10^6)

(1x10^6)

1.5 0.6
4
1.0 0.4
2
0.5 0.2
0 0.0 0.0
0

4
0

m) Day n) Day o) Day

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Macrophage mRNA Profile

p = 0.10
p = 0.003
1.5
α

Relative mRNA
IL-6 TNF

content
1000 2500 * 1.0 *
b

pg/mL

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800
pg/mL

2000
600 1500 0.5
400 1000 a *
200 500
0 0 0.0

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2

12 Y γ
2

4
a) Day b) e)

1
36

P OS
Day

/1 m2
iN -6

LO
R
rg

IL

A
D

5
A
C
SC
IL-10 IL-1 β Macrophage

Phagocytosis
80 a 200 1.5

Relative
pg/mL
pg/mL

60 150 1.0

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40 100
b 0.5
20 50
0
0
Day
AN 0.0
2

4
2

4
c) d) Day f) Day
M
D
TE
C EP
AC
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RI
PGE2 R/P ratio
40 6
RvD1/PGE2
*

SC
30 *
pg/mL

4 *
20
2
10
0 0

U
0

4
0

a) Day d) Day

LTB4
AN
R/L ratio
80 b 6 *
RvD1/LTB4

M
60
pg/mL

4
40 a * a *
*
a* b b*
2
20
D

0 0
0

4
0

b) Day e) Day
TE
RvD1/(PGE2+LTB4)

RvD1 R/(P+L) ratio

100 3 *
80
EP
pg/mL

60 2 a *
40 b b*
1
20
0 0
C
0

c) Day f) Day
AC