The Journal of Infectious Diseases
MAJOR ARTICLE
Dietary Lipid Type, Rather Than Total Number of Calories,
Alters Outcomes of Enteric Infection in Mice
Daniella DeCoffe,a Candice Quin,a Sandeep K. Gill, Nishat Tasnim, Kirsty Brown, Artem Godovannyi, Chuanbin Dai, Nijiati Abulizi, Yee Kwan Chan,
Sanjoy Ghosh, and Deanna L. Gibson
Department of Biology, University of British Columbia, Kelowna, Canada
Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious disease susceptibility remains unclear.
Deciphering lipid-induced immunity is critical to understanding the balance required for protecting against pathogens while avoiding chronic inflammatory diseases. To understand how specific lipids alter susceptibility to enteric infection, we fed mice isocaloric,
high-fat diets composed of corn oil (rich in n-6 polyunsaturated fatty acids [n-6 PUFAs]), olive oil (rich in monounsaturated fatty
acids), or milk fat (rich in saturated fatty acids) with or without fish oil (rich in n-3 PUFAs). After 5 weeks of dietary intervention,
mice were challenged with Citrobacter rodentium, and pathological responses were assessed. Olive oil diets resulted in little colonic
pathology associated with intestinal alkaline phosphatase, a mucosal defense factor that detoxifies lipopolysaccharide. In contrast,
while both corn oil and milk fat diets resulted in inflammation-induced colonic damage, only milk fat induced compensatory protective responses, including short chain fatty acid production. Fish oil combined with milk fat, unlike unsaturated lipid diets, had a
protective effect associated with intestinal alkaline phosphatase activity. Overall, these results reveal that dietary lipid type, independent of the total number of calories associated with the dietary lipid, influences the susceptibility to enteric damage and the benefits
of fish oil during infection.
Keywords. Citrobacter rodentium infection; milk fat; corn oil; fish oil; olive oil; intestinal alkaline phosphatase; inflammation;
high-fat diets; colitis; nutrition.
Dietary lipids influence the progression and severity of chronic
inflammatory diseases [1–3], but their role in altering susceptibility to infection is unknown. The saturation index between
fatty acids has a profound effect on host inflammation [1].
Thus, deciphering which fatty acids alter immunity is critical
to understanding the balance required for protecting against
pathogens while avoiding the development of chronic inflammatory diseases.
Saturated fatty acids (SFAs) contain no double bonds and are
considered to be proinflammatory. SFAs such as laurate and
palmitate activate Toll-like receptor 2 (TLR-2) and TLR-4 [4]
and induce infiltration of immune cells in the intestines [5].
In contrast, monounsaturated fatty acids (MUFAs) contain
only 1 double bond and are generally recognized as antiinflammatory. Polyunsaturated fatty acids (PUFAs) are a family of
fats with proinflammatory or antiinflammatory properties,
depending on location of the final carbon-carbon double
bond relative to the methyl end. Linoleic acid, an n-6 PUFA,
Received 29 September 2015; accepted 18 February 2016; published online 10 April 2016.
Presented in part: Keystone symposia: Inflammation and Therapeutics, Vancouver, BC, Canada, February 2014. Canadian Digestive Disease Week, Victoria, BC, Canada, February 2013.
a
D. D. and C. Q. contributed equally to this work.
Correspondence: D. L. Gibson, ASC 368, 3187 University Way, Department of Biology, Irving
K. Barber School of Arts and Sciences, University of British Columbia Okanagan, Kelowna,
British Columbia V1V 1V7, Canada ([email protected]).
The Journal of Infectious Diseases® 2016;213:1846–56
© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail [email protected].
DOI: 10.1093/infdis/jiw084
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drives inflammatory responses through the synthesis of proinflammatory eicosanoids such as prostaglandin E2 (PGE2) [6]. In
contrast, n-3 PUFAs such as docosahexaenoic acid and eicosapentaenoic acid decrease levels of proinflammatory eicosanoids
[7], cytokines, antigen presentation, and lymphocyte proliferation [8]. While it is believed that n-3 PUFA decreases chronic
inflammation [1], an impaired immune response increases the
susceptibility to Mycobacterium tuberculosis [9], Helicobacter
hepaticus [10], and Citrobacter rodentium [11]. While it is
clear that dietary lipids modulate immunity, the impact of
fatty acid type on defensive immune responses during infection
remains unclear.
Globalization has changed the profile of dietary lipid ingestion among individuals worldwide [12]. This highlights the importance of understanding the effect of dietary lipids on
infectious diseases outcomes, particularly diarrheal diseases,
which are major global killers [13]. Enteropathogenic Escherichia coli (EPEC) causes diarrheal diseases by attaching to the
intestinal epithelial cells and effacing the brush border microvilli [14]. C. rodentium, a model for EPEC infection [14], shows
similar pathology in mice [15]. Since we have shown that
high-fat diets alter the outcomes of C. rodentium infection
[11], we wanted to clarify whether these effects were due to
the total lipid load or, instead, to a specific type of lipid. To approach this, we fed mice isocaloric, high-fat diets rich in either
n-6 PUFAs (corn oil), MUFAs (olive oil), or SFAs (milk fat)
with or without fish oil (rich in n-3 PUFA), followed by challenge with C. rodentium. Overall, our results reveal that dietary
lipid type, independent of the total number of calories associated with the dietary lipid, dictates both susceptibility to enteric
damage and the benefits of fish oil during infection.
MATERIALS AND METHODS
Mice and Diets
C57BL/6 female mice (Jackson Laboratories) were housed in
specific-pathogen-free conditions at the University of British
Columbia (Vancouver, Canada). Animal protocols were approved by animal care guidelines at the University of British
Columbia. For 5 weeks, 3-week-old mice were fed irradiated
chow from Harlan Teklad (TD.88232; Supplementary Table 1–
2) blended with 20% w/w of either olive oil (TD.120023), anhydrous milk fat (TD.120021), or corn oil (TD.120025) with or
without 1% w/w fish oil (TD.130129), as previously described
[16]. In this study, we used the high-fat diet containing n-6
PUFA as the relevant control to negate the effects of higher
total number of calories and a lack of micronutrients, which
are characteristic features of all high-fat feeding regimens.
While the use of soybean oils is universal in all normal chow,
our primary research objective of delineating the effects of various
dietary fatty acids in gut immunity could not be achieved with by
using normal chow, which is rich in n-6 PUFAs. Thus, this diet
was not included for comparisons here. Food and water was provided ad libitum, and intake of both was measured weekly.
C. rodentium Infection and Tissue Collection
C. rodentium DBS100 was cultured, and mice were infected via
oral gavage and monitored for mortality and morbidity as described previously [17]. Mice were euthanized on or before day
10 after infection, and the distal colon was excised and immersed in RNAlater (Qiagen). Colon pieces were stored at
−20°C for quantitative polymerase chain reaction analysis, or
immersed in 10% formalin (Fisher) for histological analysis
and immunofluorescence.
Histopathological Scoring and Immunofluorescence
Histopathological findings were assessed by 2 blinded observers
as described previously [18]. For immunofluorescence, paraffinembedded tissue sections were prepared and incubated with antibodies as previously reported [8, 19]. Tissue sections were
mounted using DAPI (Sigma Aldrich) and imaged using MetaMorph software on an Olympus IX81 fluorescent microscope.
For inflammatory cell counts, 2 blinded observers quantified
positively stained cells by counting florescent cells in the submucosal region of the colon section for each mouse. The total
number of positive cells for each mouse was averaged to represent the mean number of positive cells found in each section.
Cell Culture
Caco-2 cells (ATCC) were cultured as described previously [20] in
Dulbecco’s modified Eagle’s medium (DMEM; ThermoScientific)
with 20% fetal bovine serum, 2% penicillin/streptomycin, 1 mM
sodium pyruvate, 4 mM Glutagro, and nonessential amino acids
(ThermoScientific) and were then incubated at 37°C in 5% CO2.
A total of 50 000 cells were plated in each well. At 80% confluency,
the media was replaced with bovine serum albumin complexed (at
a 4:1 ratio) with 0.25 mM linoleic acid, oleic acid (Sigma), or palmitic acid (Sigma), with or without docosahexaenoic acid plus
eicosapentaenoic acid (at a ratio of 1.36:1; Cayman Chemical).
Intestinal Alkaline Phosphatase Activity Assay
The lipopolysaccharide (LPS)–dephosphorylation assay was
performed as previously described [19]. LPS (Sigma L2880)
was incubated for 2 hours before the addition of 0.01% malachite green in 16% sulfuric acid, 1.5% ammonium molybdate,
and 0.18% Tween-20, as previously described [21]. For colon
tissues, the lysate was prepared using 25-mg pieces homogenized in RIPA buffer (150 mM NaCl, 1% Trition-X-100, 0.5%
sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50
mM Tris; pH 8.0) with protease inhibitor (abm). For cell culture, after 18 hours of fatty acid stimulation, LPS was added,
cells were incubated for 8 hours, and lysate was collected.
Pathogen Virulence and Attachment Assays
Caco-2 cells were washed and infected with preactivated EPEC
that had been subcultured from an overnight LB medium culture into DMEM and allowed to grow for 1 hour at 37°C in 5%
CO2. Infection proceeded at 37°C in 5% CO2 for 1 hour. For
virulence gene expression studies, QIAzol (Qiagen) was added
directly to the well for RNA isolation. For attachment assays,
EPEC-infected Caco-2 cells were washed with DMEM and
scraped into phosphate-buffered saline. Serial dilutions were
plated onto LB plates, and colony-forming units were counted
after incubation at 37°C for 18 hours.
Pathogen Quantification and Imaging
For bacterial imaging, fluorescent in situ hybridization was performed using a fluorescein isothiocyanate–labeled Gammaproteobacteria probe, as previously described [11]. For systemic
pathogen counts, the spleen and mesenteric lymph nodes
were homogenized in phosphate-buffered saline, serially diluted, and incubated on McConkey plates for 24–48 hours at 37°C.
Messenger RNA (mRNA) Analysis
RNA was extracted from tissues by using the RNA Fibrous kit
(Qiagen) and from culture cells by using QIAzol (Qiagen).
Complementary DNA was reverse transcribed from RNA,
using iScript cDNA synthesis (Bio-Rad) followed by quantitative polymerase chain reaction analysis, as described previously,
using specific primers (Supplementary Table 3) [20]. CFX manager 2.1 software (Biorad) was used to calculate relative gene expression, using 18S ribosomal RNA as a reference gene for the
host and rrsB for the pathogen [22].
Short-Chain Fatty Acid Analysis
By use of gas chromatography, acetic, propionic, and butyric
acid in fecal samples were analyzed from as previously described
[23]. Cecal samples were homogenized in isopropyl alcohol, and
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the supernatant was injected into a Trace 1300 gas chromatograph equipped with flame-ionization detector in split-less
mode. Data were analyzed with Chromeleon 7 software and
are presented as mass percentages.
Statistical Analysis
The results are expressed as mean values±standard errors of the
mean, with a sample size of 8–10 mice or 6–12 Caco-2 replicates
in each group. The log-rank test was used to determine the statistical significance of differences in survival data. For host gene
expression data, minitab was used to test for normalcy. When
comparing all diet groups, 1-way analysis of variance (ANOVA)
with the Tukey post hoc test was performed for parametric data,
and the Kruskal–Wallis test with the Dunn post hoc test
was performed for nonparametric data. To compare fish oil–
including diets to the parent diet, Student t tests were used.
For weight loss, histological scores, intestinal alkaline phosphatase (IAP) activity, and pathogen quantification in vivo, data
were analyzed via 1-way ANOVA with the Tukey post hoc
test. For pathogen attachment assays, Student t tests were
used. All analyses were performed using GraphPad Prism 5,
with P values of <.05 considered statistically significant.
RESULTS
Olive Oil Diets Protect Against Infection-Induced Pathology
Mice were fed isocaloric diets with corn oil, olive oil, or milk fat
for 5 weeks and then infected with C. rodentium. All mice, regardless of the type of lipid diet, survived infection (Figure 1A),
and none lost >15% of their initial body weight within 10 days
(Figure 1B). Infected mice fed corn oil or milk fat experienced
similar colonic damage, whereas infected mice fed olive oil were
Figure 1. Mice fed high-fat olive oil diets are protected against Citrobacter rodentium infection–induced morbidity and pathology. A, C57BL/6 mice fed corn oil, olive oil, and
anhydrous milk fat diets had a 100% survival rate during C. rodentium infection. B, Both corn oil and olive oil diets resulted in similar weight changes during infection with C.
rodentium. In contrast, mice fed milk fat diets had a significant reduction in weight by day 9 of infection, but these mice were able to recover by day 10. C, Mice fed corn oil and
milk fat diets during C. rodentium infection had the highest levels of histopathological severity as compared to mice fed olive oil diets. D, Hematoxylin-eosin staining of
representative colonic sections from the diet groups were taken at 200× original magnification. These images focus on the epithelium (top panel) and immune cell infiltration
in the submucosae (bottom panel). *P < .05.
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Figure 2. Mice fed high-fat corn oil or anhydrous milk fat diets induce robust proinflammatory responses in the colon during Citrobacter rodentium infection. A–C, C57BL/6
mice fed corn oil and milk fat diets had increased colonic immune cell infiltration as compared to monounsaturated fatty acids fed mice during C. rodentium infection. Colonic
sections from the mice were embedded, and sections were mounted on slides and stained with antibodies specific for F4/80 (macrophages; A), MPO (neutrophils; B), and
prostaglandin E2 (PGE2)–positive inflammatory cells (C) and quantified. D–I, Relative messenger RNA (mRNA) expression of inflammatory markers 10 days after infection.
*P < .05. Abbreviations: IFN-γ, interferon γ; IL-17a, interleukin 17a; IL-23a, interleukin 23a; MCP-1, monocyte chemotactic protein 1; TNF-α, tumor necrosis factor α.
protected from damage (Figure 1C and 1D). These results indicate that the type of fatty acid, independent of the total number
of calories associated with the fatty acid, alters C. rodentium infection–induced pathology and that olive oil is a protective lipid.
Corn Oil and Milk Fat Diets Induce Robust Proinflammatory Responses
in the Colon During Infection
The colonic damage observed during C. rodentium infection is
often a result of specific inflammatory responses [17, 24–26]. To
determine which inflammatory responses were associated with
the damage seen in infected mice that were fed corn oil or milk
fat, we examined infiltrating immune cells and the expression of
inflammatory markers induced during C. rodentium infection
[17, 25, 26]. Corn oil and milk fat diets resulted in greater infiltration of submucosal macrophages (Figure 2A). In addition,
milk fat diets induced the infiltration of submucosal neutrophils
and prostaglandin E2–positive cells (Figure 2B and 2C). During
infection, both corn oil–fed mice and milk fat-fed mice exhibited
strong yet distinct proinflammatory cytokine responses (Figure 2D and 2H). The milk fat diet resulted in higher tumor
necrosis factor α, interferon γ, interleukin 17a, and interleukin
23 expression, whereas the corn oil diet resulted in increased expression of Relm-β. In contrast, infected mice fed olive oil had
low expression of proinflammatory cytokines in colon tissues,
although there was a strong monocyte chemotactic protein 1
chemokine response (Figure 2I). Akin to histopathologic severity, these results suggest that alterations in inflammatory responses during enteric infection are associated with dietary fat
type, with corn oil and milk fat exacerbating C. rodentium
colitis.
Milk Fat Promotes the Induction of Protective Responses in the Colon
During Infection
To understand what protective responses are induced by lipids
during enteric infection, we examined the infiltration of Foxp3–
expressing T-regulatory cells, as well as the induction of the
antiinflammatory cytokine interleukin 10 (IL-10), the alarmin
interleukin 33 (IL-33), and the antimicrobial peptide Reg3γ
and found that these responses are uniquely induced by milk
fat (Figure 3A–D). Additionally, the milk fat-fed group had
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Figure 3. Mice fed high-fat anhydrous milk fat diets induce several protective responses involved in inflammation resolution during Citrobacter rodentium infection. A, Mice
fed milk fat diets had increased infiltration of Foxp3-expressing T-regulatory cells (Tregs) during infection with C. rodentium. Colonic sections from the mice were embedded,
and sections were mounted on slides and stained with an antibody specific for Foxp3. Infected mice fed milk fat diets had increased relative messenger RNA (mRNA) expression
of interleukin 10 (IL-10; B), interleukin 33 (IL-33; C), Reg3γ (D), GPR41 (E ), and GPR40 (F ). G–I, The fecal content of infected mice fed milk fat contained more short-chain fatty
acids during infection than the other diet groups. *P < .05.
the highest expression of GPR40 and GPR41, fatty acid receptors on epithelial cells known to play a role in inflammation resolution [27] (Figure 3E and 3F ). We examined the production
of short-chain fatty acids (SCFAs) because they protect against
chronic intestinal inflammation [28, 29] and found that the milk
fat diet resulted in the largest induction of SCFAs, whereas corn
oil resulted in the smallest induction (Figure 3G and 3I). Overall, milk fat diets uniquely induced several protective responses.
Olive Oil Diets Induce LPS Dephosphorylation During Infection
We have demonstrated that IAP activity is associated with protection against sepsis-induced mortality during C. rodentium
infection [11]. Thus, we examined IAP activity and found that
LPS dephosphorylation was highest in olive oil–fed mice following infection (Figure 4A). IAP is a mucosal defense factor expressed at the mucosal surface, but it has also been found in
the submucosa [30]. To determine which cells were expressing
IAP in the olive oil–fed group, we stained the colonic tissues
with an antibody specific to IAP and found high expression at
the luminal side of the intestinal epithelial cells in mice fed olive
oil but not those fed corn oil or milk fat (Figure 4B). We verified
these results by culturing colonic epithelial cells (Caco-2) in
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medium containing oleic acid (a MUFA), linoleic acid (an n-6
PUFA), or palmitic acid (a SFA), which revealed induction of
IAP activity in cells grown in MUFAs but not those grown in
n-6 PUFAs or SFAs (Figure 4C). These results demonstrate
that MUFA induces IAP activity in intestinal epithelial cells.
Olive Oil Diets Promote Bacterial Clearance
Our results demonstrate that olive oil induces IAP activity,
which is known to prevent bacterial translocation [31, 32]. To
determine whether IAP activity correlated with C. rodentium
clearance, we examined the location of colonic bacteria in
situ, as well as the number of colony-forming units in mesenteric lymph nodes (MLNs) and the spleen, indicative of translocated bacteria and systemic infection. Olive oil–fed mice and
milk fat-fed mice had no visible pathogens in colonic tissue
sections by 10 days after infection, whereas corn oil–fed mice
had visible C. rodentium interacting with the intestinal crypts
(Figure 5A). Furthermore, by 10 days after infection, the olive
oil–fed mice had cleared much of the infection from the spleen
and MLNs, whereas both milk fat-fed mice and corn oil–fed
mice had signs of bacterial translocation (Figure 5B). To understand whether pathogen virulence was decreased in the presence
Figure 4. Mice fed high-fat olive oil diets induce intestinal alkaline phosphatase activity during Citrobacter rodentium infection. A, Mice fed olive oil diets had increased
intestinal alkaline phosphatase (IAP) lipopolysaccharide (LPS) dephosphorylation during C. rodentium infection. Colonic tissues were homogenized and analyzed using the LPSdephosphorylation assay and malachite green solution. B, Mice fed olive oil diets had increased expression of IAP in the epithelium during C. rodentium infection as compared
to mice fed corn oil and anhydrous milk fat diets. Colonic sections were stained with an antibody specific for IAP. Representative colonic sections were photographed at 200×
original magnification, with red staining in the epithelium representing the presence of IAP. C, Caco-2 epithelial cells had increased IAP activity in the oleic acid (OA) group
during LPS stimulation. The fatty acid groups analyzed were linoleic acid (LA), OA, and palmitic acid (PA), with bovine serum albumin (BSA) as a control. The zero time point
represents control values (cells stimulated with fatty acids only). Caco-2 cells were stimulated with LPS for 8 hours, and only the OA group resulted in increased IAP activity. The
activity of IAP was determined by measuring the LPS-dephosphorylation activity per milligram of protein at 620 nm. *P < .05.
of MUFAs, we infected Caco-2 cells grown in fatty acids with
EPEC and examined gene expression of early virulence factors
[22]. While expression of ler, the product of which regulates the
type III secretion system, was unaffected, expression of espA and
tir, the products of which are involved in pathogen attachment,
was decreased (Figure 5C). We verified that fatty acids did not
affect pathogen growth (data not shown). To determine whether
pathogen attachment to intestinal epithelial cells was decreased,
we performed an attachment assay and found that approximately 40% fewer pathogens were attached in the presence of oleic
acid, compared with the presence of linoleic acid (Figure 5D).
Overall, these results suggest that MUFAs promote efficient
bacterial clearance during infection and that this may be
partly due to a preventive effect on pathogen attachment to
the gut wall.
Fish Oil Combined With Milk Fat Improves Infection Outcomes
Fish oil is a common dietary supplement that has antiinflammatory properties thought to be beneficial during chronic inflammatory disease, but its effects in acute gut infection
remains unclear. We previously demonstrated that fish oil combined with corn oil aggravated C. rodentium infection [11]. To
determine whether fish oil combined with milk fat or olive oil
had similar negative effects on infection susceptibility, we examined morbidity and mortality to C. rodentium in mice fed milk
fat plus fish oil or olive oil plus fish oil. We found that fish oil
did not result in mortality among infected mice fed milk fat or
olive oil diets, unlike the 30% mortality reported when fish oil
was combined with corn oil [11] (Figure 6A). Correspondingly,
fish oil combined with milk fat or olive oil diets resulted in mice
experiencing less weight loss during infection, which is
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Figure 5. Olive oil diets promote bacterial clearance. A, Colonic tissue sections were hybridized with a Gammaproteobacteria probe (green) to examine the location of
Citrobacter rodentium and costained with DAPI to detect the nuclei (blue). The pathogen was located within the colonic crypts (left panel; 100× original magnification) in the
mice fed corn oil and was not found mice fed olive oil and anhydrous milk fat diets. B, Corn oil–fed mice and milk fat-fed mice had increased levels of colony-forming units
(CFUs) in the spleen and mesenteric lymph nodes (MLNs) after 10 days of C. rodentium infection. CFUs were enumerated from tissues taken from mice fed various high-fat diets
after 10 days of infection with C. rodentium, homogenized, and plated in serial dilution on bacterial agar. C, The gene expression of virulence factors was examined from
enteropathogenic Escherichia coli (EPEC)–infected Caco-2 cells stimulated with the fatty acids linoleic acid (LA), oleic acid (OA), and palmitic acid (PA). OA decreased the
expression of espA, compared with LA. D, Pathogen adherence was examined from Caco-2 cells stimulated with fatty acids, and OA reduced the number of CFU when compared
to LA. *P < .05. Abbreviations: mRNA, messenger RNA; ND, non detected.
indicative of less morbidity (Figure 6B). To determine the effect
of fish oil combined with milk fat or olive oil on colonic damage, we calculated the percentage change in histopathological
score with the addition of fish oil, compared with the nonsupplemented lipid diet. We found that fish oil did not change the
damage associated with the milk fat diet but increased the damage associated with the olive oil diet (Figure 6C). This damage
was associated with an increase in bacteria in the intestinal tissues and spleen of the olive oil–fed mice, whereas the bacterial
load was significantly decreased in the milk fat-fed mice (Figure 6D and 6E ). While fish oil decreased immune cell infiltration in both corn oil–fed mice and milk fat-fed mice
(Supplementary Table 4), for the most part fish oil had little effect on local inflammatory cytokine responses in any diet
(Table 1). In contrast, IAP induction was impaired with the
addition of fish oil to the diet of the olive oil–fed mice, yet
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induction was unimpaired in the mice fed milk fat (Figure 6F ).
Overall, these results show that fish oil supplementation of
high-fat diets composed of different fatty acids has differential
effects, such that diets rich in unsaturated fatty acids impair IAP
activity and have little effect on local immune responses. This is
unlike high-fat milk fat diets, in which fish oil induced IAP activity that ultimately promoted bacterial clearance in this group
of mice. Overall, these results show that the beneficial effects of
fish oil require the presence of SFA in a combined diet.
DISCUSSION
While high-fat diets, particularly those involving SFAs, are advised against by the North America food guide [33], several European countries, including Sweden, recommend a full-fat diet
[34]. Here, we showed that not all high-fat diets result in detrimental outcomes of enteric infection and that the type of lipid
Figure 6. Fish oil (FO) combined with anhydrous milk fat improves infection outcomes. A, While C57BL/6 mice fed a corn oil diet supplemented with FO had a 30% reduction
in survival rates during Citrobacter rodentium infection, mice fed milk fat or olive oil diets survived at 100%. B, Mice fed milk fat or olive oil with FO diets did not experience
weight loss during infection, unlike the group that received corn oil plus FO. C, The histopathological scores were averaged from mice fed each high-fat diet with the addition of
FO and then divided by those same high-fat diets without FO, and the ratio was graphed. D, Colonic tissue sections were hybridized with a Gammaproteobacteria probe (green)
to examine the location of C. rodentium and costained with DAPI to detect the nuclei (blue). Mice fed olive oil supplemented with FO during C. rodentium infection had
increased infiltration of this pathogen into the crypts; these images were taken at 100× original magnification. E, Mice fed corn oil diets supplemented with FO had increased
colony-forming units (CFUs) in the mesenteric lymph node (MLN), while mice fed olive oil diets supplemented with FO had an increased number of colony-forming units (CFUs)
in the spleen after 10 days of C. rodentium infection. CFUs were enumerated from tissues taken from mice fed various high-fat diets after 10 days of infection with C. rodentium,
homogenized, and plated in serial dilution on bacterial agar. F, Intestinal alkaline phosphatase (IAP)–associated lipopolysaccharide (LPS) dephosphorylation was increased in
mice fed milk fat diets supplemented with FO during C. rodentium infection. Colonic tissues were homogenized and analyzed using the LPS-dephosphorylation assay and
malachite green solution. The activity of IAP was determined by measuring the LPS-dephosphorylation activity per milligram of protein at 620 nm. *P < .05.
consumed results in distinctive pathological, immunological,
and homeostatic responses following C. rodentium infection
in mice. Mice fed high-fat diets composed of olive oil were protected against enteric infection; these mice exhibited IAP activity corresponding to efficient pathogen clearance without
causing damage to host tissues. In contrast, mice fed high-fat
diets composed of either corn oil or milk fat had strong proinflammatory responses to enteric infection, resulting in colonic
damage. However, milk fat induced compensatory responses involved in the resolution of inflammation, concurrent with early
pathogen clearance. Finally, we showed that fish oil was beneficial
only when consumed with SFAs in a combined diet. Overall, our
study reveals lipid type–specific alterations in the susceptibility to
enteric infection.
While inflammatory responses are critical for protecting the
host from invading pathogens, their protective characteristics
and resolution after acute inflammation are critical to suppress
overactivation of the inflammatory response, which can lead to
chronic diseases such as inflammatory bowel disease. We showed
that high-fat diets composed of either corn oil or milk fat resulted
in the induction of robust inflammatory responses during acute
inflammation. When infected with C. rodentium, mice fed milk
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Table 1. Levels of Expression of Genes Encoding Inflammatory Mediators
Among Infected Mice Fed High-Fat Diets (HFDs), Compared With Levels
Among Mice Fed HFDs Plus Fish Oil (FO)
Relative Gene Expression,
Mean ± SEM, by Diet
Response, HFD
HFD
HFD Plus FO
P Valuea
.4841
TNF-α
Corn oil
11.8 ± 2.5
17.6 ± 4.3
Olive oil
24.0 ± 10.2
7.8 ± 1.9
.1622
Milk fat
121.6 ± 38.8
116.9 ± 29.2
.9188
Corn oil
91.2 ± 44.0
67.1 ± 18.9
.5969
Olive oil
192.5 ± 101.4
8.1 ± 2.0
.0983
Milk fat
842.8 ± 314.1
629.7 ± 175.9
.5401
Corn oil
9.0 ± 5.7
18.0 ± 5.5
.2456
Olive oil
3.0 ± 0.9
3.7 ± 0.7
.5326
Milk fat
11.6 ± 2.8
10.5 ± 2.7
.7761
Corn oil
165.5 ± 67.6
112.0 ± 44.3
.4879
Olive oil
514.2 ± 294.5
11.7 ± 2.8
.1295
Milk fat
1859.4 ± 780.1
3941.8 ± 719.8
.5366
IFN-γ
IL-17a
iNOS
MCP-1
Corn oil
0.3 ± 0.3
0.5 ± 0.3
.7042
Olive oil
17.2 ± 6.7
5.5 ± 1.3
.1305
Milk fat
7.8 ± 1.9
2.4 ± 0.5
.0080
Corn oil
332.8 ± 166.9
280.8 ± 88.3
.7714
Olive oil
52.4 ± 23.4
32.7 ± 10.4
.4685
Milk fat
114.8 ± 31.2
58.5 ± 14.9
.0938
Relm-β
IL-23a
Corn oil
3.7 ± 2.8
5.0 ± 2.0
.6933
Olive oil
4.7 ± 1.0
11.6 ± 3.7
.0641
Milk fat
14.2 ± 6.7
2.7 ± 0.3
.0736
Corn oil
2.2 ± 0.5
2.3 ± 0.4
.9180
Olive oil
5.7 ± 1.3
5.3 ± 1.4
.8639
Milk fat
10.0 ± 1.4
5.4 ± 0.7
.0076
Corn oil
3.4 ± 1.8
2.4 ± 1.3
.6178
Olive oil
6.4 ± 2.9
7.8 ± 2.6
.7013
Milk fat
14.8 ± 5.4
2.2 ± 0.9
.0391
Corn oil
287.6 ± 104.6
144.8 ± 86.5
.3002
Olive oil
128.1 ± 65.4
365.2 ± 172.1
.1710
Milk fat
819.1 ± 329.4
447.5 ± 82.6
.2432
IL-10
IL-33
Reg3γ
Abbreviations: IFN-γ, interferon γ; IL-10, interleukin 10; IL-17a, interleukin 17a; IL-23a,
interleukin 23a; IL-33, interleukin 33; iNOS, inducible nitric oxide synthase; MCP-1,
monocyte chemotactic protein 1; SEM, standard error of the mean; TNF-α, tumor necrosis
factor α.
a
By the Student t test. A value of <.05 was considered statistically significant.
fat and those fed corn oil experienced exacerbated gut immune cell
infiltration and prototypical colitic responses. Unlike mice fed
corn oil, mice fed milk fat were able to induce T-regulatory cell
infiltration and produce IL-10, the alarmin IL-33, and SCFAs.
These responses are interconnected: SCFAs [35] and IL-33 [36]
are important for stimulating T-regulatory cells, which maintain
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DeCoffe et al
intestinal homeostasis and regulate balanced immune responses
by protecting against overactive inflammation [37]. Milk fat diets
also increased the expression of colonic fatty acid receptors, including GPR41 and GPR40, which are known to be important
for resolution of inflammation [27]. In addition to these responses,
Reg3γ was induced in the milk fat-fed mice in response to infection, and this antimicrobial peptide has been shown to block
bacterial translocation, resulting in mucosal protection [38].
Indeed, the milk fat-fed mice did not have visible bacteria present
at the mucosal surface or in the submucosa, unlike the corn oil–fed
mice, in which the pathogen load was much higher in the colon,
the MLNs, and spleen. Overall, while mice fed milk fat diets experienced inflammation, this diet also promoted protective immunity involved in inflammation resolution, suggesting that SFAs are
more protective than n-6 PUFAs during colitis.
The Mediterranean diet, high in olive oil intake, is known to
have major health benefits [39]. Olive oil, rich in MUFAs, is
known to reduce inflammation and colonic damage in rodent
models of chemical colitis [40, 41], but its effect on infection
susceptibility is unknown. Here, we showed that high-fat diets
composed of olive oil resulted in few inflammatory cells and low
cytokine responses during infection yet, remarkably, were associated with complete bacterial clearance in the colon and spleen
10 days after infection. This may be partly due to the ability of
MUFAs to decrease pathogen attachment to the mucosal surface
through the downregulation of espA, a pathogen-attachment
factor [42]. In addition, mice fed high-fat diets composed of
olive oil responded to infection with C. rodentium through amplified IAP activity at the mucosal surface. Considering that IAP
regulates mucosal homeostasis by dephosphorylating LPS [43] and
can prevent sepsis [31], our results suggest that olive oil, through
enhanced IAP activity, may promote pathogen clearance.
Fish oil, rich in long chain n-3 PUFAs, has potent antiinflammatory effects and has been predicted to decrease chronic inflammatory diseases [44–46]. However, a few studies have
indicated that these lipids may also increase susceptibility to infectious diseases [16]. Similarly, we have previously found that
mice fed high-fats diets consisting of corn oil and fish oil combined experienced sepsis during C. rodentium infection [19]. In
the present study, we showed that fish oil combined with milk
fat did not have the same fatal effect. Interestingly, fish oil had a
negligible effect on inflammatory responses when combined
with any dietary lipid. Instead, the mechanism of fish oil action
in this model seems to involve induction of IAP activity when
combined with milk fat but impaired IAP activity when combined with unsaturated fatty acids. Thus, fish oil supplementation results in differential effects depending on the dominance
of either saturated or unsaturated fatty acids present in background diets. Overall, these results suggest that fish oil combined with unsaturated fatty acids poses a threat to enteric
infectious disease susceptibility but is beneficial when combined
with a diet richer in SFAs.
In summary, this study demonstrates key differences in the
susceptibility to colitis conferred by dietary lipid types. We
showed that not all high-fat diets aggravate colitis, as evident
by the reduced susceptibility of infected, olive oil–fed mice to
acute colitis. Additionally, we showed that milk fat diets promote protective responses involved in inflammation resolution
following a bacterial infection, which may help prevent a chronic inflammatory state after the acute inflammatory phase. Finally, we demonstrated that fish oil has a beneficial effect when
supplementing a diet composed of milk fat, through the induction of IAP activity, which promotes pathogen clearance, but
that it has a detrimental effect when combined with unsaturated
fatty acid diets. Based on these results, a diet rich in a combination of MUFAs, SFAs, and n-3 PUFAs, with limited n-6 PUFAs,
is recommended to maintain defensive inflammatory and mucosal responses to enteric infection while mitigating the damaging effects of inflammation.
Supplementary Data
Supplementary materials are available at http://jid.oxfordjournals.org.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Notes
Financial support. This work was supported by the Canadian Institutes
of Health Research (Frederick Banting and Charles Best Canada Graduate
Scholarship to C. Q.), the Canadian Association of Gastroenterology (award
to S. K .G.), the Natural Science and Engineering Research Council of Canada (award to S. K. G.), the Michael Smith Foundation of Health Research
(to S. G.), Dairy Farmers of Canada (operating grants to S. G.), the Natural
Science and Engineering Research Council (to D. L. G.), and Crohn′s and
Colitis Canada (to D. L. G.).
Potential conflicts of interest. S. G. is funded through the Dairy Farmers of Canada, although this work was not supported financially by this
funding agency. All other authors report no potential conflicts. All authors
have submitted the ICMJE Form for Disclosure of Potential Conflicts of
Interest. Conflicts that the editors consider relevant to the content of the
manuscript have been disclosed.
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