R E S E A R C H Open Access
Cooperation between host immunity and
the gut bacteria is essential for helminth-
evoked suppression of colitis
, Blanca E. Callejas
, ShuHua Li
, Arthur Wang
, Timothy S. Jayme
, Christina Ohland
, Ian A. Lewis
Brian T. Layden
, André G. Buret
and Derek M. McKay
Background: Studies on the inhibition of inflammation by infection with helminth parasites have, until recently,
overlooked a key determinant of health: the gut microbiota. Infection with helminths evokes changes in the
composition of their host’s microbiota: one outcome of which is an altered metabolome (e.g., levels of short-chain
fatty acids (SCFAs)) in the gut lumen. The functional implications of helminth-evoked changes in the enteric
microbiome (composition and metabolites) are poorly understood and are explored with respect to controlling
Methods: Antibiotic-treated wild-type, germ-free (GF) and free fatty-acid receptor-2 (ffar2) deficient mice were
infected with the tapeworm Hymenolepis diminuta, then challenged with DNBS-colitis and disease severity and gut
expression of the il-10 receptor-αand SCFA receptors/transporters assessed 3 days later. Gut bacteria composition
was assessed by 16 s rRNA sequencing and SCFAs were measured. Other studies assessed the ability of feces or a
bacteria-free fecal filtrate from H. diminuta-infected mice to inhibit colitis.
Results: Protection against disease by infection with H. diminuta was abrogated by antibiotic treatment and was
not observed in GF-mice. Bacterial community profiling revealed an increase in variants belonging to the families
Lachnospiraceae and Clostridium cluster XIVa in mice 8 days post-infection with H. diminuta, and the transfer of feces
from these mice suppressed DNBS-colitis in GF-mice. Mice treated with a bacteria-free filtrate of feces from H.
diminuta-infected mice were protected from DNBS-colitis. Metabolomic analysis revealed increased acetate and
butyrate (both or which can reduce colitis) in feces from H. diminuta-infected mice, but not from antibiotic-treated
H. diminuta-infected mice. H. diminuta-induced protection against DNBS-colitis was not observed in ffar2
Immunologically, anti-il-10 antibodies inhibited the anti-colitic effect of H. diminuta-infection. Analyses of epithelial
cell lines, colonoids, and colon segments uncovered reciprocity between butyrate and il-10 in the induction of the
il-10-receptor and butyrate transporters.
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* Correspondence: email@example.com
Gastrointestinal Research Group, Inflammation Research Network and
Host-Parasite Interaction Group, Calvin, Phoebe & Joan Snyder Institute for
Chronic Diseases, Department of Physiology and Pharmacology, Cumming
School of Medicine, University of Calgary, Calgary, Alberta, Canada
Full list of author information is available at the end of the article
Shute et al. Microbiome (2021) 9:186
Conclusion: Having defined a feed-forward signaling loop between il-10 and butyrate following infection with H.
diminuta, this study identifies the gut microbiome as a critical component of the anti-colitic effect of this helminth
therapy. We suggest that any intention-to-treat with helminth therapy should be based on the characterization of
the patient’s immunological and microbiological response to the helminth.
Despite significant increases in therapeutics for chronic
inflammatory disease, even the best of these (e.g., anti-
TNFαantibody) is ineffective in a substantial number of
patients. The rapidity of the emergence and increase in
incidence of idiopathic auto-inflammatory disease sup-
ports a role for environmental factors in the pathogen-
esis of these conditions [1,2]: an awareness that can
direct the search for new therapeutic approaches. The
inverse correlation between the geographical distribution
of inflammatory bowel disease (IBD), diabetes, and mul-
tiple sclerosis with endemic parasitic helminth-infections
has led to the hypothesis that infection with helminths
could confer protection against auto-inflammatory dis-
ease . A position supported by the fact that helminths
have evolved to manipulate their hosts’immune system
[4,5]. Indeed, analyses of animal models show that in-
fection with helminth parasites reduces the severity of
inflammatory disease [6–11], in which interleukin (il)-
10, transforming growth factor (tgf)-β, and regulatory T
cells, B cells, and macrophages were critical host factors
in the inhibition of inflammation [12–16].
This immune-centric view of the host-parasite inter-
action overlooks the possible, if not probable, participa-
tion of the microbiome in a tripartite relationship.
Descriptions of increased bacterial species richness or
diversity in helminth-infected rodents and people are
common [17–21], but the functional consequences of
these changes in the microbiome to gut homeostasis are
not well understood. The juxtaposition of helminth and
bacteria in the gut allows for the possibility that the
anti-inflammatory effect that follows infection with the
parasite could, at least in part, be via the microbiota.
This postulate is supported by data showing that re-
duced airways inflammation in mice infected with the
nematode Heligmosomoides polygyrus was abrogated by
antibiotic treatment [22,23].
The mouse is a non-permissive host for the rat-
tapeworm, Hymenolepis diminuta. Lacking hooks or
teeth, this helminth does minimal damage to the host
and seeks to establish in the small intestine (it does not
migrate through the host): the mouse mounts a Th2-
dominated immune response and expels the worm
within 8–11 days of a primary infection . Infection
with H. diminuta reduces the severity of dinitrobenzene
sulphonic acid (DNBS)-induced colitis in mice, and il-10
is important in this event . H. diminuta-infection
caused subtle, yet distinct, changes in the composition of
the mouse colonic microbiota, but the bacteria were not
required for expulsion of the worm . This presents a
model to address the issue of the intersection of hel-
minths and gut bacteria in the regulation of colitis. The
data herein, show that host immunological and micro-
biota responses (i.e., increased short-chain fatty acids
(SCFAs) synthesis) are essential to the suppression of
colitis initiated by infection with H. diminuta. Thus, in
the development of new approaches to inflammatory
disease, these data suggest that helminth therapy may be
rendered ineffective in an individual with a reduced cap-
acity to make il-10 (which may be a rare occurrence) or
with gut dysbiosis.
Antibiotic (Abx) treatment abrogates H. diminuta-evoked
suppression of colitis
The possibility that the gut bacteria participated in H.
diminuta-evoked suppression of colitis was tested with
broad-spectrum antibiotics (Fig. 1A). As assessed by
body weight, colon length and disease and histopath-
ology scores, the suppression of DNBS-induced colitis
evoked by infection with H. diminuta was absent in mice
co-treated with antibiotics (ABX) (Fig. 1B–E and Suppl.
Fig. 1). ConA-stimulated splenocytes (used as a marker
of systemic immunity and a surrogate to confirm suc-
cessful infection) from H. diminuta+DNBS-treated mice
produced more il-10 than those from non-infected or
DNBS-only treated mice (Fig. 1F). The magnitude of the
splenic il-10 production from ABX+H. diminuta+
DNBS-treated mice was reduced, yet was significantly
greater than that produced by splenocytes for ABX+
DNBS-treated mice (Fig. 1F).
Profiling of the bacterial composition revealed a lower
Shannon index in colon-associated bacteria from DNBS-
treated mice compared to control, with H. diminuta+
DNBS-treated mice having an intermediate phenotype,
statistically different from the other two groups (Fig.
2A). A similar pattern was noted for β-diversity, with the
exception that two mice in the DNBS group clustered
with controls; these mice had the lowest disease scores
in the DNBS group (Fig.2B). Differential abundance ana-
lysis revealed significant increases in ASVs in the family
Lachnospiraceae (p=2.92 × 10
) and the Clostridium
clusters XIVa (p= 1.24 × 10
)andXIVb (p=1.94 ×
)inH. diminuta+DNBS treated mice compared to
Shute et al. Microbiome (2021) 9:186 Page 2 of 18
DNBS-only treatment (Fig. 2C). DNBS-only treated mice
had increased variants belonging to the families Bacter-
oidaceae (p=3.05 × 10
), Staphylococcaceae (p=1.60 ×
), Enterococcaceae (p= 3.39 × 10
), and Erysipelo-
trichaceae (p= 1.15 × 10
) compared to the H. dimin-
uta+DNBS group (Fig. 2C). As expected, mice treated
with ABX (broad spectrum, vancomycin only, or poly-
myxin B + neomycin ± DNBS ± H. diminuta) displayed
severe disruption of their microbiota (Fig. 2A, D–F),
with a general shift away from Firmicutes and to Bacter-
oidetes (Fig. 2F). Differential abundance analysis
identified significant increases in ASVs belonging to the
genus Akkermansia (p= 1.11 × 10
), Enterococcus (p=
6.46 × 10
), and Bacteroides (p= 3.96 × 10
), as well
as the phylum Proteobacteria (p= 2.16 × 10
diminuta+DNBS+ABX compared to H. diminuta+
DNBS-treated mice (Fig. 2F). Sequence variants belong-
ing to Lachnospiraceae were significantly (p= 4.02 ×
) depleted in ABX+DNBS-treated mice.
Use of different antibiotics (vancomycin to target
Gram-positive bacteria, polymyxin B+neomycin to target
Gram-negative bacteria) to modulate microbiota
Fig. 1 Broad-spectrum antibiotic treatment prevents H. diminuta-evoked inhibition of colitis. Male BALB/c mice were treated as shown in panel A
(H. dim,H. diminuta 5 cysticercoids orally; DNBS, 3 mg ir.; ABX-drinking water ad libitum), and 3 days after DNBS, disease severity was assessed by
Bchange in body weight, Ccolon length, Ddisease activity score, and Ehistopathology score (representative H&E images in suppl. Fig. 1). Panel
Fshows il-10 production by conA-stimulated splenocytes (2 μg/mL, 5 × 10
cell/mL, 48 h) (data are mean ± SEM combined data from 3 to 4
experiments (data in panel Fare from 2 experiments); ABX, antibiotic cocktail of kanamycin (40 mg/L), gentamicin (3.5 mg/L), colistin (4.2 mg/L),
and metronidazole (21.5 mg/L); vancomycin (Van), 200 μL of 0.5 mg/mL by intraperitoneal injection; *, #,†p< 0.05 compared to control, DNBS
and DNBS+ABX, respectively; pi, post-infection)
Shute et al. Microbiome (2021) 9:186 Page 3 of 18
composition significantly reduced the richness of the
murine gut microbiota (Fig. 2A; Suppl. Fig. 2). Treating
H. diminuta-+DNBS mice with vancomycin resulted in
the reduction of several ASVs, specifically those belong-
ing to the family Lachnospiraceae (p= 2.53 × 10
the Clostridia cluster XIVb (p= 9.47 × 10
to the H. diminuta+DNBS group (some Lachnospiraceae
ASV were increased in the H. diminuta+DNBS+vanco-
mycin group and so additional sequencing will be
needed to identify the species that differ in the two
groups). Similarly, polymyxin B+neomycin-H. diminuta+
DNBS-treated mice lacked variants belonging to the
Lachnospiraceae family (p= 7.34 × 10
) and the Clos-
tridium cluster XIVa (p= 2.67 × 10
) compared to the
H. diminuta+DNBS group (Suppl. Fig. 2). The impact of
either vancomycin or polymyxin B+neomycin on the
ability of H. diminuta to suppress DNBS-induced colitis
was variable, such that disease and histopathology scores
Fig. 2 H. diminuta preservation of the gut microbiota in DNBS-treated mice is overcome by broad-spectrum antibiotic treatment (ABX). Male
BALB/c mice were treated as shown in Fig. 1A (5 cysticercoids of H. diminuta (H. dim) 8 days prior to di-nitrobenzene sulphonic acid (DNBS; 3 mg,
ir) with necropsy 3 days later ± ABX) and colon-associated bacteria assessed by 16s rRNA sequence analysis. AReduced Shannon index (α-
diversity) caused by DNBS-colitis was significantly prevented by H. diminuta-infection, while ABX-treatment, independent of DNBS or H. diminuta
had the greatest impact on α-diversity. Bβ-diversity (PCoA; Weighted UniFrac distance) reveals separation of the groups with control and H.
dim+DNBS clustering away from DNBS-only treated mice and characterized by increased ASVs for Lachnospiraceae, Clostridales, and Clostridium-
XIVa (C). D–FUniFrac distance and relative abundance analyses show the impact of ABX on colonic microbiota of DNBS ± H. diminuta-treated
mice. (ABX, antibiotic cocktail in drinking water ad libitum, kanamycin (40 mg/L), gentamicin (3.5 mg/L), colistin (4.2 mg/L), metronidazole (21.5
mg/L), and ip. vancomycin (van) at 200 μL of 0.5 mg/mL; panel A: Pmx/Neo, polymyxin B (1 g/L) and neomycin (500 mg/L) in drinking water;
horizontal line, median; black diamond, mean; box plots, 25–75% quartiles; vertical line, minimum and maximum value; Mann-Whitney Utest; see
suppl. Fig. 3A and D for treatment protocol)
Shute et al. Microbiome (2021) 9:186 Page 4 of 18
were not statistically different from the DNBS or H.
diminuta+DNBS groups (Suppl. Fig. 3), and likely re-
flects the composition of the microbiota in individual
mice at the start of the experiment combined with the
variable response to DNBS. Splenocytes from H. dimin-
uta+DNBS+vancomycin or H. diminuta+DNBS+poly-
myxin B+neomycin-treated mice produced levels of il-10
that were not different from control, and in contrast to
significantly increased output of il-10 from conA-
stimulated splenocytes for H. diminuta+DNBS-treated
mice (Suppl. Fig. 4).
Increased splenocyte il-10 production from GF-mice
confirmed a response to infection with H. diminuta
(Suppl. Fig. 5A) . GF-mice infected with H. diminuta
had increased colonic il-10 mRNA compared to control,
while il-10rαmRNA levels were not different from unin-
fected GF-mice (Suppl. Fig. 5B). While the severity of
DNBS-induced colitis was variable in GF-mice, infection
with H. diminuta did not elicit a significant anti-colitic
effect in these mice (Suppl. Fig. 5C, D).
Fecal microbial transplants from H. diminuta-infected
mice inhibits colitis
Fresh feces were collected from control specific
pathogen-free (SPF)-mice and mice infected with H.
diminuta 8 days previously, processed under anaerobic
conditions, and gavaged into separate groups of GF-mice
(Fig. 3A); animals that received feces from H. diminuta-
infected donor mice had less severe colitis when chal-
lenged with DNBS 4 weeks later (Fig. 3B–F).
Analysis revealed bacterial community compositions
in feces from control and H. diminuta-infected mice
consistent with our previous observations (data not
shown) , with a small increase in α-diversity in the
infected mice (Suppl. Fig. 6A). Four weeks post-
colonization, α-diversity was not different between the
groups, whereas taxonomic β-diversity as determined by
weighted Unifrac distance showed distinct separation of
the groups (Suppl. Fig. 6B), that was still apparent on
necropsy 72 h after DNBS-treatment (Suppl. Fig. 6A,B).
Differential abundance analysis revealed greater abun-
dance of ASVs belonging to the family Lachnospiraceae
(p= 1.33 × 10
) and Clostridia cluster XIVa (p= 4.94
) in feces from H. diminuta-infected donors com-
pared to that from naïve-donor SPF-mice (Suppl. Fig.
6C). At 4 weeks post-colonization, mice that received
feces from H. diminuta-infected mice had a higher
abundance of Lachnospiraceae (p= 8.97 × 10
nococcus (p= 8.46 × 10
), and Clostridium cluster
XIVa (p= 6.32 × 10
), while ASVs assigned to the
families Clostridiaceae_1 (p= 3.67 × 10
) and Rumino-
coccaceae (Flavonifractor;p= 2.83 × 10
), and Clos-
tridium cluster IV (p= 5.62 × 10
) were increased in
mice that received control donor feces (Suppl. Fig. 6D).
Finally, differential abundance analysis showed signifi-
cant increases in ASVs belonging to the families Lach-
nospiraceae (p= 2.12 × 10
) and Ruminococcaceae (p
= 3.11 × 10
), as well as Clostridium cluster XIVa (p=
1.22 × 10
) in DNBS-treated mice that received feces
from H. diminuta-donors compared to DNBS-treated
mice that received feces from naïve-donors; the latter
demonstrated substantial increases within the families
Enterobacteriaceae (p= 4.75 × 10
) and Bacteroida-
ceae (p= 2.89 × 10
) (Suppl. Fig. 6E).
Feces from H. diminuta-infected mice have increased
Initial NMR analyses revealed increased acetate, propion-
ate, and butyrate in feces from 8-day H. diminuta-infected
mice compared to non-infected mice (Fig. 4A). The in-
creases were transitory and were not seen with this tech-
nique when feces from 11-dpi with H. diminuta were
assessed (Fig. 4A). These increases in SCFAs were con-
firmed by paired LC-MS analyses on feces from individual
mice collected prior to and 8 days post-infection with H.
diminuta (Fig. 4B–D). The increased levels of acetate, bu-
tyrate, and propionate in feces from H. diminuta-infected
mice were ablated by antibiotic co-treatment, particularly
the cocktail with broad-spectrum activity (Fig. 4E–G). The
anti-colitic effects of butyrate and acetate were confirmed
by enema delivery or continuously in the drinking water,
respectively (Suppl. Fig. 7).
Bacteria-free filtrate of feces from H. diminuta-infected
mice reduces DNBS-induced colitis
Intra-rectal delivery of a fecal filtrate (FF) from day-8 H.
diminuta-infected mice four times over the course of
DNBS-induced colitis (Fig. 5A) significantly reduced the
severity of disease (Fig. 5B–F), as gaged by disease and
histopathology scores, but not body weight. Mice that
received control FF or FF from H. diminuta-infected do-
nors had longer controls than DNBS-only treated mice,
suggesting a mild benefit of fecal filtrate in this model
system: the benefit was most pronounced with the FF
from infected mice. Colonic tissue from mice that re-
ceived the FF from H. diminuta-infected mice had in-
creased il-10rαmRNA compared to those receiving FF
from naïve donor mice, but il-10 mRNA was not statisti-
cally significantly increased (Fig. 5G). The FF from H.
diminuta-infected mice contained more acetate (4.2±
0.81 mM*) and butyrate (467±90 μM*) compared to FF
from naïve mice (acetate = 2.3±0.53 mM; butyrate =
272±52 μM; n=3;*,p< 0.05 unpaired ttest).
DNBS-induced colitis in ffar2
mice is not affected by
infection with H. diminuta
C57/Bl6 free-fatty acid (ffar)-2
mice infected with H.
diminuta were protected from DNBS-induced colitis,
Shute et al. Microbiome (2021) 9:186 Page 5 of 18
Fig. 3 (See legend on next page.)
Shute et al. Microbiome (2021) 9:186 Page 6 of 18
whereas infected ffar2
littermates displayed colitis
that was not significantly different from DNBS-only
mice as assessed by disease activity
scores, histopathology scores, and colon length (Fig. 6A–
C). The anti-colitic effect of infection with H. diminuta
observed in ffar-2
was accompanied by increased il-10
production by conA-stimulated splenocytes compared to
splenocytes from naïve ffar-2
mice and H. diminuta+
DNBS treated ffar2
mice (Fig. 6D).
Anti-il10 antibodies eliminate the anti-colitic effect of
infection with H. diminuta
Consistent with previous findings , the inhibition of
DNBS-induced colitis by infection with H. diminuta was
abrogated in mice treated with neutralizing anti-il-10
antibodies, as assessed by disease activity and histopath-
ology scores (Suppl. Fig. 8).
Reciprocal regulation of il-10 receptor and butyrate
Colonic tissue excised from H. diminuta+DNBS treated
mice displayed increased il-10 and il-10rαmRNA and
decreased IFNγmRNA compared to DNBS-only treated
mice: these changes were abrogated by antibiotic treat-
ment of H. diminuta+DNBS treated mice (Fig. 7A). Cor-
roborating and extending data with human gut-derived
cell lines , we find that butyrate increases il-10rα
mRNA expression in a mouse rectal epithelial cell line
and in primary mouse colonoids in a dose- and time-
dependent manner (Fig. 7B, C). Immunostaining re-
vealed il-10rα-immunoreactivity in the colon of control
mice that was most prominent on the apical epithelium
with minimal positivity on lamina propria cells (as dem-
onstrable by this technique), whereas tissue from DNBS-
treated mice was largely devoid of il-10rα-immunoreac-
tivity (Fig.7D). Colon from H. diminuta+DNBS treated
mice had widespread il-10rα-immunoreactivity in the
epithelium, extending deep into the crypts, and in the
lamina propria (Fig.7D). Sections of colon from mice
that received butyrate enemas displayed il-10rα-immu-
noreactivity that was subtly increased over that observed
in control mice and was predominantly evident on the
apical epithelial cells (Fig. 7D).
Analysis of mRNA for SCFA transporters and recep-
tors revealed consistent induction of MCT1 mRNA in
the HT-29 (Fig. 8A) and CMT-93 (Fig. 8B) epithelial cell
lines and primary mouse organoids (Fig. 8C) by il-10
(HT-29 had a subtle increase in MCT1 protein) (Fig.
8A). ABCG2 mRNA was increased in il-10-treated HT-
29 and CMT-93 epithelia. Il-10 treatment increased
mRNA expression for the SCFA receptor HCAR
(GPR109A) in HT-29 cells (Fig. 8A), while the increase
in ffar2 in CMT-93 cells failed to reach statistical signifi-
cance (Fig. 8B).
Enthusiasm for helminth-therapy for inflammatory dis-
ease based on numerous animal model studies  and
small clinical trials [28–31] is tempered by a lack of effi-
cacy of Trichuris suis ova in larger trials [32–34]. We hy-
pothesized that the anti-colitic effect of infection with a
helminth parasite could be influenced by the gut micro-
biota and so its effectiveness would be reduced in IBD
patients with dysbiosis . The novel data herein reveal
helminth, host, and gut bacteria interaction in the sup-
pression of disease, and in untangling this tripartite
mechanism of the control of enteric inflammation we
note reciprocity in il-10 and butyrate signaling in the
regulation of short-chain fatty acid transporter and il-10
receptor expression, respectively.
Mechanistic studies to understand how infection with
helminth parasites inhibits inflammatory disease have
implicated suppression of Th1 immunity or production
of immunoregulatory cells and mediators [4,13,14,36].
This focus on host immunological processes, while intui-
tive, has, until recently, overlooked the potential involve-
ment of the host microbiota as a regulator of mucosal
immunity and gut homeostasis [22,37–39]. Following
identification that infection with H. diminuta signifi-
cantly increased bacterial species richness in mice (e.g.,
increased relative abundance of Lachnospiraceae 
and reduced Bacteroidaceae, members of which may
exert a pro-colitigenic effect ), treatment with broad-
spectrum antibiotics was found to prevent the inhibition
of colitis evoked by H. diminuta-infection. Moreover,
splenocytes from the antibiotic+H. diminuta+DNBS-
treated mice produced substantial amounts of il-10, sug-
gesting that lack of inhibition of colitis in the antibiotic-
treated mice was linked to the microbiota and not a by-
stander effect on the host immune response to H. dimin-
uta-infection. This supposition is supported by similar
severities of DNBS-induced colitis in GF-mice ± H.
diminuta-infection. In accordance with these data, H.
polygyrus-evoked suppression of airways inflammation
(See figure on previous page.)
Fig. 3 Feces from H. diminuta-infected mice protects mice from DNBS-treated mice. AExperimental paradigm of treatment of male BALB/c
germ-free (GF)-mice with feces from mice infected with 5 H. diminuta 8 days previously. Di-nitrobenzene sulphonic acid (DNBS: 3 mg, ir.)-induced
colitis evoked 4 weeks after fecal microbial transplant was assessed by body weight (B), colon length (C), and disease (D) and histopathology
scores (E). Panel Fshow representative H&E stained sections of mid-colon (data are mean ± SEM combined from 2 experiments; *, p< 0.05
compared to mice receiving feces from naive control mice; SPF, specific pathogen-free)
Shute et al. Microbiome (2021) 9:186 Page 7 of 18
Fig. 4 (See legend on next page.)
Shute et al. Microbiome (2021) 9:186 Page 8 of 18
or obesity induced by a high-fat diet was abrogated in
mice co-treated with antibiotics [22,41].
Dissecting the role of the microbiota in the anti-colitic
evoked by helminth-infection, transfer of feces from
mice infected 8 days previously with H. diminuta into
GF-mice conferred partial, but significant, protection
from DNBS-colitis. While the transfer of feces is a
promising approach for some conditions , there are
safety concerns, and microbiota from Schistosoma man-
soni- and H. polygyrus-infected mice exaggerated dextran
sodium sulfate- and Citrobacter rodentium-induced col-
itis, respectively . The latter studies illustrate the
specificity of host-parasite interaction, that infection
with worms that do not inhabit the gut (i.e., S. mansoni)
can affect the composition of the gut microbiota, and
that helminth therapy for a inflammatory disease is un-
likely to be via a single species of helminth [we note that
differential effects of feces from helminth-infected mice
may also be due to differences in the gut bacteria due to
source of the animal, housing or food, and be influenced
by the helminth-specific mucosal immune response].
Subsequently, enemas of filtered feces from H. dimin-
uta-infected mice, but not that from uninfected mice,
were found to inhibit DNBS-colitis in SPF-mice,
prompting analysis of the feces for molecules that could
suppress DNBS-induced colitis.
Feces from H. diminuta-infected mice had increased
levels of the short-chain fatty acids (SCFAs), acetate and
butyrate compared to uninfected mice, compatible with
the increased abundance of actinobacteria and Clostrid-
ium cluster XIVa. Some, not all, individuals with IBD
have benefited from butyrate enemas  and acetate
and butyrate can be anti-inflammatory in murine models
of colitis [44–46]; findings we recapitulated with the
DNBS-model of colitis. While many bacteria-derived
products affect the host, the finding that the ffar2
(or G-protein coupled receptor (GPR)-43 found on
colonic epithelium and immune cells ) mice were
not protected from DNBS-induced colitis by infection
with H. diminuta supports further a role for SCFA in
the anti-colitic effect. Similarly, fecal transplants from
infected mice recapitulated the reduced hypersensitiv-
ity to house dust mite in H. polygyrus-infected mice;
in this instance acetate and ffar3 (GPR41) mediated
the protective effect .
The data support a mechanism whereby infection with
H. diminuta causes increased abundance of SCFA-
producing bacteria, and that increased butyrate and acet-
ate, via ffar2,mediates the suppression of colitis. How
then to reconcile this with immunoneutralization of il-
10 blocking the anti-colitic effect of infection with H.
diminuta (6) (Suppl. Fig. 8)? Positing interaction via il-
10 and butyrate, H. diminuta-infection evoked increased
il-10rαimmunoreactivity in the colon was absent in
antibiotic co-treated mice, and infected GF-mice dis-
played increased colonic il-10, but not il-10rα, mRNA.
Moreover, enemas with fecal-filtrate from H. diminuta-
infected mice or butyrate into SPF-mice both resulted in
an increase in colonic il-10-rαmRNA or protein. Butyr-
ate directly increased il-10rαmRNA expression in a
murine rectal epithelial cell line and primary epithelia,
extending similar observations in human colon-derived
epithelial cells lines . Reciprocally, il-10 increased
mRNA expression for one or more butyrate transporter/
receptor in the human colonic HT-29 epithelial cell line,
and murine CMT-93 epithelial cells and colonoids.
Butyrate and il-10 exert a range of anti-inflammatory
effects [48,49]. Thus, we speculate that infection with H.
diminuta creates a positive feedback loop whereby
bacteria-derived butyrate and host-derived il-10 cooper-
ate to drive the anti-colitic effect: absence of either ne-
gates the beneficial effect of infection with the helminth.
In accordance, the recruitment of il-10
cells to the lungs of H. polygyrus-infected mice was
dependent on ffar3, and H. polygyrus-evoked changes in
the gut microbiome that reduced obesity in high-fat
diet-fed mice were dependent of signal transducer and
activator of transcription (STAT)-6 (i.e., il-4/il-13 signal-
ing) . These findings combined with the current data
illustrate the intertwined nature of the helminth-host-
bacteria relationship and the interplay between host im-
mune factors and bacteria-derived molecules in the sup-
pression of disease.
The present study advances understanding of helminth-
regulation of inflammatory disease, providing evidence
for a critical role of bacteria-derived SCFAs operating
via ffar2 in H. diminuta-amelioration of colitis, the es-
sential requirement of il-10 that can up-regulate
(See figure on previous page.)
Fig. 4 Feces from H. diminuta-infected mice contain increase amounts of short-chain fatty acids (SCFA). AHeat-map of NMR results shows
increased acetate, propionic acid and butyric acid in feces from mice infected 8 days previously with H. diminuta.B–DSeparate analyses
confirmed increased SCFA in feces of infected mice (paired ttest, day 0 vs. 8 days post-infection). E–GBroad-spectrum antibiotic treatment (ABX)
(see Fig. 1A) prevented the H. diminuta (H. dim) evoked increase in fecal acetate or butyrate, and a similar pattern was observed in mice treated
with vancomycin (Van) or polymyxin B and neomycin (Pmx/Neo) (see Suppl. Fig. 3A,D) (data are mean ± SEM; *, p< 0.05 compared to control or
between indicated groups; #, p< 0.05 compared to DNBS (di-nitrobenzene sulphonic acid, 3 mg, ir., necropsy 72 post-DNBS; H. dim,5
cysticercoids 8 days prior to DNBS; pi, post-infection)
Shute et al. Microbiome (2021) 9:186 Page 9 of 18
Fig. 5 (See legend on next page.)
Shute et al. Microbiome (2021) 9:186 Page 10 of 18
expression of SCFA transporters/receptors, and butyrate
regulation of il-10 receptor expression. Moreover, it pro-
vides one possible explanation for the lack of efficacy of
helminth-therapy in recent IBD trials, such that patients
who lack SCFA-producing bacteria , lack butyrate
transporters or receptors , or with a diminished cap-
acity to express il-10 or the il-10-receptor  would be
contraindicated for this novel treatment. Extrapolating
from this model system, we suggest that for helminth-
therapy to be beneficial it needs to be coupled to a pre-
cise knowledge of the immunological profile of the mal-
ady to be treated and the composition of the patients’
microbiome. Furthermore, we speculate that reduced ef-
ficacy of helminth therapy could be enhanced by
(See figure on previous page.)
Fig. 5 Bacteria-free filtrate of feces from H. diminuta-infected mice reduces the severity of DNBS-induced colitis. Feces was collected from H.
diminuta-infected mice and passed through a 0.2 μm filter (FF) and administered to specific-pathogen-free male BALB/c mice as shown in panel
A. Seventy-two hours after di-nitrobenzene sulphonic acid (DNBS: 3 mg, ir.), disease severity was assessed by Bbody weight, Ccolon length, D
disease activity, and Ehistopathology scores. Representative H&E stained sections of mid-colon are shown in panel F. Panel Gshows q-PCR for il-
10 and il-10rαin colonic segments from FF-treated mice (data are mean ± SEM combined data from 2 experiments; * and #, p< 0.05 compared
to control and control fecal filtrate from naive non-infected mice, respectively; H. dim FF, fecal filtrate from H. diminuta-infected (5 cysticercoids)
mice; pi, post-infection)
Fig. 6 Free-fatty acid repector-2 knock-out mice are not protected from DNBS-induced colitis by infection with H. diminuta. Male ffar2
littermates were infected with 5 cysticercoids of H. diminuta (H. dim) and 8 days later were challenged with di-nitrobenzene
sulphonic acid (DNBS, 3 mg, ir). At necropsy 72 h post-DNBS, disease was assessed by Adisease activity and Bhistopathology scores and colon
length (C). DIsolated splenocytes (5 × 10
/mL) were stimulated with concanavalin-A (2 μg/ml) for 48 h and il-10 production measured by ELISA
(data are mean ± SEM combined data from 2 experiments; * and #, p< 0.05 compared to control and DNBS-only in the matched mouse strain)
Shute et al. Microbiome (2021) 9:186 Page 11 of 18
Fig. 7 (See legend on next page.)
Shute et al. Microbiome (2021) 9:186 Page 12 of 18
combination with a probiotic matched to compensate
for dysbiosis in a particular individual.
Mice and H. diminuta life-cycle
All experimental procedures were approved by the Univ.
Calgary Animal Care Committee under protocol AC17-
0115 in compliance with the Canadian Council on Ani-
mal Care guidelines.
Male BALB/c and C57BL/6 mice (7–9 weeks old,
Charles River Laboratories, Quebec, Canada) were
housed in HEPA filtered micro-isolator cages with free
access to rodent chow (Pico-Vac Mouse Diet 20: 5062)
and water in a 22 °C-controlled facility on a 12 h:12 h
light:dark cycle. Breeding pairs of C57Bl/6 Ffar2
were provided by Dr. B.T. Layden (University of Illinois,
Chicago)  and maintained at the Univ. of Calgary.
Germ-free (GF) BALB/c and C57BL/6 mice were bred
and maintained in flexible-film sterile isolators in the
International Microbiome Center at the Univ. Calgary.
Germ-free status was tested by Sytox Green nucleic acid
staining (Invitrogen) of caecal contents . Mice were
humanely euthanized prior to necropsy.
Adult H. diminuta were maintained in Sprague-
Dawley Rats (Charles River) as a reservoir host and
gravid proglottids passaged through flour beetles to ob-
tain the infective cysticercoids. Mice, under mild manual
restrain, were infected with five cysticercoids in 100 μL
of 0.9% NaCl with a round-tipped oral gavage needle .
For GF mice, cysticercoids were incubated in antibiotics
(300 μL: kanamycin (400 mg/L), gentamicin (35 mg/L),
colistin (42 mg/L), and metronidazole (215 mg/L)) for 2
h at 37 °C. Cysticercoid viability after antibiotics treat-
ment was confirmed by excystment in vitro and ability
to infect il4ra
mice ). Each GF-mouse received 8–
10 cysticercoids by oral gavage.
Induction and assessment of DNBS-colitis
Colitis was induced in anesthetized mice with 3 mg di-
nitrobenzene sulphonic acid (DNBS: MP Biomedicals,
Santa Ana, CA) in 100 μL of 50% ethanol in PBS via a
polyethylene catheter inserted 3 cm into the colon .
Bodyweight was recorded daily over 72 h and on
necropsy, colon length was measured and a macroscopic
disease activity score (DAS) was calculated (maximum 5
points) . Portions of mid-colon were excised, fixed in
10% neutral-buffered formalin, dehydrated, and embed-
ded in paraffin wax. Seven μm sections were collected
on coded slides, stained with hematoxylin and eosin, and
histopathology scored in a blinded fashion on a validated
12-point scale . Additional histological sections were
immunostained for il-10 receptor-αchain using a detec-
tion rabbit-anti-mouse il-10 antibody (1:100 in PBS;
Abcam ab225820). After 24 h 4
C incubation, sections
were washed, secondary goat-anti-rabbit HRP-
conjugated antibody (1:500 in PBS, 30 min room
temperature) applied, washed, and then DAB (3,3′Di-
aminobenzidine) substrate (Abcam: ab64238) added.
Representative images were captured on an Olympus
BX41 microscope fitted with a U-TMAD T mount
adapter, using cell Sens standard software (Olympus).
Images were processed using ImageJ (version 1.80
Approximately 0.5 cm of tissue immediately distal to
that taken for histology was collected and total RNA iso-
lated using the Aurum Total RNA Mini Kit (Bio-Rad La-
boratories, Hercules, CA) as per the manufacturer’s
protocol, quantified with the Nanodrop 1000 Spectro-
photometer (Thermo Fisher Scientific, Wilmington, DE),
and 0.5 μg of RNA was converted to cDNA using an
iScript kit (Bio-Rad Lab). Quantitative real-time poly-
merase chain reaction (qPCR) of murine colonic tissue
was performed as previously described [25,55] using
primer sequences shown in Suppl. Table 1.
Interleukin-10 production by concanavalin-A (2 μg/
mL, 48 h)-stimulated spleen cells (5 × 10
/mL) was de-
termined by sandwich ELISA using paired antibodies
(R&D Systems Inc.) in accordance with the manufac-
turer’s instructions .
Antibiotic treatment of mice
Mice were treated with a broad-spectrum cocktail of an-
tibiotics (ABX: drinking water, kanamycin (40 mg/L),
gentamicin (3.5 mg/L), colistin (4.2 mg/L), and metro-
nidazole (21.5 mg/L) and ip. injections of vancomycin
(200 μL of 0.5 mg/mL)  vancomycin only, or
(See figure on previous page.)
Fig. 7 Helminth infection and butyrate upregulates IL-10 receptor expression. qPCR reveals increased in il-10 and il-10 receptor-αand reduced
interferon (ifn)-γin mid-colon of H. diminuta and DNBS treated mice compared to control and DNBS only treated mice. Co-treatment with broad-
spectrum antibiotics (ABX), vancomycin (Van), or a mixture of polymyxin B (Pmx) and neomycin (Neo) prevented the increase in il-10 or il-10
receptor-α(rα) mRNA. Exposure to butyrate (1-10 mM; 16–24 h) significantly increased the expression of il-10rαmRNA in Ba murine rectal
epithelial cell line and Cprimary murine colonoids. Panel Dis a qualitative assessment of il-10rαexpression on sections of mid-colon as detected
by immunocytochemistry, with representative images depicted (data are mean ± SEM; *, p< 0.05 compared to control; H. dim, mice infected with
5H. diminuta 8 days before intra-rectal di-nitrobenzene sulphonic acid (DNBS, 3 mg, 72 h); butyrate (500 micro-L of 100 mM enema was delivered
24 and 3 h before DNBS and 24 and 48 h after DNBS) (ABX, antibiotic cocktail in drinking water ad libitum, kanamycin (40 mg/L), gentamicin (3.5
mg/L), colistin (4.2 mg/L), metronidazole (21.5 mg/L); ip. Van. at 200 μL of 0.5mg/mL; Pmx/Neo, 1 g/L and 500mg/L in drinking water (see Fig. 1A
and suppl. Fig. 3A,D for treatment protocols)
Shute et al. Microbiome (2021) 9:186 Page 13 of 18
Fig. 8 (See legend on next page.)
Shute et al. Microbiome (2021) 9:186 Page 14 of 18
polymyxin B (PMB; 1 g/L) + neomycin sulfate (Neo; 500
mg/L) in drinking water (see figures for treatment regi-
16S rRNA analysis of bacterial communities
Feces (100 mg) was homogenized using 0.2 g of 2.8 mm
ceramic beads (Mo Bio Laboratories, #13114-50) in a
Bullet Blender (Next Advance) and DNA isolated follow-
ing the method of Surette et al.,  and bacterial com-
munity profiling performed via 16S rRNA V3-V4 region
(341F-785F) amplicon sequencing via Illumina MiSeq
(25). Analysis was performed using Rstudio (R version
3.5.0). Prior to processing the raw fastq files, adapter and
primer sequences were removed using the Cutadapt pro-
gram (version 1.17). Once non-biological nucleotides
were removed, the paired-end fastq files were processed
using the dada2 pipeline (version 1.12.1; dada2 workflow
http://benjjneb.github.io/dada2/tutorial.html). Using the
dada2 “filterAndTrim”function, the truncation lengths
were set to 270 and 200 and the maximum number of
expected errors was set at 2. After learning the error
rates (“learnErrors”in dada2) for denoising the amplicon
data of non-biological errors (“dada”in dada2), forward
and reverse reads were merged for full-denoised se-
quences (“mergePairs”in dada2) and an amplicon se-
quence variant (ASV) table generated
(“makeSequenceTable”in dada2). Taxonomic classifica-
tions were assigned to the ASV table (“assignTaxonomy”
in dada2) using the Silva 132 database (arb-silva.de/
documentation/release-132/) as a reference training set.
Community analysis of the data was performed using
Phyloseq version 1.24.2. Alpha diversity was determined
using the “plot_richness”function in Phyloseq and Wil-
coxon rank sum test assessed statistical significance.
Using the “Unifrac”function in Phyloseq, weighted Uni-
frac distances of each sample was determined and plot-
ted using Principal Coordinate Analysis (PCoA). A
permanova test using the “Adonis”function (Vegan ver-
sion 2.5-6.) tested for statistically significant compos-
itional differences between groups (β-diversity).
Following a positive permanova test, a permutation test
for homogeneity of multivariate dispersions was per-
formed, for which a non-significant test would indicate
that the permanova test is a real result and not due to
differences in group dispersion. Differential abundance
(identifying taxa within a sample/group that are
significantly increased or decreased when compared to
another sample) was performed with R program DeSeq2
(v. 3.11). Data are displayed as a log-fold change. The
raw fastq sequencing files used within this study have
been uploaded to the short reads archive (SRA) database
Short-chain fatty acid and metabolite measurement
Five hundred milligrams of fresh feces was mixed with
500 μL 100% HPLC grade methanol and 500 μL of HPLC
O, vortexed and then centrifuged at 13,000×g
for 5 min at 4 °C. Then 700 μL of the supernatant was
mixed with an equal volume of 50% HLPC grade metha-
nol, vortexed (2 min), and spun down (13,000×g, 10 min,
4 °C). The supernatant was collected, divided into two,
filtered (0.2 μm), and dried at 4 °C. One of the duplicate
dried samples was reconstituted in 800 μL of deuterium
oxide, titrated to a pH of 7.400 ± 0.005, and subjected to
nuclear magnetic resonance (NMR) analysis . NMR
data were acquired on a 600 MHz Bruker Advance III
instrument. Metabolites were assigned by
nuclear single quantum coherence (HSQC). Data were
collected using the hsqcetgpsp (Bruker) pulse program.
Spectra were acquired in 8190 points in a 12.01 ppm
sweep width in the direct dimension and 1024 incre-
ments, 110 ppm sweep width in the indirect dimension.
Data were processed in Burker TopSpin and analyzed in
rNMR. Metabolites were assigned using the Madison
Metabolomics Consortium Database reference spectra
available from the BMRB. Once the spectra had been
assigned, metabolites were quantified using 1D
with NOSEY water suppression (Bruker noesygppr1d
pulse program). Data were acquired in 65,536 points
with 32 scans and a sweep width of 12.01 ppm. Metabo-
lites were quantified following established methods .
In other experiments, 100 mg of feces was assessed by
liquid chromatography-mass spectrometry (LC-MS) for
SCFA . Samples were dissolved in ice-cold extraction
solvent containing 100 μLofH
O/acetonitrile (50:50) so-
lution containing 5 mM, 200 μM and 500 μMof
beled acetic acid (1,2-
C2, 99 atom%: #CLM-113,
Cambridge Isotope Lab.) propionic acid (99 atom %:
#589586, Sigma-Aldrich) and butyric acid (-1,2-
C: #491993, Sigma-Aldrich), respectively [for
fecal samples from antibiotic-treated mice the internal
SCFA standards were 2.5 mM, 200 μM and 50 μM,
(See figure on previous page.)
Fig. 8 IL-10 increases short-chain fatty acid (SCFA) transporter expression. The human colon-derived HT-29 epithelial cell line treated with IL-10
(10 or 100 ng/mL, 24 h) displayed significant increases in the SCFA transporters MCT1, ABCG2, and HCAR2 mRNA and a reduction in MCT4 mRNA
(A). The increased MCT1 mRNA was matched by a subtle increase in MCT1 protein (representative blot shown), as shown by densitometry and
statistical comparisons. Panel Bshows increased expression of mct1 and Abcg2 mRNA in the murine rectal epithelial CMT-93 cell line treated
with il-10 (10 ng/ml, 24 h). IL-10 treatment evoked increased mct1 mRNA in primary murine colonoids (C) (10 ng/ml, 24 h) (data are mean ± SEM;
data from 1 to 2 experiments; *, p< 0.05 compared to control; kDa, kilodaltons)
Shute et al. Microbiome (2021) 9:186 Page 15 of 18
respectively]. Samples were vortexed, then centrifuged
(10,000×g, 10 min, 4 °C), and when clear 50 μL of super-
natant was cooled to 0 °C and derivatized in an extrac-
tion solvent containing 2.5 μL of 2.4 M aniline (dissolved
in acetonitrile), followed by 2.5 μL of 1.2 M 1-ethyl-3(3-
dimethylaminopropyl) carbodiimide (dissolved in H
The mixture was vortexed for 15 s and placed on ice for
2 h (vortexing every 30 min), diluted 1:20 with 50%
methanol, vortexed for 15 s, and the samples subjected
LC-MS analysis. Data were analyzed as previously de-
scribed . Briefly, metabolites were separated on a re-
verse phase chromatographic gradient (Thermo Fisher
Hypersil GOLD TMC18 column) and metabolites were
quantified by selected reaction monitoring (SRM). Con-
centrations were calculated based on the ratio of
isotope-labeled fragments from standard compounds
relative to the corresponding fragments from microbial
Fecal microbial transplants (FMT) and bacteria-free fecal
Feces were collected from control male C57BL/6 mice
or those infected with H. diminuta 8 days previously and
immediately placed in 10 mL of pre-reduced sterile PBS
in a Ruskinn anaerobic chamber. Samples were vortexed
(2 min), centrifuged (5 min at 1000×g) and 400 μL of the
fecal supernatant was given to GF-mice by oral gavage.
Four weeks later, fecal samples were collected for 16S
rRNA analysis, and mice were challenged with DNBS (5
mg in 100 μL 50% etoh.). In other experiments, fecal
samples (500 mg) were collected from control mice and
those infected with H. diminuta 8 days previously, solu-
bilized in 10 mL of sterile PBS and passed through a
0.45 μm and then 0.2 μm pore-size filter . This sterile
filtrate was then administered as a 200 μL of 50 mg/mL
enema to naïve specific pathogen-free (SPF) mice, that
subsequently received DNBS.
Treatment of mice with short-chain fatty acids (SCFAs)
Adopting published methodologies for treatment with
SCFAs, male BALB/c mice were supplemented with 200
mM of sodium acetate (Sigma-Aldrich #S2889) in
their drinking water 7 days prior to DNBS and main-
tained on sodium acetate-drinking water throughout the
experiment. Another cohort of animals received butyrate
enemas (500 μL of 100 mM 98% sodium butyrate;
Sigma-Aldrich #B5887)  or PBS (500 μL) 24 h and 3
h before DNBS and again at 24 h and 48 h post-DNBS
Anti-IL-10 antibody treatment of mice
Following a protocol we applied previously , mice re-
ceived intraperitoneal injections of either a neutralizing
anti-il-10 antibody (clone JES5-2A5; Biolegend #504909)
or an isotype matched irrelevant IgG
#400432) at day-3 (50 μg), day-7 (100 μg), and day-9
(50 μg) post-infection with H. diminuta for a total of
200 μg of antibody. DNBS was administered at 8 days
post-infection and mice were necropsied 3 days later.
Data presentation and statistical analysis
Results are expressed as the mean ± standard error of
the mean (SE) and nis the number of mice. Data are an-
alyzed using Graph Pad Prism 8.0 in which statistical
comparisons for parametric data were performed via
one-way ANOVA with Tukey’s post-test and the
Kruskal-Wallis test with Dunn’s post-test was applied to
non-parametric data. P< 0.05 was set as the level of ac-
ceptable statistical difference.
The online version contains supplementary material available at https://doi.
Additional file 1: Suppl. Figure 1. Representative images of H&E
stained sections of mid-colon from mice treated with broad-spectrum
and DNBS. Suppl. Figure 2. Vancomycin or a combination of polymyxin
B + neomycin impact gut bacteria in H. diminuta ± DNBS-treated mice.
Suppl. Figure 3. Treatment with selected antibiotics interferes with H.
diminuta-evoked inhibition of colitis. Suppl. Figure 4. Treatment with
selected antibiotics affects stimulated splenic il-10 output. Suppl. Figure
5. H. diminuta-infected germ-free (GF) mice are not protected from
DNBS-induced colitis. Suppl. Figure 6. Germ-free (GF) mice colonized
with feces from H. diminuta-infected retain a distinct bacterial compos-
ition. Suppl. Figure 7. Short-chain fatty acids (SCFA) suppress DNBS-
induced colitis. Suppl. Figure 8. Neutralizing anti-il-10 antibody block H.
diminuta-evoked inhibition of DNBS-induced colitis.
Additional file 2: Suppl. Table 1. Primer sequences used throughout
A. Shute was supported by Beverly Phillips Snyder Institute University of
Calgary, NSERC CREATE Host Parasite Interactions, and the Canadian
Association of Gastroenterology studentships. This work was enabled by the
services of the International Microbiome Centre (IMC), which is supported by
the University of Calgary’s Cumming School of Medicine and the province of
Alberta. Metabolomics data were acquired at the Calgary Metabolomics
Research Facility (CMRF) (supported by the IMC and the Canada Foundation
for Innovation CFI-JELF 34986). I.A.L. is supported by an Alberta Innovates
Translational Health Chair.
Methodology: AS, DMM. Conceptualization: AS, DMM, AGB. Investigation: AS,
BEC, SL, AW, TSJ, CO. Writing—original draft: AS, DMM. Writing—review and
editing: AS, BEC, SL, AW, TSJ, CO, AGB, BTL, IAL, DMM. Funding acquisition:
DMM. Resources: IAL, BTL. Supervision: DMM, AGB. The authors read and
approved the final manuscript.
Funding provided by a Canadian Institutes for Health Research (CIHR) grant
(#201903PJT-418112-HDK-CBBA-3463) to D.M. McKay.
Availability of data and materials
The raw fastq sequencing files used within this study have been uploaded
to the short reads archive (SRA) database (BioProjectID:PRJNA690571).
Shute et al. Microbiome (2021) 9:186 Page 16 of 18
Ethics approval and consent to participate
All experimental procedures were approved by the Univ. Calgary Animal
Care Committee under protocol AC17-0115 in compliance with the Canadian
Council on Animal Care guidelines.
Consent for publication
The authors declare that they have no competing interests.
Gastrointestinal Research Group, Inflammation Research Network and
Host-Parasite Interaction Group, Calvin, Phoebe & Joan Snyder Institute for
Chronic Diseases, Department of Physiology and Pharmacology, Cumming
School of Medicine, University of Calgary, Calgary, Alberta, Canada.
International Microbiome Center, Cumming School of Medicine, University
of Calgary, Calgary, Canada.
Department of Biological Sciences, Faculty of
Science, University of Calgary, Calgary, Canada.
Division of Endocrinology,
Diabetes, and Metabolism, University of Illinois at Chicago, Chicago, IL, USA.
Jesse Brown Veterans Affairs Medical Center, Chicago, IL, USA.
Received: 6 March 2021 Accepted: 30 July 2021
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