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Am J Physiol Gastrointest Liver Physiol 314: G461–G470, 2018.

First published January 4, 2018; doi:10.1152/ajpgi.00295.2017.

RESEARCH ARTICLE Inflammation, Immunity, Fibrosis, and Infection

Young mice expel the tapeworm Hymenolepis diminuta and are protected
from colitis by triggering a memory response with worm antigen
Toshio Arai, Fernando Lopes, Adam Shute, Arthur Wang, and Derek M. McKay
Gastrointestinal Research Group and Inflammation Research Network, Department of Physiology and Pharmacology, Calvin,
Joan, and Phoebe Snyder Institute for Chronic Disease, Cumming School of Medicine, University of Calgary, Calgary,
Alberta, Canada
Submitted 20 September 2017; accepted in final form 27 December 2017

Arai T, Lopes F, Shute A, Wang A, McKay DM. Young mice Crohn’s disease and ulcerative colitis) over the last 50 yr (4, 6).
expel the tapeworm Hymenolepis diminuta and are protected from Pediatric IBD represents 7–20% of the total disease burden
colitis by triggering a memory response with worm antigen. Am J (14) and is typically more severe than adult-onset IBD, with
Physiol Gastrointest Liver Physiol 314: G461–G470, 2018. First
affected children being more susceptible to disease-associated
published January 4, 2018; doi:10.1152/ajpgi.00295.2017.—Infection
with helminth parasites reduces the severity of concomitant inflam- complications (1). For example, lack of calorie/nutrition intake
matory disease in adult mice. There is an alarming increase of and/or absorption in pediatric IBD often results in reduced
inflammatory bowel disease (IBD) in children. It is important to growth and can be the cause of developmental delays (8).
determine whether helminth therapy would be of value in pediatric Therapeutic options largely mirror those applied to adult-onset
IBD and whether triggering immunological memory to the worm IBD with the same caveats (19), which can be exaggerated in
would be anticolitic. Three-week-old (young) and eight-week-old the pediatric population, including, for instance, corticosteroid-
(adult) Balb/c mice were infected with H. diminuta, and infectivity induced growth impairment and susceptibility to infection (14).
and T helper 2 (Th2) immunity were assessed. Other mice received H.
diminuta with or without a crude worm extract (HdE) 28 – 42 days
In addition, IBD will be a lifelong concern, so the need to
postinfection (dpi) with or without dinitrobenzene sulphonic acid develop better treatments, identify the cause(s) of IBD, and
[DNBS, 1.5 mg (young) or 3 mg (adults), ir], and colitis was assessed formulate cures for IBD is readily apparent.
72 h later. Infected young mice developed Th2 immunity and expelled H. Epidemiological studies juxtaposed to the hygiene hypoth-
diminuta; expulsion was delayed by ~2 days compared with adult mice. esis have led to the consideration that an absence of exposure
Colitis, as gauged by macroscopic disease and histopathology scores, was to helminth parasites could predispose an individual to develop
less severe in young mice infected 10 days, but not 8 days, before DNBS. autoinflammatory disease, and the corollary of this is that
Protection against DNBS-induced colitis was accompanied by an in- infection with parasitic helminths could be used to treat disease
creased capacity to make interleukin (IL)-4 and IL-10. Mice infected with
H. diminuta were not protected from DNBS-colitis when challenged 28
(31). There is considerable support for the latter from animal
days later; however, injection of these mice with HdE coincident with models of disease, especially colitis (21). Small trials per-
DNBS resulted in less disease and increased splenic IL-4 and IL-10. formed ~10 yr ago with ova of the parasitic whipworm of pigs,
Using a boost (500 ␮g HdE, 28 dpi) and repeat HdE (100 ␮g, 42 dpi) Trichuris suis, provided provocative data on the potential of
regimen with infected mice suppressed DNBS-colitis, as did adoptive helminth therapy in IBD (37); however, these studies used
transfer of splenic CD4⫹ T cells from infected mice with low-dose HdE adult patients, and indeed the analysis of a range of helminth
challenge. Should these data translate to IBD, then helminth therapy parasites in animal models of colitis have focused on adult (i.e.,
could be of value in pediatric-onset IBD, and defining the antigen(s) that 7 wk or older) mice (21).
elicit antihelminth immunological memory could serve as an anticolitic
approach in previously infected individuals. As is typical of infection with helminth parasites (12), mice
(a nonpermissive host) infected with the rat tapeworm, Hy-
NEW & NOTEWORTHY This study demonstrates that juvenile menolepis diminuta (H. diminuta), develop a T helper 2 (Th2)-
mice are protected from colitis by infection with the tapeworm
dominated immune response (23). Moreover, infection with
Hymenolepis diminuta and that using worm antigen to trigger an
immunological memory response in previously infected mice can be this parasite or systemic administration of a crude extract of the
used to limit the severity of colitis. worm protects adult mice from chemical-induced colitis (15,
29). Using this established model system, we tested the hy-
colitis; helminth; hymenolepis; memory; young mice pothesis that infection with H. diminuta will exert an anti-
inflammatory benefit in young mice. We asked three pertinent
questions. First, will young (3 wk old, immediately postwean-
INTRODUCTION ing) mice respond to infection and expel H. diminuta with the
same kinetics as adult mice? This is important because the
There has been a dramatic and alarming increase in the young mice may not have a fully developed immune system to
prevalence of inflammatory bowel disease (IBD, including appropriately deal with infections and, in areas where hel-
minth-infections are endemic, infection in childhood is com-
Address for reprint requests and other correspondence: D. M. McKay, Dept.
mon. Second, will young mice infected with H. diminuta be
of Physiology and Pharmacology, Univ. of Calgary, HSc 1877, 3330 Hospital protected from colitis induced by intrarectal instillation of
Dr. NW, Calgary, AL, Canada T2N4N1 (e-mail: dmckay@ucalgary.ca) dinitrobenzene sulphonic acid (DNBS)? Third, will immuno-
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G462 YOUNG MICE INFECTED WITH H. DIMINUTA

logical memory to H. diminuta develop in young mice that can A 5


young
be exploited in later life as an anticolitic therapy?

% Blood eosinophils
adult
Supporting the possibility that infection with a parasitic 4 #
helminth could be therapeutic in pediatric- or juvenile-onset * * *
IBD, the data herein show that 1) young mice effectively expel 3 # #
H. diminuta (albeit slightly slower than adult mice), 2) young 2
mice can be protected from DNBS-induced colitis by infection
with H. diminuta, and 3) triggering an immunological memory 1
response against the worm can reduce the severity of colitis.
0
uninfected
MATERIALS AND METHODS
days post-infection
Mice, helminth, and worm extract. This study was conducted in
accordance with the Canadian Guidelines for Animal Welfare as B

Goblet cells (per 20 VCU)


administered and approved by the University of Calgary Health 300 #
Science Animal Care Committee (protocol AC13-0015). *
Balb/c mice were bred at the University of Calgary and housed in
filter-topped cages in specific pathogen-free conditions, with free 200 *
access to food and water. Mice were used at 3 wk (young, just
100

A 5
0
Male uninfected
4 Female
days post-infection
Parasites / young mice

C
3
small intestine IgG1 (ng/mL)

30
*
2
20

1
10

0
0
uninfected
Days after infectio n
days post-infection
Fig. 2. Young mice mount a similar immune response to Hymenolepis
B 5 diminuta as adult mice. Young (3 wk old) and adult (8 wk old) Balb/c mice
were infected with H. diminuta by oral gavage with 5 cysticercoids, and
changes in blood eosinophils (n ⫽ 3–11) (A), mid-small intestine goblet cells
(n ⫽ 3–5) (B), and intestinal IgG1 (n ⫽ 3– 4) (C) were assessed (data are
4 means ⫾ SE). vcu, villus-crypt unit; *P ⬍ 0.05 compared with uninfected
Parasites / adult mice

young mice; #P ⬍ 0.05 compared with uninfected adult mice.

3
postweaning) (25) or 8 wk (adult) (15) of age. The H. diminuta
lifecycle was maintained by cyclical passage through flour beetles
2 (intermediate host) and rats (definitive hosts). As reported previously,
mice received five infective H. diminuta cysticercoids via oral gavage
in 100 ␮l of 0.9% NaCl (15).
1 H. diminuta was flushed from the small intestine of rats, homog-
enized in PBS, and centrifuged (2,450 g, 30 min, 4°C), and the soluble
component was retained and subjected to a second centrifugation (17).
0
Protein content was measured in the PBS-soluble H. diminuta extract
(HdE) by the Bradford assay, and aliquots were stored at ⫺80°C.
Three separate antigen preparations were used and were standardized
Days after infection by their ability to suppress TNF-␣ release from LPS-treated THP1-
Fig. 1. Young mice effectively expel Hymenolepis diminuta (H. diminuta).
macrophages; extracts that suppressed TNF-␣ production by ⱖ40%
Young (3 wk old) (A) and adult (8 wk old) (B) Balb/c mice were orally infected were used (17) (⬍100 pg LPS/1 mg HdE) (ToxinSensor Chromogenic
with 5 cysticercoids of H. diminuta, and at defined time points mice were LAL kit, Genscript), and trace amounts of bacterial 16S rRNA were
euthanized, the small intestine removed and flushed with PBS; the number of detected by Q-PCR. The HdE was administered by intraperitoneal
worms retrieved was recorded (n ⫽ 3–11). injection in 500 ␮l of sterile PBS.

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YOUNG MICE INFECTED WITH H. DIMINUTA G463
Table 1. Infection with H. diminuta increases the capacity To detect circulating H. diminuta-specific IgG, 8 mg protein/ml of
of splenocytes from young and adult mice to produce Th2- HdE was diluted in 0.1 M sodium carbonate buffer (pH 9.6), and 100
type cytokines ␮l were used to coat each well of flat-bottomed 96-well plates
overnight at 4°C. Serum from control and treated mice was diluted
IL-4 IL-5 IL-10 1:10 with PBS-1% BSA, added to the plates, and incubated overnight
at 4°C, at which point goat anti-mouse IgG conjugated to horseradish
Young mice
Uninfected 717 ⫾ 110 183 ⫾ 29 378 ⫾ 67 peroxidase (1:5,000; Santa Cruz Biotechnology) was added for 2 h
5 dpi 568 ⫾ 176 134 ⫾ 35 813 ⫾ 180 followed by tetramethylbenzidine; absorbance values were read at 405
8 dpi 2,231 ⫾ 109* 1,210 ⫾ 325* 1,332 ⫾ 189* nm. Data are presented as OD units minus the blank background well.
9 dpi 2,308 ⫾ 63* 1,682 ⫾ 173* 1,383 ⫾ 176* DNBS-induced colitis. Colitis was induced by intrarectal (3 cm
10 dpi 1,013 ⫾ 520 1,090 ⫾ 385* 1,573 ⫾ 343* from the anal verge) delivery of 1.5 mg (young mice) or 3 mg (adult
11 dpi 973 ⫾ 264 256 ⫾ 34 702 ⫾ 155 mice) of DNBS (ICN Biomedicals) in 100 ␮l 50% ethanol (pilot
35 dpi 191 ⫾ 28 220 ⫾ 78 748 ⫾ 24 studies revealed that 3 mg DNBS resulted in an unethically severe
Adult mice disease in young mice, and the dose was empirically adjusted to elicit
Uninfected 70 ⫾ 3 19 ⫾ 1 200 ⫾ 15 a severity of disease/colitis that resembled that in 3 mg DNBS-treated
5 dpi 564 ⫾ 142* 79 ⫾ 69 2,266 ⫾ 575* adult mice) (15). Young mice were infected with H. diminuta 8 or 10
8 dpi 372 ⫾ 48* ND 513 ⫾ 97
days before DNBS, and adults were infected 8 days before DNBS
9 dpi 608 ⫾ 191* 284 ⫾ 190 926 ⫾ 77*
10 dpi 1,787 ⫾ 156* 2,096 ⫾ 388* 2,728 ⫾ 437* (15). Controls consisted of age-matched uninfected mice exposed to
11 dpi 713 ⫾ 170* ND 398 ⫾ 50 DNBS only.
35 dpi 180 ⫾ 15 ND 253 ⫾ 45 Mice were examined daily for signs of ill health, wet/feces-stained
anal area, anal bleeding, altered behavior, weight change, and fur
Values are means ⫾ SE (in pg/ml); n ⫽ 4 (n ⫽ 3 for 35 dpi in adult mice); ruffling. Upon necropsy at 72 h post-DNBS, the colon was excised
dpi, days postinfection with 5 cysticercoids of Hymenolepis diminuta; ND, not
(ileal-cecal junction to the anus), measured, and examined for signs of
determined. *P ⬍ 0.05 compared with uninfected (ANOVA followed by
Dunnett’s test). loose stool, fluid accumulation, bleeding, or macroscopic ulceration.
A disease activity score (maximum ⫽ 5) was assigned based on 10%
loss of body weight (0 or 1); wet anus, soft stool, or empty colon
Assessment of worm infectivity and host response. Mice were (0 –1); anal bleeding/occult blood (0 or 1); macroscopic ulcers present
necropsied at 5, 8, 9, 10, 11, 12, and 35 days postinfection (dpi). The (0 or 1); early euthanasia attributable to severe disease (e.g., drop of
small intestine was removed, flushed with 5 ml of PBS (4°C), opened weight ⬎20% of original weight) was scored 5. H and E-stained
longitudinally, and gently rinsed, and retrieved worms were counted. sections of mid-colon on coded slides were examined for signs of
A blood drop was obtained from tail snips, and smears were stained histopathology [deranged architecture (0 –3), inflammatory cell infil-
with hematoxylin and eosin (H and E) for eosinophil enumeration in trate (0 –3), goblet cell depletion (0 or 1), edema (0 or 1), ulceration
a blinded fashion (40). Serum was collected from heavily anesthetized (0 –2), muscle thickening (0 or 1), or crypt abscess (0 or 1)] and scored
mice for detection of anti-H. diminuta immunoglobulin. on a 12-point scale, as before, in a blinded fashion (15).
An ~2-cm piece of mid-small intestine was excised, formalin fixed Memory response. Injections of HdE (1 mg ip) into naïve mice
(72 h), and paraffin embedded, and 5-␮m sections were collected on reduced the severity of DNBS-induced colitis and dextran sodium
coded slides and stained with periodic-acid Schiff’s (PAS) reagent to sulfate-induced colitis (17, 28). Mice develop immunological memory
identify goblet cells that were counted in 20 randomly selected to H. diminuta and rapidly expel secondary infections (23); thus we
villus-crypt units (VCU) using light microscopy (24). posited a therapeutic benefit for HdE when used in a memory
Spleens were excised and sterilely separated into single cell sus- protocol. Three-week-old mice were infected with 5 H. diminuta and
pensions. Five million splenocytes were left nonstimulated or treated 28 days later received HdE (100 or 500 ␮g in 500 ␮l PBS ip or boiled
with concanavalin A (conA, 2 ␮g/ml, 48 h) or HdE (500 ␮g/ml, 96 h), HdE). DNBS (3 mg ir) was given 5 h later, and colitis was assessed
and cytokine levels in the supernatant were determined by ELISA 72 h post-DNBS. The effects of a booster injection of HdE was tested.
(Duo-Set kits from R&D Systems) following the manufacturer’s Thus 3-wk-old mice were infected with H. diminuta, given HdE (500
instructions (28). ␮g ip) 28 days later, and at 42 dpi received HdE (100 ␮g ip) and
Determination of serum anti-H. diminuta IgG. Segments of mid- DNBS (3 mg ir, 5 h after HdE); colitis was assessed 72 h post-DNBS.
small intestine were excised, weighed, and homogenized in PBS All salient time-matched controls were included, naïve mice, DNBS-
containing 0.1% DMSO, 0.04% EDTA, 0.5% BSA, 0.05% Tween-20, only-treated mice, H. diminuta-only-infected mice (28 days before
and a protease inhibitor cocktail (Roche Diagnostics) in the ratio of 1 DNBS, no HdE), and HdE-only treatment of noninfected mice.
ml buffer/100 mg of tissue. Following centrifugation (10,000 g, 10 In a final experiment, mice (donors) were infected with 5 cystic-
min), total IgG1 was measured in the supernatant using the Mouse ercoids of H. diminuta and 28 days later received HdE (500 ␮g ip).
IgG-1 Ready-Set-Go! ELISA assay according to the manufacturer’s Two days later these mice were euthanized, the spleens were re-
instructions (eBioscience). moved, and CD4⫹ splenic T cells were purified magnetically by

Table 2. Three-week-old Balb/c mice infected with H. diminuta 8 days before DNBS are not protected from disease
Disease Activity Score Histopathology Score

Young Adult Young Adult

Control 0 ⫾ 0 (17) 0 ⫾ 0 (10) 0.5 ⫾ 0.2 (7) 0.5 ⫾ 0.3 (6)


H. diminuta 0 ⫾ 0 (6) ND 0.3 ⫾ 0.3 (3) ND
DNBS 3.9 ⫾ 0.1* (23) 3.7 ⫾ 0.3* (12) 9.4 ⫾ 0.8* (11) 11.0 ⫾ 0.4* (10)
H. diminuta ⫹ DNBS 3.5 ⫾ 0.2* (30) 2.1 ⫾ 0.3*† (12) 8.5 ⫾ 0.7* (14) 7.5 ⫾ 0.8*† (12)
Values are means ⫾ SE with n values in parentheses; mice were infected with 5 Hymenolepis diminuta cysticercoids by oral gavage and challenged with
dinitrobenzene sulphonic acid (DNBS) [1.5 mg (young) or 3 mg (adult) ir] 8 days later. Mice were assessed 3 days post-DNBS. *P ⬍ 0.05 compared with
age-matched control (Kruskal-Wallis tests with Dunn’s posttest). †P ⬍ 0.05 compared with DNBS-only (Kruskal-Wallis tests with Dunn’s posttest).

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G464 YOUNG MICE INFECTED WITH H. DIMINUTA

A B
5 12

Histopathology score
Disease activity score
9

3
Fig. 3. Young mice are protected from dinitro-
benzene sulphonic acid (DNBS)-induced colitis 6
by infection with Hymenolepis diminuta (H.
diminuta). 3-wk-old Balb/c mice were infected 2
with 5 cysticercoids of H. diminuta and 10 days
later received DNBS (1.5 mg ir). Mice were 3
necropsied 3 days post-DNBS when macro- 1
scopic disease activity score (A) and a histopa-
thology damage score (B) were calculated
based on criteria outlined in MATERIALS AND 0 0
METHODS. C: representative hematoxylin and control DNBS H. diminuta control DNBS H. diminuta
eosin images, where damage, edema, and an + DNBS + DNBS
inflammatory infiltrate are obvious in tissue
from the DNBS treatment (M, external muscle
layers; L, lumen of the colon; C, epithelial
C
crypts; scale bar ⫽ 100 ␮m). Data are means ⫾ M
SE; n ⫽ 6 –10. *P ⬍ 0.05 compared with M L
control; #P ⬍ 0.05 compared with DNBS. C
M

L
C L

control DNBS H. diminuta + DNBS

negative selection (Easysep mouse CD4⫹ T cell enrichment kit, Stem navalin-A stimulation of splenocytes was observed with cells
Cell Technologies). Ten million of these splenic CD4⫹ cells (a from young and adult mice (Table 1). Time-course analyses
population that should have H. diminuta effector and memory cells) revealed that the kinetics of the effector cell responses to
were injected intraperitoneally into naïve Balb/c mice (recipi- infection with H. diminuta were very similar between young
ents) with or without HdE challenge (100 ␮g ip) and then treated
immediately with DNBS (3 mg ir). Mice were assessed 72 h post-
and adult mice, with the peak response slightly delayed or
DNBS. reduced in magnitude in the young mice.
Data presentation and statistical analysis. Unless stated otherwise, Young mice infected with H. diminuta are protected from
data are presented as the means ⫾ SE. Statistical comparisons for two colitis induced by DNBS. As expected, mice treated with
groups was with Student’s t-test. Multiple group comparisons for DNBS showed significant loss of body weight, reduced activity
parametric data were performed using one-way ANOVA followed (often with a hunched posture), ruffling of the fur, and short-
by Tukey’s test (i.e., cytokine and IgG levels) or Kruskal-Wallis ening of the colon. Examination of H and E-stained sections
statistics followed by Dunn’s posttest for nonparametric data (i.e., revealed loss of crypt architecture, edema, significant inflam-
macroscopic disease and histopathology scores) using GraphPad matory cell infiltrate, and ulceration that ranged from punctate
Prism 5.0 (GraphPad Software), with P ⬍ 0.05 accepted as a and focal to, in severe cases, almost circumferential lesions
statistically significant difference.
with massive tissue destruction. Previous studies showed that
RESULTS adult mice infected with H. diminuta 8 days before intrarectal

Young mice reject H. diminuta and develop immunological


memory for the parasite. On the basis of the supposition that Table 3. Young mice infected with H. diminuta, challenged
3-wk-old mice would not have a fully developed immune 10 days later by DNBS and mitogen-activated splenocytes
system, we hypothesized that they would not expel H. diminuta assessed
or that rejection of the parasite would be significantly delayed
IL-4 IL-10
compared with adult mice (8-wk-old males). However, young
mice, male and female, effectively expelled a five-worm bur- Control 692 ⫾ 117(3) 1,007 ⫾ 181(3)
den with temporal characteristics that were only ~2 days DNBS 8 ⫾ 8 (3) 543 ⫾ 106(4)
H. diminuta ⫹ DNBS 359 ⫾ 82 (6) 1,108 ⫾ 337(6)
delayed compared with adult mice (Fig. 1). Paralleling expul-
sion of H. diminuta, the young mice (male and female data Values are means ⫾ SE (in pg/ml) with n values in parentheses; splenocytes
combined) mobilized antihelminth effector events that in- (5 ⫻ 106) were activated with concanavalin A (2 ␮g/ml) for 48 h. Data are
from 1 representative experiment; 2 separate experiments were performed.
cluded blood eosinophilia (Fig. 2A), intestinal goblet cell Splenocytes were isolated 72 h post- dinitrobenzene sulphonic acid (DNBS).
hyperplasia (Fig. 2B), and increased intestinal levels of IgG1 DNBS, at 1.5 mg ir; H. diminuta, 5 cysticercoids of Hymenolepis diminuta
(Fig. 2C). Similarly, Th2 polarization as assessed by conca- given to 3-wk-old mice by gavage 10 days before DNBS.

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YOUNG MICE INFECTED WITH H. DIMINUTA G465
instillation of DNBS develop significantly less severe colonic Triggering immunological memory against H. diminuta pro-
inflammation (15). Following an identical protocol, it was tects against DNBS-induced colitis. We have reported that
observed that 3-wk-old mice infected 8 days before DNBS had repeated injections of HdE (1 mg) into the peritoneum of mice
no amelioration of their disease (Table 2). However, on the significantly reduces DNBS- and DSS-induced colitis (28). To
basis of the 2-day delay in expulsion of a five-worm burden avoid this direct effect of HdE and focus on a memory
from young mice, the impact of infection with H. diminuta 10 response, mice were infected with H. diminuta and 28 days
days before DNBS treatment was tested and found to confer later were treated with HdE (100 or 500 ␮g ip) and 5 h later
significant protection from colitis, as determined by macro- with DNBS (ir). Colitis was assessed 72 h post-DNBS admin-
scopic and microscopic analysis of the colon (Fig. 3, A–C). istration.
This protection from the colitic effects of DNBS was asso- Mice infected 28 days before DNBS were not protected from
ciated with increased production of IL-4 and IL-10 from colitis; however, challenge of infected mice with 500 ␮g (ip),
mitogen-activated splenocytes from H. diminuta-positive but not 100 ␮g, of HdE on the day of DNBS administration
DNBS-treated mice compared with DNBS-only-treated resulted in significantly less severe colitis (Fig. 4A). Analysis
mice (Table 3). of conA-stimulated splenocytes from these mice showed in-

A native HdE
5 15
Histopathology score
Disease activity score

* *
4
* * *
* * 10 *
3 * # *
# *
2 *
5
1

0 0
- - + + + - - - - + + + - - H. diminuta
- + + + + + + - + + + + + + DNBS
- - - + - + - - - - + - + - HdE (100 μ g)
Fig. 4. Hymenolepis diminuta (H. diminuta)
- - - - + - + - - - - + - + HdE (500 μ g)
[n = 15 16 17 10 8 11 8] extract (HdE) in a memory protocol protects
againstdinitrobenzenesulphonicacid(DNBS)-
B #
induced colitis. Male 3-wk-old Balb/c mice
were infected with 5 cysticercoids of H.
1.0 *
4 #
* diminuta or left uninfected (naïve). 28 days
0.8 later naïve or previously infected mice were
3
IL-10 (ng/ml)

challenged with 100 or 500 ␮g of a PBS-


IL-4 (ng/ml)

0.6 soluble crude HdE by intraperitoneal injec-


#
2 tion followed 5 h later by DNBS (3 mg ir).
0.4
* #
Mice were necropsied 72 h post-DNBS, and
1
disease severity was assessed by macro-
0.2 scopic disease and histopathology scores
(A). Isolated splenocytes (5 ⫻ 106/ml) were
0 0 stimulated with concanavalin A (2 ␮g/ml),
- - + + + - - - - + + + - - H. diminuta and cytokines were measured 48 h later (B)
- + + + + + + - + + + + + + DNBS (n ⫽ 6 – 8). A final series of experiments
assessed the impact of boiling the HdE
- - - + - + - - - - + - + - HdE (100 μ g) (bHdE) on its ability to confer protection
- - - - + - + - - - - + - + HdE (500 μ g) against colitis (C) (n ⫽ 6 – 8). Data are
means ⫾ SE; *P ⬍ 0.05 compared with
control, #P ⬍ 0.05 compared with DNBS.
C boiled HdE
5 15
Histopathology score
Disease activity score

4 * *
* * * *
* 10 *
3 # # #
# * * *
2 *
5
1

0 0
- - + + + - - - - + + + - - H. diminuta
- + + + + + + - + + + + + + DNBS
- - - + - + - - - - + - + - HdE (500 μ g)
- - - - + - + - - - - + - + bHdE (500 μ g)

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G466 YOUNG MICE INFECTED WITH H. DIMINUTA

creased production of IL-4 and IL-10 (Fig. 4B). In separate testine, and reduced maturation of CD4⫹ T cell receptor ␣/␤-T
experiments, protein in the HdE was denatured by boiling, and cells have been reported in juvenile mice (27, 39). Further-
this preparation was as effective as native HdE in suppressing more, early-life events (e.g., infection, antibiotic treatment, and
DNBS-induced colitis in mice infected 28 days earlier with H. toxin exposure) can have long-lived consequences for the
diminuta (Fig. 4C). Moreover, stimulation of splenocytes in individual, often increasing susceptibility to inflammatory dis-
vitro with HdE (100 ␮g/ml) supported the induction of mem- ease (3, 20, 26, 30). In addition, juveniles can display more
ory in the infected mice (Table 4). severe disease than adults (2, 13, 34), which may reflect a
Noninfected mice cotreated with HdE (100 or 500 ␮g ip.) degree of immaturity in their immune system, although this
and DNBS displayed, on average, subtly less disease and may be offset by a greater recuperative capacity in young
colonic histopathology, but these findings did not reach statis- compared with aged individuals (32).
tical significance (Fig. 4A). In parallel, mitogen stimulation of Age of the host at the time of infection can be an important
splenocytes from these mice resulted in IL-4 and IL-10 levels factor in the outcome of the response to infection with helminth
that were not statistically different from control mice (Fig. 4B). parasites. For instance, young Rhesus monkeys infected with the
The impact of repeated systemic HdE treatment on the trematode Schistosoma mansoni harbor a greater number of par-
outcome of DNBS was tested. As shown in Fig. 5A, mice were asites concomitant with reduced Th2 immunity compared with
infected with H. diminuta, challenged with HdE 28 days later adult monkeys (9). We speculated that absence of a fully mature
(500 ␮g ip) and then again 2 wk later (100 ␮g HdE ip), at immune system would render 3-wk-old mice more vulnerable to
which time they were given DNBS (5 h after the last HdE H. diminuta. Contrarily, we find that young mice expel H.
treatment). Immunological memory was confirmed in infected diminuta (although delayed by ~2 days compared with adults) and
mice challenged with HdE by serum antiworm IgG (Fig. 5B). mount Th2 cytokine and effector cell (i.e., eosinophils and goblet
Mice infected with H. diminuta and challenged with either cells) responses that are only slightly delayed or marginally
dose of HdE displayed less severe DNBS-induced colitis (Fig. reduced compared with adults. Intriguingly, similar events were
5C); treatment with two doses of HdE resulted in the greatest observed when juvenile and adult mice were infected with the
diminution of colonic histopathology (Fig. 5C, right). Simi- nematode Nippostrongylus brasilienses; worm expulsion was de-
larly, conA-stimulated splenocyte production of IL-4 and layed by ~2 days in the juveniles, correlating with activation of
IL-10 was greatest by cells from infected mice challenged with Th2 immunity (25). In that study, adoptive transfer of basophils to
worm antigen compared with those from naïve mice or those juveniles resulted in a recapitulation of the kinetics of the adult
treated with DNBS only (Fig. 5D). mouse response to infection with N. brasiliensis. Where helminth
Transfer of CD4⫹ splenic T cells from H. diminuta-infected parasites are endemic, childhood exposure to infective larvae/eggs
mice to naïve recipients (Fig. 6A) resulted in a degree of is frequent and hence the likely evolutionary pressure for the
protection from DNBS-induced colitis that was most signifi- immune system to rapidly develop to counter these metazoan
cant when the recipient mice were simultaneously treated with parasites in childhood, albeit possibly with slightly less efficacy
HdE (100 ␮g ip) (Fig. 6, B–E). One of five mice, while than in adults.
showing moderate macroscopic signs of disease, had signifi- The ability of juvenile mice to expel H. diminuta suggested
cant histopathology (11 on the 12-point scale), and, because of that, like adults (15), they could be protected from colitis by
this and the nonparametric data, the benefit of T-cell transfer helminth therapy (noting disease can be exaggerated by infec-
plus HdE failed to reach a statistically significant difference tion with helminths) (36, 40). Coinciding with worm expulsion
compared with DNBS-only-treated mice (Fig. 6E). and the development of Th2 immunity, mice infected with H.
diminuta 10 days before DNBS treatment displayed signifi-
DISCUSSION cantly less severe inflammation. While not tested, it is reason-
Development of the immune system occurs in utero and able to assume that the mechanism of protection mirrors that in
postnatally with colonization by microbiota and exposure to adults (15, 16), especially given that worm expulsion was
food antigens (11, 27). Shortly, 1-2 wk, after birth many delayed by 2 days in the juvenile mice and that infection 10
aspects of the immune system resemble that in the adult, but days, but not 8 days, before DNBS inhibited the inflammation
development/maturation continues postweaning; juveniles dis- (adult mice are protected from DNBS 8 dpi). In addition, the
play specific deficiencies in immunity (38). For example, increased splenocyte synthesis of IL-4 and IL-10 observed in
reduced numbers of IgA-producing plasma cells, limited num- young mice infected with H. diminuta occurs in infected adult
bers of antimicrobial-producing Paneth cells in the small in- mice, where both have been implicated in the amelioration of
DNBS-induced colitis (15, 22).
These findings complement epidemiological case study data
Table 4. Splenocytes from H. diminuta-infected mice show from South Africa, where childhood exposure to helminth
increased responses to in vitro HdE challenge parasites correlated with protection from IBD (5). Others have
Control DNBS H. diminuta H. diminuta ⫹ Boiled HdE Boiled HdE
shown that infection with H. diminuta can prevent cognitive
dysfunction in later life (41). Placing helminths in the context
IL-4 ND ND 75 ⫾ 34 626 ⫾ 88 120 ⫾ 41 of the hygiene hypothesis (7, 35), we suggest that infection in
IL-10 ND ND 142 ⫾ 42 1,708 ⫾ 332 782 ⫾ 90
n 3 3 7 7 7 childhood and adulthood contributes to the reduced incidence
of inflammatory disease, including IBD, in areas where para-
Values are means ⫾ SE (in pg/ml). Splenocytes (5 ⫻ 106) were isolated sitic helminths are endemic (18). Indeed, Stiemsma and col-
from treated mice and activated with H. diminuta extract (HdE, 100 ␮g/ml) for
96 h. DNBS, dinitrobenzene sulphonic acid (3 mg ir); H. diminuta, 5 cystic- leagues (33) commented that therapeutics aimed at decreasing
ercoids of Hymenolepis diminuta; boiled HdE, HdE (500 ␮g ip) alone or 28 the prevalence of autoinflammatory disorders will likely “in-
days after H. diminuta infection. volve personalized helminth treatments used early in life.”

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YOUNG MICE INFECTED WITH H. DIMINUTA G467

B
1.5
IgG (Δ OD normalized
to non-infected mice)

1.0

0.5

0
- - + + + H diminuta
- + + + + DNBS
- - - + - HdE (500 μ g; d28)
- - + - - HdE (100 μ g, d42)
- - - - + HdE (500 & 100 μg)
C
5 15
Histopathology score
Disease activity score

4
10
3

2
5
1

0 0
- - + + + - - + + + H diminuta
- + + + + - + + + + DNBS
- - - + - - - - + - HdE (500 μg; d28)
- - + - - - - + - - HdE (100 μg, d42)
- - - - + - - - - + HdE (500 & 100 μg)
D 2.5 5

2.0 4
IL-10 (ng/ml)
IL-4 (ng/ml)

1.5 3

1.0 2

0.5 1

0 0
- - + + + - - + + + H diminuta
- + + + + - + + + + DNBS
- - - + - - - - + - HdE (500 μ g; d28)
- - + - - - - + - - HdE (100 μ g, d42)
- - - - + - - - - + HdE (500 & 100 μ g)
Fig. 5. Repeated worm antigen treatment protects Hymenolepis diminuta (H. diminuta)-infected mice from dinitrobenzene sulphonic acid (DBNS)-induced colitis.
Male 3-wk-old Balb/c mice were infected with 5 cysticercoids of H. diminuta or left uninfected (naïve), followed by intraperitoneal injection of a PBS-soluble
crude extract of H. diminuta (HdE) at the dose, interval, and frequency depicted in A. 5 h after HdE injection on day 42 postinfection all mice received DNBS
(3 mg ir), followed by necropsy 72 h later. B: level of serum anti-H. diminuta IgG at the time of necropsy (n ⫽ 3–5). Colitis was assessed by macroscopic disease,
and histopathology scores (C) and isolated splenocytes (5 ⫻ 106/ml) were stimulated with concanavalin A (2 ␮g/ml). Cytokines were measured 48 h later (D)
(n ⫽ 6 – 8). Data are means ⫾ SE; n ⫽ 6 –9. *P ⬍ 0.05 compared with control, #P ⬍ 0.05 compared with DNBS, and †P ⬍ 0.05 compared with DNBS ⫹ H.
diminuta.

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G468 YOUNG MICE INFECTED WITH H. DIMINUTA

A day
0
donor mice 28 30

500 μ g isolate splenic


5 H.d
HdE ip. CD4+ T cells

30 recipient mice 33

107 CD4+ DNBS necropsy


±100 μ g HdE ip. (3 mg, ir.)

Fig. 6. Adoptive transfer of splenic CD4⫹ T B 5


C 12
cells from Hymenolepis diminuta (H. diminuta)-
infected mice protects against dinitrobenzene 10 * # *#

Colon length (cm)


sulphonic acid (DNBS)-induced colitis. Do- % weight change 0
nor Balb/c mice were infected with 5 cystic- 8
ercoids of H. diminuta by oral gavage and 28 -5 *
days later received an intraperitoneal injection 6
of H. diminuta extract (HdE) (500 ␮g) (A). 2 -10 *#
days later the mice were humanely eutha- 4
nized, CD4⫹ T cells were purified from the -15
spleen, and 107 cells injected intraperitoneally * 2
into naïve mice ⫾ 100 ␮g HdE, immediately *
followed by DNBS (3 mg ir). Mice were -20 0
weighed daily (B), and, on necropsy at 3 days con DNBS DNBS DNBS, con DNBS DNBS DNBS,
post-DNBS, colon length was measured (C), a +T cells T cells +T cells T cells
macroscopic disease score (DAS) calculated + HdE ip. + HdE ip.
(D), and, after tissue processing, a histopa-
thology score calculated from hematoxylin D E 15
and eosin-stained sections on coded slides 5
(E). Data are means ⫾ SE; n ⫽ 5. *P ⬍ 0.05

Histopathology score
compared with control, #P ⬍ 0.05 compared
*
4 *
with DNBS only.
10
DAS

2
5
1

0 0
con DNBS DNBS DNBS, con DNBS DNBS DNBS,
+T cells T cells +T cells T cells
+ HdE ip. + HdE ip.

Mice develop immunological memory to H. diminuta and anticolitic effect and that triggering immunological memory
rapidly expel a secondary infection (23), raising the possibility with worm antigen can be of therapeutic benefit. Thus we add
that a worm-antigen-triggered memory response could be an- a fourth possibility to the three-pronged approach to helminth
ticolitic. The conundrum is that like molecules from other therapy (21), that of triggering immunological memory in
parasites that display immunosuppressive or immunoregula- previously infected individuals (an infection that might be
tory activity (10), a crude extract of H. diminuta (⬎500 ␮g/ml) prescribed in childhood) (33). We note the caveats that the
is anticolitic in naïve adult mice (28). Serum antiworm IgG nature of the antigen(s) has not been determined and that
levels and HdE-evoked cytokine production from splenocytes development of antibodies against the helminth antigen with
confirmed infection with H. diminuta and the development of repeated HdE delivery could limit the effectiveness of this
immunological memory to the worm. Previously infected, but anticolitic therapy.
not naïve, mice were significantly protected from DNBS- In conclusion, we provide some of the first proof-of-concept
induced colitis by low-dose HdE as a single treatment or in a data in support of the potential of developing helminth therapy
boost and rechallenge regimen. As with the protective effect to prevent or treat inflammatory disease in children and that a
observed in young mice infected with H. diminuta, the anti- history of infection opens the possibility of using immunolog-
colitic effect afforded by HdE challenge of previously infected ical memory against the helminth to treat inflammation.
mice was accompanied by an increased capacity of splenocytes
GRANTS
to produce IL-4 and IL-10.
Additionally, adoptive transfer of CD4⫹ splenic T cells from This work was supported by a Natural Sciences and Engineering Research
mice infected with H. diminuta 30 days previously resulted in Council (NSERC) of Canada Discovery Grant and a Crohn’s Colitis Canada
Grant-in-Aid (D.M. McKay), by Yamanashi Prefecture Fund (Japan) (T. Arai),
less severe DNBS-induced colitis, especially in the context of postdoctoral fellowships from the Canadian Institutes for Health Research
a concomitant low-dose HdE challenge; data show that T cells (CIHR)/Canadian Association of Gastroenterology (CAG)/Allergan and the
from H. diminuta-infected mice are capable of exerting an Alberta Innovates-Health solutions (AI-HS) (F. Lopes), studentships from

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Copyright © 2018 American Physiological Society. All rights reserved.
YOUNG MICE INFECTED WITH H. DIMINUTA G469
Beverly Phillips Award though the Snyder Institute and from the NSERC 13. Hamada K, Goldsmith CA, Kobzik L, Hamada K, Goldsmith
CREATE Host-Parasite Interactions (HPI) program grant at the University of CA. Increased airway hyperresponsiveness and inflammation in a
Calgary (A. Shute), and a Canada Research Chair (CRC: Tier 1) in Intestinal juvenile mouse model of asthma exposed to air-pollutant aerosol [Part
Immunophysiology in Health and Disease (D.M. McKay). A]. J Toxicol Environ Health A 58: 129 –143, 1999. doi:10.1080/
009841099157340.
DISCLOSURES 14. Heuschkel R. Inflammatory bowel disease in children. Clin Med (Lond) 8:
297–298, 2008. doi:10.7861/clinmedicine.8-3-297.
No conflicts of interest, financial or otherwise, are declared by the authors. 15. Hunter MM, Wang A, Hirota CL, McKay DM. Neutralizing anti-IL-10
antibody blocks the protective effect of tapeworm infection in a murine
AUTHOR CONTRIBUTIONS model of chemically induced colitis. J Immunol 174: 7368 –7375, 2005.
T.A., F.L., and D.M.M. conceived and designed research; T.A., F.L., A.S., doi:10.4049/jimmunol.174.11.7368.
and A.W. performed experiments; T.A., F.L., A.S., A.W., and D.M.M. ana- 16. Hunter MM, Wang A, Parhar KS, Johnston MJ, Van Rooijen N, Beck
lyzed data; T.A., F.L., and D.M.M. interpreted results of experiments; T.A. and PL, McKay DM. In vitro-derived alternatively activated macrophages
D.M.M. prepared figures; T.A. and D.M.M. drafted manuscript; T.A., F.L., reduce colonic inflammation in mice. Gastroenterology 138: 1395–1405,
A.S., A.W., and D.M.M. edited and revised manuscript; T.A., F.L., A.W., and 2010. doi:10.1053/j.gastro.2009.12.041.
D.M.M. approved final version of manuscript. 17. Johnston MJ, Wang A, Catarino ME, Ball L, Phan VC, MacDonald
JA, McKay DM. Extracts of the rat tapeworm, Hymenolepis diminuta,
suppress macrophage activation in vitro and alleviate chemically induced
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