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Infection with Hymenolepis diminuta Blocks Colitis and Hastens Recovery While Colitis Has Minimal Impact on Expulsion of the Cestode from the Mouse Host

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Two experimental paradigms were adopted to explore host–helminth interactions involved in the regulation of colitis and to understand if colitis affects the outcome of helminth infection. First, male BALB/c mice infected with H. diminuta were challenged 4 days later with dinitrobenzene sulphonic acid (DNBS) and necropsied 3 days later. Second, mice were infected with H. diminuta 3 days after DNBS treatment and necropsied 11 or 14 days post-DNBS. Mice were assessed for colitic disease severity and infectivity with H. diminuta upon necropsy. Supporting the concept of helminth therapy, mice are protected from DNBS–colitis when infected with H. diminuta only 4 days previously, along with parallel increases in splenic production of Th2 cytokines. In the treatment regimen, H. diminuta infection produced a subtle, statistically significant, enhanced recovery from DNBS. Mice regained body weight quicker, had normalized colon lengths, and showed no overt signs of disease, in comparison to the DNBS-only mice, some of which displayed signs of mild disease at 14 days post-DNBS. Unexpectedly, colitis did not affect the hosts’ anti-worm response. The impact of inflammatory disease on helminth infection is deserving of study in a variety of models as auto-inflammatory diseases emerge in world regions where parasitic helminths are endemic.
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pathogens
Article
Infection with Hymenolepis diminuta Blocks Colitis and
Hastens Recovery While Colitis Has Minimal Impact on
Expulsion of the Cestode from the Mouse Host
Shuhua Li 1,2 , Sruthi Rajeev 1,2, Arthur Wang 1,2 and Derek M. McKay 1,2, *


Citation: Li, S.; Rajeev, S.; Wang, A.;
McKay, D.M. Infection with
Hymenolepis diminuta Blocks Colitis
and Hastens Recovery While Colitis
Has Minimal Impact on Expulsion of
the Cestode from the Mouse Host.
Pathogens 2021,10, 994. https://
doi.org/10.3390/pathogens10080994
Academic Editor: Helena Helmby
Received: 5 July 2021
Accepted: 2 August 2021
Published: 6 August 2021
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Copyright: © 2021 by the authors.
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This article is an open access article
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Gastrointestinal Research Group, Inflammation Research Network and Host-Parasite Interaction Group,
Calvin, Department of Physiology and Pharmacology, Phoebe & Joan Snyder Institute for Chronic Diseases,
Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4A1, Canada;
shuhua.li@ucalgary.ca (S.L.); sruthi.rajeev1@ucalgary.ca (S.R.); fhawang@ucalgary.ca (A.W.)
2
Inflammation Research Network and Host-Parasite Interaction Group, Calvin, Department of Physiology and
Pharmacology, Phoebe & Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine,
University of Calgary, Calgary, AB T2N 4A1, Canada
*Correspondence: dmckay@ucalgary.ca; Tel.: +1-403-220-7362
Abstract:
Two experimental paradigms were adopted to explore host–helminth interactions involved
in the regulation of colitis and to understand if colitis affects the outcome of helminth infection.
First, male BALB/c mice infected with H. diminuta were challenged 4 days later with dinitrobenzene
sulphonic acid (DNBS) and necropsied 3 days later. Second, mice were infected with H. diminuta
3 days after DNBS treatment and necropsied 11 or 14 days post-DNBS. Mice were assessed for
colitic disease severity and infectivity with H. diminuta upon necropsy. Supporting the concept of
helminth therapy, mice are protected from DNBS–colitis when infected with H. diminuta only 4 days
previously, along with parallel increases in splenic production of Th2 cytokines. In the treatment
regimen, H. diminuta infection produced a subtle, statistically significant, enhanced recovery from
DNBS. Mice regained body weight quicker, had normalized colon lengths, and showed no overt
signs of disease, in comparison to the DNBS-only mice, some of which displayed signs of mild
disease at 14 days post-DNBS. Unexpectedly, colitis did not affect the hosts’ anti-worm response. The
impact of inflammatory disease on helminth infection is deserving of study in a variety of models as
auto-inflammatory diseases emerge in world regions where parasitic helminths are endemic.
Keywords:
colitis; helminth therapy; cestode immunomodulation; DNBS colitis therapeutic; Th2
cytokines
1. Introduction
The increased prevalence of auto-inflammatory conditions, such as diabetes, arthritis,
multiple sclerosis, and inflammatory bowel disease (IBD), coupled with a lack of cures for
these conditions underscores the need for innovative approaches to manage idiopathic
disease [
1
]. Epidemiological studies demonstrating an inverse relationship between the
global distribution of endemic parasitic helminths and regions of high incidence of auto-
inflammatory disease, suggest the possibility of novel helminth-based therapy for IBD
and other allergic autoimmune disorders [
2
]. Indeed, use of animal models of disease,
especially colitis, has repeatedly shown that infection with a variety of parasitic helminths
can reduce the severity of inflammatory disease [37].
The rat tapeworm, Hymenolepis diminuta, is an intriguing candidate as a ‘therapeutic
helminth’. Infection is by ingestion and the worm does not migrate through the host;
rather, it seeks to establish in the small intestine. Bearing no teeth or hooks, it does no
obvious abrasive damage to the host. It is not auto-infective and its life-cycle requires an
invertebrate host, so there is no direct person-to-person spread. Natural infection is rare in
humans, typically restricted to malnourished or immunocompromised individuals and can
Pathogens 2021,10, 994. https://doi.org/10.3390/pathogens10080994 https://www.mdpi.com/journal/pathogens
Pathogens 2021,10, 994 2 of 14
be treated with anthelmintics [
8
]. Studies with H. diminuta in the dextran sodium sulphate
(DSS) [
9
] and di-nitrobenzene sulphonic acid (DNBS) [
3
] murine models of colitis were
among the first to provide proof-of-concept data in support of helminth therapy to reduce
the severity of colonic inflammation and concomitant signs of disease. While ongoing
studies have confirmed the anti-colitic effect in the DNBS-model and revealed some of the
nuances of the helminth–host interaction [
10
12
], two major questions remain unaddressed:
both germane to helminth therapy: First, what is the temporal window of opportunity for
H. diminuta to suppress colitis? Elucidation of the kinetics of infection in the context of
inflammation can provide insight into host–parasite interaction and potentially reveal new
targets for therapeutic intervention. Second, does intestinal inflammation affect the immune
response to the helminth? This is an important, yet largely ignored issue. For instance, as
the incidence of IBD increases in under-developed countries [
13
], will this counter helminth
infection or render the population more vulnerable to parasitic helminths?
Using an acute and spontaneously resolving colitis model, we address these two
questions. The results herein show that mice challenged with DNBS four days after
infection with H. diminuta developed less severe colitis. Reciprocally, DNBS–colitis had
negligible effects on the outcome of infection; mice still mobilized a Th2 response and
expelled a five-worm burden by 11 days post-infection (dpi). These data are consistent
with the speculation that the emergence of IBD in under-developed nations, as they adopt
a ‘westernized lifestyle’, could be slowed by the presence of parasitic helminths, and that
the immune response against these ‘old friends’ [14] may not be deleteriously affected.
2. Results
2.1. H. diminuta Infection Four Days Prior to DNBS Suppresses Colitis
Previous studies on the impact of infection with H. diminuta on DNBS-induced colitis
have focused on 8-dpi based on the rationale that at this time point anti-worm immune
responses have been mobilized (e.g., upregulation of Th2 cytokines) and a bystander effect
of this would be modulation of colitis [
3
,
15
]. Mice that received H. diminuta 4 days prior
to DNBS (Figure 1A) displayed less colitis upon necropsy at 72 h post-DNBS (i.e., 7-dpi
with H. diminuta) (successful worm infection, current or recently expelled, was confirmed
by blood eosinophilia (Figure 1B)). Typical of this model, mice challenged with DNBS
displayed decreased activity and ruffled fur and disease in 6 of 13 mice (i.e., 46%) was
so severe it required humane euthanization, whereas H. diminuta + DNBS-treated mice
appeared healthier with only 2 of 14 mice (14%) reaching a disease severity pre-determined
end-point requiring euthanasia. The drop in body weight and shortening of the colon
caused by DNBS were significantly less in H. diminuta-infected mice compared to DNBS-
only treated mice (Figure 1C,D). The average disease activity score was reduced by ~50%
in the
H. diminuta + DNBS
group, although this was not statistically significant compared
to the DNBS-only group, possibly due to inclusion of mice that required euthanization
in this overall assessment of disease (Figure 1E). Inspection of colon sections revealed
loss of crypt architecture, significant inflammatory cellular infiltration, edema, ulcera-
tion, and circumferential tissue damage in the colon of mice given DNBS. While 7 of
12 H. diminuta + DNBS
-treated mice had mild histopathology, 2 mice in the group had
major colonic ulceration, resulting in a lack of a statistically significant improvement in
histopathology in this group as compared to the DNBS-only group (Figure 1F,G). While
we think it unlikely since H. diminuta seeks to lodge in the small intestine and DNBS is
given intra-rectally (i.e., limited chance of direct contact), the possibility that the stunted
H. diminuta or enteric changes evoked in response to the infection affects the haptenizing
ability of DNBS has not been excluded.
Pathogens 2021,10, 994 3 of 14
1
Figure 1.
Infection with H. diminuta four days prior to DNBS alleviates the severity of colitis. Male BALB/c mice were
gavaged with 5 cysticercoids of H. diminuta, challenged with DNBS (3 mg, ir.) 4 days later and necropsied at 3 days
post-DNBS (
A
), when blood eosinophilia confirmed helminth infection (
B
). Disease was assessed by body weight (
C
).
(
i
), daily change post-DNBS; (
ii
), final change, colon length (
D
), disease activity score (
E
), and histopathology scores (
F
)
(representative H & E images shown in (
G
)) (data are mean
±
SEM; n= 7–12 mice pooled from 3 experiments; parametric
data analyzed by one-way ANOVA and Tukey’s multiple comparisons test and non-parametric data analyzed by a Kruskal–
Wallis test with Dunn’s post-test; * and #, p< 0.05 compared to control and DNBS only, respectively; H. d,H. diminuta; panel
G, sections (M: muscle; L: gut lumen; U: ulcer; arrow, inflammatory infiltrate)).
Pathogens 2021,10, 994 4 of 14
Using mitogen-stimulated splenocytes as a marker of systemic immunity revealed
increased IL-4, IL-5, and IL-10 production by cells from H. diminuta-infected
mice ±DNBS
(Figure 2A–C). These data confirmed successful infection with the helminth and cor-
roborated earlier studies that implicated IL-10 in the anti-colitic effect [
3
]. In contrast,
splenocytes from DNBS-treated mice had reduced IL-10 output following challenge with
concanavalin-A (Figure 2C). Production of the pro-inflammatory cytokine, TNF
α
, was
not different between the groups (Figure 2D). It is important to supplement these data
with a consideration of local (i.e., mesenteric lymph node, lamina propria lymphocytes)
immunity in this model paradigm as well as an assessment of immunoregulatory factors
(e.g., argianse1+regulatory macrophages, Foxp3+regulatory T cells).
Pathogens 2021, 10, x FOR PEER REVIEW 4 of 14
Using mitogen-stimulated splenocytes as a marker of systemic immunity revealed
increased IL-4, IL-5, and IL-10 production by cells from H. diminuta-infected mice ± DNBS
(Figure 2A–C). These data confirmed successful infection with the helminth and corrobo-
rated earlier studies that implicated IL-10 in the anti-colitic effect [3]. In contrast, spleno-
cytes from DNBS-treated mice had reduced IL-10 output following challenge with conca-
navalin-A (Figure 2C). Production of the pro-inflammatory cytokine, TNFα, was not dif-
ferent between the groups (Figure 2D). It is important to supplement these data with a
consideration of local (i.e., mesenteric lymph node, lamina propria lymphocytes) immun-
ity in this model paradigm as well as an assessment of immunoregulatory factors (e.g.,
argianse1+ regulatory macrophages, Foxp3+ regulatory T cells).
Figure 2. Infection with H. diminuta evokes increased splenic production of Th2 cytokines. Male BALB/c mice were ga-
vaged with 5 cysticercoids of H. diminuta, challenged with DNBS (3 mg, ir.) 4 days later and on necropsy 3 days later (see
Figure 1A), splenocytes were isolated and stimulated with concanavalin-A (2 µg/5 × 106 splenocytes; 48 h) and IL-4 (A),
IL-5 (B), IL-10 (C), and TNFα (D) measured by ELISA (data are mean ± SEM; n = 7–12 mice pooled from 3 experiments;
one-way ANOVA and Tukey’s multiple comparisons test; * and #, p < 0.05 compared to control and DNBS only, respec-
tively; H. d, H. diminuta).
2.2. DNBS–Colitis Prior to H. diminuta Infection Subtly Affected Worm Expulsion
Few studies have examined the effects of colitis on the kinetics of infection or expul-
sion of a helminth parasite. Thus, mice were treated with DNBS, infected with H. diminuta
72 h later and assessed 8- and 11-dpi (Figure 3A). Prior treatment with DNBS had a neg-
ligible effect on rejection of H. diminuta, with one exception: at 8-dpi, 6 of 15 mice (40%)
retained 5 worms (i.e., the full infective burden, although small and stunted (Figure 3B)),
and this was not observed in the H. diminuta-only group (0/15 mice) (Figure 3B). There
Figure 2.
Infection with H. diminuta evokes increased splenic production of Th2 cytokines. Male BALB/c mice were
gavaged with 5 cysticercoids of H. diminuta, challenged with DNBS (3 mg, ir.) 4 days later and on necropsy 3 days later (see
Figure 1A), splenocytes were isolated and stimulated with concanavalin-A (2
µ
g/5
×
10
6
splenocytes; 48 h) and IL-4 (
A
),
IL-5 (
B
), IL-10 (
C
), and TNF
α
(
D
) measured by ELISA (data are mean
±
SEM; n= 7–12 mice pooled from 3 experiments;
one-way ANOVA and Tukey’s multiple comparisons test; * and #, p< 0.05 compared to control and DNBS only, respectively;
H. d,H. diminuta).
2.2. DNBS–Colitis Prior to H. diminuta Infection Subtly Affected Worm Expulsion
Few studies have examined the effects of colitis on the kinetics of infection or expulsion
of a helminth parasite. Thus, mice were treated with DNBS, infected with H. diminuta 72 h
later and assessed 8- and 11-dpi (Figure 3A). Prior treatment with DNBS had a negligible
effect on rejection of H. diminuta, with one exception: at 8-dpi, 6 of 15 mice (40%) retained
5 worms (i.e., the full infective burden, although small and stunted (Figure 3B)), and this
Pathogens 2021,10, 994 5 of 14
was not observed in the H. diminuta-only group (0/15 mice) (Figure 3B). There was no
significant difference in average length of the worms retrieved between the two groups of
mice (Figure 3C). By 11-dpi, as is typical of immunocompetent mice [
16
], the small intestine
of DNBS + H. diminuta treated mice harbored no worms (Figure 3B).
Pathogens 2021, 10, x FOR PEER REVIEW 5 of 14
was no significant difference in average length of the worms retrieved between the two
groups of mice (Figure 3C). By 11-dpi, as is typical of immunocompetent mice [16], the
small intestine of DNBS + H. diminuta treated mice harbored no worms (Figure 3B).
Figure 3. DNBS–colitis subtly affects expulsion of H. diminuta from mice. Male BALB/c mice were
treated with DNBS (3 mg, ir.) 3 days before infection with 5 cysticercoids of H. diminuta, and nec-
ropsied 8 or 11 days post-infection (dpi) (A). Panel (B) shows worm recovery at 8- and 11-dpi and
panel (C) the length of the worms recovered in intestinal washings at 8-dpi (data are mean ± SEM;
n = 15 mice at 8-dpi (pooled from 3 experiments) and n = 5 mice at 11-dpi from 1 experiment; H. d,
H. diminuta).
From a Th2 immunological perspective, a 72-h pre-treatment with intra-rectal DNBS
had minimal impact on the systemic murine response to H. diminuta. Blood eosinophils
and jejunal goblet cell numbers were increased at 8- and 11-dpi in both H. diminuta and H.
diminuta + DNBS-treated mice (Figure 4A–C). However, variation in cell counts in this
experiment meant that control mice and 8-dpi H. diminuta-mice were not statistically dif-
ferent in blood eosinophil numbers; a finding that underscores the importance of includ-
ing time-matched controls in murine experiments. Spleen cells from H. diminuta-infected
Figure 3.
DNBS–colitis subtly affects expulsion of H. diminuta from mice. Male BALB/c mice
were treated with DNBS (3 mg, ir.) 3 days before infection with 5 cysticercoids of H. diminuta, and
necropsied 8 or 11 days post-infection (dpi) (
A
). Panel (
B
) shows worm recovery at 8- and 11-dpi and
panel (
C
) the length of the worms recovered in intestinal washings at 8-dpi (data are mean
±
SEM;
n= 15 mice
at 8-dpi (pooled from 3 experiments) and n= 5 mice at 11-dpi from 1 experiment; H. d,
H. diminuta).
From a Th2 immunological perspective, a 72-h pre-treatment with intra-rectal DNBS
had minimal impact on the systemic murine response to H. diminuta. Blood eosinophils
and jejunal goblet cell numbers were increased at 8- and 11-dpi in both H. diminuta and
H. diminuta + DNBS-treated mice (Figure 4A–C). However, variation in cell counts in this
experiment meant that control mice and 8-dpi H. diminuta-mice were not statistically differ-
Pathogens 2021,10, 994 6 of 14
ent in blood eosinophil numbers; a finding that underscores the importance of including
time-matched controls in murine experiments. Spleen cells from H. diminuta-infected mice
(8- and 11-dpi) or DNBS + H. diminuta treated mice challenged with Con-A produced
increased amounts of IL-4, IL-5, IL-10, and IL-13 compared to splenocytes from control
mice or those given DNBS-only (Figure 5).
Pathogens 2021, 10, x FOR PEER REVIEW 6 of 14
mice (8- and 11-dpi) or DNBS + H. diminuta treated mice challenged with Con-A produced
increased amounts of IL-4, IL-5, IL-10, and IL-13 compared to splenocytes from control
mice or those given DNBS-only (Figure 5).
Figure 4. Colitis does not affect the hosts’ effector response to H. diminuta. Male BALB/c mice were treated with DNBS (3
mg, ir.) 3 days before infection with 5 cysticercoids of H. diminuta, and necropsied 8 or 11 days post-infection (dpi) (see
Figure 3A), when blood eosinophils (A) and goblet cells in a section of mid-jejunum (B) were enumerated (data are mean
± SEM; n = 7–15 mice (pooled from 3 experiments) for 8-dpi and n = 4–5 mice for 11-dpi; one-way ANOVA and Tukey’s
multiple; * and #, p < 0.05 compared to control and DNBS only, respectively; VCU, villus crypt unit; H. d, H. diminuta).
Panel (C) presents representative photomicrographs of goblet cell (magenta) staining in jejunal villi.
Figure 4.
Colitis does not affect the hosts’ effector response to H. diminuta. Male BALB/c mice were treated with DNBS
(3 mg, ir.) 3 days before infection with 5 cysticercoids of H. diminuta, and necropsied 8 or 11 days post-infection (dpi)
(see Figure 3A), when blood eosinophils (
A
) and goblet cells in a section of mid-jejunum (
B
) were enumerated (data are
mean ±SEM;
n= 7–15 mice (pooled from 3 experiments) for 8-dpi and n= 4–5 mice for 11-dpi; one-way ANOVA and
Tukey’s multiple; * and #, p< 0.05 compared to control and DNBS only, respectively; VCU, villus crypt unit; H. d, H. diminuta).
Panel (C) presents representative photomicrographs of goblet cell (magenta) staining in jejunal villi.
Pathogens 2021,10, 994 7 of 14
Pathogens 2021, 10, x FOR PEER REVIEW 7 of 14
Figure 5. Spleen cell production of TH2 cytokines following infection with H. diminuta is not affected
by colitis. Male BALB/c mice were treated with DNBS (3 mg, ir.) 3 days before infection with 5 cys-
ticercoids of H. diminuta, and necropsied 8 or 11 days post-infection (dpi) (see Figure 3A). Spleno-
cytes were isolated and stimulated with concanavalin-A (2 g/5 × 106 splenocytes; 48 h) and IL-4
(A), IL-5 (B), IL-10 (C), and IL-13 (D) measured by ELISA (data are mean ± SEM; n = 4–11 mice
(pooled from 2 experiments) for 8-dpi and n = 4–5 mice for 11-dpi; one-way ANOVA and Tukey’s
multiple comparisons test; * and #, p < 0.05 compared to control and DNBS only, respectively; H. d,
H. diminuta).
Figure 5.
Spleen cell production of TH2 cytokines following infection with H. diminuta is not affected
by colitis. Male BALB/c mice were treated with DNBS (3 mg, ir.) 3 days before infection with
5 cysticercoids of H. diminuta, and necropsied 8 or 11 days post-infection (dpi) (see Figure 3A).
Splenocytes were isolated and stimulated with concanavalin-A (2
µ
g/5
×
10
6
splenocytes; 48 h) and
IL-4 (
A
), IL-5 (
B
), IL-10 (
C
), and IL-13 (
D
) measured by ELISA (data are mean
±
SEM; n= 4–11 mice
(pooled from 2 experiments) for 8-dpi and n= 4–5 mice for 11-dpi; one-way ANOVA and Tukey’s
multiple comparisons test; * and #, p< 0.05 compared to control and DNBS only, respectively; H. d,
H. diminuta).
Pathogens 2021,10, 994 8 of 14
2.3. In the DNBS Model of Colitis Infection with H. diminuta Hastens Recovery
The experimental set-up shown in Figure 3A, while designed to assess if DNBS-
induced colitis affected H. diminuta infectivity, afforded the opportunity to assess if H. dimin-
uta in a treatment regimen hastened recovery from DNBS-induced colitis. Mice sponta-
neous recover from DNBS-induced colitis, as demonstrated by a return to normal behavior
and recovery of body weight (pers. observation; Figure 6A). There is natural variability in
the murine response to DNBS, and on necropsy at 11 days post-DNBS, mice had returned
to their pre-treatment body weight, whereas those treated with
DNBS + H. diminuta
not
only gained back the weight, but also continued to thrive and were not different from
control naïve mice at 14 days post-DNBS (Figure 6A). Similarly, while mice treated 14 days
previously with DNBS displayed mild signs of disease, these were not observed in the
DNBS + H. diminuta group of mice (Figure 6A–C). Mice gavaged with H. diminuta, 3 days
after DNBS, were indistinguishable from controls when disease was assessed 14 days after
DNBS as gauged by recovery of body weight, colon length, and a zero-disease activity
score (Figure 6).
Pathogens 2021, 10, x FOR PEER REVIEW 8 of 14
2.3. In the DNBS Model of Colitis Infection with H. diminuta Hastens Recovery
The experimental set-up shown in Figure 3A, while designed to assess if DNBS-in-
duced colitis affected H. diminuta infectivity, afforded the opportunity to assess if H.
diminuta in a treatment regimen hastened recovery from DNBS-induced colitis. Mice
spontaneous recover from DNBS-induced colitis, as demonstrated by a return to normal
behavior and recovery of body weight (pers. observation; Figure 6A). There is natural
variability in the murine response to DNBS, and on necropsy at 11 days post-DNBS, mice
had returned to their pre-treatment body weight, whereas those treated with DNBS + H.
diminuta not only gained back the weight, but also continued to thrive and were not dif-
ferent from control naïve mice at 14 days post-DNBS (Figure 6A). Similarly, while mice
treated 14 days previously with DNBS displayed mild signs of disease, these were not
observed in the DNBS + H. diminuta group of mice (Figure 6A–C). Mice gavaged with H.
diminuta, 3 days after DNBS, were indistinguishable from controls when disease was as-
sessed 14 days after DNBS as gauged by recovery of body weight, colon length, and a
zero-disease activity score (Figure 6).
Figure 6. Infection with H. diminuta enhances recovery from colitis. Male BALB/c mice were treated with DNBS (3 mg, ir.)
3 days before infection with 5 cysticercoids of H. diminuta, and necropsied 8 or 11 days post-infection (dpi) (see Figure 3A).
Body weight was recorded daily (A), and on necropsy colon length was measured (B) and disease activity score calculated
(C) (data are mean ± SEM; n = 7–15 mice (pooled from 3 experiments) for 8-dpi; n = 4–5 mice for 11-dpi; parametric data
analyzed by one-way ANOVA and Tukey’s multiple comparisons test and non-parametric data analyzed by a Kruskal–
Wallis test with Dunn’s post-test; *, p < 0.05 compared to control; H. d, H. diminuta).
Figure 6.
Infection with H. diminuta enhances recovery from colitis. Male BALB/c mice were treated with DNBS (3 mg,
ir.) 3 days before infection with 5 cysticercoids of H. diminuta, and necropsied 8 or 11 days post-infection (dpi) (see
Figure 3A). Body weight was recorded daily (
A
), and on necropsy colon length was measured (
B
) and disease activity
score calculated (
C
) (data are mean
±
SEM; n= 7–15 mice (pooled from 3 experiments) for 8-dpi; n= 4–5 mice for 11-dpi;
parametric data analyzed by one-way ANOVA and Tukey’s multiple comparisons test and non-parametric data analyzed
by a Kruskal–Wallis test with Dunn’s post-test; *, p< 0.05 compared to control; H. d,H. diminuta).
Pathogens 2021,10, 994 9 of 14
3. Discussion
The concept of helminth-therapy to treat idiopathic auto-inflammatory disease is
intriguing because it seeks to harness eons of host–parasite co-evolution—that is, the hosts’
natural immune response to infection with a parasitic helminth has the bystander effect
of affecting the course of concomitant disease [
2
,
17
,
18
]. While seemingly counterintu-
itive, Desowitz (1980) elegantly presented the concept of the “Harmonious Parasite” [
19
]
and numerous studies with helminth–rodent model systems have shown that deliberate
infection with parasitic helminths can reduce inflammation [
20
22
]. Furthermore, a va-
riety of helminth-derived molecules have immunoregulatory, immunosuppressive, and
anti-inflammatory effects [2325].
The consensus on the mechanism of helminth therapy is essentially two-fold: Infection
with parasitic helminths drives Th2-dominated immunity and this reduces disease caused
by Th1 immunopathology, or mobilization of immunoregulatory cells/factors inhibits
inflammation (e.g., Foxp3
+
regulatory T cells (Tregs), alternatively activated macrophages
(AAMs), IL-10, transforming growth factor
β
(TGF
β
)) [
21
,
26
]. We showed that infection of
the non-permissive murine host with H. diminuta 8 days prior to intra-rectal DNBS resulted
in significantly less colitis, and that systemic administration of neutralizing IL-10 antibodies
negated the anti-colitic effect of infection with this helminth [
3
]. H. diminuta is particularly
potent in this system, where gavage with a single cysticercoid led to substantial inhibition of
colitis [
27
]. Typically, much larger worm burdens (e.g., Schistosoma mansoni,Heligmosomoides
polygyrus) have been used to alleviate colitis or other inflammatory conditions [5,28].
Three-week-old mice infected with H. diminuta were also protected from DNBS-
induced colitis; however, the kinetics of this response differed from adult mice: young
mice displayed less colitis when infected 10 days, but not 8 days, prior to treatment
with DNBS [
10
]. This delay in the anti-colitic effect could be due to immaturity of the
immune system in the young mouse, and it draws attention to the importance of the
kinetics of the infection-colitis regimen. Having rationalized that the anti-colitic effect of
infection with H. diminuta could be prominent at the peak time of worm expulsion from
the mouse host, we have previously focused on 8-dpi when systemic and local immune
Th2 responses are apparent [
3
,
29
]. In this paradigm, significant and consistent suppression
of DNBS-induced colitis is observed; moreover, we noted that protection against this form
of colitis occurred in mice infected 14 days prior to DNBS treatment, although to a lesser
extent [
3
]. The current study shows that DNBS–colitis is less severe in mice infected with
H. diminuta 4 days previously; however, the effect is less pronounced than that observed
in mice infected 8 days previously [
3
]. While we did not pursue the mechanism of the
protection against colitis, the similarities in the splenic response at 4- and 8-dpi imply a
common mode of action involving IL-4 and IL-10 signaling in the blockade of colitis, as we
previously determined [
30
]. Collectively, these findings show that mice challenged with
DNBS between 4–14 dpi with H. diminuta develop less severe disease.
While underscoring the capacity of helminth therapy to ameliorate colitis, the current
study, like the majority of studies in this area, used a prophylactic protocol. Small clinical
trials in which individuals with IBD were treated with ova of the pig whipworm, Trichuris
suis, have reported improvement in objective and subjective measures of colitis and disease
symptoms [
31
]. Despite the spontaneous resolution of colitis in the DNBS model, we
showed that H. diminuta given two days after DNBS hastened recovery from disease over a
seven-day period [
3
]. Corroborating and extending these data we find that while some mice
displayed signs of mild disease 14 days post-DNBS, this was not observed in mice infected
with H. diminuta 3 days post-DNBS (i.e., 11 days prior to necropsy). Although these data
support the concept of a therapeutic helminth, a recent ~250 IBD-patient trial with T. suis
ova concluded that while safe the treatment was ineffective [
32
], potentially sounding the
death knell for helminth therapy. However, Crohn’s disease is a heterogeneous condition
and with broad inclusion criteria for enrolling patients in the trial it is perhaps naïve
to think that a single species of parasitic helminth could benefit all patients: There was
also a high placebo effect in the trial [
32
]. Similarly, helminth therapy has been tested
Pathogens 2021,10, 994 10 of 14
in multiple sclerosis and found to be safe; however, any beneficial “effect was modest”
with considerable variation between subjects [
33
,
34
], with an inter-individual variability
also seen in T. suis-specific T and B cell responses [
35
]. In addition, it is increasingly
apparent that infection with enteric parasitic helminths affects the composition and activity
of the gut microbiota [
5
,
16
,
36
], the production of short chain fatty acids by which has
been implicated as a mediator of helminth-initiated anti-inflammatory effects [
28
,
37
].
Furthermore, dysbiosis is a common feature of IBD [
38
] and how this would affect helminth-
therapy is unknown. Thus, we suggest it is premature to abandon the potential of helminth
therapy and that in-depth analyses of helminth infection in murine models of disease
will aid in unraveling the complexity of immunoregulation, with the potential to identify
targets for therapeutic intervention in auto-inflammatory disease.
Using H. diminuta in a treatment regimen afforded the opportunity to ask if enteric
inflammation affected the course of infection/rejection of the worm. The expectation
that DNBS-induced colitis that can be Th1 immune skewed [
21
] would render mice more
susceptible to H. diminuta, allowing a longer duration of infection and/or increased biomass
was incorrect. While the fact that a number of DNBS + H. diminuta-treated mice bore five
worms at 8-dpi is noteworthy, and something we have never seen in immunocompetent
mice, there was a negligible impact on the Th2 response to infection, as gauged by blood
eosinophilia, goblet cell hyperplasia, and spleen cell production of IL-4, -5, -10, and -13, and
worms were not detected at 11-dpi. While many studies have sought to dissect the interplay
of co-infection, in both helminth–helminth [
39
] and helminth–microbe [
40
42
] there is a
paucity of data on how a pre-existing inflammatory condition would affect the outcome of
infection with helminth parasites. This understudied area is more than an intriguing issue
of complex immune crosstalk, it is likely to emerge as a clinically relevant topic should
the incidence of IBD and other auto-inflammatory diseases increase in global regions of
endemic helminth infections. Furthermore, while we found that DNBS–colitis did not
affect the hosts’ ability to expel the worm, it is critical that these findings be complemented
by other rodent–helminth model systems to support or counter these data, and human
epidemiological studies performed to assess relationships between inflammatory disease
and subsequent susceptibility to helminth infection.
In summary, in advancing our understanding of host–parasite interactions we found
that: (1) Mice challenged with DNBS 4-dpi with H. diminuta developed less severe disease;
(2) H. diminuta hastened the recovery from DNBS–colitis when delivered in a therapeutic
regimen; and, (3) colitis did not appreciably affect the ability of the mouse to mount Th2
immune responses and effectively expel the helminth. In total, the findings herein offer
additional support for helminth therapy to ameliorate enteric inflammation. However,
further studies with this, and other model systems, are needed to fully delineate the anti-
inflammatory mechanism(s) evoked following infection with helminth parasites, responses
that may be mediated or fine-tuned by participation of the gut microbiota.
4. Materials and Methods
4.1. Mice, Helminth and DNBS-Induced Colitis
Male Balb/c mice (7–9 weeks of age; Charles River Animal Supplies, QC, Canada)
were housed in filter-topped cages with free access to food and water on a 12:12 h light:
dark cycle. Hymenolepis diminuta lifecycle was maintained by cyclic passage through rats
(permissive host) and the flour beetle, Tribolium confusum (intermediate host). Mice received
five infective cysticercoids of H. diminuta via oral gavage in 100 µL of 0.9% NaCl [3,43].
Colitis was induced in anesthetized mice by intra-rectal (ir.) instillation of 3 mg
of di-nitrobenzene sulphonic acid (DNBS; Sigma Chemical Co., St. Louis, MI, USA) in
100
µ
L of 50% ethanol (EtOH), delivered 3 cm into the colon via a polyethylene catheter [
3
].
In the first experimental paradigm, mice were infected with H. diminuta 4 days prior
to DNBS treatment, followed by necropsy 72 h later. In a second paradigm, mice were
challenged with DNBS 72 h before H. diminuta infection, followed by necropsy at either 8
Pathogens 2021,10, 994 11 of 14
or 11 days post-infection (dpi). Controls consisted of naïve, H. diminuta-infected only, and
DNBS-treated only mice.
4.2. H. diminuta Infectivity, Goblet Cells, and Eosinophils
Following humane euthanasia, the entire small intestine was excised from previously
infected mice and was flushed with 10 mL PBS at room temperature. This intestinal wash
was collected in a Petri dish and extensively examined for the presence of H. diminuta
(typically stunted and damaged in the mouse). The number of worms was counted and
each helminth was measured [44].
A blood drop was smeared onto a coded slide (VWR Micro Slides (frosted selected
precleaned)), allowed to air dry, stained with Wright–Giemsa hematology solution, and
eosinophils counted in a blinded fashion in a random selection of 200 white blood cells [
45
].
A ~2-cm segment of mid jejunum was excised, immersion-fixed in 10% neutral
buffered formalin for 72 h, then dehydrated and embedded in paraffin wax for trans-
verse sectioning. Five-
µ
m sections were collected onto coded slides and stained by the
Schiff’s periodic acid (Sigma Chemical Co., St. Louis, MI, USA) method for goblet cell
enumeration. Goblet cells were counted on a per villus-crypt unit (VCU) basis, composed
of a crypt opening to the lumen and a round, intact villus tip [43].
4.3. DNBS-Induced Colitis
Mice were examined daily following treatment with DNBS for signs of ill health:
wet/feces-stained or bloody anus, weight loss, ruffled fur, and hunched, inactive posture.
Upon necropsy, the colon was excised, measured, and inspected for signs of inflamma-
tion/dysfunction: loose stool, fluid accumulation, bleeding, or ulceration. A disease
activity score (0–5 points) was determined based on the following criteria: >10% loss of
body weight (0 or 1); wet anus, soft stool, or empty colon (0–1); anal bleeding (0 or 1);
macroscopic ulcers present in the colon (0 or 1) [
46
]. Mice that reached a predetermined
end-point (e.g., >20% loss of body weight, rectal prolapse) prior to 72 h post-DNBS (the
experimental endpoint) were euthanized and if this occurred 24–72 h post-DNBS, mice
were allocated a score of 5 [
46
]. The excised colon was subsequently divided based on
length and the 10–30% segment distant from the anus was formalin-fixed for further
histological analysis.
4.4. Colonic Histopathology Assessment
Mid-colonic sections were excised, fixed, paraffin-embedded, cut into 5-
µ
m sections
on coded slides and stained with hematoxylin and eosin (H & E). The histopathology score
is determined on a 12-point scale and considers degree of loss of architecture, goblet cell
depletion, inflammatory cells infiltrate, muscle thickening, edema, and ulceration [3].
4.5. Cytokine Production
Spleens were removed and aseptically separated into single-cell suspensions. Five
million splenocytes were incubated with concanavalin A (conA, 2
µ
g/mL) for 48 h at 37
C,
and cytokine levels (IL-4, -5, -10, -13, TNF
α
) in the supernatants were measured by ELISA
(Duo-set kits, R&D Systems) following the manufacturer’s instructions [47].
4.6. Statistical Analysis
Data are presented as the mean
±
the standard error of the mean (SEM), where
nis the number of mice. Statistical comparisons for two groups were conducted with
a Students’ unpaired t-test. Multiple group comparisons were performed via one-way
ANOVA followed by Tukey’s test for parametric data or Kruskal–Wallis statistics followed
by Dunn’s post-test for non-parametric data, with p< 0.05 set as the level of acceptable
statistical difference [46].
Pathogens 2021,10, 994 12 of 14
Author Contributions:
Conceptualization, S.L. and D.M.M.; investigation, S.L., S.R. and A.W.; formal
analysis, S.L., S.R. and A.W.; writing—original draft preparation, S.L. and D.M.M.; writing—review
and editing, S.L., S.R., A.W. and D.M.M.; funding acquisition, D.M.M. All authors have read and
agreed to the published version of the manuscript.
Funding:
This work was supported by a Natural Science and Engineering Research Council of
Canada (NSERC) Discovery Grant to D. McKay. S. Li holds an NSERC studentship and a Beverley
Phillips Rising Star Studentship (Snyder Institute, University of Calgary). S. Rajeev is a recipient
of an Alberta Graduate Excellence Studentship (International Award) and Eyes High International
Doctoral Scholarship from the University of Calgary.
Institutional Review Board Statement:
The study was conducted in accordance with the guide-
lines of the Canadian Council on Animal Care, and approved by the Animal Care Committee of
UNIVERSITY OF CALGARY (protocol code AC17-0115, date of approval: 5 July 2017).
Informed Consent Statement: Not applicable.
Data Availability Statement:
Original data are available from authors. There are no database data
in this paper.
Acknowledgments:
The histological analysis was supported by the core facilities of the Calvin,
Phoebe & Joan Snyder Institute for Chronic Diseases, University of Calgary.
Conflicts of Interest: The authors declare no conflict of interest.
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Helminth parasites are effective in biasing Th2 immunity and inducing regulatory pathways that minimize excessive inflammation within their hosts, thus allowing chronic infection to occur whilst also suppressing bystander atopic or autoimmune diseases. Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory lesions within the central nervous system; there are very limited therapeutic options for the progressive forms of the disease and none are curative. Here, we used the experimental autoimmune encephalomyelitis (EAE) model to examine if the intestinal helminth Heligmosomoides polygyrus and its excretory/secretory products (HES) are able to suppress inflammatory disease. Mice infected with H. polygyrus at the time of immunization with the peptide used to induce EAE (myelin-oligodendrocyte glycoprotein, pMOG), showed a delay in the onset and peak severity of EAE disease, however, treatment with HES only showed a marginal delay in disease onset. Mice that received H. polygyrus 4 weeks prior to EAE induction were also not significantly protected. H. polygyrus secretes a known TGF-β mimic (Hp-TGM) and simultaneous H. polygyrus infection with pMOG immunization led to a significant expansion of Tregs; however, administering the recombinant Hp-TGM to EAE mice failed to replicate the EAE protection seen during infection, indicating that this may not be central to the disease protecting mechanism. Mice infected with H. polygyrus also showed a systemic Th2 biasing, and restimulating splenocytes with pMOG showed release of pMOG-specific IL-4 as well as suppression of inflammatory IL-17A. Notably, a Th2-skewed response was found only in mice infected with H. polygyrus at the time of EAE induction and not those with a chronic infection. Furthermore, H. polygyrus failed to protect against disease in IL-4Rα−/− mice. Together these results indicate that the EAE disease protective mechanism of H. polygyrus is likely to be predominantly Th2 deviation, and further highlights Th2-biasing as a future therapeutic strategy for MS.
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