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Ajpgi 00295 2017 PDF
Ajpgi 00295 2017 PDF
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-
http://www.ajpgi.org 0193-1857/18 Copyright © 2018 the American Physiological Society G461
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G462 YOUNG MICE INFECTED WITH H. DIMINUTA
% 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
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
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.
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
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
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
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)
4 * *
* * * *
* 10 *
3 # # #
# * * *
2 *
5
1
0 0
- - + + + - - - - + + + - - H. diminuta
- + + + + + + - + + + + + + DNBS
- - - + - + - - - - + - + - HdE (500 μ g)
- - - - + - + - - - - + - + bHdE (500 μ g)
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.”
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.
A day
0
donor mice 28 30
30 recipient mice 33
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
31. Smallwood TB, Giacomin PR, Loukas A, Mulvenna JP, Clark RJ, 37. Summers RW, Elliott DE, Urban JF Jr, Thompson RA, Weinstock
Miles JJ. Helminth immunomodulation in autoimmune disease. Front JV. Trichuris suis therapy for active ulcerative colitis: a randomized
Immunol 8: 453, 2017. doi:10.3389/fimmu.2017.00453. controlled trial. Gastroenterology 128: 825–832, 2005. doi:10.1053/j.
32. Staatz WD, Van Horn DL. The effects of aging and inflammation on gastro.2005.01.005.
corneal endothelial wound healing in rabbits. Invest Ophthalmol Vis Sci 38. Torow N, Marsland BJ, Hornef MW, Gollwitzer ES. Neonatal mucosal
19: 983–986, 1980. immunology. Mucosal Immunol 10: 5–17, 2017. doi:10.1038/mi.2016.81.
33. Stiemsma LT, Reynolds LA, Turvey SE, Finlay BB. The hygiene 39. Torow N, Yu K, Hassani K, Freitag J, Schulz O, Basic M, Brennecke
hypothesis: current perspectives and future therapies. ImmunoTargets A, Sparwasser T, Wagner N, Bleich A, Lochner M, Weiss S, Förster
R, Pabst O, Hornef MW. Active suppression of intestinal
Ther 4: 143–157, 2015. doi:10.2147/ITT.S61528.
CD4(⫹)TCR␣(⫹) T-lymphocyte maturation during the postnatal period.
34. Stolp HB, Johansson PA, Habgood MD, Dziegielewska KM, Saunders
Nat Commun 6: 7725, 2015. doi:10.1038/ncomms8725.
NR, Ek CJ. Effects of neonatal systemic inflammation on blood-brain 40. Wang A, Fernando M, Leung G, Phan V, Smyth D, McKay DM.
barrier permeability and behaviour in juvenile and adult rats. Cardiovasc Exacerbation of oxazolone colitis by infection with the helminth Hy-
Psychiatry Neurol 2011: 469046, 2011. doi:10.1155/2011/469046. menolepis diminuta: involvement of IL-5 and eosinophils. Am J Pathol
35. Strachan DP. Hay fever, hygiene, and household size. BMJ 299: 1259 – 177: 2850 –2859, 2010. doi:10.2353/ajpath.2010.100537.
1260, 1989. doi:10.1136/bmj.299.6710.1259. 41. Williamson LL, McKenney EA, Holzknecht ZE, Belliveau C, Rawls
36. Su C, Su L, Li Y, Long SR, Chang J, Zhang W, Walker WA, Xavier JF, Poulton S, Parker W, Bilbo SD. Got worms? Perinatal exposure to
RJ, Cherayil BJ, Shi HN. Helminth-induced alterations of the gut helminths prevents persistent immune sensitization and cognitive dysfunc-
microbiota exacerbate bacterial colitis. Mucosal Immunol 11: 144 –157, tion induced by early-life infection. Brain Behav Immun 51: 14 –28, 2016.
2018. doi:10.1038/mi.2017.20. doi:10.1016/j.bbi.2015.07.006.