ARTICLE
Auteur(s) : Akane Tanaka1, Yoichi
Fukushima2, Jalil Benyacoub3, Stephanie
Blum3, Hiroshi
Matsuda1
1Laboratory of Veterinary Molecular Pathology and
Therapeutics, Division of Animal Life Science, Graduate School,
Institute of Symbiotic Science and Technology, Tokyo University of
Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo
183-8509, Japan
2Nestlé Japan Ltd. Nutrition Business Group, NYK Tennoz
Building, 2-2-20, Higashi-Shinagawa, Shinagawa-Ku, Tokyo, 140 0002,
Japan
3Nestlé Research Center, Vers-chez-les-Blanc, PO Box 44,
CH 1000, Lausanne 26, Switzerland
accepté le 12 Octobre 2007
Atopic dermatitis (AD) is a chronic and relapsing inflammatory
skin disease characterized by pruritic, typically distributed,
eczematous skin lesions; frequently found in infants and children
[1-3]. The development of AD is mainly attributed to a failure of
regulation of the immune system, which may be deficiencies in
innate and adaptive immunity based on a genetic predisposition,
resulting in skin barrier dysfunction with hyperreactivity to
environmental stimuli and susceptibility to skin infections that
influence the severity of AD [2]. Over-activation of immune cells,
particularly Th2 cells, promotes the production of inflammatory
cytokines, inducing proliferation and activation of inflammatory
cells and enhanced immunoglobulin (Ig) E production, resulting in
inflammatory reactions in the affected skin [2-5]. Recent studies
showed that not only Th2 type cytokines but also subsequent
elevation of Th1 type cytokines contributed to the development of
the chronic inflammation recognized in AD [4-8], and
anti-inflammatory cytokines, TGF-β and IL-10, may play
immunosuppressive functions on both Th1 and Th2 responses [9, 10]
and down-regulate inflammatory responses [11]. Micro-organisms play
an important role in the development of immune responses in infancy
[12, 13]. In infancy, Th2 polarized immune responses are
manifested, and bacterial exposure through intestinal microflora
contribute to the development of normal immune responses by
initiating Th1 responses [14-16]. Recently, the frequency of
infections in infancy has been shown to be negatively correlated to
AD morbidity [15, 17], indicating that bacterial exposure in
infancy may modulate the development of AD. Probiotics confer
health benefits to the host when administered in adequate amounts
[18]. A probiotic strain of Lactobacillus johnsonii NC533 (La1),
screened for its high adherence property onto intestinal epithelial
cells [19], confers beneficial effects on the host [20-24] through
reinforcing natural immunity and/or improving gastrointestinal
conditions. In vitro studies showed that La1 promotes TGF-β mRNA
expression in human intestinal cell lines. Moreover, it was
reported that this strain was able to decrease house dust
mite-induced dermatitis in mice [25]. More recently, oral
supplementation of SKh: hr1 mice with La1 was shown to modulate
skin immunity and antagonize UV-induced skin immune-suppression
[26]. The capacity of La1 to modulate immunity at the mucosal,
systemic, and skin levels led us to speculate that La1 might have
the potential to have suppressive effects on AD. Intake of a
probiotic strain Lactobacillus rhamnosus GG (L. GG) reduces the
risks of AD morbidity in infants by oral administration to their
mothers during pregnancy and lactation and by oral administration
to the infants themselves [27, 28]. Some researchers figured that
the beneficial effects of probiotics may be different in each
strain [29]. The NC/NgaTnd mouse is a well-known animal model of AD
[30-32], and the preventive effects of L.GG on the onset of AD has
also been shown in this model [33]. In this study, we conducted an
animal study using NC/NgaTnd mice in order to elucidate the effects
of oral La1 on AD development, and found that supplementation of
La1 from the weaning period suppressed the aggravation of clinical
symptoms. Although we did not find a statistical significance in
scratching frequency, scratching duration (seconds) in mice fed
with La1 became shorter than control mice. These results indicate
that early supplementation of La1 may have some effects on
abatement of an itch, resulting in the reduction of clinical
severity of AD.
Materials and methods
Bacteria preparation
A lyophilized form of La1 (Nestlé culture collection NCC533,
Lausanne, Switzerland) was dissolved in sterilized water just
before application. La1-containing water was given ad libitum at
3.16 × 108 colony forming unit/ml and it was replaced
every 2 days. For a control, sterilized water alone was
administrated in the same manner.
Animals
Pregnant specific pathogen-free (SPF) NC/NgaTnd mice were moved to
the air uncontrolled conventional room in Tokyo University of
Agriculture and Technology. Twenty newborn mice from several
females were enrolled into the study from 4 weeks of age (male: 10,
female: 10). They were housed in the room maintained at 24 ±
1 °C under 12/12 h light/dark cycle during the study.
Food and sterilized water were given ad libitum. All experiments
with animals complied with the standards in the guidelines of
University Animal Care and Use Committee in Tokyo University of
Agriculture and Technology.
Study protocol
Four-week-old NC/NgaTnd mice were randomly divided into 2 groups
and either La1 dissolved in sterilized water or sterilized water
alone (n = 10 in each group, 5 males and 5 females per group) was
applied for 4 weeks. The application was started from 4 weeks
of age, and was continued until 8 weeks of age. We set this
period as the stage of AD onset following the weaning period in
NC/NgaTnd mice [25, 30]. After the end of the administration
period, just sterilized water was freely given as drinking water
for both groups until they were sacrificed at 16 weeks of age.
Clinical skin severity scores and scratching behaviour were
examined at 4, 8, 12, 14, and 16 weeks of age. Clinical features of
dermatitis were evaluated according to the criteria described
previously [30]. Scratching behaviour was recorded and analyzed
using a SCLABA system [34] (Noveltec Inc., Kobe, Japan). Blood
samples were collected from a tail vein at 4, 8, 12, and 16 weeks
of age for the measurement of plasma total IgE concentrations by an
ELISA as previously described [31]. Dorsal skin samples were
collected at 16 weeks of age and used for histological examination
with hematoxylin & eosin staining for evaluating general
pathological findings and with acidic toluidine blue (pH 4.0)
staining for counting mast cells. Spleens were collected at 16
weeks of age for mRNA analysis of cytokines by real time RT-PCR
methods with the sense and antisense primers for regulatory or
inflammatory cytokines. Relative expression level of the target
gene was normalized using that of β-actin as an endogenous
reference using the comparative CT (threshold cycle) method [35].
The amount of gene expression that was normalized to endogenous
reference is given by: 2-ΔΔCT.
Statistical analysis
Results were expressed as mean ± standard error (SE). The multiple
comparison of Dunnet or a two-tailed Student’s t test was performed
for statistical analyses of the data, and P < 0.05 was taken as
the level of significance.
Results
Oral application of La1 delayed development of AD in NC/NgaTnd
mice
In control NC/NgaTnd mice administered with sterilized water alone,
clinical severity of dermatitis and scratching behaviour were
elevated from 7-8 weeks of age and exacerbated with aging in all
mice (figures 1A and
B). In contrast, a mild clinical manifestation also
developed in 9 of 10 La1-fed mice at 7-8 weeks but did not further
increase afterwards (figure 1A). Aggravation of
dermatitis was suppressed in mice fed with La1, and they maintained
lower clinical skin severity scores than controls (figure 1A) (P < 0.05).
Clinical skin severity scores at 14 and 16 weeks of age were
significantly lower than that of control mice (P < 0.005).
Scratching frequency in mice fed with La1 tended to decrease when
compared to that of control mice, but there was no statistical
significance (figure
1B). On the other hand, the total scratching duration in
mice fed with La1 was significantly shorter than in control mice at
12 and 16 weeks of age (figure 1C) (P < 0.05).
Typical clinical features of mice in each group at 16 weeks of age
are shown in figure
2. The dermatitis consisted of eczema, erythema,
hemorrhage, erosion, excoriation, scaling, and dryness, which were
widely observed in all control mice at 12-16 weeks of age, while
the clinical symptoms were very mild or less in mice fed with La1
(figure 2). As
reported in the previous studies [30, 31], concentrations of plasma
total IgE were elevated with aging both in control mice and mice
fed with La1 from 4 weeks of age (data not shown). Unexpectedly,
there were no significant differences between the two groups during
the study.
Histological examination was performed using dorsal skin samples
at 16 weeks of age. Typical features of histological findings were
shown in figure
3A. In control mice, skin lesions such as thickening of the
epidermis with hyperplasia, marked hyperkeratosis with
parakeratosis, and dermal edema were widely observed. Infiltration
of eosinophils, mononuclear cells, and mast cells were obvious in
control mice. In contrast, less pathological features of the skin
were noticed in mice fed with La1 when compared to controls.
Infiltration of inflammatory cells was not observed in the skin of
mice fed with La1. The mast cell numbers in the dorsal skin of mice
fed with La1 was significantly lower than in controls (figure 3B). Next we
conducted semi-quantitative real-time PCR analysis to examine
cytokine production in the spleens of NC/NgaTnd mice at 16 weeks of
age. The expression of mRNA of immunosuppressive (TGF-β and IL-10)
or inflammatory (IL-4 and IFN-γ) cytokines was not different in
mice fed with La1 when compared to control mice (data not
shown).
Discussion
In this study, we have shown that the administration of live
probiotic La1 during the weaning period suppressed the aggravation
of clinical symptoms in an AD animal model. Since the neonatal
period is one of the most important periods for the prognosis of
allergic diseases [36], immune modulation by probiotics may work as
a modulator for the immune system of infants [37]. In a previous
study, L. GG decreased the rates of development of AD in high-risk
children when it was administrated to both to the mother and
neonates before delivery [27]. Our findings clearly showed that
exposure to La1 from the weaning period may have suppressive
effects on AD in NC/NgaTnd mice. Since we fed La1 just for 4 weeks
before and after the weaning period, the exposure of young mice
with a high-risk of AD to La1 during the initial stage of
dermatitis, might contribute to the suppression of disease severity
in AD later in life. Early supplementation of La1 shortened the
scratching duration with a statistically significant difference
compared to control mice. In human studies, scratching duration,
which is considered to be the best determinant of skin damage, is
more seriously involved in disease severity than scratching
frequency [38]. Therefore, our results indicate that exposure to
La1 might reduce the exacerbation of AD by reducing itch-provoked
scratching damage on the skin. Our results also confirm previous
findings that La1 could decrease house dust mite-induced AD in the
same strain of mice housed in different conditions [25]. It was
recently shown that feeding SKh: hr1 mice with La1 could modulate
skin immunity and antagonize UV-induced skin immune-suppression
[26]. More specifically, it was demonstrated that La1
supplementation maintained Langerhans cell density, morphology and
accelerated functionality recovery upon UV challenge [26],
reflecting a way how La1 might interfere with AD development
through a reinforcement of skin immune defences.
Even in mice fed with La1, mRNA expression of anti-inflammatory
cytokines in spleens was not different when compared to controls,
suggesting that it is not significantly influencing the systemic
immune system in NC/NgaTnd mice. Administration of La1 in the
weaning period has been reported to be effective in preventing or
inhibiting the development of AD after maturation, by modulating or
accelerating the gut immune response [25]. La1 is shown not to
strongly change the Th1/Th2 cytokine balance in newborn mice [39],
implying that the main mechanism of the preventive effect of La1
may be activation of the local immune system in the gut rather than
a systemic modulation of Th1/Th2 balancing. Since microbial stimuli
during the weaning period play a crucial role in the programming
and development of immune responses [14, 15], La1 may contribute to
the acquisition of anti-allergic immune responses during the
weaning period. Although no changes in plasma total IgE were
observed between the two groups in this study, our results are
consistent with human studies of L. GG [27].
In conclusion, our results indicated that oral supplementation
of La1 before and after the weaning period suppressed the
development of severe AD in NC/NgaTnd mice. These results indicated
that La1 might have properties to delay the exacerbation of AD in
later life by regulating immune responses at an early stage in
high-risk children. Lactobacillus is not a medical product, but can
be used to support action as a natural resource without side
effects.
Acknowledgements
Financial support: none. Conflict of interest: none.
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