Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse

Article (PDF Available)inDiabetologia 48(8):1565-75 · September 2005with228 Reads
DOI: 10.1007/s00125-005-1831-2 · Source: PubMed
Abstract
Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-gamma and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer's patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

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Available from: Francesco Dotta
Diabetologia (2005) 48: 15651575
DOI 10.1007/s00125-005-1831-2
ARTICLE
F. Calcinaro
.
S. Dionisi
.
M. Marinaro
.
P. Candeloro
.
V. Bonato
.
S. Marzotti
.
R. B. Corneli
.
E. Ferretti
.
A. Gulino
.
F. Grasso
.
C. De Simone
.
U. Di Mario
.
A. Falorni
.
M. Boirivant
.
F. Dotta
Oral probiotic administration induces interleukin-10 production
and prevents spontaneous autoimmune diabetes in the non-obese
diabetic mouse
Received: 6 December 2004 / Accepted: 18 April 2005 / Published online: 29 June 2005
# Springer-Verlag 2005
Abstract Aims/hypothesis: Recent observations suggest
the involvement of the gastrointestinal tract in the path-
ogenesis of islet autoimmunity. Thus, the modulation of
gut-associated lymphoid tissue may represent a means to
affect the natural history of the disease. Oral administra-
tion of probiotic bacteria can modulate local and systemic
immune responses; consequently, we investigated the ef-
fects of oral administration of the probiotic compound
VSL#3 on the occurrence of diabetes in non-obese dia-
betic (NOD) mice. Methods: VSL#3 was administered to
female NOD mice three times a week starting from 4
weeks of age. A control group received PBS. Whole blood
glucose was measured twice a week. IFN-γ and IL-10
production/expression was evaluated by ELISA in culture
supernatants of mononuclear cells isolated from Peyers
patches and the spleen, and by real-time PCR in the pan-
creas. Insulitis was characterised by immunohistochemis-
try and histomorphometric studies. Results: Early oral
administration of VSL#3 prevented diabetes development
in NOD mice. Protected mice showed reduced insulitis
and a decreased rate of beta cell destruction. Prevention
was associated with an increased production of IL-10 from
Peyers patches and the spleen and with increased IL-10
expression in the pancreas, where IL-10-positive islet-in-
filtrating mononuclear cells were detected. The protective
effect of VSL#3 was transferable to irradiated mice receiv-
ing diabetogenic cells and splenocytes from VSL#3-treat-
ed mice. Conclusions/interpretation: Orally administered
VSL#3 prevents autoimmune diabetes and induces im-
munomodulation by a reduction in insulitis severity. Our
results provide a sound rationale for future clinical trials
of the primary prevention of type 1 diabetes by oral
VSL#3 administration.
Keywords Autoimmunity
.
Gut-associated lymphoid
tissue
.
IL-10
.
Immunomodulation
.
NOD mouse
.
Probiotics
.
Type 1 diabetes
Abbreviations HBSS: Hanks balanced salt solution
.
HE: haematoxylineosin
.
GALT: gut-associated
lymphoid tissue
.
MAdCAM-1: mucosal addressin cell
adhesion molecule
.
MNCs: mononuclear cells
.
NOD:
non-obese diabetic
.
PPs: Peyers patches
F. Calcinaro
.
P. Candeloro
.
S. Marzotti
.
A. Falorni
Department of Internal Medicine, University of Perugia,
Perugia, Italy
S. Dionisi
.
V. Bonato
.
U. Di Mario
.
F. Dotta
Department of Clinical Sciences,
University of Rome La Sapienza,
Rome, Italy
M. Marinaro
.
F. Grasso
.
M. Boirivant (*)
Immune-mediated Diseases Section, Department of Infectious,
Parasitic and Immune-mediated Diseases,
Istituto Superiore di Sanità,
Viale Regina Elena, 299,
00161 Rome, Italy
e-mail: monboir@iss.it
Tel.: +39-06-49902976
Fax: +39-06-49902709
R. B. Corneli
Department of Experimental Medicine
and Biochemical Sciences, University of Perugia,
Perugia, Italy
E. Ferretti
.
A. Gulino
Department of Experimental Medicine and Pathology,
University of Rome La Sapienza,
Rome, Italy
C. De Simone
Department of Experimental Medicine,
University of LAquila,
LAquila, Italy
F. Dotta
Diabetes Unit, Department of Internal Medicine,
Endocrine and Metabolic Sciences and Biochemistry,
University of Siena,
Siena, Italy
Introduction
Type 1 diabetes mellitus is an organ-specific autoimmune
disease that develops in genetically predisposed individ-
uals. Several attempts have been made to identify envi-
ronmental trigger factors, the role of beta cell antigens in
inducing and maintaining the autoimmune response, and
the nature of the pathogenic immune mechanisms involved
[1, 2]. Due to the chronic complications of the disease, the
development of strategies aiming to delay or prevent au-
toimmune beta cell loss would result in a major benefit to
public health. As 90% of type 1 diabetes patients have no
affected relatives, primary prevention in the general pop-
ulation is the ultimate goal. This can only be achieved via
a strategy that combines efficacy, safety, specificity of ac-
tion, low cost and adequate compliance by children.
The non-obese diabetic (NOD) mouse develops a spon-
taneous form of autoimmune diabetes that mimics many
features of the human disease, thus representing a model
for investigating possible therapeutic approaches [3, 4].
Recent observations in humans and animal models have
drawn attention to a possible involvement of the gastro-
intestinal tract in the pathogenesis of autoimmune diabetes,
mainly as a possible way-in site of putative trigger factors.
This is in light of the reported association of some dietary
antigens, such as cows milk proteins, gliadin and other
cereal components, and of enteric infections with the dis-
ease [59]. A link between gut-associated lymphoid tis-
sue (GALT) and autoimmune diabetes has already been
shown in animal models; indeed, mesenteric lymphocytes
from 3-week-old NOD mice had a high diabetogenic po-
tential [10], and diet manipulations were able to modify
disease incidence and the pattern of islet-infiltrating lym-
phocytes and cytokine production [11]. Thus, the modula-
tion of GALT may represent a means to affect the natural
history of autoimmune diabetes.
Probiotics are defined as mono- or mixed cultures of
live micro-organisms which, when applied to animal or
man, beneficially affect the host by improving the proper-
ties of the indigenous microflora [ 12]. Administration of
probiotics in humans and animal models has been shown to
be beneficial in the treatment and prevention of intestinal
infections and to reduce mucosal inflammation [1319].
This effect probably results from the ability of probiotics to
adhere to mucosal surfaces and inhibit the attachment of
other pathogenic bacteria, to secrete factors that enhance
barrier integrity, and to modulate cells of the immune
system [18, 2024]. Their ability to deviate tissue cytokine
secretion from a pro-inflammatory to an anti-inflammatory
profile has been specifically described [18, 19, 23].
It has been reported that oral administration of heat-
killed Lactobacillus casei to NOD mice reduces the in-
cidence of diabetes, but the mechanism underlying this
finding has not been elucidated [25].
The aim of this study was to investigate the effect of oral
administration of VSL#3, a clinically safe mixture of dif-
ferent strains of viable lyophilised probiotic bacteria, on the
occurrence of spontaneous autoimmune diabetes in NOD
mice.
Materials and methods
Mice Four-week-old female NOD mice were obtained
from Charles River Laboratories (Calco, Milan, Italy) and
housed under specific pathogen-free conditions in the
animal facility at the University of Perugia; these mice
were used for all experiments. Animals had free access to
water and food. All studies were approved by the Animal
Care and Use Committee of the University of Perugia and
by the Italian Ministry of Health.
Probiotic preparation VSL#3 (VSL Pharmaceuticals, Ft
Lauderdale, FL, USA) is a probiotic compound containing
3×10
11
/g viable lyophilised bacteria, including bifidobac-
teria (B. longum, B. infantis and B. breve), lactobacilli
(L. acidophilus, L. casei, L. delbrueckii subsp. L. bulgaricus
and L. plantarum)andStr epto coccus salivarius sub sp.
thermophilus.
Experimental design The study was designed as sum-
marised in Fig. 1. To evaluate the effect of VSL#3
administration on the onset of diabetes, NOD mice were
randomly subdivided into two groups (Fig. 1a). In group
1, VSL#3 (3 mg/mouse, re-suspended in 100 μl PBS) was
administered orally by gavage three times a week from 4
to 32 weeks of age. In group 2, an equal volume of PBS
was administered orally by gavage three times a week
from 4 to 32 weeks of age. Mice were monitored for the
appearance of clinical signs of diabetes and were killed at
disease occurrence.
At 32 weeks of age, the mice of group 1 (VSL#3-
treated) that remained diabetes free (n=15) were divided
into three subgroups (Fig. 1b). Subgroup 1a continued
VSL#3 treatment under the same conditions. Subgroup 1b
stopped VSL#3 treatment and started PBS administration
as for group 2. Subgroup 1c continued VSL#3 treatment
and received a single i.p. cyclophosphamide injection (250
mg/kg body weight).
This study was stopped at 280 days of age and all
remaining non-diabetic mice from all groups were killed
for the evaluation of insulitis.
In a parallel study, containing a separate series of VSL#3-
or PBS-treated animals, mice were killed at 8 and 12 weeks
of age (after 4 and 8 weeks of treatment) respectively. The
pancreas, the spleen and Peyers patches (PPs) were
collected from each mouse for the evaluation of insulitis
and cytokine production.
To evaluate the effect of VSL#3 administration later
in the disease process, 36 additional female NOD mice,
housed as previously described, were randomly subdivided
into two groups. In group 1 (n=18), VSL#3 (3 mg/mouse,
re-suspended in 100 μl PBS) was administered orally by
gavage three times per week from 10 to 32 weeks of age. In
group 2 (control; n=18), an equal volume of PBS was ad-
ministered orally by gavage three times per week from 10
to 32 weeks of age. The mice in these groups were mon-
itored for the appearance of clinical signs of diabetes and
were killed at the occurrence of the disease. Finally, we
evaluated the effect of VSL#3 when administered at di-
1566
abetes onset. As such, 16 additional female NOD mice,
housed as previously described, were monitored twice a
week for the occurrence of diabetes. At diagnosis, NOD
mice were treated with VSL#3 (n=8) or PBS (n=8) fol-
lowing the same protocol as previously described. Blood
glucose was checked thereafter twice a week for up to 8
weeks of treatment.
Adoptive transfer In a separate series of animals treated
with VSL#3 or PBS, mice were killed at 12 weeks of age
(8 weeks of treatment) to prepare single-cell splenocyte
suspensions. Splenocytes were i.v. injected (3×10
7
/mouse)
into two separated groups of 775-rad irradiated NOD
female mice at 8 weeks of age. The day after, both groups
received 3.5×10
6
mononuclear splenocytes isolated from
13- to 15-week-old female NOD mice with newly onset
diabetes. Mice were monitored for the occurrence of
diabetes twice a week for up to 11 weeks after transfer.
Diagnosis of diabetes Non-fasting whole blood glucose
was measured in all animals twice a week using a gluco-
meter (Medisense; Abbott Laboratories, Abbott Park, IL,
USA) and reagent strips. In NOD mice, non-fasting blood
glucose ranges from 3 to 8 mmol/l (95% CI). Diabetes was
defined as two consecutive readings above 12 mmol/l.
Histology and immunohistochemistry The pancreases from
killed mice were removed and divided into two halves.
One half was fixed in 10% buffered formalin for 20 h and
embedded in paraffin. Sections of 4 μm were cut 40 μm
apart throughout the gland and stained with haematoxylin
eosin (HE; Merck, Whitehouse Station, NJ, USA) for the
evaluation of the insulitis score using the following scale:
0, intact islet; 1, peri-insulitis; 2, moderate insulitis (<50%
of the islet infiltrated); 3, severe insulitis (50% of the islet
infiltrated). At least 30 islets per pancreas were analysed
by two independent examiners.
The second half of the pancreas was snap-frozen in
liquid nitrogen and subsequently used for immunohisto-
chemical studies or for mRNA expression analyses by
real-time PCR.
Staining of IL-10 and IFN-γ was performed on cryostat
acetone-fixed pancreatic sections by incubation for 1 h
with either rat anti-mouse-IL-10 primary monoclonal anti-
body (Endogen, Woburn, MA, USA; distributed by Tema
Ricerca, Bologna, Italy) diluted 1:400 in 3% BSAPBS, or
Fig. 1 Experimental design for the evaluation of the effect of
VSL#3 administration on the onset of diabetes in female NOD mice.
a Female NOD mice were randomly divided into two groups: group
1, VSL#3-treated group (n=19); and group 2, PBS-treated group
(n=21). In both groups, treatment was started at 4 weeks of age.
VSL#3 (3 mg/mouse per administration, re-suspended in 100 μl
PBS; group 1) or 100 μl PBS alone (group 2) was administered
orally by gavage three times per week from 4 to 32 weeks of age. In
a separate series of animals, mice treated with VSL#3 or PBS were
killed at 8 and 12 weeks of age (after 4 and 8 weeks of treatment),
respectively. b At 32 weeks of age, mice remaining diabetes free in
the VSL#3-treated group 1 (n=15) were divided into three sub-
groups: subgroup 1a continued VSL#3 treatment under the same
conditions (n=5); subgroup 1b stopped VSL#3 treatment and started
PBS administration as for group 2 (n=5); and subgroup 1c continued
VSL#3 treatment and received a single i.p. cyclophosphamide
injection (n=5). The study was stopped at 40 weeks (280 days) of
age and all non-diabetic remaining mice from all groups were killed.
When a mouse was killed, the spleen, the pancreas and PPs of each
mouse were collected for evaluation of insulitis and assessment of
cytokine production
1567
with rat anti-mouse IFN-γ primary monoclonal antibody
(Abcam, Cambridge, UK) diluted 1:400 in 3% BSAPBS.
This step was followed by 1 h of incubation with per-
oxidase-conjugated rabbit anti-rat antibody (Sigma-Al-
drich, St Louis, MO, USA). The colour was revealed using
the 3,3-diaminobenzidine revelation system (Vector kit;
Vector Laboratory, Burlingame, CA, USA).
Insulin staining and histomorphometric analysis Insulin
staining was performed on 4-μm paraffin-embedded pan-
creatic sections by incubation for 1 h with guinea pig
anti-porcine insulin primary polyclonal antibody (Dako,
Carpinteria, CA, USA) diluted 1:100 in PBS, followed by
1 h of incubation with peroxidase-conjugated rabbit anti-
guinea pig secondary antibody (Dako) diluted 1:200 in
PBS. The colour was revealed using the 3,3-diamino-
benzidine revelation system (Vector kit). Histomorpho-
metric analysis of insulin-stained pancreatic sections was
carried out on two to three 4-μm sections from each ani-
mal using an interactive image analyser (IAS 2000; Delta
Sistemi, Rome, Italy). The sections were cut at intervals
of approximately 40 μm, and the area of the Langerhans
islets occupied by beta cells (cells stained positive for in-
sulin) was measured as a percentage ratio of the total area of
the same islets.
Cell isolation and cultures Spleens were removed, minced
and filtered through sterile 100-μm filters (Falcon; Becton
Dickinson, Franklin Lakes, NJ, USA). Cell suspensions
were then washed in Hanks balanced salt solution (HBSS;
BioWhittaker Europe, Verviers, Belgium), and erythro-
cytes were removed by osmotic lysis (ACK lysing buffer;
BioWhittaker).
The intestines of the mice were isolated and PPs were
carefully excised and incubated at 37°C for 15 min in 5
mmol/l EDTAHBSS. After this step, mechanical disso-
ciation of PPs through a nylon mesh grid was performed.
The resulting cell suspension was then washed in HBSS
and the pellet re-suspended in 30% Percoll (Sigma-Aldrich)
with 1 mmol/l EDTA, and centrifuged at 400 g for 25 min
at 20°C. Mononuclear cells (MNCs) were recovered, washed
twice in HBSS, counted and cultured as follows.
Cells were re-suspended at the concentration of 1×10
6
cells/ml in complete medium consisting of RPMI 1640
(BioWhittaker) supplemented with 2 mmol/l
L-glutamine,
25 mmol/l HEPES buffer (BioWhittaker) and 5 μg/ml
Fig. 2 Delayed onset and reduced incidence of diabetes in NOD
mice administered with VSL#3. a Life-table analysis of NOD mice
treated with PBS or VSL#3 starting from 4 weeks of age (arrow)
showing the percentage of non-diabetic NOD mice plotted against
age. The analysis shows a delay in the first diagnosis of diabetes at
12 weeks of age in PBS-treated mice (triangles) and at 16 weeks of
age in VSL#3-treated mice (circles). At 32 weeks of age, 81%
(17/21) of PBS-treated NOD mice were diabetic, in comparison with
21% (4/19) of mice in the VSL#3-treated group (p<0.001 by
KaplanMeier analysis curve with log-rank test). b Life-table
analysis of NOD mice treated with PBS (triangles) or VSL#3
(circles) starting from 10 weeks of age (arrow) showing the
percentage of non-diabetic NOD mice plotted against age. The
analysis shows a significant reduction in the frequency of diabetes in
VSL#3-treated animals at 190 days of age (p=0.028 by Kaplan
Meier analysis curve with log-rank test) but not at the end of the
study
1568
gentamicin, 50 U/ml penicillin and 50 μg/ml streptomy-
cin. Cells were cultured in complete medium plus either
10% FBS (HyClone Europe, Cramlington, UK) for eval-
uation of IFN-γ, IL-4 and IL-10 production, or 1% Nu-
tridoma SP (Roche Diagnostics, Mannheim, Germany) for
evaluation of TGF-β production. Cells were cultured in
24-well plates (Costar Corporation, Cambridge, MA, USA),
coated or not with anti-CD3 antibody (clone 145-2C11;
PharMingen, San Diego, CA, USA). Coating was accom-
plished by pre-exposure of individual wells to 10 μg/ml
murine anti-CD3 antibody in carbonate buffer (pH 9.6) for
1 h at 37°C. Soluble anti-CD28 antibody (1 μg/ml; clone
37.51; PharMingen) was also added to the coated wells.
After 48 h of culture under these conditions (or 72 h for
TGF-β), culture supernatants were collected and stored at
80°C until tested.
ELISA Cytokine concentrations in culture supernatants
(IL-10, IL-4 and IFN-γ) were measured using commer-
cially available specific ELISA kits (Endogen), while TGF-β
concentration was measured using the commercially avail-
able TGF-β quantikine kit (R&D Systems, Abingdon,
UK). Optical densities were measured using a Bio-Rad
Novapath ELISA reader (Bio-Rad, Hercules, CA, USA) at
a wavelength of 450 nm.
Fig. 3 Reduced insulitis score in non-diabetic VSL#3-treated NOD
mice vs PBS-treated NOD mice. ac Insulitis score in non-diabet-
ic NOD mice treated with PBS or VSL#3 at 8 (a), 12 (b) and 40
(c) weeks of age (4 and 8 weeks of treatment and at the end of the
study). In VSL#3-treated mice, 108, 386 and 105 islets were
evaluated at the indicated time points. In PBS-treated mice, 92, 207
and 91 islets were evaluated at the indicated time points. For the
evaluation of insulitis score the following scale was used: 0 (white),
intact islet; 1 (horizontal lines), peri-insulitis; 2 (oblique lines), mod-
erate insulitis; 3 (black), severe insulitis. p=0.002 for VSL#3
treatment vs PBS treatment at 8 weeks of age; p<0.001 for VSL#3
treatment vs PBS treatment at 12 and 40 weeks of age. di Rep-
resentative microphotographs (×400) of histological H E-stained
paraffin pancreatic sections from PBS-treated (df) or VSL#3-treated
(gi) NOD mice at 8 (d, g), 12 (e, h) and 40 (f, i) weeks of age
1569
RNA extraction and real-time PCR Total RNA was ex-
tracted from frozen pancreatic tissue using Trizol (Gibco-
BRL, Grand Island, NY, USA) and cDNA prepared as
previously described [26]. Quantitative analysis of mouse
IL-10, IFN- γ and β-actin mRNA expression was per-
formed in triplicate by real-time PCR as previously de-
scribed [27] using TaqMan pre-developed assay reagents
(Applied Biosystems, Foster City, CA, USA) and the ABI
Prism 7700 sequence detection system (Applied Biosys-
tems). Results were normalised for β-actin content and
expressed as arbitrary units.
Cyclophosphamide treatment Cyclophosphamide (Endox-
an-Asta; Asta Medica, Frankfurt, Germany) was prepared
according to manufacturers instructions immediately be-
fore injection by adding sterile distilled water to ly-
ophilised cyclophosphamide to a final concentration of
20 mg/ml. Mice randomised in the cyclophosphamide
treatment group (Fig. 1b, group 1c) received a single i.p.
injection (250 mg/kg body weight) of the drug. All mice
were tested as non-diabetic before treatment and blood
glucose was monitored twice a week after treatment.
Statistical analysis Diabetes incidence in the two groups
was compared by the KaplanMeier analysis curve with a
log-rank test. The MannWhitney U-test was used to
compare cytokine production, insulitis score and percent-
age ratio of cells stained positive for insulin among dif-
ferent groups. A p value of less than 0.05 was considered
significant.
Results
VSL#3 administration delays the onset of and reduces the
incidence of diabetes in NOD mice The effect of VSL#3
on the development of autoimmune diabetes in NOD mice
was studied by evaluating the time of onset and the
incidence of diabetes.
As shown in Fig. 2a, beginning the treatment at wean-
ing resulted in a delay in the first diagnosis of diabetes in
VSL#3-treated mice (16 weeks of age) as compared with
the PBS-treated group (12 weeks of age). In addition,
VSL#3-treated mice showed a significantly lower inci-
dence of diabetes than controls (p<0.001 by KaplanMeier
analysis). To further characterise the mechanism of VSL#3-
induced protection, we evaluated the persistence of pro-
tection and the possibility of abrogating the protective
effect. T o this end, non-diabetic VSL#3-treated mice were sub-
divided into three subgroups at 32 weeks of age (Fig. 1b).
None of the five mice with suspended VSL#3 treatment
(group 1b), and none of the five mice with continued
VSL#3 treatment (gro up 1a) developed overt diabetes during
a subsequent follow-up period of 8 weeks. In contrast, all
five mice treated with a single injection of cyclophospha-
mide (group 1c) developed overt diabetes within 2 weeks,
Fig. 4 Reduced rate of beta cell destruction in non-diabetic VSL#3-
treated NOD mice at 12 weeks of age. a, b Histomorphometric
analysis of beta cells after insulin staining in pancreatic paraffin-
embedded sections at 8 (a) and 12 (b) weeks of age. In VSL#3-
treated mice, 54 and 72 islets were evaluated at the indicated time
points. In PBS-treated mice, 68 and 43 islets were evaluated at the
indicated time points. *p=0.019 for VSL#3-treated vs PBS-treated
mice (b). cf Representative microphotographs (×400) of immuno-
peroxidase insulin staining on pancreatic paraffin-embedded sec-
tions from non-diabetic PBS- (c, d) or VSL#3- (e, f) treated NOD
mice at 8 (c, e) and 12 (d, f) weeks of age
1570
in spite of concomitant VSL#3 treatment. When VSL#3 or
PBS treatment was started at 10 weeks of age, we did not
observe any delay in the first diagnosis of diabetes in
animals treated with probiotics (Fig. 2b). At 190 days,
VSL#3-treated mice showed a significantly lower inci-
dence of diabetes than controls (p=0.028 by KaplanMeier
analysis). However, at the end of the study, frequency of
diabetes was not significantly lower in VSL#3-treated
mice than in PBS-treated mice (Fig. 2b).
In addition, no effect in terms of reversal of the hy-
perglycaemic state was observed when VSL#3 treatment
was started at diabetes onset.
VSL#3-treated mice show reduced insulitis and a decreased
rate of beta cell destruction In the group of mice treated
from 4 to 32 weeks of age we evaluated the degree of
insulitis and the rate of beta cell destruction.
Histological analysis of the pancreas of non-diabetic
animals showed the presence of insulitis in both groups of
mice, with significant differences in the degree of infil-
tration between the mice treated with VSL#3 and those not
treated (Fig. 3). In VSL#3-treated mice, prevalence of
different grades of insulitis was always lower than that
observed in PBS-treated mice. Moreover, in VSL#3-treat-
ed mice that were switched to PBS treatment at 32 weeks of
Fig. 5 Increased IL-10 produc-
tion by MNCs isolated from PPs
(a, c , e) and the spleen (b, d, f)
of VSL#3-treated NOD mice.
IL-10, IFN- γ and IL-4 produc-
tion by PPs and spleen MNCs
from VSL#3- or PBS-treated
animals was evaluated in non-
diabetic NOD mice and diabetic
NOD mice at diagnosis. Culture
supernatants (48 h) from unstim-
ulated cells (white columns)or
anti-CD3/CD28-stimulated cells
(black columns) were assayed by
specific ELISA for IL-10 (a, b),
IFN-γ (c, d) and IL-4 (e, f).
Columns represent mean values
obtained from at least five ani-
mals per group. Bars represent
standard errors. *p<0.05 for
non-diabetic VSL#3-treated vs
non-diabetic PBS-treated mice
and vs diabetic PBS- or VSL#3-
treated mice. ND not detectable
1571
age, histological examination of the pancreas showed the
absence of insulitis in 87% of islets, with 12% of islets
showing only peri-insulitis. Furthermore, at 12 (but not at 8)
weeks of age, the pancreas of VSL#3-treated non-diabet-
ic mice showed a reduced rate of beta cell destruction,
as evidenced by a minimal loss of insulin-positive cells
compared with PBS-treated mice (Fig. 4d, f). As shown
in Fig. 4a, b, quantification of islet beta cell content by
histomorphometric analysis confirms this observation. In
particular, at 12 weeks of age (week 8 of treatment), insulin-
positive beta cells represented 57.9±10.8% of the islet mi-
croscopic surface section in VSL#3-treated NOD mice, and
24.9±17.6% in PBS-treated NOD mice (p=0.019; Fig. 4b).
As expected, all the pancreata removed from the dia-
betic animals, irrespective of the treatment group, showed
a very low number of islets that were severely infiltrated
and showed no insulin staining (data not shown).
Protection from diabetes in VSL#3-treated mice is
associated with increased IL-10 production by isolated
PPs and spleen MNCs To assess the immunomodulatory
role of VSL#3 administration, cytokine production by
MNCs isolated from PPs and the spleen was evaluated in a
separate series of mice treated from 4 weeks of age and
killed at 8 and 12 weeks of age, and in diabetic mice at the
time of diagnosis. Since cytokine production was com-
parable in each group at the various time points, we pooled
the data from the different time points. We show here the
average level of each evaluated cytokine. We found a sig-
nificant increase (p<0.05) in IL-10 production by stim-
ulated MNCs isolated from PPs and from the spleen of
mice treated with VSL#3 when compared with PBS-
treated mice (Fig. 5a, b). This increase was not observable
in the diabetic mice of each group (Fig. 5a, b). Slightly
higher IFN-γ production from MNCs isolated from PPs
was detected in VSL#3-treated mice as compared with
control animals (NS). However, the increased IFN-γ pro-
duction was restricted to PP-derived cells from non-
diabetic animals (Fig. 5c) and was not detectable in culture
supernatants of MNCs isolated from the spleen (Fig. 5d).
IL-4 production by PPs and spleen MNCs in VSL#3-
treated mice was not statistically different from that ob-
served in PBS-treated mice (Fig. 5e, f). TGF-β production
was only occasionally detectable and not significantly dif-
ferent between VSL#3- (spleen: 37.3±40 pg/ml; PP: 3.9±
2.3 pg/ml) and PBS- (spleen: 22.9±40.8 pg/ml; PP: 6.6±
5.3 pg/ml) treated mice.
Protection from diabetes in VSL#3-treated mice is
associated with increased IL-10 mRNA expression and
with the presence of IL-10-positive infiltrating MNCs in
the pancreas To further characterise the VSL#3-induced
protection from diabetes, we quantified and compared by
real-time PCR IL-10 and IFN-γ mRNA expression in the
pancreas of mice treated or not, from 4 weeks of age, with
VSL#3. As shown in Fig. 6a, a statistically significant
increase in IL-10 mRNA expression level was observed in
the pancreas of VSL#3-treated mice as compared with that
of PBS-treated animals (p=0.02). IFN-γ mRNA expres-
sion levels were comparable in VSL#3-treated and PBS-
Fig. 6 Increased IL-10 mRNA expression in the pancreas, and IL-
10 positivity in the infiltrated islets of VSL#3-treated NOD mice.
a, b Quantitative analysis by real-time PCR of mRNA expression
for IL-10 (a) and IFN-γ (b) in the pancreas of non-diabetic mice
treated with PBS or VSL#3. Columns represent mean values ob-
tained from four animals per group. Bars represent standard errors.
*p=0.021 for VSL#3-treated vs PBS-treated mice (a); p=0.563 for
VSL#3- vs PBS-treated mice (b). cf Representative microphoto-
graphs (×400) of immunoperoxidase IL-10 (c, e) and IFN-γ (d, f)
staining on pancreatic cryostatic sections of PBS- (c, d) and VSL#3-
(e, f) treated NOD mice at 12 weeks of age. IL-10-positive cells were
only detected in VSL#3-treated mice and not in the PBS-treated
group. No difference in IFN-γ staining was observed between the
VSL#3- and the PBS-treated groups. Arrows point at IL-10-positive
and IFN-γ-positive cells
1572
treated mice (Fig. 6b). Finally, we evaluated the presence
of IL-10- or IFN-γ-producing MNCs in islet infiltrates. As
shown in Fig. 6, IL-10-positive cells were only detected in
islets of VSL#3-treated mice (Fig. 6e), while IFN-γ-
positive cells were detected in the islets of both VSL#3-
and PBS-treated mice (Fig. 6d, f).
Adoptive transfer of splenocytes from VSL#3-treated mice
into naïve mice is associated with protection from diabetes
in the recipient mice We tested the ability of splenocytes
isolated from VSL#3-treated mice to transfer protection to
irradiated naïve NOD mice when co-transferred with spleen
cells isolated from newly diabetic NOD mice (see Materials
and methods). As shown in Table 1, 11 weeks after cell
transfer we observed full protection from diabetes in mice
that received splenocytes from VSL#3-treated mice. In
contrast, the mice that received splenocytes from PBS-
treated mice all developed diabetes 4 to 5 weeks after cell
transfer.
Discussion
Our study shows that oral administration of VSL#3 can
delay the onset of, and reduce the incidence of, autoim-
mune diabetes in NOD mice. In the VSL#3-treated group,
diabetes prevention was associated with less destructive
islet-specific autoimmunity and significantly increased pro-
duction of IL-10 by isolated PPs and spleen MNCs. More-
over, in VSL#3-treated mice we were able to show the
presence, in pancreatic islet infiltrates, of an increased level
of IL-10 mRNA expression and an increased number of IL-
10-positive MNCs. It has been observed that mucosal
addressin cell adhesion molecule (MAdCAM-1) becomes
expressed on islet vessels of NOD mice in the early phase of
lymphocyte accumulation in islets, and that it preferentially
mediates the homing of β7-integrin
high
, L-selectin-positive
mucosal lymphocytes [2831]. Therefore, MAdCAM-1
islet expression could favour early accumulation of mu-
cosal-associated lymphocytes in pancreatic islets. In ad-
dition, α4β7-integrin-positive, GAD-specific, circulating
lymphocytes have been detected in type 1 diabetic patients
[32]. Both of these observations suggest that GALT may
play a critical role in favouring islet-specific autoimmunity
in diabetes-prone individuals, even in humans. Our data
show that oral VSL#3 administration in NOD mice induces
IL-10-producing cells in GALT. This finding, together with
the observation of IL-10-producing lymphocytes in the
spleen and in the islet infiltrates, suggests that VSL#3-in-
duced IL-10-producing lymphocytes may recirculate from
GALT and home to the pancreatic islet where they down-
modulate destructive insulitis and preserve beta cells.
Previous studies in NOD mice show that IL-10 admin-
istration [33] and adeno-associated virus vector-mediated
IL-10 gene delivery prevent the development of autoim-
mune diabetes [3436]. In addition, low incidence of dia-
betes in BDC2.5/NOD transgenic mice has been shown to
be caused by T-cell regulation involving endogenous IL-10
[37]. Moreover, it has also been shown that IL-10 has a
protective effect on the function and survival of isolated
human islets exposed to Th1 inflammatory cytokines [26].
IL-10 is generally considered to be an anti-inflammatory
cytokine that acts mostly on antigen-presenting cells by
inhibiting antigen presentation and inflammatory cytokine
production [38]. It has recently been shown that repeated
antigen stimulation in the presence of high levels of this
cytokine leads to the generation of regulatory T-cells that
produce IL-10 and TGF-β [39]. These cells can inhibit
experimental colitis and allograft rejection [4042].
In addition, recent in vivo studies suggest that IL-10
production and regulatory T-cells participate in the process
of immune tolerance towards intestinal resident flora [43,
44]. In previous studies, the administration of different
lactobacilli resulted in the modulation of local and sys-
temic immune responses in humans and animal models
[19, 23, 45, 46]. This effect is thought to be accomplished
through the occurrence of several different biological
events [18, 4749]. In particular, probiotic-soluble products
have been shown to influence epithelial ubiquitination
by inhibiting the production of inflammatory cytokines
[18, 47, 48]. Moreover, probiotics are able to modulate
in vitro expression of cytokines and surface maturation
markers in murine dendritic cells [50]. However, different
species of lactobacilli induce very different dendritic cell
activation patterns, and, furthermore, one bacterial species
may be able to inhibit the biological activities of other spe-
cies in the genus. In particular, while the ability to induce
IL-12 production is linked to a number of species, all the
species tested were able to induce IL-10 production when
used at high concentrations [50]. VSL#3 contains, in ad-
dition to bifidobacteria, a combination of several species
of lactobacilli, in different proportions but at very high
concentrations. It has been shown that VSL#3 adminis-
tration at a high dose can maintain antibiotic-induced re-
mission in patients with pouchitis [17] by increasing local
production of IL-10 [23].
Table 1 Occurrence of diabetes after the adoptive transfer of 3×10
7
splenocytes per animal from VSL#3-treated or PBS-treated mice into
irradiated NOD females
Donor cells Recipient
mice (n)
Diabetic mice
(5 weeks after transfer)
Diabetic mice
(11 weeks after transfer)
Group A Splenocytes from PBS-treated mice 5 5 NA
Group B Splenocytes from VSL#3-treated mice 5 0 0
Both groups (A and B) received 3.5×10
6
splenocytes from newly diabetic NOD females (see Materials and methods)
NA not applicable because all animals became diabetic within 5 weeks of transfer
1573
Our study also suggests that the protection from auto-
immune diabetes by VSL#3 oral administration is dom-
inant and is achieved mostly through the induction of
regulatory cells. In fact, we were able to prevent diabetes in
mice receiving diabetogenic lymphocytes by transferring
splenocytes from VSL#3-treated mice. In addition, cyclo-
phosphamide administration at 32 weeks of age promptly
resulted in the development of overt diabetes in all the mice
treated. As cyclophosphamide is thought to delete reg-
ulatory T-cells [51], our experiments suggest that immu-
nocompetent diabetogenic cells are indeed still present
in VSL#3-treated mice, but they are actively regulated by
the regulatory cells induced by probiotic treatment. Active
regulation is effective in preventing complete islet de-
struction and the onset of clinical signs of diabetes when
VSL#3 treatment is started early in the disease process,
before the appearance of histological signs of insulitis, but
not when it is started when insulitis has already occurred or
when clinical diabetes is overt. Stimulation of GALT at the
time of weaning may be critical in the natural history of
anti-islet autoimmunity in diabetes-prone individuals.
In conclusion, our study provides evidence that oral
VSL#3 treatment, started at weaning in diabetes-prone
NOD mice, induces a change in the cytokine secretion
pattern by GALT which is associated with a qualitative
modification of the islet infiltrates, the down-regulation of
islet-specific destructive autoimmunity and, eventually,
diabetes prevention. Given the absence of side-effects in
VSL#3 treatment in NOD mice in our study and the dem-
onstrated safety of the use of this compound in humans [17,
5254], our results provide a sound rationale for future
clinical trials of the primary prevention of type 1 diabetes
by oral VSL#3 administration.
Duality of interest . The authors declare that no duality
of interest exists. C. De Simone is a shareholder of VSL
Pharma. No funding source had any role in the study de-
sign, collection or analysis, in data interpretation, or in the
writing of the report.
Acknowledgements This study was supported by the Italian
Ministry of Health (research project Inflammatory bowel disease
and autoimmune disease; mucosal immune regulation in the path-
ogenesis and prevention art 12 del D.L.gs 502/1992), the Italian
Ministry of Research (grant number 2001-063815), the Promoter
Foundation ONLUS, and Consorzio Italiano Biotecnologie (CIB).
F. Calcinaro and S. Dionisi contributed equally to this study. A.
Falorni, M. Boirivant and F. Dotta share senior authorship.
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