Parasite Immunology, 2016, 38, 326–330 DOI: 10.1111/pim.

12314

Brief Definitive Report

Modulation of IL-12 and IFNc by probiotic supplementation

promotes protection against Toxocara canis infection in mice

L. F. D. C. DE AVILA,1 P. M. M. DE LEON,2 M. Q. DE MOURA,1 M. E. A. BERNE,1 C. J. SCAINI3 & F. P. LEIVAS LEITE1,2

1
Post-Graduate Program in Parasitology, Universidade Federal de Pelotas (UFPel), Pelotas, Brazil, 2Center for Technological Development
– Biotechnology, UFPel, Pelotas, Brazil, 3Post-Graduate Program in Health Science,Universidade Federal do Rio Grande (FURG), Rio
Grande, Rio Grande do Sul, Brazil

SUMMARY the nematode Toxocara canis, which is an intestinal para-

site of dogs and cats, is among the most widespread zoo-
In this study, supplementation with the probiotic Saccha-
noses in the world and has a high prevalence in Latin
romyces boulardii promoted a reduction in intensity of
America (3). Many cases of toxocariasis are asymp-
infection by Toxocara canis and modulates cytokines mRNA
tomatic; however, severe disease can occur. The transmis-
expression in experimentally infected mice. IL-12 gene tran-
sion of this parasite to humans and other paratenic hosts
scription had 40-fold increase in S. boulardii supplemented
occurs primarily via the ingestion of T. canis eggs (4).
uninfected mice and sevenfold increase in supplemented
After ingestion, larvae emerge from the eggs and penetrate
infected mice comparing with not supplemented group.
the small intestine, enter the circulation, and then invade
Regarding IFNc, similar results were observed, since probi-
several organs (5). According to Maizels (6), improved
otic supplementation induced approximately 43-fold
knowledge concerning the mechanisms by which this
increase, but only in uninfected mice (P < 0
05). T. canis
nematode suppresses immunity and colonizes the host
infection upregulated IL-10 expression while S. boulardii
would make it possible to promote immune protection
downregulated it and no change was observed for IL-4.
against T. canis.
Thus, based in these findings; we suggest that one possible
The use of probiotic microorganisms has revealed the
mechanism responsible for S. boulardii protection effect
importance of immunomodulation for the prevention and
against T. canis infection is by the modulation of cytokines
treatment of parasitic disease (7, 8). In the search for new
expression, especially IL-12.
alternatives for the control of toxocariasis, study with Sac-
charomyces boulardii supplementation demonstrated reduc-
Keywords immunomodulatory, innate immunity, Saccha-
tion of T. canis larvae in mice 48 h post-inoculation (9). It
romyces boulardii, toxocariasis
is important to clarify that the time points of 24 and 48 h
were selected based on prior study in which S. boulardii
INTRODUCTION action in just 48 h of infection (9). In the same study, a
Helminths act by modulating the immune response to group of animals was kept receiving probiotic up to
reduce the inflammatory process and ensure their survival 60 days after infection and was observed that the reduc-
in the host for long periods of time. Studies that used a tion rate was similar to the observed at 48 h. This result
murine experimental model improved our understanding suggests that the probiotic action might occur in the first
of how the host immune system regulates susceptibility 48 h. The mechanism of action of this probiotic is
and resistance to infections (1, 2). The infection caused by unknown, what is known so far is that S. boulardii does
not exert a direct effect on the parasite larvae (10). Con-
Correspondence: Luciana Farias da Costa de Avila, Post-Graduate sidering that S. boulardii modulates the structure and the
Program in Parasitology, Universidade Federal de Pelotas (UFPel), activity of adhesions of the intestinal mucosa and these
Campus Universitário, S/N, Caixa Postal 354, CEP: 96010-900, effects could contribute to the enhancement of intestinal
Pelotas, RS, Brazil (e-mail: lucostaavila@hotmail.com). cell restitution (11), this study aimed to understand the
Disclosures: The authors declare no conflict of interest.
possible immunomodulatory mechanism of S. boulardii in
Received: 29 October 2015
Accepted for publication: 11 March 2016 mice experimentally infected by T. canis.

326 © 2016 John Wiley & Sons Ltd

Volume 38, Number 5, May 2016 IL-12 and IFNc protect against Toxocara canis

(synthesized from 300 ng/lL of RNA), 6
25 lL of SYBR

MATERIALS AND METHODS
Green PCR Master Mix (Applied Biosystems), 0
5 lM of
Toxocara canis eggs were collected and incubated accord- each primer, and 4
25 lL of RNAse-free water (Sigma
ing to Avila et al. (9). The extraction and maintenance of Aldrich) in a total volume of 12
5 lL. The temperatures
T. canis larvae was performed according to De Savigny were as follows: denaturation at 95°C for 5 min, followed
(12) with brief modification. Saccharomyces boulardii yeast by 40 cycles of denaturation at 95°C for 30 s, annealing at
were cultivated in YPD (Yeast Peptone Dextrose) medium 60°C for 60 s and extension at 72°C for 60 s and a final
and quantified by determining colony forming units extension at 72°C for 5 min. All samples were analyzed in
(CFU). The supplemented feed consisted of commercial duplicate. The comparative threshold cycle (ΔΔCt) was
mice feed mixed with S. boulardii at 107 CFU/g (9). used to determine the relative amount of mRNA for each
Two groups of 15 female Swiss mice, at 6 weeks of age, gene that was normalized using b-actin reference gene
were formed; one group received supplemented feed for (18). Data regarding the differences in mRNA expression
15 days, and the other received feed without probiotic. and the recovery of larvae from the organs of mice were
After that, five mice from both groups were inoculated subjected to an analysis of variance, and the means were
intragastrically with 100 T. canis eggs; 24 h later, another compared by Student’s t-test, with a significance level of
five mice were inoculated the same way. The remaining 95%. The results of the relative mRNA levels are
five mice from both groups were maintained without infec- expressed graphically using the means and standard devia-
tion (inoculated with saline only). The infective dose of tions.
100 T. canis eggs was based on previous studies that
observed protective effect of probiotics against this para-
RESULTS
site (9, 13). All mice received food and water ad libitum
and were euthanized on the same day according to the It was possible to observe a significant reduction of the
experimental period (i.e., without infection or 24 or 48 h intensity of infection by T. canis in mice supplemented
post-inoculation). A tissue digestion technique was per- with S. boulardii. The mean number of larvae recovered
formed to quantify T. canis larvae in the liver, lungs, from mice supplemented with S. boulardii was 35
8% and
brain, heart, eyes, kidneys and carcass of all animals (14). 40
4% lower, at 24 and 48 h post-inoculation, respectively
This study was approved by the Ethics Committee in (Table 1). In both groups (i.e., supplemented and not with
Research at the Federal University of Pelotas (CEEA the probiotic) the greater number of larvae was recorded
6554) and all the experiments were carried out following in the liver, which is consistent with the acute phase of
the Federal Government legislation on animal care. toxocariasis (19). In the other analyzed organs, no differ-
Splenocytes (2 9 106 cells) were cultivated at 37°C with ence was observed.
5% CO2 in 1 mL of RPMI 1640 medium supplemented IL-12 gene transcription was increased in mice supple-
with 10% foetal bovine serum (Invitrogen) and antibiotic mented with S. boulardii in both infected and uninfected
antimycotic solution (A5955; Sigma-Aldrich, St. Louis, groups. In mice supplemented with the probiotic, the gene
MO, USA). After 24 h, the splenocytes were stimulated transcription of IL-12 was 7
2 and 6
5 times higher
with the following: (a) two T. canis larvae per mL; (b) (P < 0
05), after 24 and 48 h respectively, compared to
107 CFU/mL of S. boulardii; or (c) culture medium alone infected not supplemented mice. In uninfected mice, probi-
(control). Stimulation with 2
5 lg/mL of Concanavalin A
(Sigma-Aldrich) was used as a control for cell viability. Table 1 Mean number of T. canis larvae recovered from mice
supplemented with S. boulardii and control mice
The splenocytes were collected in TRI Reagentâ (Sigma-
Aldrich) and stored at 70°C. Mean number
RNA extraction was performed according to TRI Mean number of of larvae
Reagentâ protocol and cDNA synthesis according to the Groups larvae (liver) (all tissues)a Amplitude
manufacturer’s instructions of the High Capacity cDNA
24 h post-inoculation
Reverse Transcription Kit (Applied Biosystems, Foster S. boulardii 2
4* (1
5) 2
5* 1–5
City, CA, USA). A quantitative polymerase chain reaction Control 3
7 (1
9) 3
9 1–7
(qPCR) was performed on a Stratagene M93005P real- Reduction 35
1% 35
8%
time PCR system (Agilent Technologies) using specific pri- 48 h post-inoculation
S. boulardii 9
4* (3
5) 10
3* 4–18
mers for IL-12, IL-10, IFN-c, IL-4 (15–17). b-actin and
Control 16
3 (5
1) 17
3 10–25
GAPDH were tested as a reference gene, and, based on Reduction 42
3% 40
4%
M-value of 0
9 and 1
5, respectively, b-actin was selected.
The qPCR reactions were performed using 1 lL of cDNA *P < 0
05. aLiver, lungs, brain, heart, eyes, kidneys and carcass.

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 38, 326–330 327
L. F. d. C. de Avila et al. Parasite Immunology

otic supplementation promoted a 42-fold increase of IL-12 increased transcription of IL-10 (P < 0
05) (Figure 1b).
mRNA in splenocytes analyzed ex vivo (without stimula- The same modulation tendency occurred with splenocytes
tion) (P < 0
05), and after in vitro stimulation with from uninfected mice, in which T. canis stimulation
S. boulardii, the expression of this cytokine was even increased IL-10 mRNA levels while S. boulardii decreased
higher, increase by 51-fold. When the splenocytes from it (P < 0
05) (Figure 1f). IFNc gene transcription was
uninfected mice were stimulated with T. canis larvae a low down regulated in T. Canis-infected mice (below the
level of IL-12 mRNA was observed (Figure 1e). Similar threshold 1). However, after in vitro stimulation with
effect was observed in splenocytes from infected mice S. Boulardii, we can see a blockade in IFNc suppression
when stimulated in vitro with T. canis larvae (P < 0
05) (Figure 1c). In uninfected S. boulardii supplemented mice,
(Figure 1a). the levels of IFNc mRNA were 43 times increased
Regarding IL-10 mRNA, T. Canis-infected mice (Fig- (P < 0
05) (Figure 1g). The IL-4 gene transcription
ure 1b) showed an increase of this cytokine when com- levels were low in all periods analysed and there was no sig-
pared to non-infected mice (Figure 1f). After 48 h of nificant variation after T. canis infection as well as in mice
infection, only mice without supplementation showed supplemented with S. boulardii (Figure 1d and h).

S· boulardii Control
(a) (e) Figure 1 Gene transcription of IL-12, IL-10,
10 60 *
IFNc and IL-4 in splenocytes of mice
mRNA relative levels

mRNA relative levels

*
8 * 50 * supplemented or not with S. boulardii.
* 40
6 Letters a (IL-12), b (IL-10), c (IFNc) and d
IL-12

IL-12

30 (IL-4) are from T. canis infected mice.

4
20 Letters e (IL-12), f (IL-10), g (IFNc) and h
2 10 (IL-4) are from uninfected mice. Splenocytes
0 0 were analyzed ex vivo (without stimulation)
ex vivo Tc Sb ex vivo Tc Sb ex vivo Tc Sb con A
and after in vitro stimulation with two
24 h 48 h T. canis larvae/well (Tc) or 107 CFU of
S. boulardii (Sb). The normalized mRNA of
(b) (f) supplemented mice was expressed relative to
60 4
mRNA relative levels

mRNA relative levels

* the respective cytokine in not supplemented

50
3 mice. Mice supplemented with S. boulardii
40
*
IL-10

IL-10

are represented by grey bars and not

30 2
supplemented with black bars – control.
20
* 1 Asterisks (*) means difference, by Student’s
10
* t-test of P < 0
05.
0 0
ex vivo Tc Sb ex vivo Tc Sb ex vivo Tc Sb con A

24 h 48 h
(g)
(c)
3 50 *
mRNA relative levels

mRNA relative levels

2·5 * 40
2
IFNγ

IFNγ

30
1·5
20
1
0·5 10
0 0
ex vivo Tc Sb ex vivo Tc Sb ex vivo Tc Sb con A

24 h 48 h

(d) (h)
2·5 2
mRNA relative levels

mRNA relative levels

2 1·5
1·5
IL-4

IL-4

1
1
0·5 0·5

0 0
ex vivo Tc Sb ex vivo Tc Sb ex vivo Tc Sb con A

24 h 48 h

328 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 38, 326–330
Volume 38, Number 5, May 2016 IL-12 and IFNc protect against Toxocara canis

McClemens et al. (28) While in T. canis infection, the

DISCUSSION
increase of IL-10, with consequent reduction of Th1
The results of this study suggest the involvement of cells, may cause immunosuppression and favour infec-
innate immunity in the protection mediated by tion.
S. boulardii against T. canis. We could observe a ten- As in others studies (20, 21), our results suggest that
dency of reduction in the number of larvae at 24/48 h T. canis down regulates IFNc expression. Instead,
after infection, and an increase in the gene transcription S. boulardii supplementation increases IFNc levels, mostly
of IL-12 in mice supplemented with S. boulardii. IL-12 in uninfected mice. This IFNc down regulation promoted
suppression is an important immunomodulatory mecha- by T. canis probably inhibited the stimulatory effect of
nism utilized by Toxocara canis. After T. canis penetrates S. boulardii on this cytokine in infected mice. Further-
into the intestinal mucosa of the host, the suppression of more, we suggest that the increase of IFNc observed in
IL-12 promoted by parasite hinders the recruitment of uninfected supplemented mice (ex vivo) might have been
macrophages and impedes parasite death (20). In con- produced by immune cells of the intestinal mucosa, where
trast, the increase of IL-12 mRNA that was observed in S. boulardii developed its action (14) and then migrated
mice supplemented with S. boulardii suggesting that this to the spleen. It seems to be necessary the contact of the
probiotic blocks the inhibitory effect of T. canis on IL-12 yeast with the intestinal mucosa of the host because even
and participates in the protection against infection. This with a secondary S. boulardii stimulation in vitro the
result is consistent with other researches, who found that splenocytes showed low levels of IFNc mRNA. This
an increase in IL-12, TNF-a and IFN-c may enhance might explain why splenocytes from uninfected mice when
the protective host response against T. canis (21). In stimulated in vitro with S. boulardii (Sb) did not promote
other helminthosis such as Schistosoma mansoni, Trichi- IFNc increase like observed in supplemented mice
nella spiralis and Trichuris suis infection, the importance (ex vivo) (Figure 1g). In a study that assessed the stimula-
of IL-12 for the development of a protective immune tion of splenocytes with b-glucan derived from Saccha-
response was also demonstrated (22–24). In all these romyces cerevisiae (30) was also observed reduced IFNc
examples, the reduction of IL-12 promotes worm’s levels after in vitro stimulation when compared to in vivo
survival. pre-treatment. These authors attributed the differences
CD4 Foxp3+ cells (Treg cells) are contributors to between in vivo and in vitro profiles to the distinct mecha-
immune homeostasis and acting on the expression of the nisms involved.
anti-inflammatory cytokine IL-10. These cells are The IL-4 mRNA levels showed no changes after T. canis
detected inside and around granulomas as well as in iso- infection or S. boulardii supplementation (P > 0
05). The
lated inflammatory foci, participating of the development low levels of this cytokine observed in this study may be due
and extension of toxocariasis pathology (25). In this to the low infective dose (only 100 eggs) or the early phase
study, we observed a significant increase of IL-10 gene of infection, since the predominance of Th2 cells and IL-4
transcription provoked by T. canis, while S. boulardii increase are more commonly found in animals infected with
supplementation reduced this cytokine. The reduction of higher doses or after 10 days of infection (2, 20). Similar
IL-10 mRNA promoted by S. boulardii may have helped results were observed with peripheral blood mononuclear
to maintain the higher IL-12 levels and hold back the cells from healthy dogs stimulated with T. canis antigens, in
establishment of infection in supplemented group, since which IL-4 levels showed no modifications from the control
the increase of IL-10 act by antagonizing the effects of group after 48 h (31).
IL-12 (26) that could favour T. canis infection (27). In Despite the immunomodulatory effects exercised by
another study, the probiotic Lactobacillus rhamnosus pro- S. boulardii it is important to note that, in immunocom-
moted increase of IL-10 levels, which was related to petent hosts, this probiotic does not increase susceptibil-
increase of Trichuris muris expulsion from the gut of ity to other infections (32). Several studies characterize
mice (28). In both studies, we noted a protective role of this as a good probiotic biotherapeutic agent who acts
the probiotics tested, although in T. canis experiment in the prevention and treatment of gastrointestinal dis-
IL-10 reduction was beneficial whereas in T. muris eases by regulation of intestinal homeostasis (32) and
experiment the opposite was observed. This may be jus- contribute to the enhancement of intestinal cell restitu-
tified because different probiotics promote different tion (11). In the present study, it was possible to expand
effects in their hosts (29). Furthermore, the beneficial the knowledge of how the immune system behaves dur-
effect of IL-10 on T. muris expulsion is caused by the ing the early stage of T. canis infection in mice supple-
fact that parasites present in the intestinal lumen are mented or not with probiotic. We observed that the
eliminated by the action of Th2 cytokine and IL-10 parasite increased the mRNA of the regulatory cytokine

© 2016 John Wiley & Sons Ltd, Parasite Immunology, 38, 326–330 329
L. F. d. C. de Avila et al. Parasite Immunology

IL-10 and down regulated IL-12 and IFNc, while the

ACKNOWLEDGEMENTS
probiotic increased the mRNA of these pro-inflamma-
tory cytokines (i.e. IL-12 and IFNc). The increase of We thank the Fundacß~ao de Amparo a  Pesquisa do Estado
IL-12 promoted by S. boulardii was very significant and do Rio Grande do Sul for available resources (FAPERGS
sustained even after T. canis infection. From these – PqG 001/2013), Coordenacß~ ao de Aperfeicßoamento de
results, we suggest that the up regulation of pro-inflam- Pessoal de Nıvel Superior (CAPES) for a scholarship
matory cytokines, especially IL-12, is the basis for the grant that funded this study, the Post-Graduate Program
protective mechanism of action of this probiotic against in Parasitology (UFPel), Professor Odir Dellagostin and
T. canis. colleagues Fernando Leivas Leite, Caroline Rizzi and
Helena Thurow for collaboration in this study.

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330 © 2016 John Wiley & Sons Ltd, Parasite Immunology, 38, 326–330