Microbes and Infection 14 (2012) 1144e1151

www.elsevier.com/locate/micinf

Original article

Dermatophyteehost relationship of a murine model of experimental

invasive dermatophytosis
James Venturini a,b, Anuska Marcelino Álvares c, Marcela Rodrigues de Camargo a,b,
Camila Martins Marchetti a, Thais Fernanda de Campos Fraga-Silva a, Ana Carolina Luchini d,
Maria Sueli Parreira de Arruda a,*
a
Faculdade de Cieˆncias, UNESP e Univ Estadual Paulista, Departamento de Cieˆncias Biológicas, Laboratório de Imunopatologia Experimental,
Av. Eng. Luiz Edmundo C. Coube 14-01, 17033-360 Bauru, SP, Brazil
b
Faculdade de Medicina de Botucatu, UNESP e Univ Estadual Paulista, Distrito de Rubião Junior s/n, 18618-970 Botucatu, SP, Brazil
c
Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP e Universidade Federal de São Paulo, R. Botucatu 862, 04023-900 São Paulo, SP, Brazil
d
Instituto de Cieˆncias Biológicas e Naturais, UFTM, Av. Frei Paulino 30, 38025-180 Uberaba, MG, Brazil
Received 15 March 2012; accepted 16 July 2012
Available online 27 July 2012

Abstract

Recognizing the invasive potential of the dermatophytes and understanding the mechanisms involved in this process will help with disease
diagnosis and with developing an appropriate treatment plan. In this report, we present the histopathological, microbiological and immunological
features of a model of invasive dermatophytosis that is induced by subcutaneous infection of Trichophyton mentagrophytes in healthy adult
Swiss mice. Using this model, we observed that the fungus rapidly spreads to the popliteal lymph nodes, spleen, liver and kidneys. Similar to the
human disease, the lymph nodes were the most severely affected sites. The fungal infection evoked acute inflammation followed by a granu-
lomatous reaction in the mice, which is similar to what is observed in patients. The mice were able to mount a Th1-polarized immune response
and displayed IL-10-mediated immune regulation. We believe that the model described here will provide valuable information regarding the
dermatophyteehost relationship and will yield new perspective for a better understanding of the immunological and pathological aspects of
invasive dermatophytosis.
Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Dermatophytes; Invasive dermatophytosis; Trichophyton mentagrophytes; Cellular immune response; Experimental dermatophytosis

1. Introduction common skin disorder among children younger than 12 and

Dermatophytes are a group of fungi that are able to utilize
keratin as a food source. These fungi are universally distrib-
uted and may be categorized as geophilic, zoophilic or
anthropophilic based on their natural habitats [1]. Dermato-
phytes are among the most common zoonotic agents. Der-
matophytosis affects approximately 20e25% of the world’s
population and are responsible for 30% of all skin fungal
infections; in addition, it is considered to be the third most
* Corresponding author. Tel.: þ55 14 31036078; fax: þ55 14 31036092.
E-mail address: sueli@fc.unesp.br (M.S.P. de Arruda).
the second most common skin disorder in adults [2,3].
Dermatophytosis is not an opportunistic infection and
frequently infects the skin, hairs and nails of healthy humans
and animals, causing superficial infections [4,5]. The clinical
manifestations of dermatophytosis range from mild to severe
based on the host reaction to the fungal metabolic products,
the species of the fungus, the virulence of the fungus and the
anatomic site of infection [6]. Rarely, in immunocompromised
hosts, the infection can disseminate into deeper skin layers and
other organs, resulting in invasive dermatophytosis [7e10].
Experimental models of dermatophytosis have played
a crucial role in assessing the efficacy of antimicrobial agents
[11,12] and in obtaining a better understanding of disease

1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.micinf.2012.07.014
 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151
pathogenesis [13e15]. Guinea pigs and rabbits have been the
most frequently employed model animals in studies of derma-
tophytosis [16]. However, several points remain to be elicited.
One of them is that these experimental models have not been
used to explore the mechanisms involved in immune response
against dermatophytes, mainly due to the limited specific tolls
for these species such as species-specific recombinant protein
and monoclonal antibodies targeting to these species. The other
one refers the question of fungal dissemination to internal
organs. According to Hay & Baran [17], ‘the mechanisms
limiting spread of dermatophyte fungi, under normal circum-
stances, to the epidermis remain elusive. The hypothesis linking
a specific clinical form of dermatophyte nail infection with
lymphatic spread, although intriguing, needs verification’.
Cutaneuos dermatophytosis in guinea pigs and rabbits are self-
limiting, which restricts their usefulness in the investigation of
the aspects of the disseminated infection. In order to overcome
these claims, this study presents a model of invasive dermato-
phytic infection using Swiss mice inoculated subcutaneously
with Trichophyton mentagrophytes. The model mimics the
human condition by presenting the following aspects: 1) spread
of the fungus from prior subcutaneous lesion, 2) involvement of
various internal organs, 3) resolution of infection associated
with development of cellular immune response, 4) in the deep
layer of skin, the acute inflammatory lesion progresses to
granulomatous formations. Once it is well characterized, this
model will allow studies of underling conditions that exhibit
increased susceptibility to dermatophytosis such as diabetes.
2. Material and methods
2.1. Mice
Two-month-old male Swiss mice from the UNESP Animal
House at the Laboratorio de Imunopatologia Experimental
(LIPE) of UNESP e Univ Estadual Paulista, Bauru, SP, Brazil
were housed in groups of three to five mice and were provided
food and water ad libitum. All protocols used were in accor-
dance with the ethical principles for animal research adopted
by the Brazilian College of Animal Experimentation
(COBEA). This study was approved by the Ethical Committee
of Faculdade de Ciências, UNESP e Univ Estadual Paulista.
2.2. T. mentagrophytes inoculum
The T. mentagrophytes strain (2118/99-ILSL) was origi-
nally obtained from the fungal collection of the Lauro de
Souza Lima Institute, Bauru, SP, Brazil. The fungi were
maintained on MycoselÒ agar slants (Difco Laboratories,
Detroit, Michigan, USA) and cultured in the same media for
10 days at 25  C. The fungi were washed carefully by sterile
saline solution. Afterward, the suspension were mixed twice
for 10 s on a vortex-mixer and decanted off for 5 min. The
supernatants were collected and washed twice. Fungal
viability was determined by cotton blue staining, and the
concentrations were adjusted to 5  108 viable conidia of
T. mentagrophytes/ml.
1145
2.3. Experimental design
In the present study we employed the Swiss mice because
this strain is more susceptible to dermatophytes [18], partic-
ularly to T. mentagrophytes [19]. Thus, the mice were injected
in the footpad, subcutaneously, with 0.04 ml of T. menta-
grophytes inoculum (TM group) or sterile phosphate-buffered
saline (PBS), pH 7.4, alone (CTL group). The mice were
sacrificed at 6, 24 and 48 h and 7, 14 and 30 days after the
inoculation.
2.4. Collection of the biological material
Mice were sacrificed via CO2 asphyxiation. Fragments of
the footpad, popliteal lymph nodes, liver, spleen and kidneys
were dissected and submitted to microbiological analyses. The
footpads were submitted to histological analyses by routine
procedures for embedding in paraffin.
2.5. Colony-forming unit (CFU) determination and
frequency of fungal dissemination
Fragments of the footpad, popliteal lymph nodes, liver,
spleen and kidneys were weighed and homogenized in 1 ml of
PBS, and 0.1 ml of the homogenate was cultured on
15  90 mm MycoselÒ agar plates at 25  C for 14 days. Each
sample was assessed in duplicate. Total colonies were counted,
and the results were expressed as the number (log10) of T.
mentagrophytes per gram of tissue.
The frequencies of fungal dissemination were also reported
as the frequencies of T. mentagrophytes-positive mice which
had one or more internal organ affected and exclude footpad/
total number of mice analyzed.
2.6. Histological procedures
Histological sections (4 mm) of mouse footpads were
stained with hematoxylin-eosin (HE) and periodic acid-Schiff
staining (PAS).
2.7. Spleen cell culture
Fragments of the spleen were collected and homogenized in
ice-cold sterile PBS. The red blood cells were lysed with
0.15 M ammonium nitrate. After washing, the cellular
suspension was centrifuged, and the cells were resuspended in
1 ml RPMI-1640 (Nutricell, Campinas, SP, Brazil) containing
10% heat-inactivated fetal calf serum (Gibco BRL, Grand
Island, NY, USA). The cell concentrations were adjusted to
2.0  106 cells/ml, as determined by 0.1% trypan blue stain-
ing. A total of 2.0  105 spleen cells were placed into each
well of 96-well flat-bottom microtiter plates (Costar, Cam-
bridge, MA, USA) and incubated at 37  C in a 5% CO2
humidified chamber. The cells were cultured with or without
10 mg/ml Concanavalin A (Sigma, St. Louis, MO, USA) as an
internal control (data not shown). After 48 h, the cell-free
1146 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151

supernatants were harvested and stored at 80  C for cytokine

analysis.

2.8. Cytokine analyses

The levels of TNF-a, IFN-g and IL-10 were measured in

the cell-free supernatants of the spleen cell cultures using
a cytokine Duo-Set Kit (R&D Systems, Minneapolis, MI,
USA), according to the manufacturer’s instruction. Each
sample was analyzed in duplicate.

2.9. Footpad test

The development of a specific cellular immune response

was evaluated using the footpad test (FPT) as described by
Arruda et al. [20]. We used the exoantigens produced from the
same strain of T. mentagrophytes inoculated in the mice
according Venturini et al. [21]. On the 6th, 13th and 29th days
after inoculation, the mice were injected with the specific
antigen (50 mg of protein /ml, previously standardized e data
not showed) in the opposite footpad. After 24 h, the footpads
were collected and evaluated histologically. Mice were
considered FPT-positive if they showed mononuclear cell
infiltration.

2.10. Ouchterlony double immunodiffusion

The humoral immune response was evaluated based on the

protocol described by Camargo et al. [22] using the T. men-
tagrophytes exoantigens [21].

2.11. Statistical analyses

The microbiological analyses and cytokine production

data were analyzed using the non-parametric KruskaleWallis
test and Dunn’s post-test. All statistical tests were conducted
using GraphPad InStat version 3.0 for Windows (GraphPad
Software, San Diego, California, USA), and a two-tailed P-
value of less than 0.05 was considered to be statistically
significant.
Fig. 1. Microbiological evaluation of the footpads (A), popliteal lymph nodes
3. Results (B) and spleen (C). Swiss mice were inoculated with T. mentagrophytes and
evaluated from 6 h to 30 days after fungal inoculation. The various letters
3.1. Microbiological evaluation indicate the statistical differences between the experimental time points.
KruskaleWallis test; P < 0.05, n ¼ 6.

All TM-mice contained fungi at their sites of inoculation

until day 7. The fungal load began to decrease on day 7,
culminating in fungal clearance by day 30 (Fig. 1AeC).
The data regarding the frequency of mice with fungal
dissemination are summarized in Table 1. Six hours after
fungal inoculation, we observed dissemination of the fungus to
the popliteal lymph nodes, spleen, liver and kidneys. The
fungal dissemination was more frequent in the popliteal lymph
nodes and spleen. The number of mice with affected organs
decreased with time until complete clearance was observed on
day 14 after fungal inoculation.
3.2. Histopathological evaluation
Changes in the footpad inoculation sites were observed as
early as the first few hours after fungal inoculation. These
lesions were characterized by acute inflammatory reactions
leading to suppurative abscess formation. edema and lym-
phohistiocytic infiltrates, with polymorphonuclear leukocytes
being observed at the limit between the abscess and the upper
layer of the skin (Fig. 2B). In the abscesses, hyphae and
conidia were detected by PAS staining (Fig. 3A).
 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151 1147

Table 1 incidence of invasive dermatophytic infection is increased in

Fungal dissemination frequencies in mice inoculated with T. mentagrophytes. immunocompromised patients; however, the infection is often
Microbiological analyses of the popliteal lymph nodes, spleen, liver and
kidneys from 6 h to 30 days after fungal inoculation. The values are reported
undiagnosed, and thus its occurrence may be underestimated.
as the frequencies of T. mentagrophytes-positive mice which had one or more Consequently, little is known about the pathophysiology of
internal organ affected and exclude footpad/total number of mice analyzed. invasive dermatophytosis and its associated immune events.
(n ¼ 6). Because there are considerable difficulties associated with
Organ Infection progress conducting studies in humans, the use of experimental animal
Hours Days models has facilitated researchers’ understanding of the
6 24 48 7 14 30
phenomena involved in infectious diseases. The experimental
fungal spread in dermatophytosis was documented by Cutsen
Popliteal lymph nodes 6/6 4/6 5/6 2/6 0/6 0/6
Spleen 4/6 3/6 3/6 0/6 0/6 0/6
and Jensen [15], and these authors reported that guinea pigs
Liver 2/6 1/6 0/6 0/6 0/6 0/6 and rabbits inoculated intravenously with T. mentagrophytes
Kidneys 1/6 1/6 1/6 0/6 0/6 0/6 contained fungi in their lungs, kidneys and liver. In contrast
with these findings, the spread seen in humans stems from
a pre-existing superficial dermatophyte infection, and there are
After 24 h, the interstitial infiltration was more intense, and no published data indicating unexpected fungemia due to
the epidermis exhibited hyperkeratosis (Fig. 2B). During this dermatophytes [8,17]. In order to better mimic the human
time period, the hyphae and conidia decreased (Fig. 3B). condition, we studied the behavior of the fungus in Swiss mice
At 48 h, the reaction had spread, and the perilesional after introduction into the subcutaneous footpad tissue. We
infiltrates were composed of fibroblasts and mononuclear cells observed that the fungus reached the popliteal lymph nodes,
(Fig. 2C). The fungal pattern at this time point was the same as spleen, liver and kidneys soon after T. mentagrophytes footpad
that observed at 24 h. inoculation. In a review of 41 human cases with invasive
On day 7 and 14, acanthosis was observed in the epidermis, dermatophytosis, the lymph nodes were the most infected sites
and the infiltrated area were enlarged with central necrosis and (17%); however, several other organs and tissues were also
a peripheral granulomatous reaction (Fig. 2D), which was affected by invasive dermatophyte infection, including the
composed of lymphocytes, plasma cells and a small number of bones (7.3%), brain (7.3%), liver (4.9%) and other sites, such
modified macrophages (Fig. 2D). Fungal fragments were seen as spleen, muscle, testis and vertebral joints (2.4% each) [8].
inside the modified macrophages on day 14 (Fig. 3C). Thus, our model more closely mimics what occurs in patients
On day 30, the lesions showed well-formed granulomas with invasive dermatophytosis.
composed of modified macrophage and rare epithelioid cells Histologically, the introduction of subcutaneous T. menta-
surrounded by mononuclear cell infiltrates (Fig. 2E and F). No grophytes in Swiss mice initiates an acute inflammatory
fungal elements were detected by PAS at this time point process followed by a granulomatous reaction. In the human
(Fig. 3D). disease, approximately 44% of the histopathological findings
included granuloma formation [8]. In our experimental model,
3.3. Specific cellular and humoral immune responses the number of mice with affected tissues decreased with time,
culminating in complete clearance by day 14 after fungal
TM-mice showed positive FPTs to specific antigens at 7, 14 inoculation. While spontaneous clearance of fungi from the
and 30 days after infection. Sera from these mice were non- organs has not been observed in the human disease, this
reactive for the IDD test. Both specific cellular and humoral clearance cannot be ruled out because little is known regarding
responses were negative in the CTL-mice. the biology of the dermatophytes in the deeper tissues. Inter-
estingly, several authors have suggested that internal organs
3.4. Cytokine determination by spleen cells could act as reservoirs of fungi in recurrent dermatophytic
infections [15]. Our results do not confirm this premise, as the
In the TM-mice, the TNF-a and IFN-g levels were only internal organs were the first ones to exhibit fungal clearance
lower at 48 h after fungal inoculation (Fig. 4A and Fig. 4B). in our model. Also, it is important to highlight that the normal
Interestingly, the levels of IFN-g were higher after fungal body temperature is one of the factors involved in the death of
clearance than during earlier periods. Conversely, we observed dermatophytes in deep tissue.
high levels of IL-10 soon after introduction of the fungus, and The immunological mechanisms involved in the initiation
over time, the levels of this cytokine decreased to baseline and clearance of systemic dermatophytosis remain poorly
levels (Fig. 4C). understood. Several studies have suggested that the cellular
immune response participates in modulating the disease, as
4. Discussion this response could cause increased epidermal proliferation
and facilitate dermatophyte elimination [review in [24]]. The
Several recent studies have reported that superficial der- data from our study support this premise once that we
matophytic infection may result in complications such as observed epidermal hyperplasia in the early stages of infection
bacterial superinfection in diabetic patients [23] and in inva- and became more expressive with the development of the
sive dermatophytosis [review in 8]. Although rare, the infection and delayed-type hypersensitivity (DTH).
1148 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151

Fig. 2. Histopathological evaluation of the footpad. (A) Normal footpads of Swiss mice inoculated with sterile saline solution and evaluated after 6 h (HE). (B) 6/
24 h. Hyperkeratosis, suppurative abscess formation, edema and lymphohistiocytic infiltrates with polymorphonuclear leukocytes (HE). (C) 48 h. The reaction
spread, and the perilesional infiltrates were composed of fibroblasts and mononuclear cells (HE). (D) Day 7. Acanthosis and enlarged infiltrated areas were
observed with central necrosis and peripheral granulomatous reaction (HE). (E) Day 30. Well-formed granulomas composed of modified macrophages and rare
epithelioid cells (HE). This observation is depicted in detail in Fig. 2F (HE). (F) Details of the a well-formed granuluma (HE).

Despite our findings, clinical and experimental data have
suggested that the immune response to dermatophytes is not
exclusively DTH-dependent. Among patients with AIDS, for
example, the characteristic decrease in CD4þ T lymphocytes
was expected to lead to higher incidences of invasive derma-
tophytosis, but in reality, the frequency is not increased in
these patients. Conversely, invasive dermatophytosis is more
frequently observed in non-AIDS immunocompromised
patients, such as those suffering from atopic disease, leukemia,
diabetes, systemic lupus erythematous, psoriasis and myas-
thenia gravis [8,17], suggesting that the immunological
mechanisms underlying invasive dermatophytosis are
different. Furthermore, patients with chronic dermatophytosis
may present DTH against trichophytin [25]. These findings
have also been confirmed experimentally. While athymic nude
BALB/C mice were resistant to T. mentagrophytes infection
[26], in others strains of mice, the ablation of T-cell mediated
pathways leads to chronic and extensive but not internally
disseminated dermatophytic infection [27].
In order to expand the discussion about this question, in our
experimental model we carried out other immunological
approaches. Thus, we investigated the production of the Th1
 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151
Fig. 3. Microscopic examination of fungi in the footpad. (A) 6 h. Hyphae and conidia were detected in the abscess (PAS). (B) 24 h. Decreased hyphae and conidia
elements (PAS). (C) Day 14. Fungal fragments were seen inside modified macrophages (PAS). (D) Day 30. No detection of fungal elements (PAS).
cytokine IFN-g, the Th2/regulatory IL-10, and the pro-
inflammatory cytokine TNF-a in the cell-free supernatants
of cultured splenocytes. We did not observe altered IFN-g
levels during the early phase of the infection, but it is possible
that these results may be associated with the high levels of IL-
10, which is a downregulator of the Th1 response. Over time,
the response changed, and IL-10 production decreased, while
the levels of IFN-g increased. In the human disease, IFN-g-
positive cells were observed in the upper dermis in lesions in
situ [28]. Interestingly, in our study, the levels of IL-10 were
notably high during the early phase of the infection when the
fungus was present in the viscera, including the spleen. These
results are similar to the findings of Campos et al. [29], who
reported that the interaction of Trichophyton rubrum with
phagocytic peritoneal cells induced production of IL-10. This
finding was also supported by our microbiological analyses of
the infections in our mice, which showed that when the fungal
load decreased, the levels of IL-10 returned to basal levels.
The role of IL-10 in this process remains unclear. In addition
to inducing a Th2-type response, this cytokine plays an
important role in both innate immunity and the regulation of
the immune response. By preventing excessive production of
TNF-a and cytotoxic metabolites, IL-10 prevents the devel-
opment of a destructive inflammatory response and facilitates
the proper development of a specific immune response [30].
With respect to the humoral immune response, we were
unable to determine the presence of specific antibodies in the
sera of our mice. Even considering the low sensitivity of the
1149
method used, the presence of IgG and IgM antibodies specific
for the fungus had been previously demonstrated [30e33].
Similar to other pathologies, the consensus among most
researchers is that specific humoral immunity does not play
a large role in the resistance to fungal nfections. According to
the authors, the humoral response is weak and directed
predominantly against polysaccharide antigens. The fact that
antibody titers were higher in chronic infections suggests that
these antibodies are not effective at controlling these infec-
tions [33e35].
Taken together, the subcutaneous inoculation of Swiss
mice with T. mentagrophytes triggers fungal dissemination to
the internal organs, where the fungus remains until approxi-
mately the seventh day. During this period, the mice assem-
bled a Th1-type immune response and IL-10-mediated
immune regulation. Our lab has initiated studies using this
experimental model to evaluate dermatophytic infection
during a hypoinsulinemia-hyperglycemic (HH) state, a model
for diabetes, as this metabolic disease is associated with
a high incidence of dermatophytic infection [36]. We report
that HH mice exhibited disseminated dermatophytosis
following a delay in the infection outcome and that this der-
matophytosis was associated with a lower percentage of
peripheral blood CD4þ T cells [36]. The data obtained from
our experimental model may provide more information
regarding immunological mechanisms to help researchers
better understand the immunological and pathological aspects
of invasive dermatophytosis.
1150 J. Venturini et al. / Microbes and Infection 14 (2012) 1144e1151
Fig. 4. Evaluation of TNF-a (A), IFN-g (B) and IL-10 (C) levels in the
supernatants of spleen cells maintained in culture for five days. Swiss mice
inoculated with T. mentagrophytes were evaluated from 6 h to 30 days after
fungal infection. The CTL group was composed of animals inoculated with
sterile saline. The various letters indicate the statistical differences between the
experimental time points. KruskaleWallis test; P < 0.05, n ¼ 6.
Acknowledgments
Supported by FAPESP and FUNDUNESP grants.
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