IAI Accepted Manuscript Posted Online 16 February 2016 Infect. Immun. doi:10.1128/IAI.01544-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
IAI01373-15- Revised
2 3 4
Antonio Cassone1* and Jack D Sobel2
5 6
Experimental models of vaginal candidiasis and their relevance to human
7
candidiasis.
8 9 10
1
Polo d’innovazione della genomica, genetica e biologia, Università di 2
11
Perugia, Perugia, Italy;
Detroit Medical Center , Wayne State University
12
School of Medicine, Detroit, US
13
14 15 16 17 18 19 20 21
*Corresponding Author Prof. Antonio Cassone Polo innovazione GGB Università di Perugia, via Gambuli, Perugia tel 0755858255 [email protected]
22 23
Short title ; Human vs animal models of vaginal candidiasis
24 25 1
26
ABSTRACT
27
Vaginal candidiasis (VVC) is a high incident disease seriously affecting the
28
quality of life of women worldwide, particularly in its chronic, recurrent forms
29
(RVVC), and with no definitive cure or preventive measure. Experimental
30
studies in currently used rat and mouse models of vaginal candidiasis have
31
generated a large mass of data on pathogenicity determinants, inflammation
32
and immune responses of potential importance for the control of human
33
pathology. However, reflection is necessary about the relevance of these
34
rodent models to RVVC. Here we examine the chemical, biochemical and
35
biological factors that determine or contrast the disease in rodent models and
36
in women, and highlight the differences between them. We also appeal for
37
approaches to improve or replace the current models in order to enhance
38
their proximity to human infection.
39
INTRODUCTION
40
Amongst human diseases caused by Candida albicans , vaginal candidiasis,
41
especially in its chronic and recurrent forms (RVVC), is by far the most
42
frequent. Recent epidemiological investigations give a global estimate of
43
RVVC incidence approaching 2% that compares with the highest incidence of
44
any single infectious disease on our planet (1). Although not a lethal disease,
45
the quality of life of young women in their most socially and economically
46
productive period can be truly devastated. Past and recent reviews highlight
47
the dominant signs and symptoms of RVVC that make this chronic disease so 2
48
devastating (2,3). With the above premise, admirable is the magnitude of the
49
efforts made by a number of investigators to identify disease mechanisms
50
and host inflammatory and immune responses in vaginal candidiasis by
51
adopting animal models of the disease. In some cases, the data obtained by
52
the use of the above models directed the investigators toward the choice of
53
vaccine candidates or, more recently, to devise novel therapeutic options
54
based on the control of pathogenic vaginal inflammation (4-8). Overall,
55
research has now progressed to promising future important applications for
56
effective disease control (4). Nonetheless, it appears that some reflection is
57
needed on the true nature of these models with respect to human disease so
58
enhancing the relevance of the current data extrapolations from animal
59
models and better defining the field of potential applications to humans. In
60
this reflection, we will succinctly consider the main factors which influence
61
the success of C.albicans as a vaginal pathogen i.e. estrogens,
62
commensalism, immune-priming-tolerance axis, as well as the biochemical
63
and microbiological properties of the vaginal environment. We will also
64
provide some suggestions that may be useful to overcome the shortcomings
65
of the current models so as to improve or even replace them. A detailed
66
review of the vast literature on RVVC and animal models of vaginal
67
candidiasis is outside the scope of this paper, as is a review of studies made
68
in human reconstituted vaginal epithelial cells. We will not discuss other forms
69
of mucosal candidiasis (oral and intestinal) which differ so much from 3
70
VVC/RVVC in relation to risk factors, pathogenicity determinants and immune
71
responses to infection, although some mentions of them will be made,
72
whenever appropriate. For detailed information in the above areas a number
73
of excellent reviews are available (9-12).This review will focus upon the main
74
contributions on Candida-host relationship in the rodent models of vaginal
75
candidiasis versus clinical disease. A model of experimental vaginal
76
candidiasis in non-human primates (two species of macaques) has been
77
reported (13), but no further attempts were made to study Candida-host
78
relationship in this model (see also below). Here we start with brief historical
79
notes and description of the main features characterizing the two commonly,
80
if not exclusively used vaginal candidiasis models in rats and mice.
81
Short history of rodent models and their main results.
82
The rat model.
83
The rat model of vaginal candidiasis has been adopted since early 1960,
84
mostly with the purpose of assessing the anticandidal activity of new drugs. A
85
substantial contribution to the physiology of the model and its strong
86
dependence on pseudoestrus maintenance was provided first by Sobel and
87
Muller (14), then by Kinsman and Collard (15) who showed the chronic nature
88
of the infection and provided details about ovariectomy and estrogen usage
89
that are required for infection to occur. The burden of infection was assessed
90
by enumeration of colony forming units (cfu) in the vaginal fluid or in excised 4
91
vaginal tissue following an intravaginal Candida challenge. In the experiments
92
conducted by the above investigators there was no evidence of strong
93
leukocyte infiltration in pseudoestrus animals challenged by the fungus, and
94
there were no appreciable or measurable clinical signs of disease. Candida
95
pathogenicity and host responses at vaginal level were mostly addressed by
96
Cassone and collaborators who highlighted the nature of the various host
97
immunoeffectors involved in the acquired protection, and showed the critical
98
role exerted by the secretory aspartyl proteinases (Sap) in this model (7,16-
99
18). An intense hyphal growth with biofilm adhering to the vaginal epithelial
100
cells (VEC) was the dominant cytological aspect of the infection, with
101
evidence for macrophages rather than neutrophil infiltration (19). A detailed
102
description of the model has been reported elsewhere (20). Among the
103
immunoeffectors shown to provide anticandidal protection in this model,
104
antibodies against a major Sap (Sap2) and surface mannoproteins were
105
prevalent, although a degree of protection could be conferred by adoptive
106
transfer of both B and T cells from Candida-immunized rats to naïve animals.
107
Anti-Sap2 antibodies were shown to inhibit adherence and biofilm formation
108
by Candida on the vaginal epithelial surface (7,19). Together with some
109
clinical data from RVVC subjects (16), these studies supported the notion of
110
anti-Candida vaccination in women affected by RVVC (reviewed in 4).
111
The mouse model.
5
112
A second and more popular model of vaginal candidiasis is a murine model,
113
particularly developed and adopted by Fidel and collaborators (21-26).
114
Advantages of this model over the rat one are lower cost, easy handling and
115
large availability of genetically modified animals. In addition, no ovariectomy
116
is necessary for infection, as estrogen administration alone is sufficient for
117
pseudoestrus induction. As in rats, the infection is evaluated and monitored
118
by colony forming counts (cfu) of fungal cells in the vaginal fluid or in the
119
tissue of excised vagina following intravaginal Candida administration. Fidel
120
and collaborators showed the typical estrogen-dependence of Candida
121
infection, the changes of the surface vaginal epithelium caused by the
122
pseudoestrus, the fungal biofilm on vaginal epithelium and the partial
123
refractoriness to a subsequent re-infection, as in rats (22-25). Striking
124
differences from the rat model regarding mechanisms of immune responses
125
and inflammation have been reported in most of the above studies. In mice,
126
the fungal intravaginal challenge causes a strong inflammatory exudate with
127
dominance of polymorphonuclear cells (neutrophils) and the production of
128
neutrophil-chemoattractive chemokines and cytokines by the VEC (25). No
129
signs of induction of adaptive immune responses and protective T cells or
130
antibodies have been reported, rather, a protective state could only be
131
attributed to the absence of inflammatory response (26). Some authors, using
132
variant mouse model have reported the induction of Th17, a particular subset
133
of CD4 T cells and have associated the production of the cytokines IL-17A, 6
134
IL-22 , beta-defensin, and anti-Candida defensive peptides to at least partial
135
control of the infection, with some similarity to oral candidiasis (11,27,28).
136
More recently, the activation of a typical Nod-like receptor Protein 3 (NLRP3)
137
inflammasome-mediated cytokine response has been reported to occur (29-
138
31), and Sap production by C.albicans has been associated to the vaginal
139
inflammation indicating that one or more of these enzymes could indeed be
140
the direct or indirect cause of inflammasome activation in the epithelial cells
141
(29,30,32). Interestingly, the data on the relevant pathogenetic role exerted
142
by the above enzymes in the mouse model are probably the only ones that
143
largely match those reported in the rat model and in women (see above),
144
hence collectively suggesting that C.albicans Sap could indeed play a
145
dominant pathogenic role in vaginal candidiasis. Data by Naglik’s research
146
group, in a similar mouse vaginal infection model, also highlighted the role of
147
some Sap in contributing to infection, in keeping with SAP gene expression
148
in infected women (33-34). Overall, among the many putative virulence
149
factors of C.albicans, including dimorphic yeast to hypha transition, some
150
adhesins ( in particular those of Als family) (6,35) and biofilm formation, Sap
151
have been those most intensely studied and advocated for a role in vaginal
152
candidiasis and perhaps also in some forms of oral infection (36).
153
Factors conditioning or differentially modulating the outcome of
154
infection and host response in rat and mouse vaginal candidiasis.
7
155
Estrogens
156
The most salient, common aspect that generates confidence in the reliability
157
of the models is that rodent models and human infection are stringently
158
estrogen-dependent. Vaginal candidiasis is extremely infrequent in pre-
159
pubertal and, to some extent, in postmenopausal women, and women with a
160
history of infrequent or rare VVC episodes become susceptible following
161
hormone replacement therapy (3). Estrogens exert a multifunctional
162
permissive role for vaginal candidiasis through a number of both host- and
163
Candida-directed effects (reviewed in 4). Both rats and mice must be placed
164
into stable pseudoestrus conditions to establish experimental vaginal
165
infection. In rats this is usually achieved by ovariectomy followed by estrogen
166
treatment although recent studies shows that a unique regimen of estrogen
167
treatment may be sufficient to allow for C.albicans infection (37). Of note,
168
ovariectomy has been shown to cause reduction in the number of the
169
lactobacilli component of the vaginal microbiota (38; see also below).
170
mice, estrogen administration is the standard treatment taking into account
171
the variable susceptibility of different animal strains to estrogens (39-40). In
172
both rodents, pseudoestrus induces stage-dependent, profound changes in
173
the vaginal epithelium, with a keratinized surface to which fungal cells
174
strongly adhere and multiply by hyphal growth and eventually forming a
175
biofilm (19,24). Besides these structure-modifying properties, other estrogen-
176
induced biological activities appear to be of special relevance for VVC/RVVC.
In
8
177
In particular, estradiol has been reported to inhibit the differentiation of Th17
178
cells, a subset of CD4 cells supposedly involved in the mucosal antifungal
179
defense, with stronger evidence, however, in oral and intestinal rather than in
180
vaginal infection (11,27,41,42). Overall, there is evidence that the response to
181
estrogen has similarities in rodents and humans. However, the marked
182
differences between rodent models and humans in other relevant biological
183
aspects need to be considered, as shown below.
184
Commensalism and immune priming
185
C.albicans is a normal, though minor, component of the human microbiota
186
whereas it is absent from microbiota of rodents. Candida commensalism
187
shapes human anti-Candida immunity by priming/boosting a strong memory
188
immunity as evidenced by both T and B cell recall responses to fungal
189
antigens
190
activation of innate anti-Candida immunoeffectors (43-45). It is logical to think
191
that the above broad-spectrum immunity has a role in the resistance of
192
healthy subjects to candidiasis but how and to what degree this role is played
193
at vaginal level is unknown. Women with neutropenia or low level of CD4+ T
194
cells as in AIDS, who suffer of severe systemic or oro-oesophageal
195
candidiasis, respectively, are not at major risk of vaginal candidiasis in the
196
absence of the known typical risk factors for vaginitis such as pregnancy,
197
diabetes, hormone replacement therapy, antibiotic therapy to quote only the
in normal, healthy subjects, perhaps also involving a persistent
9
198
major ones (3,9,10). Therefore, if commensalism-induced immunity has a
199
functional role in vaginal candidiasis, this must be sought for in the unique
200
immunity of the female reproductive tract, not in systemic or other non-vaginal
201
mucosal sites-derived factors. As outlined elsewhere (4), the vagina is indeed
202
a strongly tolerant body site as it must accept both constant (the vaginal
203
microbiota) and occasional (semen and fetal) non-self-material. Hence, a
204
delicate balance is maintained that must take into account two equally
205
essential needs for health of the vagina—immune defense (resistance) and
206
immune tolerance (46). Disease may well occur by loss of tolerance rather
207
than loss of resistance. Commensalism-shaped immunity is likely to
208
participate into specific mechanisms of C.albicans vaginal immune tolerance
209
through a complex set of immune-regulatory responses. Among them, an
210
experimentally-supported concept is that tolerance is regulated by IL-22 and
211
IDO-dependent
212
tolerance can be overwhelmed or by-passed by high burden of fungal cells,
213
particularly in the hyphal forms, in the presence of the afore-mentioned typical
214
predisposing factors to candidal vaginitis. On this basis, a reasonable
215
assumption is that in women with RVVC, the tolerance ‘threshold’ is lower,
216
and more easily surpassed than in women not prone to VVC, possibly
217
because of various disease-predisposing gene polymorphisms or other
218
unknown factors (47). A demonstration that commensalism-immune priming-
219
tolerance axis of the human vagina can be overwhelmed by high burdens of
tryptophan
metabolism
(kynurenins)
(8).
Importantly,
10
220
C. albicans cells was provided by studies performed by Fidel’s research
221
group in human volunteers ( 48; see below for further consideration of these
222
studies).
223
On the other hand, the role of commensalism in vaginal candidiasis is clearly
224
appreciated when the source of infection is considered. There is sufficient
225
evidence to suggest that most of the cases of VVC/RVVC are of endogenous
226
nature, due to vaginal colonization from the intestine or the cervico-vaginal-
227
vulvar areas or skin or even the persistence of low number of fungal cells in
228
the vagina( endogenous infection in Candida-primed ) (3). This makes an
229
important, perhaps critical, difference in comparison to experimental rodent
230
models in which the infection is achieved by direct intravaginal inoculation of
231
high burdens of fungal cells, no signs of infection besides fungus burden are
232
clinically appreciable and the animals are immunologically naïve to the fungus
233
(exogenous infection in Candida-unprimed). While not impossible, it is
234
unlikely that endogenous infection occurs through the introduction of a single
235
bout of fungal cells( in some studies, millions) from the intestine/perivaginal
236
area. Exogenous infection in animals without any pre-existing immunity to
237
Candida can be naturally “pro-inflammatory” particularly under neutral pH
238
condition and in the absence of lactobacilli ( as in mice), all conditions which
239
favor rapid and extensive hyphal development. Recent data suggest that
240
lactate, an abundant product of sugar fermentation in a lactobacilli-dominated
241
vaginal environment, down-modulate pro-inflammatory cytokine production 11
242
(49) .From this point of view, the mouse model would appear to be the most
243
“pro-inflammatory” one and in fact rapid and sustained intravaginal influx of
244
neutrophils accompanied by high levels of inflammasome-dependent IL-1β is
245
typical of this model (29-31). A similar PMN influx is not seen in the rat model
246
which, under estrogen treatment, may have an acidic rather than neutral
247
vaginal pH and hyphae are formed more slowly. Moreover, lactobacilli are
248
present and can play an anti-inflammatory role (50-51). Only limited
249
macrophage infiltration is seen in these Candida-infected animals, cell-
250
mediated, Th1-type responses to C.albicans are present and both pro-
251
inflammatory and anti-inflammatory cytokines are produced during the course
252
of infection (17,18,52). This picture is somewhat closer to human infection,
253
particularly in the Th1-type response (53), provided there is no concomitant
254
bacterial infection.
255
Despite all the above, strong support to the concept that mouse rather than
256
rat models could be a proxy of vaginal candidiasis in women was derived
257
from the already mentioned studies by Fidel’s research group (47) in human
258
volunteers intravaginally challenged with live fungal cells (exogenous
259
infection). In these studies, a correlation was found between fungal
260
intravaginal burden and inflammation, as shown by leukocyte infiltration and
261
induction of a pro-inflammatory cytokine rich-environment. However, the
262
caveat remains that a direct intravaginal challenge with high fungal loads
263
would not reflect the natural source of infection nor the immune responses 12
264
that follow the naturally-acquired infection, particularly in RVVC subjects. In
265
the above-reported exogenous infection there was no influence of the stage
266
of the menstrual cycle, whereas it is well known that in naturally infected
267
women, VVC essentially occurs or worsens in the luteal phase of the cycle
268
when estrogen and progesterone levels rise up (3). Importantly, and in
269
contrast to rodent models, no keratinization of vaginal epithelial surface has
270
ever been reported in women.(3)
271
The vaginal microbiota and pH issues
272
It is generally assumed that the vaginal microbiota exerts a role in the vaginal
273
healthy state and protects against Candida infection, although it is unknown
274
to what extent and by which mechanisms this occurs. In healthy pre-
275
menopausal women, this role is dominantly played by several Lactobacillus
276
spp which produce lactic acid from sugar substrates and so render the
277
vaginal microenvironment typically acidic (54). In a recent study (55), the
278
vaginal microbiota of mice was seen to be mostly composed by
279
Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria,
280
with no evidence of dominant lactobacilli. In contrast, Lactobacillus spp have
281
been reported to be a major component of rat vaginal microflora and can
282
contribute to make the vaginal pH lower than neutral under estrus conditions,
283
although not as uniformly as in humans (56-58). Ovariectomized rats lose the
284
lactobacillus component of the vaginal microbiota but regained its full
285
composition, and its acidic condition, under estrogen treatment (38). Using an 13
286
ex-vivo model of cervico-vaginal epithelium, Abramov and collaborators (50)
287
have recently shown that L.crispatus, a major component of the vaginal
288
microbiota, binds to epithelial cells and induces NF-κB activation but does
289
not induce expression of innate immunity mediators and pro-inflammatory
290
cytokines such as IL-8, IL-1β, IL-1α and TNF-α. L- crispatus 2029 inhibited
291
IL-8 production in epithelial cells and increased production of IL-6 working as
292
anti-inflammatory cytokine, hence maintaining the homeostasis of female
293
reproductive tract.
294
Overall the studies performed so far highlight important differences between
295
rats and mice treated with estrogens with regard to both the pH of the vaginal
296
microenvironment, the vaginal microbiota and overall immune responses.
297
However, rats appear to be less distant than mice from women in some of the
298
above vaginal parameters. In particular, it is difficult to conceive that similar
299
immune-pathogenetic mechanisms are taking place at such different pH
300
values as 4.5 and 7. Nonetheless, we should recognize that the “real” pH of
301
the vaginal micro-niches where infectious foci develop and immunoeffectors
302
are recruited is not known, and could be quite different and dynamically
303
variable from the “static” one measured in the vaginal fluid.
304
Table 1 is a short summary of the main similarities and differences between
305
rat and mouse models, as well as between rodent models and human
306
candidiasis.
307 14
308
Conclusions about the relevance of rodent models to human disease
309
and suggestions for future work.
310 311
The data and considerations made above point out that experimental vaginal
312
candidiasis in rats and, too a greater extent, in mice should be cautiously
313
taken for a proxy of human VVC. This conclusion particularly applies to
314
women
315
vulnerability aspects. This specifically refers to the immune-regulation along
316
the axis of commensalism-immunity-tolerance that is present in women and
317
naturally absent, and not reproduced in rodent models. This does not mean
318
that the pathogenicity and immunological studies done with these models are
319
irrelevant to human candidiasis. There are indeed factors in rodent models
320
that are quite relevant to the control of human infection. These include the
321
expression of some fungus virulence traits (dimorphic transition, biofilm
322
formation, some adhesins and Sap, to quote the major ones) and the capacity
323
of mounting a protective immune response under estrogenic stimulation that
324
bear demonstrated or probable equivalent in human infection. Nonetheless,
325
the data also point out the existence of major differences and gaps of relevant
326
information that need to be filled in order to enhance the proximity and
327
relevance of animal models to disease in women.
328
A point that usually escapes attention is that vaginitis is also defined in
329
women by signs and symptoms such as vulvar erythema, oedema, itching,
with
RVVC
who
express
unique
immunopathogenetic
and
15
330
vaginal discharge and painful sexual intercourse. They can be quantified in
331
women and are absent or not measurable in animals. When signs and
332
symptoms of vaginal candidiasis are present, the mere presence of Candida
333
cells, not their intravaginal burden, is a clinical, diagnostic parameter of
334
infection in women, whereas fungus burden is the end-point metric of animal
335
models. It should be recognized that microbial burden and cytokine detection
336
cannot be taken as equivalent to symptoms and tissue inflammation.
337
In our opinion, the primary challenge to meet is the design of vaginal infection
338
studies in animals colonized by C.albicans, so expressing a primed immunity
339
to this opportunistic pathogen. The ideal one would be a model in non-human
340
primates, being Candida-colonized, hence immune-primed. An attempt of
341
using macaques was made by Fidel’s research group (9) but without a
342
substantial follow-up. Interestingly, a successful infection in Rhesus
343
macaques was not paralleled by disease signs (9). Investigations of vaginal
344
candidiasis in non-human primate models are cost- and labor-demanding,
345
and are not easily approved by institutional boards. In mice, a pioneering
346
study was conducted by Cutler research group showing that animals fed with
347
antibiotic-containing water were stably colonized by the fungus (59). Other
348
studies have largely confirmed and expanded on mechanisms of Candida
349
colonization in these mice, with no data, however, on vaginal colonization and
350
infection (60-63). Since antibiotic treatment is a well-known risk factor for
351
vaginal candidisis in women, this model should be characterized by some 16
352
level of spontaneous susceptibility to vaginal candidiasis .This also applies to
353
the rat model if it could be demonstrated that antibiotic treatment induces
354
persistent and stable vaginal colonization.
355
Nonetheless, the search for other laboratory animals that are naturally and
356
stably colonized without antibiotics would be desirable. Besides non-human
357
primates, a more laboratory-suitable, small animal recently reported to be
358
colonized by the fungus without antibiotic treatment is the piglet, which,
359
importantly, has an intestinal microbiota not dissimilar from the human one
360
(64). No data have been published about the possibility of achieving candidal
361
vaginitis in these animals. Other possibilities to explore are the use of germ-
362
free mice, despite the peculiar conditions imposed by the use of these
363
animals
364
A second issue quite appropriate to address in rodent models is the study of
365
the nature and mechanisms of protection acquired after resolution of the
366
primary vaginal infection by C.albicans. In both rats and mice, healing of the
367
primary infection generates a state of partial refractoriness (resistance) to a
368
second fungal challenge (5,23). This is an important similarity between the
369
two models and has implications for disease epidemiology and vaccination in
370
women. For instance, why among so many women (70-80%) who suffer
371
infrequent Candida vaginitis episodes, only a fraction (5-8%) develop
372
RVVC?. Is post-infection acquired resistance also present in women and
373
what mechanisms are involved? In rats refractoriness and protection are 17
374
highly specific, requires functional T cells and memory compartments, and
375
can be transferred by immune cells and antibodies, thus it is considered as a
376
specific post-infection acquired immunity, a sort of natural vaccination. No
377
such adaptive response appears to be present in mice although some short-
378
term protective “memory” is theoretically possible by activation of local or
379
intravaginally recruited innate immune-effectors such as the macrophages
380
(44,45). All this suggests a priming of anti-Candida immunity caused by the
381
experimental infection. It would be important to know whether this process
382
mimics the natural priming induced by the commensalism and its differences.
383
These studies may reveal important, critical information for progress of anti-
384
Candida vaccination and immunotherapy studies .
385
The third and, perhaps, the most important aspect is encouraging and
386
stimulating
387
experimental microbiologists and clinicians or clinical investigators. There are
388
relatively few data on Candida pathogenicity and anti-Candida immune
389
responses obtained by direct sampling of the vaginal cavity and tissues of
390
Candida-infected women. When feasible, materials from vaginal or cervico-
391
vaginal tissues may provide direct information to compare with the relevant
392
findings from animal studies. Improved studies with ex-vivo tissues (as, for
393
instance, taken from routinary hysterectomy) can usefully add to the already
394
used reconstituted vaginal cell lines (65). Overall, the aforementioned
395
expansion of our model studies can favor
research
programs
with
close
collaborations
between
a more comprehensive 18
396
understanding of the mechanisms controlling vaginal candidiasis and can
397
provide important insight into the relationship between the widely used rodent
398
models and human disease.
399
ACKNOWLEDGEMENTS
400
The Authors wish to thank Anna Maria Marella for secretarial help in the
401
preparation of this manuscript.
402 403
REFERENCES
404 405
1. Foxman,B. R.Muraglia, J.P.Dietz, J.D.Sobel and J.Wagner J.2013.
406
Prevalence recurrent vulvovaginal candidiasis in 5 European countries
407
and thUnited States: results from an internet panel survey. J Low Geni
408
Tract Dis 17:340-345.
409 410 411 412 413 414 415
2. Ringdahl E.N. 2006. Recurrent vulvovaginal candidiasis. Mol Med 103:165–168. 3. Sobel,J.D. 2015. Recurrent vulvovaginal candidiasis. Jul 9. pii: S00029378(15)00716-4. doi: 10.1016/j.ajog.2015.06.067. 4. Cassone,A.2015.
Vulvovaginal
Candida
albicans
infections:
pathogenesis, immunity and vaccine prospects. BJOG 122:785-794. 5. Cassone,
A.
M.Boccanera,
D.Adriani,
G.Santoni
and
F.De
416
Bernardis.1995. Rats clearing a vaginal infection by Candida albicans
417
acquire specific, antibody-mediated resistance to vaginal re-infection.
418
Infect Immun 63: 2619–2624. 19
419
6. Ibrahim, A.S., G.Luo, T. Gebremarian,H. Lee, C.S.Schmidt, J.P.
420
Hennesey, S.W.French, S.G.Filler and J.E.Edwards. 2013, NDV-3
421
protects mice from vulvovaginal candidiasis through T- and B-cell immune
422
response. Vaccine 31: 5549-5556.
423
7. De Bernardis,F., M.Amacker, S.Arancia, S.Sandini, C.Gremion,
424
R.Zurbriggen,C.Moser and A.Cassone.2012.
425
A virosomal vaccine against candidal vaginitis:
426
efficacy and safety profile in animal models. Vaccine 30:4490-4497.
immunogenicity,
427
8. De Luca, A.A. Carvalho , C.Cunha, R.G. Iannitti, L. Pitzurra ,
428
G.Giovannini , A.Mencacci, L. Bartolomei , S. Moretti , C.Massi-
429
Benedetti , D.Fuchs , F.De Bernardis , P.Puccetti P and L.Romani .
430
2013. IL-22 and IDO1 affect immunity and tolerance to murine and
431
human vaginal candidiasis. PLoS Pathog ;9: e1003486.
432 433 434 435 436 437 438 439
9. Naglik,J.R., P.L.Fidel and F.Odds. 2008. Aninal models of mucosal Candida infection. FEMS Microbiol Lett. 283 : 129-139. 10.
Fidel, P.L. 1999. Host defense against oropharyngeal and vaginal
candidiasis : site-specific differences. Rev Iberoam Micol 16 :8-15. 11.
Hernandez-Santos, N. and S.L. Gaffen. 2012. Th17 cells in
immunity to Candida albicans. Cell Host Microbe 11:425-435. 12.
Shaller,
M.
and
G.
Weindl.
2009.
Models
of
oral
and vaginal candidiasis based on in vitro reconstituted human epithelia
20
440
for the study of host-pathogen interactions..Methods Mol Biol 470. 327-
441
345.
442
13.
Steele, C., M.Ratterree
and Fidel PL Jr. 1999. Differential
443
susceptibility of two species of macaques to experimental vaginal
444
candidiasis. J Infect Dis, 180: 802-810.
445
14.
Sobel, J.D. and G. Muller. 1983. Comparison of ketoconazole,
446
Bay N7133, and Bay L9139 in the treatment of experimentalvaginal
447
candidiasis. Antimicrob Agents Chemother. 24: 434-436.
448 449 450
15.
Kinsman, M. and A.E.Collard. 1986. Hormonal Factors in
Vaginal Candidiasis in Rats. Infect Immun 53:498-504. 16.
Cassone, A., F. De Bernardis , F. Mondello , T. Ceddia and L.
451
Agatensi .1987. Evidence for a correlation between proteinase
452
secretion and vulvovaginal candidosis. J Infect Dis.156:777-783.
453
17.
De Bernardis, F., G. Santoni , M. Boccanera, E. Spreghini,
454
D.Adriani,
455
anticandidal immune responses in a rat model of vaginal infection by
456
and protection against Candida albicans. Infect Immun. 68:3297-3304.
457
18.
L.Morelli
and
A.Cassone. 2000.
Local
Santoni, G., M. Boccanera , D. Adriani , R. Lucciarini , C.
458
Amantini , S. Morrone , A. Cassone and F. De Bernardis .2002.
459
Immune cell-mediated protection against vaginal candidiasis: evidence
460
for a major role of vaginal CD4(+) T cells and possible participation of
461
other local lymphocyte effectors. Infect Immun. 70:4791-4797. 21
462
19.
De Bernardis F, H. Liu , R. O'Mahony , R. La Valle S.
463
Bartollino,
464
Basset,
465
Human domain antibodies against virulence traits of Candida albicans
466
inhibit fungus adherence to vaginal epithelium and protect against
467
experimental vaginal candidiasis. J Infect Dis 195:149-157.
468
20.
S. Sandini , S.Grant, N.Brewis, I.Tomlinson, R.C.
J.Holton
,
I.M.Roitt
and
A.
Cassone.
2007.
De Bernardis, F., R. Lorenzini and A. Cassone . 1999. Rat
469
Model of Candida Vaginal Infection. Zak,O., Sande, M.A. (ed) pp.735-
470
740, Elsevier.
471 472 473
21.
Fidel PL Jr. 2007. History and update on host defense against
vaginal candidiasis. Am J Reprod Immunol. 57:2-12. 22.
Fidel, P.L.Jr. J. Cuttright and C. Steele. 2000. Effect of
474
reproductive hormones on experimental vaginal candidiasis. Infect
475
Immun. 68: 651-657.
476
23.
Fidel P.L Jr, M.E.Lynch , D.H. Conaway , L. Tait and
477
J.D.Sobel. 1995. Mice immunized by primary vaginal Candida albicans
478
infection
479
Immun;63,547–553.
480
24.
develop
acquired
vaginal
mucosal
immunity.
Infect
Harriott M.M., E.A. Lilly, T.E. Rodriguez, P.L.Jr Fidel and M.C.
481
Noverr. 2010. Candida albicans forms biofilms onthe vaginal mucosa.
482
Microbiology 156: 3635-44.
22
483
25.
Yano, J., E. Lilly, M. Barousse and P.L.Jr Fidel. 2010. Epithelial
484
cell-derived S100 calcium-binding proteins as key mediators in the
485
hallmark acute neutrophil response during Candida vaginitis. Infect
486
Immun. 78: 5126-5137.
487
26.
Peters, B.M., J.Yano, M.C.Noverr and P.L.Fidel.2014. Candida
488
Vaginitis: When Opportunism Knocks, the Host Responds. PLoS
489
Pathog 10: e1003965.
490
27.
Pietrella, D. A. Rachini , M. Pines ,N. Pandey , P. Mosci , F.
491
Bistoni , C. d'Enfert and A., Vecchiarelli A. 2011.Th17 cells and IL-17
492
in protective immunity to vaginal candidiasis. PLoSOne.6:e22770.doi:
493
10.1371/journal.pone.0022770.
494
28.
Pietrella, D.,N. Pandey , E. Gabrielli, E. Pericolini , S. Perito ,
495
L.Kasper, F. Bistoni, A.Cassone,, B. Hube and A.,Vecchiarelli.
496
2013. Secreted aspartic proteases of Candida albicans activate the
497
NLRP3 inflammasome. Eur J Immunol. 43:679-692.
498
29.
Bruno,V.M, A.C. Shetty , J. Yano , P.L. Fidel Jr, M.C. Noverr
499
andB.M.Peters.2015
500
candidiasis identifies a role for the NLRP3 inflammasome. MBio. 6. pii:
501
e00182-15. doi: 10.1128/mBio.00182-15.
502
30.
Pericolini, E,
Transcriptomic
analysis
of
vulvovaginal
E. Gabrielli , M.Amacker ,L. Kasper , E.
503
Roselletti , E. Luciano , S. Sabbatini , M. Kaeser , C. Moser , B.
504
Hube , A. Vecchiarelli and A. Cassone . 2015. Secretory aspartyl 23
505
proteinases cause vaginitis and can mediate vaginitis caused by
506
Candida albicans in mice. mBio 6:e00724.doi:10.1129/mBio.00724-15.
507
31.
Borghi,M.,A.deLuca,
M.Puccetti,
M.Jaeger,
A.Mencacci,
508
V.Oikonomou, M.Parian, C.Garlanda, S.Moretti, A.Bartoli, J.Sobel,
509
F.L. van de Veerdonk, C.A. Dinarello, M.G.Netea and L.Romani.
510
2015. Pathogenic NLRP3 Inflammasome activity during Candida
511
infection is negatively regulated by IL-22 via activation of NLRC4 and
512
IL-1Ra. Cell Host Microb. 18 : 198-209.
513
32.
Kozik, A.,M. Gogol, O.Bochenska, J. Karlowska-Kuleta,
514
N.Wolak, W. Kamysz, W.Aoki, M. Ueda, A.Faussner and M.Rapala-
515
Kozik. 2015. Kinin release from human kininogen by 10 aspartic
516
proteases produced by pathogenic yeast Candida albicans. BMC
517
Microbiol 15 : 60-74.
518
33.
Naglik, J:R:, D. Moyes, J. Makwana, P. Kanzaria, E.Tsichlaki,
519
G.Weindl,
520
S.J.Challacombe, M.Schaller and
521
expression of the Candida albicans secreted aspartyl proteinase gene
522
family in human oral and vaginal candidiasis. Microbiology 154 : 3266-
523
3280.
524
34.
A.R.
Tappuni,
C.A:
Rodgers,
A.J.Woodman,
B-Hube. 2008. Quantitative
Naglik, J.R., C.A. Rodgers, P.J.Shirlaw, J.I. Dobbie, L.L.
525
Fernandes-Naglik, D.Greenspan, N.Agabian and S.J.Challacombe.
526
2003. Differential expression of of Candida albicans secreted aspartyl 24
527
proteinase and phospholipase B genes in humans correlates with active
528
oral and vaginal infections. J Infect Dis 188: 469-479.
529
35.
Hoyer, L.L., C.B. Green, S.H.Oh and X.Zhao.2009. Discovering the
530
secrets of the Candida albicans agglutinin-like sequence (ALS) gene
531
family--a sticky pursuit. Med Mycol 46 : 1-15.
532 533 534
36.
Kumar, R:, D.Saraswat, S.Tati and M.Edgerton. 2015. Novel
535
Aggregation
536
proteinase Sap6 Mediate Virulence in Oral Candidiasis.
537
Infect Immun 83 :2614-2626
538
37.
Properties
of
Candida
albicans
Carrara, M.A. , L. Donatti , E. Damke, T.I.
secreted
aspartyl
Svidizinski, M.E.
539
Consolaro, and M.R.Batista. 2010. A new model of vaginal infection
540
by Candida albicans in rats. Mycopathologia. 170:331-338.
541
38.
Bezirtzoglou E, C.H. Voidarou, A. P Papadaki, A.Tiuotsias,
542
O.Kotsovolou and O.Konstandi. , 2008 Hormone therapy alters the
543
composition of the vaginal microflora in ovariectomized rats. Microb
544
Ecol. 55:751-759.
545
39.
Clemons K.V, J.L. Spearow , R. Parmar , M. Espiritu and D.A. ,
546
Stevens
2004. Genetic susceptibility of mice to Candida albicans
547
vaginitis correlates with host estrogen sensitivity. Infect Immun.
548
72:4878-80.
25
549
40.
Mosci P, D. Pietrella , G. Ricci , N. Pandey , C. Monari , E.
550
Pericolini E. Gabrielli , S. Perito , F. Bistoni and A. Vecchiarelli .
551
2013. Mouse strain-dependent differences in estrogen sensitivity during
552
vaginal candidiasis. Mycopathologia. 175:1-11.
553
41.
Lasarte S,
D. Elsner ,
M. Guı´a-Gonza´lez,
Ramos-
554
Medina,
555
Fernandez and , M. Relloso M. 2013. Female sex hormones regulate
556
theTh17 immune response to sperm and Candida albicans . Human
557
Reproduction, 28, 3283–3291.
558
42.
S. Sa´nchez-Ramo´n , P. Esponda ,
R.
M.A.Mun˜oz-
Chen, R.Y., Y.M. Fan, Q. Zhang, S. Liu, Q.Li, G.L.Ke, C. Li and
559
Z.You. 2015.Estradiol inhibits Th17 cell differentiation through inhibition
560
of RORγT transcription by recruiting the ERα/REA complex to estrogen
561
response elements of the RORγT promoter. J Immunol. 194:4019-28.
562
43.
Ausiello, C.M., G.C. Spagnoli , M. Boccanera , I.Casalinuovo ,
563
F. Malavasi , C.U. Casciani and A. Cassone .1986. Proliferation of
564
human
565
by Candida albicans and its cell wall fractions. J Med Microbiol. 22:195-
566
202.
567
44.
peripheral
blood
mononuclear
cells
induced
Bistoni F, A. Vecchiarelli , E. Cenci ,P. Puccetti , P. Marconi
568
and A. Cassone A.1986.Evidence for macrophage-mediated protection
569
against lethal Candida albicans infection. Infect.Immun, 51: 668-674.
26
570
45.
Netea, M. G., J. Quintin, and J.W. & van der Meer.
571
2011.Trained immunity: a memory for innate host defense. Cell Host
572
Microbe 9:355–361.
573 574
46.
Sansonetti, P.J. 2011.To be or not to be a pathogen: that is the
mucosally relevant question. Mucosal Immunol 4:8-14..
575 576
47.
Smeekens SP, F.L. van de Veerdonk , B.J. Kullberg and M.G.,
577
Netea .2013. Genetic susceptibility to Candida infections. EMBO Mol
578
Med 5:805-813.
579
48.
Fidel P.L, M. Barousse , T. Espinosa , M. Ficarra , J.
580
Sturtevant , D.H. Martin, A.J. Quayle and K., Dunlap K.2004. An
581
intravaginal live Candida challenge in humans leads to new hypotheses
582
for the immunopathogenesis of vulvovaginal candidiasis. Infect Immun
583
72:2939–2946.
584
49.
Ene, I.V, S.C. Cheng, M.G. Netea and A.J.Brown. 2013. Growth
585
of Candida albicans cells on the physiologically relevant carbon
586
source lactate affects their recognition and phagocytosis by immune
587
cells. Infect Immun 81,238-248.
588
50.
Abramov
V,
V.
R, Suzina
Khlebnikov N, Machulin
V, Kosarev A, Sakulin
I, Bairamova
589
G, Vasilenko
V, Kulikova
590
N, Vasilenko N, Karlyshev A, Uversky V,Chikindas M.L. 2014.
591
Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic 27
592
Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity
593
to Genitourinary Pathogens. Probiotics Antimicrob Proteins. 6,:165-76.
594
51.
Iraporda
C,
A.Errea
A, Romanin
D.E, Cayet
D, Pereyra
595
E, Pignataro O, Sirard JC, Garrote GL, Abraham A.G, Rumbo M.
596
2015. Lactate and short chain fatty acids produced by microbial
597
fermentation downregulate proinflammatory responses in intestinal
598
epithelial cells and myeloid cells. Immunobiol 220:1161-1169.
599
52.
Ji H-x,
Y-l Zou, J-j, Z-r, Duan, X-j. Jia Li, Z. Wang, L.
600
Li, Y.W.LI, G.Y
601
X.R. Dai, L.He,
602
Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects
603
against Candida albicans Vaginitis via Inducing Macrophage M2
604
Polarization. PLoS ONE 8: e56004. doi:10.1371/journal.pone.0056004.
605
53.
Liu
, M.Q.
Tong, X.Y
Li, G.H.,
D.I.Zhang,
Z.Y.Li, C.Cao and Y. Yang. 2013. The Synthetic
Piccinni, M.P., A. Vultaggio, C.Scaletti, C. Livi, M.J.Gomez,
606
M.G.Giudizi, R. Biagiotti, A.Cassone, S. Romagnani and E. Maggi. ,.
607
2002. Type 1 T helper cells specific for Candida albicans antigens in
608
peripheral blood and vaginal mucosa of women with recurrent vaginal
609
candidiasis. J Infect Dis. 186: 87-93.
610
54.
Petrova M.I, E. Lievens , S. Malik , N. Imholz and S. , Lebeer .
611
2015. Lactobacillus species as biomarkers and agents that can promote
612
various aspects of vaginal health. Front. Ph,ysiol. 6 :8-87.
28
613
55.
Barfod,K.K, M.
Roggenbuck
S, Larsen
615
murine lung microbiome
616
and vaginal bacterial communities. BMC Microbiol. 13:303-311. 56.
in
S.J, Krogfelt
.LH, Schjørring
614
617
S.T, Sørensen
M, Hansen
relation
K.A to
2013. the
The
intestinal
Larsen, B. A.J. Markovetz, and R.P. Galask 1976. The bacterial
618
flora of the female rat genital tract. Proc Soc Exp Biol Med. 151:571-
619
574.
620 621 622 623 624
57.
Aisa E.A. 1950 The formation of lactic acid in the vagina of adult
rat. Acta Endocrinol 4:285-290. 58.
Baker, H.J, J.R.Russell Lindsey and S.H., Wesibroth . 1976.
The Laboratory Rat: Biology and Diseases, Volume 1, Academic Press. 59.
Cutler J.E, M. Corti , P. Lambert , M. Ferris and H. Xin. 2011.
625
Horizontal Transmission of Candida albicans and Evidence of a
626
Vaccine Response in Mice Colonizedwith the Fungus. PLoS ONE
627
6:e22030. doi:10.1371/journal.pone.0022030.
628
60.
Vautier, S, R.A. Drummond , K. Chen ,
G.I. Murray , D.
629
Kadosh , A.J.P. Brown , N.A. Gow , D.M. MacCallum , J.K.Kolls
630
and G. Brown .2015. Candida albicans colonization and dissemination
631
from the murine gastrointestinal tract: the influence of morphology and
632
Th17 immunity Cell Microbiol 17: 445–450.
29
633
61.
Andrew, Y and H. Koh
2013. Murine Models of Candida
634
Gastrointestinal Colonization and Dissemination. Eukar.Cell 12, 1416–
635
1422.
636
62.
Fan
D, L.A.
Coughlin
L.A, Neubauer
M.M, Kim
J, Kim
637
M.S, Zhan X Simms-Waldrip T.R, Xie Y, Hooper L.V, Koh A.Y. 2015.
638
Activation
639
inhibits Candida albicans colonization.Nat.Med. . doi: 10.1038/nm.3871.
640
63.
of
HIF-1α
and
LL-37
by
commensal
bacteria
Pande, K, C. Chen and S.M. Noble . 2013. Passage through
641
the mammalian gut triggers a phenotypic switch that promotes Candida
642
albicans commensalism. Nat Genet. 45: 1088–1091.
643
64.
Hoeflinger, J.L., D.A Coleman , S.H. Oh, M.J. Miller and
644
L.L. Hoyer.2014.A piglet model for studying Candida albicans
645
colonization of the human oro-gastrointestinal tract. FEMS Microbiol
646
Lett. 357,10-15.
647
65.
Schaller, M., K. Zakikhani, J.R.Naglik,G.Weindl and B.Hube.
648
2006. Models od oral and vaginal candidiasis based on in vitro
649
reconstituted human epithelia. Nat Protoc 1: 2767-2773.
650 651
30
Table 1. Major factors determining and/or regulating human , rat and mouse vaginal candidiasis Factor(s)
Human
Rat
Mouse
Estrogens dependencea
Yes
Yes
Yes
Candida colonization and immune-primingb
Yes
No
No
Induction of protective Immunity after infection Uncertain resolutionc
Yes
Yes
Induction of protective Immunity after NAl vaccinationd
Yes
Yes
Dominant Inflammatory cells during infectione
Mixed
Macrophages
PMN
Clinical signs of diseasef
Typically present
Absent
Absent
pHg
Acidic
Acidic to neutral In different reports
Neutral
Dominant microbiotah
Lactobacilli
Mixed, lactobacilli
Non-lactobacilli
Y-M transition and biofilmi
Yes
Yes
Yes
Role of Sap in infectionl
Strongly suggested
Yes
Yes
a : see Refs 3,15,20,22,41,42; b:see Refs 42,52,58,61 c: see Refs 5,23,26 d: see Refs 6,7,17,18 e: see Refs 20,51 f: see Refs 2,3,20,21 g: see Refs 3,4,21,50,55-57 h: see Refs 53-55 i: see Refs 1\9.24.26 l: see Refs 16,29,30,33, 34.
l: NA, not applicable, trial ongoing
1
IAI01373-15- Revised
2 3 4
Antonio Cassone1* and Jack D Sobel2
5 6
Experimental models of vaginal candidiasis and their relevance to human
7
candidiasis.
8 9 10
1
Polo d’innovazione della genomica, genetica e biologia, Università di 2
11
Perugia, Perugia, Italy;
Detroit Medical Center , Wayne State University
12
School of Medicine, Detroit, US
13
14 15 16 17 18 19 20 21
*Corresponding Author Prof. Antonio Cassone Polo innovazione GGB Università di Perugia, via Gambuli, Perugia tel 0755858255 [email protected]
22 23
Short title ; Human vs animal models of vaginal candidiasis
24 25 1
26
ABSTRACT
27
Vaginal candidiasis (VVC) is a high incident disease seriously affecting the
28
quality of life of women worldwide, particularly in its chronic, recurrent forms
29
(RVVC), and with no definitive cure or preventive measure. Experimental
30
studies in currently used rat and mouse models of vaginal candidiasis have
31
generated a large mass of data on pathogenicity determinants, inflammation
32
and immune responses of potential importance for the control of human
33
pathology. However, reflection is necessary about the relevance of these
34
rodent models to RVVC. Here we examine the chemical, biochemical and
35
biological factors that determine or contrast the disease in rodent models and
36
in women, and highlight the differences between them. We also appeal for
37
approaches to improve or replace the current models in order to enhance
38
their proximity to human infection.
39
INTRODUCTION
40
Amongst human diseases caused by Candida albicans , vaginal candidiasis,
41
especially in its chronic and recurrent forms (RVVC), is by far the most
42
frequent. Recent epidemiological investigations give a global estimate of
43
RVVC incidence approaching 2% that compares with the highest incidence of
44
any single infectious disease on our planet (1). Although not a lethal disease,
45
the quality of life of young women in their most socially and economically
46
productive period can be truly devastated. Past and recent reviews highlight
47
the dominant signs and symptoms of RVVC that make this chronic disease so 2
48
devastating (2,3). With the above premise, admirable is the magnitude of the
49
efforts made by a number of investigators to identify disease mechanisms
50
and host inflammatory and immune responses in vaginal candidiasis by
51
adopting animal models of the disease. In some cases, the data obtained by
52
the use of the above models directed the investigators toward the choice of
53
vaccine candidates or, more recently, to devise novel therapeutic options
54
based on the control of pathogenic vaginal inflammation (4-8). Overall,
55
research has now progressed to promising future important applications for
56
effective disease control (4). Nonetheless, it appears that some reflection is
57
needed on the true nature of these models with respect to human disease so
58
enhancing the relevance of the current data extrapolations from animal
59
models and better defining the field of potential applications to humans. In
60
this reflection, we will succinctly consider the main factors which influence
61
the success of C.albicans as a vaginal pathogen i.e. estrogens,
62
commensalism, immune-priming-tolerance axis, as well as the biochemical
63
and microbiological properties of the vaginal environment. We will also
64
provide some suggestions that may be useful to overcome the shortcomings
65
of the current models so as to improve or even replace them. A detailed
66
review of the vast literature on RVVC and animal models of vaginal
67
candidiasis is outside the scope of this paper, as is a review of studies made
68
in human reconstituted vaginal epithelial cells. We will not discuss other forms
69
of mucosal candidiasis (oral and intestinal) which differ so much from 3
70
VVC/RVVC in relation to risk factors, pathogenicity determinants and immune
71
responses to infection, although some mentions of them will be made,
72
whenever appropriate. For detailed information in the above areas a number
73
of excellent reviews are available (9-12).This review will focus upon the main
74
contributions on Candida-host relationship in the rodent models of vaginal
75
candidiasis versus clinical disease. A model of experimental vaginal
76
candidiasis in non-human primates (two species of macaques) has been
77
reported (13), but no further attempts were made to study Candida-host
78
relationship in this model (see also below). Here we start with brief historical
79
notes and description of the main features characterizing the two commonly,
80
if not exclusively used vaginal candidiasis models in rats and mice.
81
Short history of rodent models and their main results.
82
The rat model.
83
The rat model of vaginal candidiasis has been adopted since early 1960,
84
mostly with the purpose of assessing the anticandidal activity of new drugs. A
85
substantial contribution to the physiology of the model and its strong
86
dependence on pseudoestrus maintenance was provided first by Sobel and
87
Muller (14), then by Kinsman and Collard (15) who showed the chronic nature
88
of the infection and provided details about ovariectomy and estrogen usage
89
that are required for infection to occur. The burden of infection was assessed
90
by enumeration of colony forming units (cfu) in the vaginal fluid or in excised 4
91
vaginal tissue following an intravaginal Candida challenge. In the experiments
92
conducted by the above investigators there was no evidence of strong
93
leukocyte infiltration in pseudoestrus animals challenged by the fungus, and
94
there were no appreciable or measurable clinical signs of disease. Candida
95
pathogenicity and host responses at vaginal level were mostly addressed by
96
Cassone and collaborators who highlighted the nature of the various host
97
immunoeffectors involved in the acquired protection, and showed the critical
98
role exerted by the secretory aspartyl proteinases (Sap) in this model (7,16-
99
18). An intense hyphal growth with biofilm adhering to the vaginal epithelial
100
cells (VEC) was the dominant cytological aspect of the infection, with
101
evidence for macrophages rather than neutrophil infiltration (19). A detailed
102
description of the model has been reported elsewhere (20). Among the
103
immunoeffectors shown to provide anticandidal protection in this model,
104
antibodies against a major Sap (Sap2) and surface mannoproteins were
105
prevalent, although a degree of protection could be conferred by adoptive
106
transfer of both B and T cells from Candida-immunized rats to naïve animals.
107
Anti-Sap2 antibodies were shown to inhibit adherence and biofilm formation
108
by Candida on the vaginal epithelial surface (7,19). Together with some
109
clinical data from RVVC subjects (16), these studies supported the notion of
110
anti-Candida vaccination in women affected by RVVC (reviewed in 4).
111
The mouse model.
5
112
A second and more popular model of vaginal candidiasis is a murine model,
113
particularly developed and adopted by Fidel and collaborators (21-26).
114
Advantages of this model over the rat one are lower cost, easy handling and
115
large availability of genetically modified animals. In addition, no ovariectomy
116
is necessary for infection, as estrogen administration alone is sufficient for
117
pseudoestrus induction. As in rats, the infection is evaluated and monitored
118
by colony forming counts (cfu) of fungal cells in the vaginal fluid or in the
119
tissue of excised vagina following intravaginal Candida administration. Fidel
120
and collaborators showed the typical estrogen-dependence of Candida
121
infection, the changes of the surface vaginal epithelium caused by the
122
pseudoestrus, the fungal biofilm on vaginal epithelium and the partial
123
refractoriness to a subsequent re-infection, as in rats (22-25). Striking
124
differences from the rat model regarding mechanisms of immune responses
125
and inflammation have been reported in most of the above studies. In mice,
126
the fungal intravaginal challenge causes a strong inflammatory exudate with
127
dominance of polymorphonuclear cells (neutrophils) and the production of
128
neutrophil-chemoattractive chemokines and cytokines by the VEC (25). No
129
signs of induction of adaptive immune responses and protective T cells or
130
antibodies have been reported, rather, a protective state could only be
131
attributed to the absence of inflammatory response (26). Some authors, using
132
variant mouse model have reported the induction of Th17, a particular subset
133
of CD4 T cells and have associated the production of the cytokines IL-17A, 6
134
IL-22 , beta-defensin, and anti-Candida defensive peptides to at least partial
135
control of the infection, with some similarity to oral candidiasis (11,27,28).
136
More recently, the activation of a typical Nod-like receptor Protein 3 (NLRP3)
137
inflammasome-mediated cytokine response has been reported to occur (29-
138
31), and Sap production by C.albicans has been associated to the vaginal
139
inflammation indicating that one or more of these enzymes could indeed be
140
the direct or indirect cause of inflammasome activation in the epithelial cells
141
(29,30,32). Interestingly, the data on the relevant pathogenetic role exerted
142
by the above enzymes in the mouse model are probably the only ones that
143
largely match those reported in the rat model and in women (see above),
144
hence collectively suggesting that C.albicans Sap could indeed play a
145
dominant pathogenic role in vaginal candidiasis. Data by Naglik’s research
146
group, in a similar mouse vaginal infection model, also highlighted the role of
147
some Sap in contributing to infection, in keeping with SAP gene expression
148
in infected women (33-34). Overall, among the many putative virulence
149
factors of C.albicans, including dimorphic yeast to hypha transition, some
150
adhesins ( in particular those of Als family) (6,35) and biofilm formation, Sap
151
have been those most intensely studied and advocated for a role in vaginal
152
candidiasis and perhaps also in some forms of oral infection (36).
153
Factors conditioning or differentially modulating the outcome of
154
infection and host response in rat and mouse vaginal candidiasis.
7
155
Estrogens
156
The most salient, common aspect that generates confidence in the reliability
157
of the models is that rodent models and human infection are stringently
158
estrogen-dependent. Vaginal candidiasis is extremely infrequent in pre-
159
pubertal and, to some extent, in postmenopausal women, and women with a
160
history of infrequent or rare VVC episodes become susceptible following
161
hormone replacement therapy (3). Estrogens exert a multifunctional
162
permissive role for vaginal candidiasis through a number of both host- and
163
Candida-directed effects (reviewed in 4). Both rats and mice must be placed
164
into stable pseudoestrus conditions to establish experimental vaginal
165
infection. In rats this is usually achieved by ovariectomy followed by estrogen
166
treatment although recent studies shows that a unique regimen of estrogen
167
treatment may be sufficient to allow for C.albicans infection (37). Of note,
168
ovariectomy has been shown to cause reduction in the number of the
169
lactobacilli component of the vaginal microbiota (38; see also below).
170
mice, estrogen administration is the standard treatment taking into account
171
the variable susceptibility of different animal strains to estrogens (39-40). In
172
both rodents, pseudoestrus induces stage-dependent, profound changes in
173
the vaginal epithelium, with a keratinized surface to which fungal cells
174
strongly adhere and multiply by hyphal growth and eventually forming a
175
biofilm (19,24). Besides these structure-modifying properties, other estrogen-
176
induced biological activities appear to be of special relevance for VVC/RVVC.
In
8
177
In particular, estradiol has been reported to inhibit the differentiation of Th17
178
cells, a subset of CD4 cells supposedly involved in the mucosal antifungal
179
defense, with stronger evidence, however, in oral and intestinal rather than in
180
vaginal infection (11,27,41,42). Overall, there is evidence that the response to
181
estrogen has similarities in rodents and humans. However, the marked
182
differences between rodent models and humans in other relevant biological
183
aspects need to be considered, as shown below.
184
Commensalism and immune priming
185
C.albicans is a normal, though minor, component of the human microbiota
186
whereas it is absent from microbiota of rodents. Candida commensalism
187
shapes human anti-Candida immunity by priming/boosting a strong memory
188
immunity as evidenced by both T and B cell recall responses to fungal
189
antigens
190
activation of innate anti-Candida immunoeffectors (43-45). It is logical to think
191
that the above broad-spectrum immunity has a role in the resistance of
192
healthy subjects to candidiasis but how and to what degree this role is played
193
at vaginal level is unknown. Women with neutropenia or low level of CD4+ T
194
cells as in AIDS, who suffer of severe systemic or oro-oesophageal
195
candidiasis, respectively, are not at major risk of vaginal candidiasis in the
196
absence of the known typical risk factors for vaginitis such as pregnancy,
197
diabetes, hormone replacement therapy, antibiotic therapy to quote only the
in normal, healthy subjects, perhaps also involving a persistent
9
198
major ones (3,9,10). Therefore, if commensalism-induced immunity has a
199
functional role in vaginal candidiasis, this must be sought for in the unique
200
immunity of the female reproductive tract, not in systemic or other non-vaginal
201
mucosal sites-derived factors. As outlined elsewhere (4), the vagina is indeed
202
a strongly tolerant body site as it must accept both constant (the vaginal
203
microbiota) and occasional (semen and fetal) non-self-material. Hence, a
204
delicate balance is maintained that must take into account two equally
205
essential needs for health of the vagina—immune defense (resistance) and
206
immune tolerance (46). Disease may well occur by loss of tolerance rather
207
than loss of resistance. Commensalism-shaped immunity is likely to
208
participate into specific mechanisms of C.albicans vaginal immune tolerance
209
through a complex set of immune-regulatory responses. Among them, an
210
experimentally-supported concept is that tolerance is regulated by IL-22 and
211
IDO-dependent
212
tolerance can be overwhelmed or by-passed by high burden of fungal cells,
213
particularly in the hyphal forms, in the presence of the afore-mentioned typical
214
predisposing factors to candidal vaginitis. On this basis, a reasonable
215
assumption is that in women with RVVC, the tolerance ‘threshold’ is lower,
216
and more easily surpassed than in women not prone to VVC, possibly
217
because of various disease-predisposing gene polymorphisms or other
218
unknown factors (47). A demonstration that commensalism-immune priming-
219
tolerance axis of the human vagina can be overwhelmed by high burdens of
tryptophan
metabolism
(kynurenins)
(8).
Importantly,
10
220
C. albicans cells was provided by studies performed by Fidel’s research
221
group in human volunteers ( 48; see below for further consideration of these
222
studies).
223
On the other hand, the role of commensalism in vaginal candidiasis is clearly
224
appreciated when the source of infection is considered. There is sufficient
225
evidence to suggest that most of the cases of VVC/RVVC are of endogenous
226
nature, due to vaginal colonization from the intestine or the cervico-vaginal-
227
vulvar areas or skin or even the persistence of low number of fungal cells in
228
the vagina( endogenous infection in Candida-primed ) (3). This makes an
229
important, perhaps critical, difference in comparison to experimental rodent
230
models in which the infection is achieved by direct intravaginal inoculation of
231
high burdens of fungal cells, no signs of infection besides fungus burden are
232
clinically appreciable and the animals are immunologically naïve to the fungus
233
(exogenous infection in Candida-unprimed). While not impossible, it is
234
unlikely that endogenous infection occurs through the introduction of a single
235
bout of fungal cells( in some studies, millions) from the intestine/perivaginal
236
area. Exogenous infection in animals without any pre-existing immunity to
237
Candida can be naturally “pro-inflammatory” particularly under neutral pH
238
condition and in the absence of lactobacilli ( as in mice), all conditions which
239
favor rapid and extensive hyphal development. Recent data suggest that
240
lactate, an abundant product of sugar fermentation in a lactobacilli-dominated
241
vaginal environment, down-modulate pro-inflammatory cytokine production 11
242
(49) .From this point of view, the mouse model would appear to be the most
243
“pro-inflammatory” one and in fact rapid and sustained intravaginal influx of
244
neutrophils accompanied by high levels of inflammasome-dependent IL-1β is
245
typical of this model (29-31). A similar PMN influx is not seen in the rat model
246
which, under estrogen treatment, may have an acidic rather than neutral
247
vaginal pH and hyphae are formed more slowly. Moreover, lactobacilli are
248
present and can play an anti-inflammatory role (50-51). Only limited
249
macrophage infiltration is seen in these Candida-infected animals, cell-
250
mediated, Th1-type responses to C.albicans are present and both pro-
251
inflammatory and anti-inflammatory cytokines are produced during the course
252
of infection (17,18,52). This picture is somewhat closer to human infection,
253
particularly in the Th1-type response (53), provided there is no concomitant
254
bacterial infection.
255
Despite all the above, strong support to the concept that mouse rather than
256
rat models could be a proxy of vaginal candidiasis in women was derived
257
from the already mentioned studies by Fidel’s research group (47) in human
258
volunteers intravaginally challenged with live fungal cells (exogenous
259
infection). In these studies, a correlation was found between fungal
260
intravaginal burden and inflammation, as shown by leukocyte infiltration and
261
induction of a pro-inflammatory cytokine rich-environment. However, the
262
caveat remains that a direct intravaginal challenge with high fungal loads
263
would not reflect the natural source of infection nor the immune responses 12
264
that follow the naturally-acquired infection, particularly in RVVC subjects. In
265
the above-reported exogenous infection there was no influence of the stage
266
of the menstrual cycle, whereas it is well known that in naturally infected
267
women, VVC essentially occurs or worsens in the luteal phase of the cycle
268
when estrogen and progesterone levels rise up (3). Importantly, and in
269
contrast to rodent models, no keratinization of vaginal epithelial surface has
270
ever been reported in women.(3)
271
The vaginal microbiota and pH issues
272
It is generally assumed that the vaginal microbiota exerts a role in the vaginal
273
healthy state and protects against Candida infection, although it is unknown
274
to what extent and by which mechanisms this occurs. In healthy pre-
275
menopausal women, this role is dominantly played by several Lactobacillus
276
spp which produce lactic acid from sugar substrates and so render the
277
vaginal microenvironment typically acidic (54). In a recent study (55), the
278
vaginal microbiota of mice was seen to be mostly composed by
279
Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria,
280
with no evidence of dominant lactobacilli. In contrast, Lactobacillus spp have
281
been reported to be a major component of rat vaginal microflora and can
282
contribute to make the vaginal pH lower than neutral under estrus conditions,
283
although not as uniformly as in humans (56-58). Ovariectomized rats lose the
284
lactobacillus component of the vaginal microbiota but regained its full
285
composition, and its acidic condition, under estrogen treatment (38). Using an 13
286
ex-vivo model of cervico-vaginal epithelium, Abramov and collaborators (50)
287
have recently shown that L.crispatus, a major component of the vaginal
288
microbiota, binds to epithelial cells and induces NF-κB activation but does
289
not induce expression of innate immunity mediators and pro-inflammatory
290
cytokines such as IL-8, IL-1β, IL-1α and TNF-α. L- crispatus 2029 inhibited
291
IL-8 production in epithelial cells and increased production of IL-6 working as
292
anti-inflammatory cytokine, hence maintaining the homeostasis of female
293
reproductive tract.
294
Overall the studies performed so far highlight important differences between
295
rats and mice treated with estrogens with regard to both the pH of the vaginal
296
microenvironment, the vaginal microbiota and overall immune responses.
297
However, rats appear to be less distant than mice from women in some of the
298
above vaginal parameters. In particular, it is difficult to conceive that similar
299
immune-pathogenetic mechanisms are taking place at such different pH
300
values as 4.5 and 7. Nonetheless, we should recognize that the “real” pH of
301
the vaginal micro-niches where infectious foci develop and immunoeffectors
302
are recruited is not known, and could be quite different and dynamically
303
variable from the “static” one measured in the vaginal fluid.
304
Table 1 is a short summary of the main similarities and differences between
305
rat and mouse models, as well as between rodent models and human
306
candidiasis.
307 14
308
Conclusions about the relevance of rodent models to human disease
309
and suggestions for future work.
310 311
The data and considerations made above point out that experimental vaginal
312
candidiasis in rats and, too a greater extent, in mice should be cautiously
313
taken for a proxy of human VVC. This conclusion particularly applies to
314
women
315
vulnerability aspects. This specifically refers to the immune-regulation along
316
the axis of commensalism-immunity-tolerance that is present in women and
317
naturally absent, and not reproduced in rodent models. This does not mean
318
that the pathogenicity and immunological studies done with these models are
319
irrelevant to human candidiasis. There are indeed factors in rodent models
320
that are quite relevant to the control of human infection. These include the
321
expression of some fungus virulence traits (dimorphic transition, biofilm
322
formation, some adhesins and Sap, to quote the major ones) and the capacity
323
of mounting a protective immune response under estrogenic stimulation that
324
bear demonstrated or probable equivalent in human infection. Nonetheless,
325
the data also point out the existence of major differences and gaps of relevant
326
information that need to be filled in order to enhance the proximity and
327
relevance of animal models to disease in women.
328
A point that usually escapes attention is that vaginitis is also defined in
329
women by signs and symptoms such as vulvar erythema, oedema, itching,
with
RVVC
who
express
unique
immunopathogenetic
and
15
330
vaginal discharge and painful sexual intercourse. They can be quantified in
331
women and are absent or not measurable in animals. When signs and
332
symptoms of vaginal candidiasis are present, the mere presence of Candida
333
cells, not their intravaginal burden, is a clinical, diagnostic parameter of
334
infection in women, whereas fungus burden is the end-point metric of animal
335
models. It should be recognized that microbial burden and cytokine detection
336
cannot be taken as equivalent to symptoms and tissue inflammation.
337
In our opinion, the primary challenge to meet is the design of vaginal infection
338
studies in animals colonized by C.albicans, so expressing a primed immunity
339
to this opportunistic pathogen. The ideal one would be a model in non-human
340
primates, being Candida-colonized, hence immune-primed. An attempt of
341
using macaques was made by Fidel’s research group (9) but without a
342
substantial follow-up. Interestingly, a successful infection in Rhesus
343
macaques was not paralleled by disease signs (9). Investigations of vaginal
344
candidiasis in non-human primate models are cost- and labor-demanding,
345
and are not easily approved by institutional boards. In mice, a pioneering
346
study was conducted by Cutler research group showing that animals fed with
347
antibiotic-containing water were stably colonized by the fungus (59). Other
348
studies have largely confirmed and expanded on mechanisms of Candida
349
colonization in these mice, with no data, however, on vaginal colonization and
350
infection (60-63). Since antibiotic treatment is a well-known risk factor for
351
vaginal candidisis in women, this model should be characterized by some 16
352
level of spontaneous susceptibility to vaginal candidiasis .This also applies to
353
the rat model if it could be demonstrated that antibiotic treatment induces
354
persistent and stable vaginal colonization.
355
Nonetheless, the search for other laboratory animals that are naturally and
356
stably colonized without antibiotics would be desirable. Besides non-human
357
primates, a more laboratory-suitable, small animal recently reported to be
358
colonized by the fungus without antibiotic treatment is the piglet, which,
359
importantly, has an intestinal microbiota not dissimilar from the human one
360
(64). No data have been published about the possibility of achieving candidal
361
vaginitis in these animals. Other possibilities to explore are the use of germ-
362
free mice, despite the peculiar conditions imposed by the use of these
363
animals
364
A second issue quite appropriate to address in rodent models is the study of
365
the nature and mechanisms of protection acquired after resolution of the
366
primary vaginal infection by C.albicans. In both rats and mice, healing of the
367
primary infection generates a state of partial refractoriness (resistance) to a
368
second fungal challenge (5,23). This is an important similarity between the
369
two models and has implications for disease epidemiology and vaccination in
370
women. For instance, why among so many women (70-80%) who suffer
371
infrequent Candida vaginitis episodes, only a fraction (5-8%) develop
372
RVVC?. Is post-infection acquired resistance also present in women and
373
what mechanisms are involved? In rats refractoriness and protection are 17
374
highly specific, requires functional T cells and memory compartments, and
375
can be transferred by immune cells and antibodies, thus it is considered as a
376
specific post-infection acquired immunity, a sort of natural vaccination. No
377
such adaptive response appears to be present in mice although some short-
378
term protective “memory” is theoretically possible by activation of local or
379
intravaginally recruited innate immune-effectors such as the macrophages
380
(44,45). All this suggests a priming of anti-Candida immunity caused by the
381
experimental infection. It would be important to know whether this process
382
mimics the natural priming induced by the commensalism and its differences.
383
These studies may reveal important, critical information for progress of anti-
384
Candida vaccination and immunotherapy studies .
385
The third and, perhaps, the most important aspect is encouraging and
386
stimulating
387
experimental microbiologists and clinicians or clinical investigators. There are
388
relatively few data on Candida pathogenicity and anti-Candida immune
389
responses obtained by direct sampling of the vaginal cavity and tissues of
390
Candida-infected women. When feasible, materials from vaginal or cervico-
391
vaginal tissues may provide direct information to compare with the relevant
392
findings from animal studies. Improved studies with ex-vivo tissues (as, for
393
instance, taken from routinary hysterectomy) can usefully add to the already
394
used reconstituted vaginal cell lines (65). Overall, the aforementioned
395
expansion of our model studies can favor
research
programs
with
close
collaborations
between
a more comprehensive 18
396
understanding of the mechanisms controlling vaginal candidiasis and can
397
provide important insight into the relationship between the widely used rodent
398
models and human disease.
399
ACKNOWLEDGEMENTS
400
The Authors wish to thank Anna Maria Marella for secretarial help in the
401
preparation of this manuscript.
402 403
REFERENCES
404 405
1. Foxman,B. R.Muraglia, J.P.Dietz, J.D.Sobel and J.Wagner J.2013.
406
Prevalence recurrent vulvovaginal candidiasis in 5 European countries
407
and thUnited States: results from an internet panel survey. J Low Geni
408
Tract Dis 17:340-345.
409 410 411 412 413 414 415
2. Ringdahl E.N. 2006. Recurrent vulvovaginal candidiasis. Mol Med 103:165–168. 3. Sobel,J.D. 2015. Recurrent vulvovaginal candidiasis. Jul 9. pii: S00029378(15)00716-4. doi: 10.1016/j.ajog.2015.06.067. 4. Cassone,A.2015.
Vulvovaginal
Candida
albicans
infections:
pathogenesis, immunity and vaccine prospects. BJOG 122:785-794. 5. Cassone,
A.
M.Boccanera,
D.Adriani,
G.Santoni
and
F.De
416
Bernardis.1995. Rats clearing a vaginal infection by Candida albicans
417
acquire specific, antibody-mediated resistance to vaginal re-infection.
418
Infect Immun 63: 2619–2624. 19
419
6. Ibrahim, A.S., G.Luo, T. Gebremarian,H. Lee, C.S.Schmidt, J.P.
420
Hennesey, S.W.French, S.G.Filler and J.E.Edwards. 2013, NDV-3
421
protects mice from vulvovaginal candidiasis through T- and B-cell immune
422
response. Vaccine 31: 5549-5556.
423
7. De Bernardis,F., M.Amacker, S.Arancia, S.Sandini, C.Gremion,
424
R.Zurbriggen,C.Moser and A.Cassone.2012.
425
A virosomal vaccine against candidal vaginitis:
426
efficacy and safety profile in animal models. Vaccine 30:4490-4497.
immunogenicity,
427
8. De Luca, A.A. Carvalho , C.Cunha, R.G. Iannitti, L. Pitzurra ,
428
G.Giovannini , A.Mencacci, L. Bartolomei , S. Moretti , C.Massi-
429
Benedetti , D.Fuchs , F.De Bernardis , P.Puccetti P and L.Romani .
430
2013. IL-22 and IDO1 affect immunity and tolerance to murine and
431
human vaginal candidiasis. PLoS Pathog ;9: e1003486.
432 433 434 435 436 437 438 439
9. Naglik,J.R., P.L.Fidel and F.Odds. 2008. Aninal models of mucosal Candida infection. FEMS Microbiol Lett. 283 : 129-139. 10.
Fidel, P.L. 1999. Host defense against oropharyngeal and vaginal
candidiasis : site-specific differences. Rev Iberoam Micol 16 :8-15. 11.
Hernandez-Santos, N. and S.L. Gaffen. 2012. Th17 cells in
immunity to Candida albicans. Cell Host Microbe 11:425-435. 12.
Shaller,
M.
and
G.
Weindl.
2009.
Models
of
oral
and vaginal candidiasis based on in vitro reconstituted human epithelia
20
440
for the study of host-pathogen interactions..Methods Mol Biol 470. 327-
441
345.
442
13.
Steele, C., M.Ratterree
and Fidel PL Jr. 1999. Differential
443
susceptibility of two species of macaques to experimental vaginal
444
candidiasis. J Infect Dis, 180: 802-810.
445
14.
Sobel, J.D. and G. Muller. 1983. Comparison of ketoconazole,
446
Bay N7133, and Bay L9139 in the treatment of experimentalvaginal
447
candidiasis. Antimicrob Agents Chemother. 24: 434-436.
448 449 450
15.
Kinsman, M. and A.E.Collard. 1986. Hormonal Factors in
Vaginal Candidiasis in Rats. Infect Immun 53:498-504. 16.
Cassone, A., F. De Bernardis , F. Mondello , T. Ceddia and L.
451
Agatensi .1987. Evidence for a correlation between proteinase
452
secretion and vulvovaginal candidosis. J Infect Dis.156:777-783.
453
17.
De Bernardis, F., G. Santoni , M. Boccanera, E. Spreghini,
454
D.Adriani,
455
anticandidal immune responses in a rat model of vaginal infection by
456
and protection against Candida albicans. Infect Immun. 68:3297-3304.
457
18.
L.Morelli
and
A.Cassone. 2000.
Local
Santoni, G., M. Boccanera , D. Adriani , R. Lucciarini , C.
458
Amantini , S. Morrone , A. Cassone and F. De Bernardis .2002.
459
Immune cell-mediated protection against vaginal candidiasis: evidence
460
for a major role of vaginal CD4(+) T cells and possible participation of
461
other local lymphocyte effectors. Infect Immun. 70:4791-4797. 21
462
19.
De Bernardis F, H. Liu , R. O'Mahony , R. La Valle S.
463
Bartollino,
464
Basset,
465
Human domain antibodies against virulence traits of Candida albicans
466
inhibit fungus adherence to vaginal epithelium and protect against
467
experimental vaginal candidiasis. J Infect Dis 195:149-157.
468
20.
S. Sandini , S.Grant, N.Brewis, I.Tomlinson, R.C.
J.Holton
,
I.M.Roitt
and
A.
Cassone.
2007.
De Bernardis, F., R. Lorenzini and A. Cassone . 1999. Rat
469
Model of Candida Vaginal Infection. Zak,O., Sande, M.A. (ed) pp.735-
470
740, Elsevier.
471 472 473
21.
Fidel PL Jr. 2007. History and update on host defense against
vaginal candidiasis. Am J Reprod Immunol. 57:2-12. 22.
Fidel, P.L.Jr. J. Cuttright and C. Steele. 2000. Effect of
474
reproductive hormones on experimental vaginal candidiasis. Infect
475
Immun. 68: 651-657.
476
23.
Fidel P.L Jr, M.E.Lynch , D.H. Conaway , L. Tait and
477
J.D.Sobel. 1995. Mice immunized by primary vaginal Candida albicans
478
infection
479
Immun;63,547–553.
480
24.
develop
acquired
vaginal
mucosal
immunity.
Infect
Harriott M.M., E.A. Lilly, T.E. Rodriguez, P.L.Jr Fidel and M.C.
481
Noverr. 2010. Candida albicans forms biofilms onthe vaginal mucosa.
482
Microbiology 156: 3635-44.
22
483
25.
Yano, J., E. Lilly, M. Barousse and P.L.Jr Fidel. 2010. Epithelial
484
cell-derived S100 calcium-binding proteins as key mediators in the
485
hallmark acute neutrophil response during Candida vaginitis. Infect
486
Immun. 78: 5126-5137.
487
26.
Peters, B.M., J.Yano, M.C.Noverr and P.L.Fidel.2014. Candida
488
Vaginitis: When Opportunism Knocks, the Host Responds. PLoS
489
Pathog 10: e1003965.
490
27.
Pietrella, D. A. Rachini , M. Pines ,N. Pandey , P. Mosci , F.
491
Bistoni , C. d'Enfert and A., Vecchiarelli A. 2011.Th17 cells and IL-17
492
in protective immunity to vaginal candidiasis. PLoSOne.6:e22770.doi:
493
10.1371/journal.pone.0022770.
494
28.
Pietrella, D.,N. Pandey , E. Gabrielli, E. Pericolini , S. Perito ,
495
L.Kasper, F. Bistoni, A.Cassone,, B. Hube and A.,Vecchiarelli.
496
2013. Secreted aspartic proteases of Candida albicans activate the
497
NLRP3 inflammasome. Eur J Immunol. 43:679-692.
498
29.
Bruno,V.M, A.C. Shetty , J. Yano , P.L. Fidel Jr, M.C. Noverr
499
andB.M.Peters.2015
500
candidiasis identifies a role for the NLRP3 inflammasome. MBio. 6. pii:
501
e00182-15. doi: 10.1128/mBio.00182-15.
502
30.
Pericolini, E,
Transcriptomic
analysis
of
vulvovaginal
E. Gabrielli , M.Amacker ,L. Kasper , E.
503
Roselletti , E. Luciano , S. Sabbatini , M. Kaeser , C. Moser , B.
504
Hube , A. Vecchiarelli and A. Cassone . 2015. Secretory aspartyl 23
505
proteinases cause vaginitis and can mediate vaginitis caused by
506
Candida albicans in mice. mBio 6:e00724.doi:10.1129/mBio.00724-15.
507
31.
Borghi,M.,A.deLuca,
M.Puccetti,
M.Jaeger,
A.Mencacci,
508
V.Oikonomou, M.Parian, C.Garlanda, S.Moretti, A.Bartoli, J.Sobel,
509
F.L. van de Veerdonk, C.A. Dinarello, M.G.Netea and L.Romani.
510
2015. Pathogenic NLRP3 Inflammasome activity during Candida
511
infection is negatively regulated by IL-22 via activation of NLRC4 and
512
IL-1Ra. Cell Host Microb. 18 : 198-209.
513
32.
Kozik, A.,M. Gogol, O.Bochenska, J. Karlowska-Kuleta,
514
N.Wolak, W. Kamysz, W.Aoki, M. Ueda, A.Faussner and M.Rapala-
515
Kozik. 2015. Kinin release from human kininogen by 10 aspartic
516
proteases produced by pathogenic yeast Candida albicans. BMC
517
Microbiol 15 : 60-74.
518
33.
Naglik, J:R:, D. Moyes, J. Makwana, P. Kanzaria, E.Tsichlaki,
519
G.Weindl,
520
S.J.Challacombe, M.Schaller and
521
expression of the Candida albicans secreted aspartyl proteinase gene
522
family in human oral and vaginal candidiasis. Microbiology 154 : 3266-
523
3280.
524
34.
A.R.
Tappuni,
C.A:
Rodgers,
A.J.Woodman,
B-Hube. 2008. Quantitative
Naglik, J.R., C.A. Rodgers, P.J.Shirlaw, J.I. Dobbie, L.L.
525
Fernandes-Naglik, D.Greenspan, N.Agabian and S.J.Challacombe.
526
2003. Differential expression of of Candida albicans secreted aspartyl 24
527
proteinase and phospholipase B genes in humans correlates with active
528
oral and vaginal infections. J Infect Dis 188: 469-479.
529
35.
Hoyer, L.L., C.B. Green, S.H.Oh and X.Zhao.2009. Discovering the
530
secrets of the Candida albicans agglutinin-like sequence (ALS) gene
531
family--a sticky pursuit. Med Mycol 46 : 1-15.
532 533 534
36.
Kumar, R:, D.Saraswat, S.Tati and M.Edgerton. 2015. Novel
535
Aggregation
536
proteinase Sap6 Mediate Virulence in Oral Candidiasis.
537
Infect Immun 83 :2614-2626
538
37.
Properties
of
Candida
albicans
Carrara, M.A. , L. Donatti , E. Damke, T.I.
secreted
aspartyl
Svidizinski, M.E.
539
Consolaro, and M.R.Batista. 2010. A new model of vaginal infection
540
by Candida albicans in rats. Mycopathologia. 170:331-338.
541
38.
Bezirtzoglou E, C.H. Voidarou, A. P Papadaki, A.Tiuotsias,
542
O.Kotsovolou and O.Konstandi. , 2008 Hormone therapy alters the
543
composition of the vaginal microflora in ovariectomized rats. Microb
544
Ecol. 55:751-759.
545
39.
Clemons K.V, J.L. Spearow , R. Parmar , M. Espiritu and D.A. ,
546
Stevens
2004. Genetic susceptibility of mice to Candida albicans
547
vaginitis correlates with host estrogen sensitivity. Infect Immun.
548
72:4878-80.
25
549
40.
Mosci P, D. Pietrella , G. Ricci , N. Pandey , C. Monari , E.
550
Pericolini E. Gabrielli , S. Perito , F. Bistoni and A. Vecchiarelli .
551
2013. Mouse strain-dependent differences in estrogen sensitivity during
552
vaginal candidiasis. Mycopathologia. 175:1-11.
553
41.
Lasarte S,
D. Elsner ,
M. Guı´a-Gonza´lez,
Ramos-
554
Medina,
555
Fernandez and , M. Relloso M. 2013. Female sex hormones regulate
556
theTh17 immune response to sperm and Candida albicans . Human
557
Reproduction, 28, 3283–3291.
558
42.
S. Sa´nchez-Ramo´n , P. Esponda ,
R.
M.A.Mun˜oz-
Chen, R.Y., Y.M. Fan, Q. Zhang, S. Liu, Q.Li, G.L.Ke, C. Li and
559
Z.You. 2015.Estradiol inhibits Th17 cell differentiation through inhibition
560
of RORγT transcription by recruiting the ERα/REA complex to estrogen
561
response elements of the RORγT promoter. J Immunol. 194:4019-28.
562
43.
Ausiello, C.M., G.C. Spagnoli , M. Boccanera , I.Casalinuovo ,
563
F. Malavasi , C.U. Casciani and A. Cassone .1986. Proliferation of
564
human
565
by Candida albicans and its cell wall fractions. J Med Microbiol. 22:195-
566
202.
567
44.
peripheral
blood
mononuclear
cells
induced
Bistoni F, A. Vecchiarelli , E. Cenci ,P. Puccetti , P. Marconi
568
and A. Cassone A.1986.Evidence for macrophage-mediated protection
569
against lethal Candida albicans infection. Infect.Immun, 51: 668-674.
26
570
45.
Netea, M. G., J. Quintin, and J.W. & van der Meer.
571
2011.Trained immunity: a memory for innate host defense. Cell Host
572
Microbe 9:355–361.
573 574
46.
Sansonetti, P.J. 2011.To be or not to be a pathogen: that is the
mucosally relevant question. Mucosal Immunol 4:8-14..
575 576
47.
Smeekens SP, F.L. van de Veerdonk , B.J. Kullberg and M.G.,
577
Netea .2013. Genetic susceptibility to Candida infections. EMBO Mol
578
Med 5:805-813.
579
48.
Fidel P.L, M. Barousse , T. Espinosa , M. Ficarra , J.
580
Sturtevant , D.H. Martin, A.J. Quayle and K., Dunlap K.2004. An
581
intravaginal live Candida challenge in humans leads to new hypotheses
582
for the immunopathogenesis of vulvovaginal candidiasis. Infect Immun
583
72:2939–2946.
584
49.
Ene, I.V, S.C. Cheng, M.G. Netea and A.J.Brown. 2013. Growth
585
of Candida albicans cells on the physiologically relevant carbon
586
source lactate affects their recognition and phagocytosis by immune
587
cells. Infect Immun 81,238-248.
588
50.
Abramov
V,
V.
R, Suzina
Khlebnikov N, Machulin
V, Kosarev A, Sakulin
I, Bairamova
589
G, Vasilenko
V, Kulikova
590
N, Vasilenko N, Karlyshev A, Uversky V,Chikindas M.L. 2014.
591
Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic 27
592
Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity
593
to Genitourinary Pathogens. Probiotics Antimicrob Proteins. 6,:165-76.
594
51.
Iraporda
C,
A.Errea
A, Romanin
D.E, Cayet
D, Pereyra
595
E, Pignataro O, Sirard JC, Garrote GL, Abraham A.G, Rumbo M.
596
2015. Lactate and short chain fatty acids produced by microbial
597
fermentation downregulate proinflammatory responses in intestinal
598
epithelial cells and myeloid cells. Immunobiol 220:1161-1169.
599
52.
Ji H-x,
Y-l Zou, J-j, Z-r, Duan, X-j. Jia Li, Z. Wang, L.
600
Li, Y.W.LI, G.Y
601
X.R. Dai, L.He,
602
Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects
603
against Candida albicans Vaginitis via Inducing Macrophage M2
604
Polarization. PLoS ONE 8: e56004. doi:10.1371/journal.pone.0056004.
605
53.
Liu
, M.Q.
Tong, X.Y
Li, G.H.,
D.I.Zhang,
Z.Y.Li, C.Cao and Y. Yang. 2013. The Synthetic
Piccinni, M.P., A. Vultaggio, C.Scaletti, C. Livi, M.J.Gomez,
606
M.G.Giudizi, R. Biagiotti, A.Cassone, S. Romagnani and E. Maggi. ,.
607
2002. Type 1 T helper cells specific for Candida albicans antigens in
608
peripheral blood and vaginal mucosa of women with recurrent vaginal
609
candidiasis. J Infect Dis. 186: 87-93.
610
54.
Petrova M.I, E. Lievens , S. Malik , N. Imholz and S. , Lebeer .
611
2015. Lactobacillus species as biomarkers and agents that can promote
612
various aspects of vaginal health. Front. Ph,ysiol. 6 :8-87.
28
613
55.
Barfod,K.K, M.
Roggenbuck
S, Larsen
615
murine lung microbiome
616
and vaginal bacterial communities. BMC Microbiol. 13:303-311. 56.
in
S.J, Krogfelt
.LH, Schjørring
614
617
S.T, Sørensen
M, Hansen
relation
K.A to
2013. the
The
intestinal
Larsen, B. A.J. Markovetz, and R.P. Galask 1976. The bacterial
618
flora of the female rat genital tract. Proc Soc Exp Biol Med. 151:571-
619
574.
620 621 622 623 624
57.
Aisa E.A. 1950 The formation of lactic acid in the vagina of adult
rat. Acta Endocrinol 4:285-290. 58.
Baker, H.J, J.R.Russell Lindsey and S.H., Wesibroth . 1976.
The Laboratory Rat: Biology and Diseases, Volume 1, Academic Press. 59.
Cutler J.E, M. Corti , P. Lambert , M. Ferris and H. Xin. 2011.
625
Horizontal Transmission of Candida albicans and Evidence of a
626
Vaccine Response in Mice Colonizedwith the Fungus. PLoS ONE
627
6:e22030. doi:10.1371/journal.pone.0022030.
628
60.
Vautier, S, R.A. Drummond , K. Chen ,
G.I. Murray , D.
629
Kadosh , A.J.P. Brown , N.A. Gow , D.M. MacCallum , J.K.Kolls
630
and G. Brown .2015. Candida albicans colonization and dissemination
631
from the murine gastrointestinal tract: the influence of morphology and
632
Th17 immunity Cell Microbiol 17: 445–450.
29
633
61.
Andrew, Y and H. Koh
2013. Murine Models of Candida
634
Gastrointestinal Colonization and Dissemination. Eukar.Cell 12, 1416–
635
1422.
636
62.
Fan
D, L.A.
Coughlin
L.A, Neubauer
M.M, Kim
J, Kim
637
M.S, Zhan X Simms-Waldrip T.R, Xie Y, Hooper L.V, Koh A.Y. 2015.
638
Activation
639
inhibits Candida albicans colonization.Nat.Med. . doi: 10.1038/nm.3871.
640
63.
of
HIF-1α
and
LL-37
by
commensal
bacteria
Pande, K, C. Chen and S.M. Noble . 2013. Passage through
641
the mammalian gut triggers a phenotypic switch that promotes Candida
642
albicans commensalism. Nat Genet. 45: 1088–1091.
643
64.
Hoeflinger, J.L., D.A Coleman , S.H. Oh, M.J. Miller and
644
L.L. Hoyer.2014.A piglet model for studying Candida albicans
645
colonization of the human oro-gastrointestinal tract. FEMS Microbiol
646
Lett. 357,10-15.
647
65.
Schaller, M., K. Zakikhani, J.R.Naglik,G.Weindl and B.Hube.
648
2006. Models od oral and vaginal candidiasis based on in vitro
649
reconstituted human epithelia. Nat Protoc 1: 2767-2773.
650 651
30
Table 1. Major factors determining and/or regulating human , rat and mouse vaginal candidiasis Factor(s)
Human
Rat
Mouse
Estrogens dependencea
Yes
Yes
Yes
Candida colonization and immune-primingb
Yes
No
No
Induction of protective Immunity after infection Uncertain resolutionc
Yes
Yes
Induction of protective Immunity after NAl vaccinationd
Yes
Yes
Dominant Inflammatory cells during infectione
Mixed
Macrophages
PMN
Clinical signs of diseasef
Typically present
Absent
Absent
pHg
Acidic
Acidic to neutral In different reports
Neutral
Dominant microbiotah
Lactobacilli
Mixed, lactobacilli
Non-lactobacilli
Y-M transition and biofilmi
Yes
Yes
Yes
Role of Sap in infectionl
Strongly suggested
Yes
Yes
a : see Refs 3,15,20,22,41,42; b:see Refs 42,52,58,61 c: see Refs 5,23,26 d: see Refs 6,7,17,18 e: see Refs 20,51 f: see Refs 2,3,20,21 g: see Refs 3,4,21,50,55-57 h: see Refs 53-55 i: see Refs 1\9.24.26 l: see Refs 16,29,30,33, 34.
l: NA, not applicable, trial ongoing
