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Infect Dis Obstet Gynecol. 2006; 2006: 61265. Published online 2006 March 7. doi: 10.1155/IDOG/2006/61265. | PMCID: PMC1581471 |
Efficacy of an Immune Modulator in Experimental
Chlamydia trachomatis Infection of the Female
Genital Tract Joseph M. Lyons,1* James I. Ito,1 and Servaas A. Morré2 Department of Infectious Diseases, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA Laboratory of Immunogenetics, Section Immunogenetics of Infectious Diseases, VU University Medical Center, 1007 MB Amsterdam, The Netherlands Received March 8, 2005; Revised March 31, 2005; Accepted April 30, 2005. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. The aim of this study was to
determine if vaginal application of the immune response
modifier imiquimod (Aldara cream, 3M Pharmaceuticals, St Paul,
Minn) would alter the course and/or outcome of female genital
tract infection with a human isolate of Chlamydia
trachomatis in a murine model. Methods. Groups
of CF-1 mice were treated with Aldara on three different
schedules: (1) ongoing beginning 5 days prior to and continuing
through day 5 of infection; (2) a single prophylactic dose 2
hours prior to infection; and (3) therapeutic from day 4
to day 14 of infection. Mice were infected vaginally
with a serovar D strain of C trachomatis, and
monitored by culture to determine the level of shedding and
duration of infection. Results. We observed a
significant reduction in both duration of infection and the level
of shedding during the acute phase in mice treated on an ongoing
basis commencing 5 days prior to infection. There was no effect
with respect to the other regimens.
Conclusion. These results demonstrate that
ongoing Aldara treatment has efficacy and may enhance local innate
immunity which reduces the duration of subsequent infection with
human isolates of C trachomatis in a murine
model of female genital tract infection. Imiquimod is an immune response modifier [ 1] that under the
trade name of Aldara (3M Pharmaceuticals, St Paul, Minn) has FDA
approval for the external treatment of anogenital warts. In
addition to this highly effective
application [ 2], imiquimod has been shown to be effective in
an increasing number of dermatological conditions, including common warts,
actinic keratoses, and basal cell carcinomas, as well as lentigo
malina and molluscum contagiosum [ 3]. In
two other infectious diseases with cutaneous manifestations,
genital herpes and leishmaniasis, it has demonstrated significant
efficacy in animal models [ 4, 5],
although results in humans have been
equivocal [ 6– 9].
In all of these settings,
imiquimod is thought to act by binding independently to toll-like
receptors (TLR)-7 and TLR-8 and activating the signal
transduction cascade that triggers transcription factor NK-kB,
which in turn upregulates production of cytokines and chemokines
that direct a sustained T H1 dominant cellular immune
response [ 10– 12].
Both TLR-7 and TLR-8 have been shown to
be expressed on both human and mouse cells [ 13], making mouse
models of human disease useful tools in the assessment of
the treatment potential of imiquimod in those settings
where such models exist. Chlamydia trachomatis is the most prevalent
sexually transmitted bacterial pathogen in the world, with an
estimated 89 million new cases occurring each year [ 14]. The
significant morbidity associated with the severe sequelae of
C trachomatis genital tract infection in women
has driven efforts to both control the spread of and to
effectively treat those infected with this human pathogen.
Although highly effective antibiotics are available
[ 15, 16],
the incidence has been on the increase since the mid 1990s
[ 14], and despite considerable efforts over the last 20
years, no vaccine candidates have emerged. It is generally
accepted that the immune responses made during persistent and
recurrent infections contribute to the immunopathic nature of the
severe sequelae, which includes pelvic inflammatory disease,
tubal infertility, and ectopic pregnancy
[ 17– 20].
While on the other hand, T H1 dominant immune
responses have been shown to play a role in controlling the
spread of infection within the genital tract and to provide some
level of acquired immunity as evidenced by reduced shedding of
infectious units and shorter duration of infection following
reinfection [ 21]. Taken together, these facts make the use
of immune modulators that enhance local T H1 dominant responses an appealing approach to prophylacticly and/or therapeutically treat C
trachomatis female genital tract infection. Much of our understanding of C trachomatis
female genital tract infection has been derived from
the use of a murine model of noninvasive lower genital tract
infection developed by Tuffrey and Taylor-Robinson [ 22]. In
our laboratory, we use this model and routinely infect mice with
chlamydial strains belonging to the oculogenital biovar of
C trachomatis (serovars D-K), which results in
an infection that in most of its features closely mimics human
infection, both in its course and outcome [ 23]. Using this
model, we assessed the efficacy of imiquimod to alter the course
of infection under three different vaginal application schedules:
(1) ongoing beginning 5 days prior to and continuing through day
5 of infection; (2) a single prophylactic dose 2 hours prior to
infection; and (3) therapeutic from day 4 to day 14 of
infection. Efficacy was evaluated by comparing the susceptibility
to infection, the level of shedding, and the duration of infection
between imiquimod- and placebo-treated groups of mice. Murine model Female CF-1 mice were purchased from
Charles River Laboratories (Wilmigton, Mass)
and were used at 7-8 weeks of age. In order to interrupt the
normal 4-5 day estrous cycle and induce prolonged diestrous and
thus enhance the initial infection rate, progesterone in the form
of medroxyprogesterone acetate (Depo-Proveva, Pharmacia
& Upjohn Co Peapeck, NJ), was administered subcutaneously in
2.5 mg doses, 10 and 3 days prior to infection
[ 22, 23].
Mice were inoculated intravaginally by direct instillation of
10 μL of C trachomatis (serovar D)
bacterial suspension containing 1 × 10 5 inclusion forming units (IFU). The human C trachomatis serovar D
genital isolate was propagated, titrated,
and isolated in cycloheximide-treated McCoy cell
monolayers using standard techniques [ 24]. All experiments were conducted in a BL-2 containment facility in compliance with
animal care regulations and under protocols approved by the
Institutional Research Animal Care Committee. Imiquimod treatment On the days prior to (−) and/or after (+) infection as indicated in the results table, 10 uL of
Aldara diluted 1 : 4 in saline or a placebo
similar in composition to the inactive base used in
Aldara was administered intravaginally. The
following Aldara regimens were tested: (1)
Aldara or base at days −5, −3, −1, +1, +3,
+5; (2) Aldara or base at days +4, +6, +8, +10, +12, +14; (3)
Aldara or base 2 hours prior to infection; and (4)
neither Aldara nor base. Infection monitoring The presence of Chlamydia in the lower genital
tract was determined by culturing the material obtained by
swabbing the vaginal vault and ectocervix with a dacron-tipped
swab that was stored at −70°C in 2-SPA transport medium
until tested. Specimens were plated onto McCoy monolayers in
duplicate microtiter plates, centrifuged, and incubated at
37°C for 72 hours. One plate was then fixed, stained
with iodine and inclusions were enumerated (actual IFU counts in
table), while the other plate was stored at −70°C and used to verify the status of primary culture negative specimens.
A specimen was considered culture positive if inclusions were
observed in either primary or secondary culture. Statistic analysis The duration of infection between groups was analyzed using the
Wilcoxon rank sum test. As displayed in Table 1, we observed a statistically
significant reduction in the median duration of infection in mice
treated on multiple occasions prior to and during the acute phase
of C trachomatis infection, 4 days for
imiquimod-treated animals as compared to 19 days for the placebo
group ( P < 0.5). Remarkably, although no effect was observed on
the incidence of infection, there was a suggestion that the
number of IFU shed during the first round of intracellular
replication (day 2) was reduced when compared to the placebo
group. This is especially marked when the average number of IFU
shed by treated mice with a duration of infection less than or
equal to the median duration of the group is compared to the
average number of IFU shed by the placebo group on day 2, 224 IFU
versus 2010 IFU, respectively. By day 4 the differences between
the ongoing treatment group and placebo group were unequivocal,
with 6 of 8 treated mice being culture negative in primary
culture while all four placebo control mice were primary culture
positive. | Table 1The influence of multiple intravaginal application
regimens on the course of C trachomatis genital
tract infection. |
Following the discontinuation of imiquimod treatment on day 5
postinfection, 5 of 8 treated mice were culture negative for the
duration of the study period, while the remaining 3 mice in
this group had shedding patterns and durations of infection
similar to placebo mice. The other regimens assessed, a single prophylactic application 2
hours prior to infection (data not shown) and a 6-application
therapeutic regimen commencing on day 4 postinfection, had
neither a positive nor negative effect on either the duration of
infection or the pattern of shedding during infection. Ramsey and colleagues [ 25] recently reported in this journal
that imiquimod had no efficacy in modifying the susceptibility to
or course of infection with the mouse pneumonitis biovar (MoPn) of
Chlamydia when administered either orally or
intravaginally in essentially the same murine model of
C trachomatis female genital tract infection
used in the present study. In that study, imiquimod was
administered to groups of inbred BALB/c mice either
orally on five occasions (3 days prior to infection, on the day
of infection, and 1, 3, and 6 days after infection), or
intravaginally on four occasions (2 days prior to infection, and
1, 3, and 6 days after infection). These authors
concluded that although the results do not hold promise for
imiquimod in therapy for chlamydial infection, the efficacy of
imiquimod may have been masked due to the obvious potent
T H1 response that naturally occurs during genital
tract infection with MoPn and/or that the TLR-7 expression was
absent or insufficient within the gastrointestinal and genital
tracts to allow a response to imiquimod. We, on the other hand,
observed a significant reduction in the median duration of
infection and level of shedding in outbred CF-1 mice
treated intravaginally on six occasions (5, 3, and 1 days prior
to infection and 1, 3, and 5 days after infection) with a strain
belonging to the oculogenital biovar of C
trachomatis, the biovar that actually causes human disease.
Based on this result, we would conclude that TLR-7 and/or TLR-8
is expressed in the female mouse genital tract, and that
imiquimod and/or immune modulators as a class might have some,
albeit limited, place in the therapy of chlamydial infection. In
a letter to the Editor, we questioned the utility of MoPn to
serve as a surrogate for the oculogenital biovar and referenced
our finding [ 26], the details of which form the basis of
this report. Although differences in the details of the two studies, such as
mouse strain and intravaginal application schedule, could explain
the contrary results obtained in these two studies, in our opinion
the major difference in experimental design that contributed to
the different outcomes was the selection of the biovar of
Chlamydia against which to assess the efficacy
of this potent immunomodulator. Multiple phenotypic differences
that bear directly on the ability to detect the potential
infection and immunity altering effects of imiquimod have been
reported between C muridarum, MoPn, and the 11
different serovars that comprise the human oculogenital biovar of
C trachomatis [ 27]. Most notable among
these differences are those that arise from variability in both
the unique obligate intracellular developmental cycle
[ 28]
and the degree of tryptophan auxotrophy [ 29] that exists
within the genus Chlamydia. Taken together,
phenotypes associated with these two characteristics of a given
strain define the extent to which interferon-gamma can exert a
nutritional influence on the chlamydial replication cycle via
tryptophan depletion following IDO induction. In the present
context, MoPn has been shown to be significantly less sensitive
to the tryptophan depleting effect of interferon-gamma when
compared in vitro to strains belonging to different serovars of
the oculogenital biovar, including a strain identified as serovar
D [ 30]. In addition, the course and outcome of MoPn
infection in the female mouse genital tract is essentially
independent of interferon-gamma compared to infection with
serovar D, as evidenced by uncontrolled and invasive progression
of disease during serovar D infection in interferon-gamma
deficient mice compared to the controlled and contained course
seen in interferon-gamma sufficient
mice [ 31, 32]. This
latter difference between the biovars is likely to be a
result of a mechanism that involves basic elements of the
T H1 innate response that are induced by
interferon-gamma, including events triggered in the host cell by
IDO-mediated tryptophan depletion
[ 33, 34], as well as events
associated with the significantly more rapid replication and
release kinetics characteristic of the MoPn developmental cycle
[ 28]. This latter phenotypic difference might favor the
escape of MoPn from many of the immediate consequences of the
potent T H1 responses that it induces, effects that
cannot be escaped by the slower replicating strains of the
oculogenital biovar. Imiquimod is thought to exercise its antiviral, antitumor, and
antimicrobial activity through multiple pathways that promote a
T H1 dominant response that augments the
cell-mediated immune responses of both the innate and acquired
immune systems. This is achieved through the induction, via TLR-7
and/or TLR-8, of an array of immune response modifiers, most
notably interferon-γ and tumor necrosis factor-α, as well as interferon-α, G-CSF, GM-CSF, the interleukins,
IL-1, IL-2, IL-6, IL-8, and IL-12, and chemokines such as MIP-1a,
MIP-1b, and MCP-1
[ 1, 35]. In addition,
imiquimod has been shown to induce nitric oxide synthase, enhance antigen
presentation by dendritic cells [ 36], and most recently to
induce apoptosis [ 37]. Many of these elements of innate and
adaptive immunity have been independently assessed in the murine
model used in this study, and some have been shown to play a role
in the protective immunity that follows infection
[ 38]. However, much of this knowledge has been obtained using
MoPn as a surrogate model agent for the oculogenital biovar that
actually causes human disease. Unfortunately, the translational
value of much of this work may be in question as a result of
numerous differences that have been recently reported between MoPn
and the human biovar [ 39]. Two of these differences were
drawn upon to help explain the discrepancy between our results
and those of Ramsey and colleagues, and other
differences might also play a role. In conclusion, using the murine model of human female genital
tract infection and in contrast to the results of Ramsey and
colleagues, we observed that intravaginally applied imiquimod
significantly reduced the median duration of genital tract
infection with a human isolate of C
trachomatis. When administered on multiple occasions, days prior
to and during the acute phase of infection, both the duration of
infection and the level of shedding in some mice were reduced,
perhaps through a mechanism of enhanced local innate immunity.
Understanding the mechanism of this enhanced responsiveness might
provide insight into the complex immunobiology of female genital
tract infection with C trachomatis and may lead
to new prevention and treatment methods. Also, given that acquired
protective immunity to C trachomatis shares many
elements in common with the innate immune responses made during
initial exposure [ 33], one might speculate that imiquimod
could be effective in previously infected women by
promoting an enhanced anemnestic response to
reinfection. It is also interesting to speculate if women using
the multiple application regimen of imiquimod approved for the
treatment of genital warts might realize an alteration in the
cytokine responsiveness within the genital tract and a possible
reduced risk of C trachomatis genital tract
infection. SA Morré is supported by Tramedico BV, The Netherlands, the
Falk Foundation, Germany, the Department of Internal Medicine of
the VU University Medical Center, The Netherlands, and the
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