# Toll-Like Receptors and Cytokines as Surrogate Biomarkers for Evaluating

Authors: Sadhana M. Gupta; Clara C. Aranha; Madhu C. Mohanty; K. V. R. Reddy
Journal: Mediators of Inflammation
 (2008)

---

## Abstract

Topical microbicides are intended for frequent use by women in reproductive age. Hence, it is essential to evaluate their impact on mucosal immune function in the vagina. In the present study, we evaluated nisin, a naturally occurring antimicrobial peptide (AMP), for its efficacy as an intravaginal microbicide. Its effect on the vaginal immune function was determined by localizing Toll-like receptors (TLRs-3, 9) and cytokines (IL-4, 6 , 10 and TNF-α) in the rabbit cervicovaginal epithelium following intravaginal administration of high dose of nisin gel for 14 consecutive days. The results revealed no alteration in the expression of TLRs and cytokines at both protein and mRNA levels. However, in SDS gel-treated group, the levels were significantly upregulated with the induction of NF-κB signalling cascade. Thus, TLRs and cytokines appear as sensitive indicators for screening immunotoxic potential of candidate microbicides.


---

## Body

## 1. Introduction

Topical microbicides are  being sought for preventing the transmission of human
immunodeficiency virus (HIV) infection and other sexually transmitted
infections (STIs) [1]. The success of anti-HIV/STI microbicides depends on
their ability to maintain natural vaginal defenses, since the stratified
epithelium of human vagina in unison with the microflora provides an efficient
barrier against HIV-1 [2]. It
is known that inflammatory conditions induced by
pathogens or chemical irritants may lead to a higher risk of acquiring and
transmitting HIV-1 due to epithelial disruption and recruitment of HIV-1 by
immune cells [3].During early efforts to
develop safe and effective topical microbicides to eliminate the risk of sexual
transmission of STIs/HIV-1, Nonoxynol-9 (N-9), a chemical-based product,
emerged as the lead candidate due to its potent activity against microbesin
vitro and widespread commercial use as a contraceptive [4, 5]. However,
clinical trials revealed that frequent use of N-9 resulted in inflammatory
lesions and ulceration of the genital tract mucosa creating a direct portal of
entry for HIV-1 [6, 7]. Two other products, cellulose sulfate (6%) and carraguard
also failed as microbicides despite preclinical safety trials (http://www.aidsmap.com).One of the most
promising alternatives to chemical-/detergent-based vaginal microbicides
appears to be the naturally occurring antimicrobial peptides (AMPs) [8, 9].
They represent ancient host defense effector molecules produced by the innate
immune system in response to pathogenic microbes or inflammatory stimuli. The
most extensively characterized AMP is the lantibiotic nisin, a 34 amino acid
cationic, amphiphilic peptide having a molecular mass of  3.5 kDa [10, 11]. Our earlier studies have
shown that intravaginal administration of nisin does not trigger any
inflammatory response in CVE of rabbits [8, 12].In the context of developing
a safe microbicide, it is also essential to determine its interference with the
vaginal mucosal immune function. A remarkable feature of the immune cells of
the vaginal mucosa is their ability to recognize and discriminate invading
microbes from commensal bacteria or inflammatory agents [13, 14]. The vaginal
mucosal immune cells are  equipped with different Toll-like receptors (TLRs), which
belong to a large family of highly conserved proteins that are essential
pathogen-specific recognition sensors of the innate immune system [15–18]. So far, 11
members of this family have been identified on various cells [19–21]. TLR signalling
is a key component of communication between vaginal epithelial cells and
underlying immune cells in the lamina propria [20, 22, 23]. Therefore, we
presumed  that TLRs may act as suitable biomarkers for assessing vaginal
inflammation induced by candidate microbicides.A critical gap in
microbicide development has been caused due to the absence of surrogate safety markers.
Since vaginal microbicides would be used repeatedly over decades, it would be
advantageous to evaluate their effects on vaginal innate immune defense system.
Suitable biomarkers are required to determine the changes that take place in
the mucosal environment at molecular level, including induction of
inflammation or loss of host vaginal defenses, which are not reflected by
histology [23]. A noninvasive method to detect subtle tissue inflammation would
be a helpful adjunct to the histological findings. Considering the pivotal role of TLRs
and cytokines in vaginal mucosal innate immunity, we carried out a study to
determine the vaginal immune response after intravaginal application of nisin
gel.For this study, we selected
TLRs-3 and 9, and cytokines (IL-4, 6, 10, and TNF-α) as they play an indispensable role in host
vaginal innate and adaptive immunity. TLRs-3 and 9 are known to be expressed on
plasma membrane as well as within the  endosomes and  stimulate memory B-cell development,
proliferation, and differentiation. They also induce the secretion of anti-inflammatory cytokines
from mucosal epithelium when they sense inflammatory stimuli [19]. The
objectives of the present study were to (1) examine the effect of repetitive
application of high dose of nisin gel on vaginal mucosal immunity using rabbit
model; (2) investigate changes induced by nisin gel in the TLR and
cytokine milieu of rabbit vagina; and (3) determine the role of TLRs and their
downstream signalling cascade in host vaginal defense mechanism. To the best of
our knowledge, this is the first report to
demonstrate the in vivo
distribution of TLRs and identify putative pathways driven by TLR cytokine
interactions in the CVE of rabbit after intravaginal administration of
microbicide, nisin gel.


## 2. Material and Methods

Nisin  (Sigma Aldrich, St. Louis,
USA, Lot# 075K1355) was purified by gel filtration chromatography on Sephadex G-25 and
RP-HPLC as described [24]. Briefly, after initial
purification by gel filtration chromatography, the lyophilized sample was
acidified with 0.1% trifluoroacetic acid (Merck Ltd, Mumbai, India)
and applied onto a preparative C8 column (Vydac, Ill, USA)
connected to a Dionex HPLC unit. Peptide fractions were eluted in a three-step
gradient of 0.01% trifluoroacetic acid in water and 0.01% trifluoroacetic acid
in 50% acetonitrile (Spectrochem, Mumbai, India): 0–100%
(60 minutes), held at 100% for 5 minutes and brought back to 0% (100-0%). The
individual peptide peaks were collected and their absorbance was monitored at
220 nm using spectrophotometer (Shimadzu, UV-160, and Japan). The positive fractions were
pooled and concentrated by lyophilization. Nisin gel (15.15 mM equivalent to
50 mg) was formulated using 1% polycarbophil as a gelling agent, SDS gel (56 μM
equivalent to 32 μg) in 1% polycarbophil, and placebo gel using 1% polycarbophil.


### 2.1. Animals

Sexually mature female Belgium White Rabbits (mean
age7±1 months; mean body weight, 2.70±0.40 kg) were maintained under
standard laboratory conditions (temperature 20±1C°, relative humidity 50±10%, and 12 hours light:12 hours darkness cycle). Animals were housed
individually in stainless steel cages; food and water were made available ad libitum. The study was approved by
the Ethics Committee of National Institute for Research in Reproductive Health
(NIRRH), Parel, and Mumbai. All measures taken were in accordance with approved
guidelines of Committee for the purpose of Control and Supervision of
Experiments on Animals (CPCSEA), established by Government of India  on animal care.


### 2.2. Intravaginal
Administration of Nisin Gel in Rabbits

Rabbits were divided into
three groups each consisting of six animals. First group of animals received nisin
gel formulation prepared in 1% polycarbophil (15.15 mM in 2 mL of 1%
polycarbophil/day for 14 consecutive days). The second and third groups of
rabbits received SDS gel (56μM in 2 mL of 1% polycarbophil/day/14 consecutive
days) and 2 ml of 1% polycarbophil gel, respectively. The gel formulations were
delivered into the vagina using a 12-cm flexible catheter, inserted up to its 8 cm mark. The body weights of rabbits and clinical observations were recorded
daily, including swollen vulva areas, blood stained urine, and soft stools.On
day 15, the cervicovaginal lavages (CVLs) were collected from placebo and treated rabbits by administering 500μl of saline
and animals were autopsied using CO2 asphyxiation. Samples
were centrifuged at 1000 g for 5 minutes to separate supernatant from cell
debris. The vaginal tissues were slit open ventrally between the urethral orifice
and fornix. Portions of cervicovaginal tissues were fixed in Bouin’s
solution. Paraffin sections of 5 μm thick were cut and utilized for
immunolocalization of TLRs (TLR-3 and 9) and cytokines (IL-4, 6, 10, and TNF-α). The remaining tissues were processed immediately
for flow cytometric evaluation of TLRs and RT-PCR analysis.


### 2.3. Isolation of Rabbit CVE
Cells

CVE tissue was isolated
from three groups of rabbits, cut into small fragments, and pooled (200–300 mg) groupwise
prior to being snap frozen on dry ice and stored in liquid nitrogen until
further analysis. On the day of analysis, cell suspensions were prepared by
enzymatic digestion as described previously [25]. Briefly, fragments of CVE were digested with
50 000 U/mL of trypsin (Sigma Aldrich, St. Louis, USA) in 2.5% (w/v) pancreatin
solution (Sigma) for 1 hour at 4C°, followed by 1 hour digestion
at 22C° with constant rotation (120 rpm). Digested tissues were vortexed to release the
epithelial sheets before passing cell suspension through a 20 μm nylon mesh
(Millipore Corp., Billerica, MA, USA). Cells were aspirated through
20 gauge needles to prepare isolated cells before resuspension in complete
Rosewell Park Memorial Institute-1640 (RPMI-1640) medium (Life Technologies,
Inc., Md, USA) (25 mM HEPES supplemented with 10% fetal bovine serum (Hyclone),
50 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL
penicillin) and then seeded onto Nunc cell culture inserts (diameter, 10 mm,
pore size, 0.4 μm) coated with diluted Matrigel (Sigma) at 300 μl/apical
chamber of the cell culture insert. Inserts were placed into 24 well tissue
culture plates containing 500 μl of culture media per well. The purity of
epithelial cell cultures was evaluated by using anti-CD45 antibody which showed
that CVE cells accounted for more than 90% of the cells present on each insert.


### 2.4. Immunofluourescent (IF) Localization of TLRs

The
purpose of this experiment was to determine the pattern of changes in TLR expression
in CVE of placebo, nisin, and SDS gel-treated rabbits. CVE tissue sections were
deparafinized in xylene, rehydrated in descending grades of alcohol, and
finally washed in PBS (0.1 M PBS, pH 7.4). The nonspecific binding was blocked
with 1:100 dilution of normal goat serum and incubated at 37C° for
1 hour. After washing in PBS, slides were incubated overnight at 4C°
with 1:100 dilution of goat antihuman TLR-3 and TLR-9 antibody (Imgenex)
(cat # SC-8691 for TLR-3 and cat # IMG 305 for TLR-9) in PBS. The sections were
washed in PBS, and incubated with FITC conjugated donkey antigoat secondary
antibody (Sigma Aldrich, St. Louis, USA) at
a dilution of 1:1000 at 37C° for 1 hour in dark. Immunofluorescence
signals were captured using laser scanning confocal microscopy (LSCM) (Zeiss,
510 meta, Germany).


### 2.5. Flow Cytometric Analysis

Flow cytometry was performed
on a FACS vantage flow cytometer (Becton Dickinson, USA)
to evaluate the surface expression of TLR-3 and 9 in CVE cells. Based on
initial optimization studies, the cells (1×106/mL) were washed
briefly in PBS, and nonspecific binding was blocked with 1:100 dilution of
normal goat serum at 37C° for 1 hour. For
intracellular staining, cells were first treated with fixation buffer (4%
pararformaldehyde) followed by permeabilization buffer (0.1% Triton X 100).
After washing with PBS, slides were incubated overnight at 4C° with
1:100 dilution of goat antihuman antibody to TLR-3 and TLR-9 in 1% BSA in
PBS. After washing in PBS, the cells
were incubated at 37C° for 1 hour in dark with FITC conjugated
donkey antigoat secondary antibody at a dilution of 1:1000. After washing five
times in PBS, fluorescence signals for each cell were detected through a 520 nm
argon-ion laser. We included normal goat serum as appropriate –ve control to rule
out any nonspecific binding of antibody through Fc-receptors. Data for a minimum of 10 000
events was collected for each sample. Fluorescence histograms and dot plots
were generated after gating the cell population using cell quest software (http://facs.scripps.edu/).


### 2.6. Immunohistochemical
Localization of Cytokines

The immunohistochemical localization of proinflammatory cytokines (IL-6
and TNF-α) was carried out as per the kit protocol described by
manufacturer (R&D systems, Minn, USA)
(cat # DTA00C for IL-6 and cat # DY206 for TNF-α). The localization of IL-4 and 10 was performed using goat
antihuman primary antibody (Sigma Aldrich, cat # I7526 for IL-4, I5020 for IL-10). Briefly, cervicovaginal tissue sections from
placebo, nisin, and SDS gel-treated rabbits were processed as described above.
The endogenous peroxidase activity was quenched by treating cervicovaginal tissues
with 0.03%H2O2 in methanol for 10 minutes and rinsed in
PBS for 5 minutes. To rule out nonspecific binding, sections were incubated
in normal goat serum instead of primary antibody and considered as negative
controls. After washing with PBS, slides were incubated with respective primary antibodies overnight at 4C°.
Next day, slides were washed twice in PBS and incubated for 1 hour at RT with
1:100 dilution of donkey antigoat secondary antibody conjugated to horse radish
peroxidase (HRP) (Sigma Aldrich, cat # G6638).After washing in PBS, the
sections were incubated for 10 minutes with3-3′diaminobenzidine
tetrahydrochloride (DAB) (Sigma Aldrich, St. Louis, USA)
containing Ni2+ and H2O2 in PBS. The tissue
sections were then briefly rinsed in water, counterstained with hematoxylin.
After dehydration in alcohol series,  sections were cleared in xylene,
mounted with DPX, and visualized using Olympus BX50 microscope.


### 2.7. Image Analysis

Semiquantitative analysis of
the immunoprecipitates of cytokines was performed using image analysis software
Biovis 1.42 as detailed previously [26]. Briefly, four areas from each section
were randomly selected for each animal in the three groups. Images from these
areas were grabbed by the CCD camera.
The integrated optical density (IOD) of each area was calculated for the
brown color immunoprecipitates. Negative control slides were analyzed in a
similar fashion. The mean ± SDs were calculated for
each group.


### 2.8. Measurement of Cytokine
Levels in Cervicovaginal Lavages

Levels of proinflammatory (IL-6
and TNF-α), Th-2 type IL-4, and anti-inflammatory cytokine (IL-10) in
CVLs were measured by ELISA as per the protocol discussed above. Optical densities
of the product formed were measured by using ELISA reader (ELX 800, Bio-TEK
Instruments). Cytokine levels were calculated by quadratic regression analysis
based on logarithmically transformed optical densities. Interference of gel
formulations in cytokine ELISA was ruled out by spiking known concentrations of
standards. Control and treated samples were assayed in duplicate wells and the
experiment was repeated thrice.


### 2.9. Measurement of Phospho-NF-kB
p65 in CVE Cells

The activation of NF-kB signalling cascade in CVE tissue was
determined as per the manufacturer’s protocol using Phospho-NF-kB p65 (Ser536) Sandwich ELISA kit (Cell
Signaling, MA, USA).
Briefly, cell lysates were prepared with lysis buffer (20 mM tris,150 mM NaCl,
1 mM EDTA,1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N′,N′ (EGTA), 1% Triton
X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 Vo4, 1 μg/mL leupeptin) and sonicated on ice. The lysates were centrifuged at 14 000 rpm (Eppendorf 541R) for 10 minutes at 4C° to pellet the cell debris.
The supernatants were collected, aliquoted, and stored at −80C° in
single use aliquots. Cell lysates were added to microwells coated with
Phospho-NF-kB p65 (Ser536) (93H1)
mouse mAb and incubated overnight at 4C°.Following extensive washing
with wash buffer, 100μl of Phospho-NF-kB
p65 rabbit mAb (Cell Signaling, MA, USA, cat # 3033) was added to the wells
and incubated for 1 hour at 37C° to detect the captured Phospho-NF-kB p65 protein. After washing, 100 μl
antirabbit IgG secondary antibody coupled to HRP (Cell Signaling, MA, USA, cat
#7074) was added and incubated at 37C° for 1hour to recognize the
bound antibody. 100 μl of substrate, 3′-3′tetramethylbenzidine (TMB) was added and
incubated for 10 minutes at 37C° to develop blue color. Reaction was
stopped by adding 100 μl of stop solution. The OD of yellow-colored product was
measured at 450 nm by ELISA reader. Each value is the mean ±SD of six
individual observations obtained from three experiments performed on different
days.


### 2.10. RNA Isolation and RT-PCR
Analysis of TLRs, Cytokines, and Defensin-1 Molecules

Total RNA was isolated from
CVE tissue from nisin, SDS, and placebo gel-treated rabbits by sequential
extraction using TriPure reagent according to the manufacturer’s protocol (Roche, Germany).
RNA was quantitated spectrophotometrically at 260 and 280 nm (UV-160A, Shimadzu, Japan).
The quality and integrity of RNA was determined by 1.5% denaturing formaldehyde
agarose gel electrophoresis and used for semiquantitative RT-PCR. Prior to
amplification, all RNA samples were treated with RNase-free DNase-I (Qiagen) to
preclude genomic  DNA contamination. Briefly, the reaction was carried out in a
PTC-200 thermal cycler (MJ Research, Maryland, USA) in a total volume of 50μl reaction mixture containing 10 μl 5X RT-PCR buffer, 2 μl dNTP mixture, 1 μl enzyme mix, 2 μl of 0.4 μM each primer, 1 μl total RNA (2 μg), 1 μl DTT solution, and 31 μl RNAse free water. RT PCR to synthesize cDNA
was carried out at 50C° for 30 minutes followed by amplification using gene-specific
primers (Table 1) for 30–40 cycles at 94C°
for 30 seconds, annealing at 55–64C° for 30 seconds,
extension at 68C° for 40 seconds followed by a final extension at 68C° for 7
minutes. Reaction was amplified in the absence of reverse transcriptase as
negative control. A nontemplate control reaction was also included to ensure
lack of DNA contamination. Housekeeping gene, GAPDH
served as
internal loading control. PCR products were electrophoresed through 1.5%
agarose gel, stained with EtBr (0.5 μg/mL) (Sigma Aldrich, St. Louis, USA),
visualized by UV transillumination, and imaged using
Gel Doc system (Bio-Rad, Calif, USA).Table 1
Oligonucleotide primers used for the study.
GeneAccession NoPrimer sequence(5′→3′)Product size (bp)Annealing temperatureIL-4DQ_852343F-AGTTCTACCTCCACCACAAGGT21260C°R-TCAGCTCTGACGCTTTGAGTATIL-6NM_001082064F-AGGAGCTGAGGAAAGAGATGTG22558C°R-TGTTTTCTTCGTCACTCCTGAAIL-10NM_001082045F-TTCTTTCAATCGAAGGATCAGC26759C°R-CTCATTCATGGCTTTGTAGACGTNF-αID_AB128153F-CTAGCCCACGTAGTAGCAAACC15960C°R-GCTGAAGAGAACCTGGGAGTAGTLR-3NM_003265F-GAT CTG TCT CAT AAT GGC TTG30560C°R-GAC AGA TTC CGA ATG CTT GTGTLR-9NM_017442F-TTC CCT GTA GCT GCT GTC C20759C°R-ACA GCC AGT TGC AGT TCA CCDefensin-1NM_001082299F-CTCTGCTTGCTGCCATTCTC19660C°R-AATCGTCTGCAAGTACAGGACACGAPDHNM_01082253F-CCATTCATTGACCTCCACTACA19959C°R-CGTTGCTGACAATCTTGAGAGA


### 2.11. Statistical Analysis

In
most instances, the results were expressed as mean ± standard deviations. Statistically, significant differences between placebo and
treatment groups were assessed using analysis of variance ort test, and differences were considered
significant if  p values were <.05.


## 2.1. Animals

Sexually mature female Belgium White Rabbits (mean
age7±1 months; mean body weight, 2.70±0.40 kg) were maintained under
standard laboratory conditions (temperature 20±1C°, relative humidity 50±10%, and 12 hours light:12 hours darkness cycle). Animals were housed
individually in stainless steel cages; food and water were made available ad libitum. The study was approved by
the Ethics Committee of National Institute for Research in Reproductive Health
(NIRRH), Parel, and Mumbai. All measures taken were in accordance with approved
guidelines of Committee for the purpose of Control and Supervision of
Experiments on Animals (CPCSEA), established by Government of India  on animal care.


## 2.2. Intravaginal
Administration of Nisin Gel in Rabbits

Rabbits were divided into
three groups each consisting of six animals. First group of animals received nisin
gel formulation prepared in 1% polycarbophil (15.15 mM in 2 mL of 1%
polycarbophil/day for 14 consecutive days). The second and third groups of
rabbits received SDS gel (56μM in 2 mL of 1% polycarbophil/day/14 consecutive
days) and 2 ml of 1% polycarbophil gel, respectively. The gel formulations were
delivered into the vagina using a 12-cm flexible catheter, inserted up to its 8 cm mark. The body weights of rabbits and clinical observations were recorded
daily, including swollen vulva areas, blood stained urine, and soft stools.On
day 15, the cervicovaginal lavages (CVLs) were collected from placebo and treated rabbits by administering 500μl of saline
and animals were autopsied using CO2 asphyxiation. Samples
were centrifuged at 1000 g for 5 minutes to separate supernatant from cell
debris. The vaginal tissues were slit open ventrally between the urethral orifice
and fornix. Portions of cervicovaginal tissues were fixed in Bouin’s
solution. Paraffin sections of 5 μm thick were cut and utilized for
immunolocalization of TLRs (TLR-3 and 9) and cytokines (IL-4, 6, 10, and TNF-α). The remaining tissues were processed immediately
for flow cytometric evaluation of TLRs and RT-PCR analysis.


## 2.3. Isolation of Rabbit CVE
Cells

CVE tissue was isolated
from three groups of rabbits, cut into small fragments, and pooled (200–300 mg) groupwise
prior to being snap frozen on dry ice and stored in liquid nitrogen until
further analysis. On the day of analysis, cell suspensions were prepared by
enzymatic digestion as described previously [25]. Briefly, fragments of CVE were digested with
50 000 U/mL of trypsin (Sigma Aldrich, St. Louis, USA) in 2.5% (w/v) pancreatin
solution (Sigma) for 1 hour at 4C°, followed by 1 hour digestion
at 22C° with constant rotation (120 rpm). Digested tissues were vortexed to release the
epithelial sheets before passing cell suspension through a 20 μm nylon mesh
(Millipore Corp., Billerica, MA, USA). Cells were aspirated through
20 gauge needles to prepare isolated cells before resuspension in complete
Rosewell Park Memorial Institute-1640 (RPMI-1640) medium (Life Technologies,
Inc., Md, USA) (25 mM HEPES supplemented with 10% fetal bovine serum (Hyclone),
50 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL
penicillin) and then seeded onto Nunc cell culture inserts (diameter, 10 mm,
pore size, 0.4 μm) coated with diluted Matrigel (Sigma) at 300 μl/apical
chamber of the cell culture insert. Inserts were placed into 24 well tissue
culture plates containing 500 μl of culture media per well. The purity of
epithelial cell cultures was evaluated by using anti-CD45 antibody which showed
that CVE cells accounted for more than 90% of the cells present on each insert.


## 2.4. Immunofluourescent (IF) Localization of TLRs

The
purpose of this experiment was to determine the pattern of changes in TLR expression
in CVE of placebo, nisin, and SDS gel-treated rabbits. CVE tissue sections were
deparafinized in xylene, rehydrated in descending grades of alcohol, and
finally washed in PBS (0.1 M PBS, pH 7.4). The nonspecific binding was blocked
with 1:100 dilution of normal goat serum and incubated at 37C° for
1 hour. After washing in PBS, slides were incubated overnight at 4C°
with 1:100 dilution of goat antihuman TLR-3 and TLR-9 antibody (Imgenex)
(cat # SC-8691 for TLR-3 and cat # IMG 305 for TLR-9) in PBS. The sections were
washed in PBS, and incubated with FITC conjugated donkey antigoat secondary
antibody (Sigma Aldrich, St. Louis, USA) at
a dilution of 1:1000 at 37C° for 1 hour in dark. Immunofluorescence
signals were captured using laser scanning confocal microscopy (LSCM) (Zeiss,
510 meta, Germany).


## 2.5. Flow Cytometric Analysis

Flow cytometry was performed
on a FACS vantage flow cytometer (Becton Dickinson, USA)
to evaluate the surface expression of TLR-3 and 9 in CVE cells. Based on
initial optimization studies, the cells (1×106/mL) were washed
briefly in PBS, and nonspecific binding was blocked with 1:100 dilution of
normal goat serum at 37C° for 1 hour. For
intracellular staining, cells were first treated with fixation buffer (4%
pararformaldehyde) followed by permeabilization buffer (0.1% Triton X 100).
After washing with PBS, slides were incubated overnight at 4C° with
1:100 dilution of goat antihuman antibody to TLR-3 and TLR-9 in 1% BSA in
PBS. After washing in PBS, the cells
were incubated at 37C° for 1 hour in dark with FITC conjugated
donkey antigoat secondary antibody at a dilution of 1:1000. After washing five
times in PBS, fluorescence signals for each cell were detected through a 520 nm
argon-ion laser. We included normal goat serum as appropriate –ve control to rule
out any nonspecific binding of antibody through Fc-receptors. Data for a minimum of 10 000
events was collected for each sample. Fluorescence histograms and dot plots
were generated after gating the cell population using cell quest software (http://facs.scripps.edu/).


## 2.6. Immunohistochemical
Localization of Cytokines

The immunohistochemical localization of proinflammatory cytokines (IL-6
and TNF-α) was carried out as per the kit protocol described by
manufacturer (R&D systems, Minn, USA)
(cat # DTA00C for IL-6 and cat # DY206 for TNF-α). The localization of IL-4 and 10 was performed using goat
antihuman primary antibody (Sigma Aldrich, cat # I7526 for IL-4, I5020 for IL-10). Briefly, cervicovaginal tissue sections from
placebo, nisin, and SDS gel-treated rabbits were processed as described above.
The endogenous peroxidase activity was quenched by treating cervicovaginal tissues
with 0.03%H2O2 in methanol for 10 minutes and rinsed in
PBS for 5 minutes. To rule out nonspecific binding, sections were incubated
in normal goat serum instead of primary antibody and considered as negative
controls. After washing with PBS, slides were incubated with respective primary antibodies overnight at 4C°.
Next day, slides were washed twice in PBS and incubated for 1 hour at RT with
1:100 dilution of donkey antigoat secondary antibody conjugated to horse radish
peroxidase (HRP) (Sigma Aldrich, cat # G6638).After washing in PBS, the
sections were incubated for 10 minutes with3-3′diaminobenzidine
tetrahydrochloride (DAB) (Sigma Aldrich, St. Louis, USA)
containing Ni2+ and H2O2 in PBS. The tissue
sections were then briefly rinsed in water, counterstained with hematoxylin.
After dehydration in alcohol series,  sections were cleared in xylene,
mounted with DPX, and visualized using Olympus BX50 microscope.


## 2.7. Image Analysis

Semiquantitative analysis of
the immunoprecipitates of cytokines was performed using image analysis software
Biovis 1.42 as detailed previously [26]. Briefly, four areas from each section
were randomly selected for each animal in the three groups. Images from these
areas were grabbed by the CCD camera.
The integrated optical density (IOD) of each area was calculated for the
brown color immunoprecipitates. Negative control slides were analyzed in a
similar fashion. The mean ± SDs were calculated for
each group.


## 2.8. Measurement of Cytokine
Levels in Cervicovaginal Lavages

Levels of proinflammatory (IL-6
and TNF-α), Th-2 type IL-4, and anti-inflammatory cytokine (IL-10) in
CVLs were measured by ELISA as per the protocol discussed above. Optical densities
of the product formed were measured by using ELISA reader (ELX 800, Bio-TEK
Instruments). Cytokine levels were calculated by quadratic regression analysis
based on logarithmically transformed optical densities. Interference of gel
formulations in cytokine ELISA was ruled out by spiking known concentrations of
standards. Control and treated samples were assayed in duplicate wells and the
experiment was repeated thrice.


## 2.9. Measurement of Phospho-NF-kB
p65 in CVE Cells

The activation of NF-kB signalling cascade in CVE tissue was
determined as per the manufacturer’s protocol using Phospho-NF-kB p65 (Ser536) Sandwich ELISA kit (Cell
Signaling, MA, USA).
Briefly, cell lysates were prepared with lysis buffer (20 mM tris,150 mM NaCl,
1 mM EDTA,1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N′,N′ (EGTA), 1% Triton
X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3 Vo4, 1 μg/mL leupeptin) and sonicated on ice. The lysates were centrifuged at 14 000 rpm (Eppendorf 541R) for 10 minutes at 4C° to pellet the cell debris.
The supernatants were collected, aliquoted, and stored at −80C° in
single use aliquots. Cell lysates were added to microwells coated with
Phospho-NF-kB p65 (Ser536) (93H1)
mouse mAb and incubated overnight at 4C°.Following extensive washing
with wash buffer, 100μl of Phospho-NF-kB
p65 rabbit mAb (Cell Signaling, MA, USA, cat # 3033) was added to the wells
and incubated for 1 hour at 37C° to detect the captured Phospho-NF-kB p65 protein. After washing, 100 μl
antirabbit IgG secondary antibody coupled to HRP (Cell Signaling, MA, USA, cat
#7074) was added and incubated at 37C° for 1hour to recognize the
bound antibody. 100 μl of substrate, 3′-3′tetramethylbenzidine (TMB) was added and
incubated for 10 minutes at 37C° to develop blue color. Reaction was
stopped by adding 100 μl of stop solution. The OD of yellow-colored product was
measured at 450 nm by ELISA reader. Each value is the mean ±SD of six
individual observations obtained from three experiments performed on different
days.


## 2.10. RNA Isolation and RT-PCR
Analysis of TLRs, Cytokines, and Defensin-1 Molecules

Total RNA was isolated from
CVE tissue from nisin, SDS, and placebo gel-treated rabbits by sequential
extraction using TriPure reagent according to the manufacturer’s protocol (Roche, Germany).
RNA was quantitated spectrophotometrically at 260 and 280 nm (UV-160A, Shimadzu, Japan).
The quality and integrity of RNA was determined by 1.5% denaturing formaldehyde
agarose gel electrophoresis and used for semiquantitative RT-PCR. Prior to
amplification, all RNA samples were treated with RNase-free DNase-I (Qiagen) to
preclude genomic  DNA contamination. Briefly, the reaction was carried out in a
PTC-200 thermal cycler (MJ Research, Maryland, USA) in a total volume of 50μl reaction mixture containing 10 μl 5X RT-PCR buffer, 2 μl dNTP mixture, 1 μl enzyme mix, 2 μl of 0.4 μM each primer, 1 μl total RNA (2 μg), 1 μl DTT solution, and 31 μl RNAse free water. RT PCR to synthesize cDNA
was carried out at 50C° for 30 minutes followed by amplification using gene-specific
primers (Table 1) for 30–40 cycles at 94C°
for 30 seconds, annealing at 55–64C° for 30 seconds,
extension at 68C° for 40 seconds followed by a final extension at 68C° for 7
minutes. Reaction was amplified in the absence of reverse transcriptase as
negative control. A nontemplate control reaction was also included to ensure
lack of DNA contamination. Housekeeping gene, GAPDH
served as
internal loading control. PCR products were electrophoresed through 1.5%
agarose gel, stained with EtBr (0.5 μg/mL) (Sigma Aldrich, St. Louis, USA),
visualized by UV transillumination, and imaged using
Gel Doc system (Bio-Rad, Calif, USA).Table 1
Oligonucleotide primers used for the study.
GeneAccession NoPrimer sequence(5′→3′)Product size (bp)Annealing temperatureIL-4DQ_852343F-AGTTCTACCTCCACCACAAGGT21260C°R-TCAGCTCTGACGCTTTGAGTATIL-6NM_001082064F-AGGAGCTGAGGAAAGAGATGTG22558C°R-TGTTTTCTTCGTCACTCCTGAAIL-10NM_001082045F-TTCTTTCAATCGAAGGATCAGC26759C°R-CTCATTCATGGCTTTGTAGACGTNF-αID_AB128153F-CTAGCCCACGTAGTAGCAAACC15960C°R-GCTGAAGAGAACCTGGGAGTAGTLR-3NM_003265F-GAT CTG TCT CAT AAT GGC TTG30560C°R-GAC AGA TTC CGA ATG CTT GTGTLR-9NM_017442F-TTC CCT GTA GCT GCT GTC C20759C°R-ACA GCC AGT TGC AGT TCA CCDefensin-1NM_001082299F-CTCTGCTTGCTGCCATTCTC19660C°R-AATCGTCTGCAAGTACAGGACACGAPDHNM_01082253F-CCATTCATTGACCTCCACTACA19959C°R-CGTTGCTGACAATCTTGAGAGA


## 2.11. Statistical Analysis

In
most instances, the results were expressed as mean ± standard deviations. Statistically, significant differences between placebo and
treatment groups were assessed using analysis of variance ort test, and differences were considered
significant if  p values were <.05.


## 3. Results

### 3.1. Immunofluorescent
Localization of TLRs

To determine the effect of nisin
gel on mucosal immunity, rabbits were intravaginally administered with placebo,
nisin, and SDS gels. Representative images of TLR-9 immunofluorescent localization
of TLR-9 in CVE cells are shown in Figure1. TLR-9 was localized on the cell membrane and also
within the cytoplasm of CVE cells. Similar pattern of expression was seen for
TLR-3 (data not shown). Vaginal delivery of high-dose nisin gel had no adverse
effect on the expression of TLRs-3 and 9 compared to the placebo gel-treated
group.Laser Scanning Confocal Microscope (LCMS) images of TLR-9 protein expression in rabbit CVE after intravaginal administration of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel), (c) and 2 mL of 1% placebo gel (a) for 14 consecutive days. Expression was found to be localized in the cytoplasm of cells. Note significant increase of TLR expression in SDS gel group (c) compared to placebo (a) and nisin gel administered (b) groups. Similar expression pattern was observed in TLR-5. The figures shown are the representative pictures from three independent experiments (Mag X 40; inset image X 630).
(a)(b)(c)(d)


### 3.2. Flow Cytometry
of TLRs

The
inflammatory potential of nisin gel was further evaluated by flow cytometry.
The results indicated no significant difference in the number of cells positive
for TLR-3 and 9 between placebo and nisin gel-treated groups (Figure2).
However, in the SDS gel-treated group, mean fluorescence intensity for TLR-3
and 9 increased significantly. The experiment was repeated twice in duplicates
and similar results were obtained.Figure 2
Flow cytometric analysis of TLR expression in rabbit CVE cells after intravaginal administration of placebo, nisin (15.15 mM in 2 mL of 1% polycarbophil gel) SDS (56μM in 2 mL of 1% polycarbophil gel) and 2 mL of 1% polycarbophil gel. Mean fluorescence intensity for TLR-3 and 9 were significantly increased in SDS gel-treated animals compared to their respective placebo and nisin gel-treated groups. The mean and error bars indicate standard deviation of triplicate measurements obtained from three separate experiments.


### 3.3. RT-PCR analysis of TLR
Expression

To determine whether vaginal administration of nisin gel
leads to modulation of TLRs and cytokine genes, expression of TLR and cytokine
mRNA transcripts were analyzed using RT-PCR. As shown in Figures3 and 9,
RT-PCR results demonstrated no change in the expression in nisin gel-administered
rabbits compared to the placebo group.Figure 3
RT-PCR analysis of TLR- 3 and 9 expression. Total RNA was extracted from CVE cells and subjected to RT-PCR analysis as described in the Material and Methods. No change in TLR gene expression was observed in nisin administered animals compared to placebo gel-treated group. However, in SDS gel administered animals, the expression of TLR genes was upregulated. House keeping gene, GAPDH expression confirms roughly equivalent loading of RNA samples.


### 3.4. Immunohistochemical Localization
of Cytokines

Having
demonstrated that CVE  cells express TLRs-3 and 9, we next investigated the modulation of various
cytokines  in cervicovaginal tissue
in response to microbicide gels by immunohistochemistry. The expression of cytokines
(IL-4, 6, 10, and
TNF-α) was observed in the cytoplasm as secreted products
with no change in staining intensity between placebo and nisin-treated groups.
However, the levels  were significantly
upregulated (Figures 4–7) in SDS treated animals. This increase
was not associated with enhancement in the number of leucocytes or neutrophils in CVLs
(data not shown). ELISA results of CVLs were in perfect agreement with
immunohistochemical data (Figure 8).Immunohistochemical localization of IL-4 in the CVE after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel) (c) and 2 mL of 1% polycarbophil gel (a) for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semi-quantitative comparison by measuring integrated optical density (IOD) of immunoreactive IL-4 in treated and placebo is shown (e). The IOD in each area was calculated for the brown color immunoprecipitates. Each IOD value measured is the mean ±SD of six observations obtained from three independent samples. (Mag X 20) (EC = epithelial cells, SE = Squamous epithelium, LP = lamina propria), (=* value is statistically significant at P<.05).
(a)(b)(c)(d)(e)Immunohistochemical localization of IL-6 in the cervicovaginal tissue after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel) (c) and  2 mL  1% polycarbophil gel (a) for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semiquantitative comparison by measuring integrated optical density (IOD) of immunoreactive IL-6 is shown (e). The IOD in each area was then measured for the brown color immunoprecipitates. Each value is the mean ±SD of six observations obtained from three independent samples. The figure shown is representative pictures from three independent rabbits (Mag X 20). (EC = epithelial cells, SE = squamous epithelium, LP = lamina propria) (=* value are statistically significant at P<.05).
(a)(b)(c)(d)(e)Immunohistochemical localization of IL-10 in the CVE after intravaginal application of nisin gel (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel) (c) and  2 mL of 1% polycarbophil gel for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semiquantitative comparison by measuring integrated optical density (IOD) of immunoreactive IL-10 in treated and placebo is shown (e). The IOD in each area was then measured for the brown color immunoprecipitates. Each value is the mean ±SD of six observations obtained from three independent samples. Figure shown is representative picture from three independent experiments. (Mag X 20) (EC = epithelial cells, SE = squamous epithelium, LP = lamina propria), (=* values are statistically significant at P<.05).
(a)(b)(c)(d)(e)Immunohistochemical localization of TNF-α in the cervicovaginal tissue after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56 μM in 2 mL of 1% polycarbophil gel) (c) and 2 mL of 1% polycarbophil gel (a) for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semiquantitative comparison by measuring integrated optical density (IOD) of immunoreactive TNF-α in treated and placebo is shown (e). The IOD in each area was then measured for the brown color immunoprecipitates. Each value is the mean ±SD of six observations obtained from three independent samples. Figure shown is representative picture from three independent experiments. (Mag X 20) (EC = epithelial cells, SE = squamous epithelium, LP = lamina propria) (=* values are statistically significant at P<0.05).
(a)(b)(c)(d)(e)Figure 8
Levels of cytokines (IL-4, IL-6, IL-10 and TNF-α) in CVL after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) SDS (56 μM in 2 mL of 1% polycarbophil gel) and 2 mL of 1% polycarbophil gel for 14 consecutive days. CVL are collected on day 15, separated from cell debris by centrifugation and used for cytokine determination as per the protocol given in the material and methods section. Each value is the mean ±SD of six observations from three separate experiments. No significant difference in cytokine levels was observed between placebo and nisin gel treated groups. In contrast the levels were significantly increased in SDS gel treated animals (=* values are statistically significant at P<0.05).Figure 9
RT-PCR analysis of cytokine expression following intravaginal application of nisin (15.15 mM/day/14 days), SDS (56μM/day/14 days) and 1% placebo gels in rabbits. After separation of cells, they were subjected to RNA extraction as per the protocol given in the material and methods section. The expression of IL-4, IL-6, IL-10 and TNF-α. genes in nisin treated group was comparable to the placebo group. In contrast, expression of  the cytokine genes was upregulated in SDS treated group. GAPDH blots confirmed roughly equivalent loading of RNA samples.


### 3.5. Determination of
NF-kB
P65 in CVE Cells

Modulation of NF-kB-related signals was determined after
intravaginal application of nisin gel and compared with placebo gel-administered
group. The phosphorylation state of NF-kB
and consequent changes in gene expression following NF-kB modulation were determined by ELISA by comparing relative
amounts of phosphorylated NF-kB
protein to the total NF-kB protein.
Exposure of CVE to SDS gel triggered a marked activation of NF-kB. However, such induction in NF-kB signalling was not observed in nisin
gel treated animals and the placebo gel-treated group (Figure 10).Figure 10
Determination of total and phosphorylated NF-kB p65 protein levels by ELISA as per the protocol described in materials and methods. No change was observed in total NF-kB levels. However, the phosphorylated NF-kB protein levels were significantly elevated in SDS gel administered animals.


### 3.6. RT-PCR Analysis of Rabbit Defensin-1

To examine whether the
observed increase in cytokine levels is associated
with changes  in vaginal mucosal immunity, we evaluated the
expression of defensin-1 gene in CVE cells. The results from three independent
experiments revealed that nisin gel treatment did not cause any alterations in
the mRNA expression of defensin-1 compared to placebo gel-treated group.
Whereas in SDS gel treated group expression of defensin-1 gene was markedly elevated
(Figure11).Figure 11
RT-PCR analysis of defensin-1 gene in CVE cells of rabbit. No change was seen in defensin-1 gene expression between nisin gel and placebo gel administered groups. However, significant activation of defensin-1 gene expression was observed in SDS gel administered group compared to placebo and nisin gel treated animals.


## 3.1. Immunofluorescent
Localization of TLRs

To determine the effect of nisin
gel on mucosal immunity, rabbits were intravaginally administered with placebo,
nisin, and SDS gels. Representative images of TLR-9 immunofluorescent localization
of TLR-9 in CVE cells are shown in Figure1. TLR-9 was localized on the cell membrane and also
within the cytoplasm of CVE cells. Similar pattern of expression was seen for
TLR-3 (data not shown). Vaginal delivery of high-dose nisin gel had no adverse
effect on the expression of TLRs-3 and 9 compared to the placebo gel-treated
group.Laser Scanning Confocal Microscope (LCMS) images of TLR-9 protein expression in rabbit CVE after intravaginal administration of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel), (c) and 2 mL of 1% placebo gel (a) for 14 consecutive days. Expression was found to be localized in the cytoplasm of cells. Note significant increase of TLR expression in SDS gel group (c) compared to placebo (a) and nisin gel administered (b) groups. Similar expression pattern was observed in TLR-5. The figures shown are the representative pictures from three independent experiments (Mag X 40; inset image X 630).
(a)(b)(c)(d)


## 3.2. Flow Cytometry
of TLRs

The
inflammatory potential of nisin gel was further evaluated by flow cytometry.
The results indicated no significant difference in the number of cells positive
for TLR-3 and 9 between placebo and nisin gel-treated groups (Figure2).
However, in the SDS gel-treated group, mean fluorescence intensity for TLR-3
and 9 increased significantly. The experiment was repeated twice in duplicates
and similar results were obtained.Figure 2
Flow cytometric analysis of TLR expression in rabbit CVE cells after intravaginal administration of placebo, nisin (15.15 mM in 2 mL of 1% polycarbophil gel) SDS (56μM in 2 mL of 1% polycarbophil gel) and 2 mL of 1% polycarbophil gel. Mean fluorescence intensity for TLR-3 and 9 were significantly increased in SDS gel-treated animals compared to their respective placebo and nisin gel-treated groups. The mean and error bars indicate standard deviation of triplicate measurements obtained from three separate experiments.


## 3.3. RT-PCR analysis of TLR
Expression

To determine whether vaginal administration of nisin gel
leads to modulation of TLRs and cytokine genes, expression of TLR and cytokine
mRNA transcripts were analyzed using RT-PCR. As shown in Figures3 and 9,
RT-PCR results demonstrated no change in the expression in nisin gel-administered
rabbits compared to the placebo group.Figure 3
RT-PCR analysis of TLR- 3 and 9 expression. Total RNA was extracted from CVE cells and subjected to RT-PCR analysis as described in the Material and Methods. No change in TLR gene expression was observed in nisin administered animals compared to placebo gel-treated group. However, in SDS gel administered animals, the expression of TLR genes was upregulated. House keeping gene, GAPDH expression confirms roughly equivalent loading of RNA samples.


## 3.4. Immunohistochemical Localization
of Cytokines

Having
demonstrated that CVE  cells express TLRs-3 and 9, we next investigated the modulation of various
cytokines  in cervicovaginal tissue
in response to microbicide gels by immunohistochemistry. The expression of cytokines
(IL-4, 6, 10, and
TNF-α) was observed in the cytoplasm as secreted products
with no change in staining intensity between placebo and nisin-treated groups.
However, the levels  were significantly
upregulated (Figures 4–7) in SDS treated animals. This increase
was not associated with enhancement in the number of leucocytes or neutrophils in CVLs
(data not shown). ELISA results of CVLs were in perfect agreement with
immunohistochemical data (Figure 8).Immunohistochemical localization of IL-4 in the CVE after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel) (c) and 2 mL of 1% polycarbophil gel (a) for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semi-quantitative comparison by measuring integrated optical density (IOD) of immunoreactive IL-4 in treated and placebo is shown (e). The IOD in each area was calculated for the brown color immunoprecipitates. Each IOD value measured is the mean ±SD of six observations obtained from three independent samples. (Mag X 20) (EC = epithelial cells, SE = Squamous epithelium, LP = lamina propria), (=* value is statistically significant at P<.05).
(a)(b)(c)(d)(e)Immunohistochemical localization of IL-6 in the cervicovaginal tissue after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel) (c) and  2 mL  1% polycarbophil gel (a) for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semiquantitative comparison by measuring integrated optical density (IOD) of immunoreactive IL-6 is shown (e). The IOD in each area was then measured for the brown color immunoprecipitates. Each value is the mean ±SD of six observations obtained from three independent samples. The figure shown is representative pictures from three independent rabbits (Mag X 20). (EC = epithelial cells, SE = squamous epithelium, LP = lamina propria) (=* value are statistically significant at P<.05).
(a)(b)(c)(d)(e)Immunohistochemical localization of IL-10 in the CVE after intravaginal application of nisin gel (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56μM in 2 mL of 1% polycarbophil gel) (c) and  2 mL of 1% polycarbophil gel for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semiquantitative comparison by measuring integrated optical density (IOD) of immunoreactive IL-10 in treated and placebo is shown (e). The IOD in each area was then measured for the brown color immunoprecipitates. Each value is the mean ±SD of six observations obtained from three independent samples. Figure shown is representative picture from three independent experiments. (Mag X 20) (EC = epithelial cells, SE = squamous epithelium, LP = lamina propria), (=* values are statistically significant at P<.05).
(a)(b)(c)(d)(e)Immunohistochemical localization of TNF-α in the cervicovaginal tissue after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) (b), SDS (56 μM in 2 mL of 1% polycarbophil gel) (c) and 2 mL of 1% polycarbophil gel (a) for 14 consecutive days. To rule out the nonspecific binding, sections were incubated in preimmune sera instead of primary antibody and considered as negative control (d). Semiquantitative comparison by measuring integrated optical density (IOD) of immunoreactive TNF-α in treated and placebo is shown (e). The IOD in each area was then measured for the brown color immunoprecipitates. Each value is the mean ±SD of six observations obtained from three independent samples. Figure shown is representative picture from three independent experiments. (Mag X 20) (EC = epithelial cells, SE = squamous epithelium, LP = lamina propria) (=* values are statistically significant at P<0.05).
(a)(b)(c)(d)(e)Figure 8
Levels of cytokines (IL-4, IL-6, IL-10 and TNF-α) in CVL after intravaginal application of nisin (15.15 mM in 2 mL of 1% polycarbophil gel) SDS (56 μM in 2 mL of 1% polycarbophil gel) and 2 mL of 1% polycarbophil gel for 14 consecutive days. CVL are collected on day 15, separated from cell debris by centrifugation and used for cytokine determination as per the protocol given in the material and methods section. Each value is the mean ±SD of six observations from three separate experiments. No significant difference in cytokine levels was observed between placebo and nisin gel treated groups. In contrast the levels were significantly increased in SDS gel treated animals (=* values are statistically significant at P<0.05).Figure 9
RT-PCR analysis of cytokine expression following intravaginal application of nisin (15.15 mM/day/14 days), SDS (56μM/day/14 days) and 1% placebo gels in rabbits. After separation of cells, they were subjected to RNA extraction as per the protocol given in the material and methods section. The expression of IL-4, IL-6, IL-10 and TNF-α. genes in nisin treated group was comparable to the placebo group. In contrast, expression of  the cytokine genes was upregulated in SDS treated group. GAPDH blots confirmed roughly equivalent loading of RNA samples.


## 3.5. Determination of
NF-kB
P65 in CVE Cells

Modulation of NF-kB-related signals was determined after
intravaginal application of nisin gel and compared with placebo gel-administered
group. The phosphorylation state of NF-kB
and consequent changes in gene expression following NF-kB modulation were determined by ELISA by comparing relative
amounts of phosphorylated NF-kB
protein to the total NF-kB protein.
Exposure of CVE to SDS gel triggered a marked activation of NF-kB. However, such induction in NF-kB signalling was not observed in nisin
gel treated animals and the placebo gel-treated group (Figure 10).Figure 10
Determination of total and phosphorylated NF-kB p65 protein levels by ELISA as per the protocol described in materials and methods. No change was observed in total NF-kB levels. However, the phosphorylated NF-kB protein levels were significantly elevated in SDS gel administered animals.


## 3.6. RT-PCR Analysis of Rabbit Defensin-1

To examine whether the
observed increase in cytokine levels is associated
with changes  in vaginal mucosal immunity, we evaluated the
expression of defensin-1 gene in CVE cells. The results from three independent
experiments revealed that nisin gel treatment did not cause any alterations in
the mRNA expression of defensin-1 compared to placebo gel-treated group.
Whereas in SDS gel treated group expression of defensin-1 gene was markedly elevated
(Figure11).Figure 11
RT-PCR analysis of defensin-1 gene in CVE cells of rabbit. No change was seen in defensin-1 gene expression between nisin gel and placebo gel administered groups. However, significant activation of defensin-1 gene expression was observed in SDS gel administered group compared to placebo and nisin gel treated animals.


## 4. Discussion

Topical microbicides are
being evaluated as promising strategies for the prevention of STIs/HIV while
protecting or enhancing the natural mucosal barrier [27, 28]. Nisin, a
naturally occurring AMP, is currently being developed as an intravaginal microbicide
for the prevention of STIs and unintended pregnancies [8]. To screen nisin gel
for undesirable characteristics if any in a preclinical setting, we chose the
rabbit model, since it has been documented that responsiveness of rabbit
vagina to microbicides resembles that of the human vagina [29].Immunomodulation  inflammation
has been implicated to play a role in vaginal innate immunity [30]. Inflammatory responses limit the
beneficial effect of microbicides, which could be of particular concern if the
inflammation persists beyond the time the microbicide was present [29]. Vaginal epithelial cells represent the
predominant resident innate immune cell in the vagina and may modulate the
inflammatory response to microbicides [31]. Mammalian Toll-like
receptors (TLRs), including TLR-1 to TLR11, serve an important role as sensors in
host defense mechanism [32]. On the basis of TLR role in the modulation of innate immunity and
responses to inflammatory challenge, we examined the expression of TLR-3 and 9 as
these play a crucial role in the recognition of inflammatory stimuli in host vaginal
immune defense [33, 34].Under conditions of their intended use as intravaginal/rectal
microbicides, individuals would be exposed to microbicides for shorter or
longer periods. Therefore, high dose of nisin gel (15.15 mM in 2 mL of 1% polycarbophil) per day for 14 consecutive days was administered in rabbits
to ensure whether the estimated risk/benefit profile of the gel is in
favor of its intended use. The immunofluorescent results demonstrated
localization of TLRs-3 and 9 on the plasma membrane as well as in endosomes present in
the cytoplasm of CVE cells. TLR expression did not change in the CVE cells of nisin
and placebo gel-treated groups. These results were in agreement with the flow
cytometry data where no change in percent immunoreactive cells expressing TLRs-3
and 9 was observed. However, the number of cells expressing both TLRs-3 and 9 were
significantly elevated in SDS gel-treated animals. The studies were extended
further to gain an insight into the regulation of TLR expression at the
molecular level. RT-PCR results demonstrated no change in the expression
of TLRs-3 and 9 mRNA transcripts in CVE cells of nisin gel-treated rabbits
compared to the placebo gel-treated group, suggesting that vaginal application of nisin
gel did not induce any inflammation associated alterations at the
transcriptional and translational level. The CVE cells recognize tissue
inflammation by means of TLR-3 and 9, likely through a nonspecific mechanism.
These results
tempted us to speculate that signalling through TLRs may be involved
in mechanisms provoking Th2-based immune responses and may either prevent or
promote vaginal inflammation [35].Earlier studies have suggested that many of the local inflammatory
responses initiated in the vaginal mucosa are mediated by a complex
interplay between TLRs and inflammatory agents in the human vagina [32, 36]. In
an elegant study, Fichorova et al. [33] have shown the presence of TLR 1-6 genes
in primary and immortalized endocervical, ectocervical, and vaginal epithelial
cell lines. They demonstrated mRNA for TLRs-1, 2, 3, and 6 in these cells. In
contrast, no PCR product was detected for TLR-4 in either of the cells. It has
been shown recently that agonists of TLR-2 (zymogen), TLR-3 [poly (I: C)], TLR-5
(flagellin), and TLR-9 (CpG ODN) are known to stimulate various immune cells to
secrete several proinfammatory cytokines [23]. These studies support our
present observations that nisin gel did not upregulate the levels of inflammatory cytokines secreted by CVE cells. In contrast, in SDS gel-treated
animals, the levels of  cytokines in the vaginal tissue and vaginal
fluid were upregulated in a TLR-3/9-dependent manner probably to slow down the
tissue inflammation.To further
dissect the mechanism, we next investigated the expression of NF-kB, known for its ability to regulate
cytokine and defensin-1 expression in CVE cells. As shown in Figure 10, the phospho
NF-kB p65 and total NF-kB p65 remained unchanged in placebo and nisin
gel-treated groups, suggesting that vaginal application of nisin gel did not alter
the NF-kB signalling cascade.We further evaluated the expression of defensin-1
which is the principal effector molecule of mucosal innate immunity against
infection/inflammation [37].
Considering the fact that several defensins are expressed by neutrophils,
epithelial cells, macrophages, NK cells, and T cell subsets in human [38, 39], the present results indicated that,
nisin gel  treatment did not cause any change in the expression of defensin-1 gene suggesting that the  NF-kB
signalling cascade was not affected. The
aforementioned studies demonstrated that nisin gel may either enhance or
maintain the normal immune status to assist in the resolution of inflammation,
while SDS gel appears to cause immunosuppressive effect.Therefore, we hypothesized
that TLRs or cytokines appear to be predictive biomarkers
for evaluating the inflammatory status of    vaginal mucosa upon microbicide administration.
These results are supported by the fact that soluble TLRs-2 and 4 modulate specific TLR-mediated responses,
thereby providing new biomarkers of tissue inflammation [40]. The present data
led us to postulate that TLRs appear to be crucial mediators of vaginal
defense and when perturbed cause inflammatory pathways to predominate in CVE cells
leading to mucosal damage. These findings
contribute to our understanding of mucosal immunity against STIs/HIV and
can aid in enhancing the quality of woman’s reproductive health. Establishing downstream
targets for TLR–microbicide interaction may further unravel
the intricacies of vaginal function.The present study is the first to
provide evidence of nisin gel as a safe intravaginal compound for future use in
humans. Testing additional compounds in the presently planned clinical trials
would allow validation of this approach and determine whether this safe and
inexpensive strategy provides a surrogate biomarker to predict microbicide
safety and efficacy. However, this study is too small to make a definite
conclusion. If confirmed in a larger sample, this strategy could be used to
determine which future generation microbicides should go forward in
development, possibly resulting in major cost savings. However, given the
nascent state of knowledge concerning this important area, it is clear that
more studies are needed to provide valuable insights into the biology of
TLRs and vaginal innate immunity.



---

*Source: 534532-2008-12-22.xml*