Download Stimulation of TLRs by LMW-HA induces self-defense

Immunology and Cell Biology (2011) 89, 630–639
& 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11
www.nature.com/icb
ORIGINAL ARTICLE
Stimulation of TLRs by LMW-HA induces self-defense
mechanisms in vaginal epithelium
Giuseppina F Dusio1,3, Diego Cardani1,3, Laura Zanobbio1, Martina Mantovani1, Patrizia Luchini1,
Lorenzo Battini1, Valentina Galli1, Angela Diana1, Andrea Balsari1,2 and Cristiano Rumio1
The innate immune system is present throughout the female reproductive tract and functions in synchrony with the adaptive
immune system to provide protection in a way that enhances the chances for fetal survival, while protecting against potential
pathogens. Recent data show that activation of Toll-like receptor (TLR)2 and 4 by low-molecular weight hyaluronic acid
(LMW-HA) in the epidermis induces secretion of the antimicrobial peptide b-defensin 2. In the present work, we show that
LMW-HA induces vaginal epithelial cells to release different antimicrobial peptides, via activation of TLR2 and TLR4. Further,
we found that LMW-HA favors repair of vaginal epithelial injury, involving TLR2 and TLR4, and independently from its classical
receptor CD44. This wound-healing activity of LMW-HA is dependent from an Akt/phosphatidylinositol 3 kinase pathway.
Therefore, these findings suggest that the vaginal epithelium is more than a simple physical barrier to protect against invading
pathogens: on the contrary, this surface acts as efficient player of innate host defense, which may modulate its antimicrobial
properties and injury restitution activity, following LMW-HA stimulation; this activity may furnish an additional protective activity
to this body compartment, highly and constantly exposed to microbiota, ameliorating the self-defense of the vaginal epithelium
in both basal and pathological conditions.
Immunology and Cell Biology (2011) 89, 630–639; doi:10.1038/icb.2010.140; published online 23 November 2010
Keywords: vaginal epithelial cells; low-molecular weight hyaluronic acid; Toll-like receptors; antimicrobial peptides; wound healing
The mucosal immune system in the female reproductive tract has
evolved to meet the unique requirements of dealing with sexually
transmitted bacterial and viral pathogens, allogenic spermatozoa and
the immunologically distinct fetus.1,2 This immune system regulates
transport of immunoglobulins, levels of cytokines, distribution of
various cell populations and antigen presentation in the genital tissues
during the reproductive cycle.3,4 Protection against potential pathogens in the female reproductive tract is provided by a variety of
measures that can be grouped into two broad categories: innate and
adaptive immunity. Recognized as the first line of defense, the innate
immune system functions to prevent and control the invasion of
pathogens.5 The innate immune system has evolved to recognize
foreign structures that are not normally found in the host; it relies
on conserved germline-encoded receptors that recognize conserved
pathogen-associated molecular patterns found in groups of microorganisms. The pattern recognition receptors of the host that recognize pathogen-associated molecular patterns are expressed on cells of
the innate immune system. Toll-like receptors (TLRs) are one group of
pattern recognition receptors that are expressed on macrophages,
dendritic cells, and as more recently shown, neutrophils, natural killer
cells and epithelial cells.6 Signaling through TLRs in response to
pathogen-associated molecular patterns involves a number of adapters
and other molecules that lead to recruitment of immune cells as well
as the production of intracellular and secreted anti-microbial factors,
which both kill invading microbes (bacteria, fungi and viruses) as well
as link innate and acquired immunity.7–9 The antimicrobial properties
of mucosal tissues are largely because of the contributions of bioactive
polypeptides, which additionally take part to restitution processing
following epithelial injury.10
Although much attention has been paid to innate immune function
in the lung and intestine, very few studies have investigated presence
and function of the innate immune system in the female reproductive
tract. What is becoming clear is that the innate immune system is
present throughout the reproductive tract and functions in synchrony
with the adaptive immune system to provide protection in a way that
enhances the chances for fetal survival, while protecting against
potential pathogens.
Cells throughout the female reproductive tract express TLR1–6 as
well as MD-2, an accessory molecule that complexes with TLR4.11
Epithelial cells from the lower reproductive tract have been shown to
express a different variety of TLRs, including TLR1, 3, 5 and 6 by
vaginal and cervical epithelial cell lines. In fact, the vagina makes up
the lower reproductive tract that is exposed to the external environment: therefore, epithelial cells lining the vaginal tract, which are
1iMIL, italian Mucosal Immunity Laboratory, Dipartimento di Morfologia Umana e Scienze Biomediche ‘Città Studi’, Università degli Studi di Milano, Milano, Italy and 2Istituto
Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy
3These authors contributed equally to this work.
Correspondence: Professor C Rumio, Dipartimento di Morfologia Umana e Scienze Biomediche, Università degli Studi di Milano, via Mangiagalli 31, Milano 20133, Italy.
E-mail: [email protected]
Received 19 October 2009; revised 6 October 2010; accepted 19 October 2010; published online 23 November 2010
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constantly exposed to pathogens, are equipped with a set of pattern
recognition receptors that let them recognize these microorganisms.12
We have recently shown that activation of TLR2 and4 by lowmolecular weight hyaluronic acid (LMW-HA) in the epidermis
induces secretion of the antimicrobial peptide b-defensin 2.13 In the
present work, we have investigated on the possible immune-stimulatory activity of LMW-HA in the vaginal epithelium, through in vitro
experiments using the VK2/E6E7 vaginal epithelial cell line.
RESULTS
Human vaginal epithelial cells express TLRs and respond to their
ligands
It has been reported that female reproductive tract constitutively
expresses members of TLRs family11 but little is known about the
vaginal tract in particular. In this study, we have used an in vitro model
of human vaginal epithelial cells (human cell line VK2/E6E7) based on
our previous work13 that shows the involvement of TLR2 and TLR4 in
b-defensin 2 secretion by keratinocytes following LMW-HA treatment. We have initially investigated on the expression of these two
receptors in VK2/E6E7 cells and we have shown TLR2 and TLR4
proteins by both western blot and immunofluorescence experiments
(Figure 1a). Proper controls, performed by omission of primary
antibody (Figure 1a) and by use of an isotype control antibody
(data not shown) confirmed the specificity of the immunofluorescent
staining.
TLR activation by specific ligands results in the secretion of
antimicrobial peptides, secretion of chemokines that attract phagocytic cells to the affected tissue and secretion of type 1 interferons that
attract and activate natural killer cells. In our in vitro model, an
intense increase in transcription of antimicrobial peptides human beta
defensin (HBD)1, HBD2, HBD3, LL37, secretory leukocyte protease
inhibitor (SLPI) and lactoferrin, assessed by PCR analysis, was
observed when cells were stimulated for 18 h with TLR2 and TLR4
specific bacterial ligands, peptidoglycan and lipopolysaccharide
(LPS), respectively (Figure 1b). However, these intense antimicrobial
peptides secretion was accompanied by proinflammatory cytokine
interleukin (IL)-8 production, suggesting the probable arise of an
inflammatory response (data not shown).
HA treatment induces vaginal epithelial cells to release
antimicrobial peptides, but not proinflammatory mediators,
via activation of TLR2 and TLR4
We have recently reported13 that LMW fragments of HA are able to
induce the secretion of antimicrobial peptide HBD2 in human
keratinocytes. To evaluate if LMW-HA might be able to induce
expression of various antimicrobial peptides also in vaginal epithelial
cells, VK2/E6E7 cells were incubated for 18 h with a medium containing 0.2% LMW-HA; LPS-treated cells were used as positive control.14
LMW-HA-treated cells showed a marked increase in the transcription
of HBD1, HBD2, HBD3, LL37, SLPI and lactoferrin genes, as revealed
by PCR analysis (Figure 1b).
Quantification of HBD2 secretion at different time points of
treatment (30 min, 1, 6, 18 h), assessed by enzyme-linked immunosorbent assay (ELISA), showed that the levels of secreted HBD2 were
significantly increased in LMW-HA-treated cells (P¼0.0019 LMW-HA
vs UNTREATED cells, with t¼18 h), with a progressive accumulation
of the peptide in the culture media (Figure 2a). Further experiments
assessed HBD-2 production following incubation of VK2/E6E7 cells
with a 0.2% high-molecular weight (HMW)-HA solution: ELISA
showed that HMW-HA was unable to induce HBD-2 production
(Figure 2a).
To assess whether the observed antimicrobial peptides production
had to be ascribed to the stimulation of TLR2 and TLR4 by HA, cells
were incubated for 18 h with 0.2% LMW-HA and small interfering
RNA (siRNA) for TLR2 and/or TLR4. Cells supernatants were
analyzed by ELISA for their HBD2 production: in cells incubated
with one of the TLR-siRNA, the HA-induced HBD2 production was
substantially diminished, in comparison with cells treated with HA
alone (Figure 2a). Cells incubated with HA and both siRNA for TLR2
and TLR4 showed an almost complete inhibition of HBD2 production
Figure 1 Effects of LMW-HA on secretory activity of vaginal epithelial cells via TLRs. (a) The expression of TLR2 and TLR4 in human vaginal epithelial cells
VK2/E6E7 was assessed by western blot analysis and immunofluorescence technique (red staining; nuclei are stained blue with 4¢,6¢-diamidino-2phenylindole (DAPI)) (neg.ctrl., negative control by omission of primary antibody); bars: 10 mm. Inhibition of TLR2 and TLR4 by siRNA treatment was
assessed by western blot analysis. (b) Evaluation of transcription levels of different antimicrobial peptides by PCR analysis, in VK2/E6E7 cells treated with
peptidoglycan (PGN), LPS, 0.2% LMW-HA or left untreated (UNTR); (LACTOF, lactoferrin; B-ACT, b-actin).
Immunology and Cell Biology
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Figure 2 Effects of LMW-HA on secretion of antimicrobial peptide HBD2 and cytokines production via TLRs. (a) Quantization of HBD2 peptide released by
VK2/E6E7 cells following treatment with 0.2% LMW-HA has been evaluated by ELISA on cell supernatants at different time-points from the beginning of
treatment; further experiments assessed the production of HBD2 following treatment with HMW-HA or LMW-HA plus specific siRNA for TLR2 (siTLR2),
TLR4 (siTLR4) or CD44 (siCD44). Data are presented as means±s.d.; *P¼0.0019 LMW-HA vs UNTREATED, with t¼18 h; **P¼0.0026 LMW-HA+siTLR2
and 4 vs LMW-HA, with t¼18 h. (b–e) ELISA assay on VK2/E6E7 cell supernatants revealed no production of IL-1b (b), IL-6 (c), IL-8 (d) and TNF-a (e) by
0.2% LMW-HA treatment; LPS-treated cells served as positive control. Data are presented as means±s.d. (f) Western blot analysis for NF-kB.
(Figure 2a) (P¼0.0026 LMW-HA + siTLR2 and 4 vs LMW-HA, with
t¼18 h). An unrelated scrambled siRNA showed no interference in the
LMW-HA-induced HBD2 production (data not shown). The degree
of TLR2 and TLR4 knockdown achieved in the VK2/E6E7 cells by
siRNA technique results to be around 70%, as shown by Figure 1a.
To further confirm that the observed antimicrobial peptides production was dependent only from TLR2 and TLR4, we assessed if the
classical HA receptor CD44 was implicated in the observed antimicrobial peptides production. VK2/E6E7 cells were incubated for
18 h with 0.2% LMW-HA and siRNA for CD44 and analyzed by
ELISA for their HBD2 production: in cells incubated with CD44
siRNA, the LMW-HA-induced HBD2 production was not significantly
different from the one observed with no siRNA, in the presence of
LMW-HA (Figure 2a).
Immunology and Cell Biology
In order to estimate if HA induces the release of pro-inflammatory
mediators, we evaluated IL-1b, IL-6, IL-8 and tumor necrosis factor-a
production in treated vaginal epithelial cells. ELISA assays revealed
that the levels of these molecules in LMW-HA-treated cells were
comparable to basal production of untreated VK2/E6E7 cells, after
18 h of treatment (Figures 2b–e). LPS-treated cells served as positive
control. Thus, LMW-HA exerts positive activity on antimicrobial
peptides stimulation, without any pro-inflammatory effect; to confirm
this result we evaluated nuclear translocation of NF-kB, a transcription factor involved in proinflammatory cytokines production. We
treated VK2/E6E7 cell for 18 h with LMW-HA and LPS as positive
control and we performed NF-kB western blot analysis on cytoplasmic
and nuclear extracts compared with extracts of VK2/E6E7-untreated
cell as negative control. The result demonstrated that LMW-HA
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produced an increase in cytoplasmic NF-kB levels but it did not
induce a significant nuclear NF-kB translocation (+6%) compared
with LPS-induced one (+43%), without consequent activation of
proinflammatory pathways (Figure 2f).
Extracts of vaginal epithelial cells treated with LMW-HA exert
increased antimicrobial activity
Given the observed increase in antimicrobial peptides transcription
induced by LMW-HA treatment in human vaginal epithelial cells, we
aimed to evaluate the intrinsic antimicrobial activity in protein
extracts of cells treated with LMW-HA. Therefore, we prepared
protein extracts (in 0.01% acetic acid) from LMW-HA-treated
and -untreated VK2/E6E7 cells and tested the antibacterial activity
of these extracts against Escherichia coli (E. coli) American Type
Culture Collection 4157, a bacterial strain that is reported to be
sensitive to HBD2. We evaluated the number of colony-forming unit
(CFU) in the different samples (n¼5 per treatment) after overnight
incubation of bacteria previously mixed with the protein extracts: the
extract from untreated VK2/E6E7 cells induces a very slight inhibition
of bacterial CFU formation (10.1±3.3% inhibition of CFU formation
for control cells vs 0.01% acetic acid), while the extract from cells
treated with LMW-HA exerts a significantly higher antimicrobial
activity with respect to control untreated cells (75.9±6.9% inhibition
of CFU formation for LMW-HA cells vs 0.01% acetic acid)
(P¼4.010–5 LMW-HA vs untreated cells).
LMW-HA favors repair of vaginal epithelial injury
It has been reported that different antimicrobial peptides influence
wound-healing process: given the observed induction of antimicrobial
peptides following treatment of vaginal epithelial cells with LMW-HA,
we assessed if this molecule was able to stimulate also the repair of
vaginal epithelial barrier integrity after injury.
Specific mechanisms underlying injury-associated cell ‘activation’
can be addressed using in vitro systems that recapitulate particular
aspects of post-trauma re-epithelialization.15–19 Indeed, certain events
associated with injury repair in vivo can be recreated during cell
migration into the denuded areas of a scrape-injured monolayer.20–23
Several hallmarks of epithelial injury resolution are in fact reproduced
in scrape-wounded epithelial cell monolayers, validating the use of this
culture model for the study of cellular events associated with the repair
process. For this reason, confluent monolayers of VK2/E6E7 cells were
wounded with a sterile razor blade and subsequently were incubated
with control medium or in medium containing 0.2% LMW-HA. The
rate of wound closure was determined at different time-points. As
shown by Figure 3a, LMW-HA treatment resulted in a significant
increase in migrating activity of VK2/E6E7 cells against untreated cells
with a time-dependent increase in the injury repair process: in fact, the
measure of the wounds in untreated cells shows that after 8 h the
wound closure has reached a mean value of 3.3±0.6% while in LMWHA-treated cells this measure is increased to 17.3±6.0%; after 12 h,
the wound closure has reached a mean value of 6.0±2.0% in
untreated samples, while in LMW-HA-treated cells this measure is
highly increased, with a mean wound closure of 34.0±7.6% (P¼0.020
LMW-HA vs untreated cells); finally, after 24 h the wound closure of
untreated cells has reached a mean value of 10.0±3.0%, while in
LMW-HA-treated cells this measure is highly increased, with a mean
wound closure of 51±10.5% (P¼0.018 LMW-HA vs untreated cells).
Also on this aspect of epithelial protection activity, HMW-HA did not
present the same ability observed with LMW-HA (Figure 3a): in fact
the level of wound closure activity of HMW-HA-treated cells is not
statistically different from untreated cells, at all the time-points
considered. The magnitude of wound closure in these studies may
appear relatively small, but the effect is statistically relevant after 12
and 24 h of treatment (P¼0.020 LMW-HA vs untreated cells, with
t¼12h; P¼0.018 LMW-HA vs untreated cells, with t¼24 h). Nonetheless, such apparently modest alterations may be biologically
important in determining whether a mucosal wound heals or fails
to heal in vivo.
Data of literature24,25 suggest that LMW-HA itself may be a woundhealing stimulator; to assess if the observed wound-healing activity
was dependent from LMW-HA itself or from the induced antimicrobial peptides, we performed the same experiment of wound closure
evaluation in the presence of siRNA for TLR2 and TLR4: as shown by
Figure 3b, only a slight increase in migrating activity of VK2/E6E7 cells
was observed in the presence of LMW-HA in these conditions
(P¼0.0098 LMW-HA+siTLR2 and 4 vs LMW-HA, with t¼24 h),
characterized by an almost complete inhibition of antimicrobial
peptides production (data not shown). Further, as the wound-healing
activity of LMW-HA has always been reported to be mediated by
CD44 receptor, the same experiment of wound closure evaluation was
performed in the presence of siRNA for CD44: in the absence of this
receptor the wound-healing activity of LMW-HA was maintained,
suggesting that this receptor was not involved in the LMW-HA effects
on wound closure in vaginal epithelial cells (Figure 3b). Thus, we can
confirm that the observed increase in migrating activity of VK2/E6E7
in the presence of LMW-HA may be related to the induction of
antimicrobial peptides via TLR2 and TLR4.
In order to assess whether the observed phenomenon was dependent only from an increase in cells migration and not from an higher
proliferation rate, the metabolic activity of VK2/E6E7 cells was
assessed by MTT assay: LMW-HA-treated cells presented a metabolic
activity comparable to the one of control untreated cells, confirming
the absence of cellular proliferation stimulation (data not shown). Cell
counts using Trypan blue exclusion were also performed, showing no
increase in cell proliferation by LMW-HA (data not shown).
A further control of this aspect consisted in treating LMW-HAtreated cells with mitomycin C, a toxin well-known for its ability to
block DNA synthesis and therefore cell proliferation: the stimulation
of wound closure was not influenced by the presence of this toxin,
confirming that the observed phenomenon of wound healing was
dependent only from cellular migration and not from cellular proliferation (Figure 3b). Therefore, we can assess that LMW-HA favors
wound healing of vaginal epithelial cells by exerting a motogenic effect
rather than a mitogenic effect.
LMW-HA-induced wound healing is dependent from an Akt/PI3K
pathway
Mucosal restitution is known to be enhanced by various peptides and
cytokines.26 However, the factors or driving forces required for the
migration of vaginal epithelial cells are largely unknown. Therefore, we
investigated the role of different intracellular signaling molecules in
the LMW-HA-driven stimulation of vaginal epithelial cells migration
and mucosal repair, using our in vitro model.
Some investigators have reported that a key step in mediating cell
migration is the activation of phosphatidylinositol 3 kinase (PI3K)
and the downstream effector Akt/PKB;27 additionally, it is known that
in mammalian cells PI3K regulates the actin cytoskeleton, which has
an essential role during cell movement, with the majority of the
filamentous actin at the front or leading edge.28,29 Thus, we investigated the possible involvement of phospho-Akt in the LMW-HAinduced wound-healing process of vaginal epithelial cells, through the
study of Akt phosphorylation/dephosphorylation status following
Immunology and Cell Biology
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Figure 3 Effects of LMW-HA on wound-healing activity of vaginal epithelial cells via TLRs. (a) The percentage of wound closure, with respect to the wound
width at t¼0 h, was determined at different time-points on confluent VK2/E6E7 cells, following treatment with 0.2% LMW-HA or 0.2% HMW-HA. Data are
presented as means±s.d.; *P¼0.018 LMW-HA vs UNTREATED, with t¼24 h. (b) The percentage of wound closure, with respect to the wound width at
t¼0 h, was determined at different time-points on confluent VK2/E6E7 cells, following treatment with 0.2% LMW-HA plus specific siRNA for TLR2 (siTLR2),
TLR4 (siTLR4) or CD44 (siCD44). Data are presented as means±s.d.; *P¼0.0098 LMW-HA + siTLR2 and 4 vs LMW-HA, with t¼24 h. (c) Analysis of the
signaling pathway activated by LMW-HA treatment; VK2/E6E7 protein extracts, following LMW-HA and HMW-HA treatment, were analyzed by western
blotting analysis with anti-phospho-Akt (pAkt) and anti-Akt (Akt) antibodies, or with anti-phospho-MLC (pMLC) and anti-MLC (MLC) antibodies. (d) The
percentage of wound closure, with respect to the wound width at t¼0 h, was determined at different time-points on confluent VK2/E6E7 cells, following
treatment with 0.2% LMW-HA plus specific PI3K inhibitor LY-294002. Data are presented as means±s.d. (e) Confocal microscopy imaging following
staining of VK2/E6E7 cells actin cytoskeleton with phalloidin (stained green) reveals the presence of only a few filopodia in untreated cells (arrowhead),
while samples treated with 0.2% LMW-HA present a significant formation of lamellipodia (arrows). Nuclei are counterstained with 4¢,6¢-diamidino-2phenylindole (DAPI); bars: 10 mm.
LMW-HA treatment. Western blot analysis of Akt phosphorylation
was performed by the use of two different antibodies, which recognize
the phosphorylated form (phospho-Akt) and the total kinase (Akt).
With anti-phospho-Akt Ab, VK2/E6E7 cells revealed increased Akt
Immunology and Cell Biology
activity in cells treated with LMW-HA (Figure 3c). Western blot
analysis with anti-Akt Ab showed that total Akt levels were comparable among different samples (Figure 3c). The relative protein estimation using standard scanning densitometry revealed that in untreated
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sample pAkt corresponded to 23% of total Akt, while in LMW-HAtreated sample pAkt corresponded to 82% of total Akt. Western blot
analysis for the same proteins panel, performed on HMW-HA-treated
samples, showed a substantial absence of activity for this specific form
of HA.
When VK2/E6E7 cells were incubated with the PI3K inhibitor
LY-294002, previously used as a pharmacological inhibitor of Akt
phosphorylation,30 plus LMW-HA, we observed a wound-healing
activity comparable to untreated cells (Figure 3d), confirming the
essential role of Akt phosphorylation in the pathway leading to LMWHA-induced vaginal epithelial cells migration.
A central event in the cell migratory process is active reorganization
of filamentous actin. As cells become motile, they extrude actin-rich
projections (lamellipodia/filopodia) that transiently adhere to the
underlying matrix to create traction forces necessary for forward cell
movement.31–33 It has been reported that the Akt/PI3K pathway in
wound healing is linked to lamellipodia formation at the leading
edge;34 to further assess the involvement of this pathway in the LMWHA-induced vaginal epithelial cells migration, a staining assay of
cytoskeletal actin was performed by the use of fluorescent phalloidin.
Confocal microscopy imaging (Figure 3e) revealed that cells treated
with LMW-HA presented a prevalence of lamellipodia formation
(arrows), with only a few filopodia, at the leading edge and an
increased density of F-actin bundles within lamellipodia. Untreated
cells showed the presence of only a small number of pseudopodia,
with prevalence of filopodia (arrowhead) (Figure 3e).
Data of literature indicate that pMLC has been implicated in
generation of the contractive force required to support cellular
migration35 and is essential in actin-myosin interaction, stress fiber
and contractile ring formation and ultimately cell motility; therefore,
we evaluated pMLC expression in LMW-HA-treated cells by western
blot analysis. Western blot analysis of MLC phosphorylation was
performed by the use of two different antibodies, which recognize
the phosphorylated form (pMLC) and the total protein (MLC). With
anti-pMLC Ab, VK2/E6E7 cells revealed increased MLC activity
following treatment with LMW-HA, in comparison with untreated
cells (Figure 3c). Western blot analysis with anti-MLC Ab showed that
total MLC levels were comparable among different samples
(Figure 3c). The relative protein estimation using standard scanning
densitometry revealed that in untreated sample pMLC corresponded
to 9% of total MLC, while in LMW-HA-treated sample pMLC
corresponded to 21% of total MLC. The same protein pattern
evaluation after treatment with HMW-HA showed no relevant alterations both in absolute expression and in total- vs phosphorilated-form
ratio.
Collectively, these results indicate that an intracellular pathway
involving both Akt and MLC phosphorilation is required for activating the cytoskeletal motor that moves vaginal epithelial cells following
LMW-HA treatment.
DISCUSSION
The vagina makes up the lower reproductive tract that is exposed to
the external environment as well as commensal flora: therefore,
epithelial cells lining the vaginal tract are constantly exposed to
microbiota, representing a primary site of host–microbe interactions.
Epithelial barriers represent a ‘first-line’ defense, responsible for
inactivation of microorganisms, having thereby a critical role in the
prevention of immune cell activation and infection. Remarkably,
epithelial cells possess an intrinsic capacity to survive despite the
continuous exposure to considerable amounts of microorganisms
present in the environment, potentially toxic to host cells, suggesting
that some homeostatic forces link antimicrobial strategies to mechanisms for control tissue integrity.36
Our results show that the human vaginal epithelial cell line VK2/
E6E7 expresses TLR2 and TLR4: given the roles imputed to TLR2 and
TLR4 in the regulation of innate immunity and response to bacterial
challenge, their expression by vaginal epithelium seems of note. In
fact, TLR2 and TLR4 are activated by a broad spectrum of microbeassociated molecular patterns, including peptidoglycan and LPS for
gram-positive bacteria, bacterial lipoproteins, mycobacterial cell wall
lipoarabinomannan and yeast cell walls:11 thus, their presence in the
non-sterile lower tissues of the female reproductive tract, such as the
vagina, harboring a variety of commensal bacteria, among which
Lactobacilli normally predominate, may reflect adaptation of the
innate immune response to the distinct microbial milieu encountered
by this tissue. Our data obtained in vitro are sustained by literature
data, reporting the presence of different members of TLRs family in
samples from human female reproductive tract.11,37
Although a majority of studies have focused on mucosal adaptive
immunity of the female reproductive tract, only recently the attention
has been focused on the innate immune factors. Mounting evidence
suggests that the vaginal epithelium is more than a simple physical
barrier to protect against invading pathogens.7,38 On the contrary, this
surface and its overlying fluids are replete with antimicrobial peptides
and proteins that act as effectors of innate host defense. The layer of
mucosal fluid that covers the cervical and vaginal epithelia is composed of secretions from the cervical vestibular glands, endometrial
and oviductal fluids, and plasma transudate; vaginal epithelial cells,
cervical glands and neutrophils supply the majority of antimicrobial
peptides to the milieu of the vaginal fluid, including calprotectin,
lysozyme, lactoferrin, SLPI, human neutrophil protein-1, -2 and -3,
and HBDs.
Our results show that vaginal epithelial cells treated with LMW-HA
increase their production of different antimicrobial peptides. The wide
variety of antimicrobial peptides induced by LMW-HA treatment is
certainly highly interesting as each of them is characterized by the
ability to exert a peculiar activity in the lower female reproductive
tract, including both the innate and adaptive immunity and the crosstalk between them. Lactoferrin, for example, has been reported to link
innate and adaptive immunity through induction of dendritic cells
maturation; b-defensins have been shown to rapidly bind the negatively charged surface of bacteria resulting in the disruption of the
bacterial membrane and cell lysis, and to directly induce the full
maturation of iDCs. Further, SLPI is a potent inhibitor of proteases
that kills both gram-positive and gram-negative bacteria and has also
been shown to have antifungal capabilities and to block infection by
the human immunodeficiency virus.39 Therefore, as we have demonstrated that LMW-HA is an effective inducer of many different
antimicrobial peptides, it may endow vaginal epithelial cells with a
plethora of active molecules, which provide a broad antimicrobial and
protective activity against different microbiota. Further, although
Singh et al.40 found only an additive effect between HBD2 and
lysozyme, they did find synergism with the following combinations:
lysozyme/lactoferrin, lysozyme/SLPI, lactoferrin/SLPI and lysozyme/
LL37. Thus, although individually many of the proteins may be
present in the vaginal fluid at concentrations below their active
range, in the epithelium, and all together, they may constitute an
effective barrier to microbial invasion of the epithelial surface.
Silencing experiments using siRNA for TLR2 and TLR4 have clearly
shown that LMW-HA-induced HBD2 production involves the activation of these two receptors. Additionally, silencing experiments with
siRNA for CD44, which is classically reported to bind LMW-HA, have
Immunology and Cell Biology
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excluded its involvement in this antimicrobial peptide production,
because cells depleted of CD44 activity have HBD2 levels following
LMW-HA treatment comparable to the ones of cells treated with
LMW-HA alone. Therefore, in this case LMW-HA acts as an endogenous ligand of TLR2 and TLR4, inducing an antimicrobial peptides
production, without activation of TLR-proinflammatory response
based on NF-kB nuclear translocation. This important evidence is
consistent with the ELISA results for proinflammatory cytokines
levels: LMW-HA is not able to induce a significant nuclear translocation of NF-kB and consequent IL-1b, IL-6, IL-8 and tumor necrosis
factor-a production. These results extend our previous observations
regarding the TLR-ligand role of LMW-HA.13 In fact, as HA is usually
present in the underlying connective tissue in the form of HMW
polymer, the presence of important amounts of LMW fragments
because of a tissue injury is perceived by the epithelial compartment
as a danger signal for the organism,41 which responds with antimicrobial peptides release to fight an eventual microorganism invasion.
In particular, the stimulation of antimicrobial polypeptides by
LMW-HA treatment might have a greater role in vaginal host defense
of women who have high vaginal pH and low concentrations of
lactate, as lactic acid contributes to the resistance of the normal vagina
to colonization by exogenous microbes. Future studies will be necessary to determine whether there are differences in the concentration of
antimicrobial polypeptides in the vaginal fluid of women without
chronic genital pathologies compared with women who have recurrent
episodes of diseases linked with abnormal levels of microbial colonization, such as bacterial vaginosis and candidiasis: in this situation, a
therapeutic use of LMW-HA-based products might increases levels of
antimicrobial peptides in women with a low production of these
molecules. Regarding this possible application, our in vitro data on
vaginal epithelial cells are sustained by our previous observations on
HBD2-stimulating activity of LMW-HA in whole skin in vivo:13 thus,
it is reasonably plausible that the antimicrobial peptides transcription
induction showed in in vitro experiments may be present also in vivo
in the lower female reproductive tract.
The epithelial lining of the female genital tract forms a selectively
permeable barrier separating the lumen from underlying tissues and
breakdown of this barrier by mucosal injury is often accompanies
severe inflammatory conditions and traumatic stress.
Fluid loss and influx of antigens exacerbate inflammatory responses
and preservation of epithelial barrier function sealing mucosal defects
is a pivotal point for anti-inflammatory action. A major mechanism
by which wound healing is achieved involves epithelial cells migration,
also referred to as ‘restitution’. The complexity of wound healing
requires a well-orchestrated system to control this process: recent
evidences increasingly suggest the importance of immune mechanisms
as part of this regulatory cascade. The innate immune response that
serves to eliminate infections is probably also active in restoring the
structural and functional integrity of injured epithelia. Interestingly,
accumulating evidence also suggests that TLRs have a role in cell
migration and injury repair processes, although they are not considered classical wound-healing activators.42
Our data show that treatment of vaginal epithelial cells with LMWHA ameliorates, the ability of these cells to reseal an epithelial wound
increasing their migratory activity without stimulating their proliferation rate. Silencing experiments with siRNA have shown that this
stimulation of vaginal epithelial cells migration by LMW-HA is
independent from CD44, but is strictly linked to TLR2 and TLR4
activity. Involvement of TLRs in epithelial repair has recently been
demonstrated in vitro, following stimulation of these receptors by
microbial patterns, such as Staphylococcus aureus:43 the novelty of our
Immunology and Cell Biology
work resides in the stimulatory activity exerted by LMW-HA, a safe
and endogenous molecule, on these receptors. This effect may be
induced by two different and complementary mechanisms: a direct
activity of LMW-HA as a ligand on TLR2 and TLR4, linked to the
reported contribution of these receptors to epithelial repair processes
and our experimental evidence that treatment of vaginal epithelial cells
with LMW-HA increases their production of antimicrobial peptides:
the effects of these proteins on epithelial repair is well established.44–46
Thus, both these aspects may contribute the observed wound-healing
effects of LMW-HA treatment on vaginal epithelium.
Our investigations on the intracellular pathway involved in the
observed LMW-HA-induced vaginal epithelial repair process have
shown that this effect involves activation of Akt/PI3K pathway on
activation of TLR2 and TLR4. Different works have reported that Akt
is activated at the leading edge of a migrating cell and has an essential
role in directed cell migration;34,47 additionally, Gingis-Velitski et al.48
have shown that direct activation of Akt by heparanase is able to
induce epithelial cells migration. Therefore, the use of a molecule that
directly activates Akt may be beneficial in the stimulation of woundhealing activity: this supports our hypothesis to use LMW-HA to
stimulate wounds restitution, by activating Akt pathway.
Therefore, our data show that LMW-HA in vaginal compartment
may exert a double activity: increase the natural protection of this
compartment in physiological conditions and induce resolution
of vaginal surface epithelial healing disorders, supporting a role
for LMW-HA application in loco. In particular, induction of antimicrobial peptides and wound-healing activity by LMW-HA treatment may furnish an additional protective activity in this body
compartment, which is highly and constantly exposed to microbiota,
ameliorating vaginal epithelium self-defense in both basal and pathological conditions.
METHODS
Cell treatments
The human vaginal epithelial cell line VK2/E6E7, obtained from normal human
vaginal epithelium, immortalized by expression of human papillomavirus 16/
E6E7,49 was kindly provided by Dr Raina Fichorova (Department Obstetrics
and Gynecology, Brigham and Women’s Hospital, Boston, MA, USA). Cells
were cultured in keratinocyte-SFM with 0.1 ng ml–1 EGF, 50 mg ml–1 bovine
pituitary extract, 10 ml l–1 of penicillin/streptomycin (all from Gibco, Invitrogen, Auckland, New Zealand) and 0.4 mM CaCl2 (Sigma-Aldrich, St Louis, MO,
USA).
For experiments on antimicrobial peptides stimulation, cells were cultured in
medium alone or in medium added with LPS from E. coli (5 mg ml–1, SigmaAldrich) or peptidoglycan from E. coli (10 mg ml–1, InvivoGen, San Diego, CA,
USA) or in medium containing 0.2% w/v of LMW-HA sodium salt (Soliance,
Paris, France; Mr o200 kDa, obtained by biosynthesis). LMW-HA concentration
was chosen according to literature data,50–52 accordingly with our previous doseresponse experiments on keratinocytes13 and depending on the solubility of the
sodium salt and cells viability. Incubation with a solution containing 0.2% HMWHA sodium salt served as control. All experiments were performed in triplicate.
The absence of endotoxin contamination in the LMW-HA sodium salt
solution was initially assessed by LAL test (Sigma-Aldrich), which resulted
negative for the presence of endotoxins; as LAL assay may be insensitive to
detect small amounts of LPS and does not detect lipoproteins, additional
controls included both hyaluronidase (bovine testicular hyaluronidase, SigmaAldrich) treatment of LMW-HA (3 h at 37 1C with 10 U hyaluronidase per 50 ml
LMW-HA solution) and boiling of LMW-HA solution, to denature possible
protein contaminants. All these assays showed no presence of contamination
(data not shown).
For evaluation of HBD2 release, media were collected at different time
points, starting from 30 min to 18 h after the beginning of treatment. For
evaluation of IL-8, tumor necrosis factor-a, IL-1 and IL-6 release in the
Immunomodulatory activity of LMW-HA in vagina
GF Dusio et al
637
Table 1 PCR primers and cycles used for different antimicrobial peptides transcription analysis
Gene
Primers
Cycle
HBD1
HBD1 Fw 5¢-GTCAGCTCAGCCTCCAAAGG-3¢
HBD1 Rw 5¢-CTTCTGCGTCATTTCTTCTG-3¢
Denaturation at 94 1C for 3¢; 40 cycles of denaturation at 94 1C for 30¢¢, annealing at
55 1C for 30¢¢, extension at 72 1C for 1¢; final extension at 72 1C for 5¢
HBD2
HBD2 Fw 5¢-TTTGGTGGTATAGGCGATCC-3¢
HBD2 Rw 5¢-GAGGGAGCCCTTTCTGAATC-3¢
Denaturation at 94 1C for 5¢; 35 cycles of denaturation at 94 1C for 1¢, annealing at 62 1C
for 1¢, extension at 72 1C for 2¢; final extension at 72 1C for 10¢
HBD3
HBD3 Fw 5¢-CTGTTTTTGGTGCCTGTTCC-3¢
HBD3 Rw 5¢-CTTTCTTCGGCAGCATTTTC-3¢
Denaturation at 94 1C for 3¢; 40 cycles of denaturation at 94 1C for 15¢¢, annealing at
48 1C for 20¢¢, extension at 68 1C for 30¢¢; final extension at 68 1C for 5¢
Lactoferrin
Lactoferrin Fw 5¢-CTCCAGACCGCAGACATGAA-3¢
Lactoferrin Rw 5¢-CTGGGAGGAGAAGGCACATT-3¢
Denaturation at 94 1C for 3¢; 40 cycles of denaturation at 94 1C for 15¢¢, annealing at
57 1C for 20¢¢, extension at 68 1C for 30¢¢; final extension at 68 1C for 5¢
LL37
LL37 Fw 5¢-TCGGATGCTAACCTCTACCG-3¢
LL37 Rw 5¢-GGGTACAAGATTCCGCAAAA-3¢
Denaturation at 94 1C for 5¢; 40 cycles of denaturation at 94 1C for 1¢, annealing at 52 1C
for 1¢, extension at 72 1C for 90¢¢; final extension at 72 1C for 10¢
SLPI
SLPI Fw 5¢-CTGTGGAAGGCTCTGGAAAG-3¢
SLPI Rw 5¢-GCCATAAGTCACTGGGCACT-3¢
Denaturation at 94 1C for 3¢; 40 cycles of denaturation at 94 1C for 15¢¢, annealing at
57 1C for 20¢¢, extension at 68 1C for 30¢¢; final extension at 68 1C for 5¢
b-Actin
B-ACT Fw 5¢-GCATGGAGTCCTGTCGCATCCACG-3¢
B-ACT Rw 5¢-CGTCATACTCCTGCTTGCTGATCCA-3¢
Denaturation at 94 1C for 2¢; 40 cycles of denaturation at 94 1C for 1¢, annealing at 55 1C
for 30¢, extension at 72 1C for 30¢¢; final extension at 72 1C for 7¢
medium, VK2/E6E7 supernatants were collected 18 h after the beginning of
treatment. For experiments on receptor silencing, siRNA for TLR2 and TLR4
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and for CD44 (OriGene,
Rockville, MD, USA) were used according to the manufacturer’s instructions,
during the incubation with 0.2% LMW-HA. In both cases, the silencing
efficiency of siRNA was checked by western blot analysis (data not shown) of
corresponding protein.
In order to confirm the role of Akt pathway in the signaling cascade leading
to the LMW-HA-induced wound-healing activity, VK2/E6E7 cells were incubated with the PI3K inhibitor LY-294002 (Merck, Darmstadt, Germany), at the
final concentration of 10 mM, together with the usual LMW-HA concentration.
Mitomycin C (Sigma-Aldrich) was prepared as 0.5 mg ml–1 stock solution in
water, made fresh for each experiment, and used on cell cultures at the final
concentration of 10 mg ml–1; incubation with mitomycin C started 24 h before
scratch wound induction.
PCR analysis
Transcription levels of HBD1, HBD2, HBDB3, LL37, SLPI and lactoferrin were
investigated in VK2/E6E7 cells by reverse transcriptase-PCR. Total RNA was
isolated and converted into complementary DNA. PCR was performed using
the HotGold Star Mix (Eurogentec, Explera srl, Jesi, Italy). b-Actin RNA
transcription levels were used as a reference loading control. The PCR cycles
and primers used for different antimicrobial peptides mRNA are reported in
Table 1. The PCR products were visualized on an ethidium bromide 1%
agarose gel.
Immunofluorescence
VK2/E6E7 cells were sub-cultured on cover glasses and fixed in methanol. For
TLR2 and TLR4 staining, fixed cells were incubated with the respective primary
antibodies (Santa Cruz Biotechnology) and secondary antibody donkey antigoat TRITC (Molecular Probes Inc., Eugene, OR, USA). Proper controls were
performed, by omission of primary antibody and by use of an isotype control
antibody. Nuclei were counterstained with 4¢,6¢-diamidino-2-phenylindole.
Samples were observed under a Nikon Eclipse 80i microscope equipped with
a digital Nikon DS-L1 camera (Nikon Instruments, Firenze, Italy).
For cytoskeleton staining, following fixation with formaldehyde, cells were
incubated with fluorescein isothiocyanate-conjugated phalloidin (SigmaAldrich) and nuclei were counterstained with 4¢,6¢-diamidino-2-phenylindole;
samples were observed by the use of a Nikon C1si Spectral confocal microscope
(Nikon).
ELISA
Production of human HBD2 in VK2/E6E7 cells supernatants was quantified
using the Enzyme Immuno Assay kit from Phoenix Pharmaceuticals (Belmont,
CA, USA). Concentrations of IL-8, tumor necrosis factor-a, IL-1b and IL-6 in
VK2/E6E7 supernatants were evaluated using ELISA kits obtained respectively
from GE Healthcare (Buckinghamshire, UK), Endogen (Woburn, MA, USA)
and MedSystem Diagnostic GmbH (Vienna, Austria, for both IL-1b and IL-6)
and carried out according to the manufacturer’s instructions.
Western blot analysis
Following the different treatments, proteins were extracted from cells by
incubation in lysis buffer (0.1 M NaCl, 0.01 M Tris–HCl pH 7.6, 0.001 M EDTA,
pH 8), 1% Triton X-100, 0.5 ng ml–1 leupeptin, 1 ng ml–1 pepstatin, 2 ng ml–1
aprotinin and 100 mg ml–1 phenylmethylsulfonyl fluoride (all reagents by
Sigma-Aldrich) on ice for 45 min. Proteins were quantitatively estimated using
the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein samples (15 mg)
were fractionated on a 8% acrylamide (BIO-RAD Laboratories, Hercules, CA,
USA) slab gel containing 0.1% sodium dodecyl sulfate (SDS) (Sigma-Aldrich)
and transferred onto a nitrocellulose filter (Amersham Bioscences, Little
Chalfont, UK) by electro blotting. After incubation for 1 h in TBS with 1%
Tween-20 (Sigma-Aldrich) and 5% milk powder (Merck) to block nonspecific
binding sites, the filter was incubated with primary antibodies directed to TLR2
or TLR4 (Santa Cruz Biotechnology), diluted 1:100 in TBS with 5% milk
powder, for 1 h; after three washes for 20 min each in TBS, 1% Tween-20 and
5% milk powder, the filter was incubated with secondary antibody in TBS,
0.1% Tween-20 and 5% milk powder for 1 h at room temperature. Bands were
visualized using ECL Western Blotting Detection Reagents and autoradiography
film (Amersham Biosciences, Piscataway, NJ, USA).
For determination of Akt and MLC activation, VK2/E6E7 cells treated as
previously reported were washed with cold phosphate-buffered saline (PBS)
and lysed with lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton,
10% glycerol, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg ml–1 leupeptin and 10 mg ml–1 aprotinin). The crude lysate was
centrifuged at 13 000 r.p.m. for 10 min at 4 1C. Samples were then boiled for
5 min at 95 1C. Protein concentrations were determined by BCA assay (Pierce
Biotechnology Inc., Rockford, IL, USA). Total cell lysates (50 mg per lane) were
subjected to 10% SDS-polyacrylamide gel electrophoresis, and proteins were
blotted to nitrocellulose membranes (Amersham Bioscience). After blocking
with Blot solution (5% low fat dry milk in PBS), filters were probed with antiphospho-Akt antibody (New England Biolabs Inc., Beverly, MA, USA) or antiphospho MLC antibody (Santa Cruz Biotechnology), and proteins were
visualized with peroxidase-coupled secondary antibody using the ECL detection system (Amersham Bioscience). Filters were stripped in buffer containing
62.5 mM Tris–HCl, pH 6.8, 2% SDS and 100 mM b-mercaptoethanol for 30 min
at 65 1C, washed three times in PBS, blocked, and reprobed with anti-Akt
antibody (New England Biolabs Inc.) or anti-MLC antibody (Santa Cruz
Biotechnology).
Immunology and Cell Biology
Immunomodulatory activity of LMW-HA in vagina
GF Dusio et al
638
For determination of NF-kB expression and translocation VK2/E6E7, cells
treated with LMW-HA and LPS as previously reported were washed with cold
PBS and nuclear and cytoplasmic proteic fractions were collected with EpiQuik
nuclear extraction kit (Epigentek Group Inc., Brooklyn, NY, USA) following
constructor instructions protein concentrations were determined by BCA assay
(Pierce Biotechnology Inc.). Cell lysates (20 mg per lane) were subjected to 8%
SDS-polyacrylamide gel electrophoresis, and proteins were blotted to nitrocellulose membranes (Amersham Bioscience). After blocking with Blot solution
(5% low fat dry milk in TBS), filters were probed with anti-NF-kB antibody
(Santa Cruz Biotechnology) and proteins were visualized with peroxidasecoupled secondary antibody using the ECL detection system (Amersham
Bioscience).
Scanning densitometry on the developed film was conducted using an Umax
Powerlook III Scanner and analyzed using Phoretix 1D Pro analysis software
(NonLinear Dynamics, Newcastle upon Tyne, UK). The relative expression of
each phosphorylated form is presented as a percentage of total protein
evaluated on the film following optical density analysis.
Antimicrobial assay
VK2/E6E7 cells were treated with 0.2% LMW-HA or medium alone
and proteins were extracted under gentle agitation for 2 h in 5% acetic
acid with addition of protease inhibitors (phenylmethylsulfonyl fluoride
0.02 mM, pepstatin 2 ng ml–1, leupeptin 2 ng ml–1). The soluble proteins in
the supernatant were dried under vacuum and resuspended in 0.01%
acetic acid. Protein concentrations were determined by BCA assay (Pierce
Biotechnology Inc.).
For examination of the activity of antimicrobial peptides produced by cells,
we first incubated 104 bacteria ml–1 (OD600) of mid-logarithmic-phase E. coli
American Type Culture Collection 4157 (International PBI, Milan, Italy) in
phosphate buffer pH 7.4 in a final volume of 100 ml. The E. coli suspension was
mixed with 30 mg of the cellular protein extracts or with 10 mg of HBD2
recombinant peptide (positive control) (Millipore Corp., Milan, Italy) or with
10 ml of 0.01% acetic acid and protease inhibitor cocktail (negative control) and
incubated at 37 1C for 120 min. At the end of the incubation, 10 ml of these
bacteria suspensions were plated in triplicate in Petri dishes containing Tryptic
Soy Broth (International PBI) and incubated overnight at 37 1C. At the end of
incubation, the number of CFU for each sample was determined.
Wound-healing assay
To perform a wound-healing assay, a scratch wound was made with a sterile
razor blade on confluent monolayers of VK2/E6E7 cells and the debris was
removed by washing with serum-free medium. The cells were further cultured
in fresh medium or in medium containing 0.2% LMW-HA or 0.2% HMW-HA
and photographed immediately (t¼0 h). Additional experiments included the
use of specific siRNA for TLR2, TLR4 or CD44, as previously described. The
distance of wound closure (compared with control at 0 h) was measured in
three independent wound sites per group. The percentage of wound closure was
calculated as the wound width at t¼0 h minus the wound width at t¼8 h,
t¼12 h and t¼24 h, as evaluated by phase-contrast microscopy. All measurements were performed using the software Image pro-plus (version 4.5.019;
Media Cybernetics, Bethesda, MD, USA). Values from three independent
experiments were pooled and expressed as means±s.d.
Cell proliferation assay
Following incubation of VK2/E6E7 cells with medium alone or additioned with
0.2% LMW-HA, cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, essentially as described.53
MTT was dissolved at a concentration of 5 mg ml–1 in sterile PBS. Following
18 h of treatment with LMW-HA or medium alone, 12 ml of the 5 mg ml–1
solution of MTT was added to each well, and the incubation continued for 24 h.
In all, 100 ml of the cell lysis buffer (20% SDS/50% N,N-dimethylformamide,
pH 4.7) were added at different time-points. The absorbance was read at
570 nm on a microplate reader. To assess cell viability, cell counts using Trypan
blue exclusion were also performed.
Immunology and Cell Biology
Statistical analysis
Student’s t-test (paired two-tailed) and GraphPad Prism software (GraphPad
Prism Software Inc., San Diego, CA, USA) were used for comparisons between
groups. P-values o0.05 were considered significant.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Immunology and Cell Biology