Abstract
The mucosal barrier consisting of physicochemical, immune, and
microbial components is the first line of defense against external
stimuli. Breakdown of the mucosal barrier causes the occurrences of
various diseases, while methods capable of multifacetedly restoring
mucosal barrier functions have been rarely reported. Here, we describe
the restoration of the physicochemical, immune, and microbial
homeostasis of the mucosal barrier by in situ formation of a
living-synthetic therapeutic coating (LSTC). Through metal-phenolic
complexation and π-π stacking interactions, ethyl gallate can chelate
Bi^III ions to form an adhesive coating on mucosal surfaces, which
enables further hybridization with living bacteria. Due to the
beneficial effects of Bi^III and ethyl gallate and the probiotic
characteristic of carried bacteria, LSTC increases the barrier
integrity, mitigates mucosal inflammation, and maintains normal
homoeostasis of the microbiota. In two murine models of aerobic
vaginitis and vaginal candidiasis, LSTC demonstrates the potency to
alleviate vaginal pathological injury and decrease vaginal inflammatory
infiltration.
Subject terms: Drug delivery, Drug delivery, Biosurfaces
__________________________________________________________________
Vaginal infections influence about 75% of adult women worldwide each
year. Here, the authors report a strategy to treat vaginal infection by
restoring the homeostasis of the vaginal mucosal barrier through in
situ formation of a living-synthetic therapeutic coating.
Introduction
The mucosal barrier is the first line of defense against infection and
environmental insults as most noxious stimuli enter the body at mucosal
surfaces^[42]1–[43]3. Mucosal barrier homeostasis is primarily
maintained by the combined effects of commensal microbiota, the
physicochemical barrier, and mucosal immune responses^[44]4,[45]5. The
breakdown of the mucosal barrier may lead to the pathogenesis and
progression of a variety of diseases, including infection, allergy, and
inflammation^[46]6–[47]8. Restoration of mucosal barrier functions has
emerged as a promising approach to protect the host from exogenous
stimuli. For example, immunoregulatory agents, such as anti-tumor
necrosis factor (TNF) antibody drugs, are currently prevailing
treatments to revert the dysregulated mucosal barrier through blocking
a specific inflammatory pathway^[48]9,[49]10. While, inhibition of
immune functions often results in an increased risk of severe or even
life-threatening infections^[50]11. Furthermore, several small molecule
kinase inhibitors have been exploited to directly promote mucosal
healing by reinforcing barrier integrity or increasing epithelial
regeneration^[51]12. Unfortunately, the promotion of mucosal healing
may cause abnormal cell proliferation and thus induce oncogenic
transformation^[52]13. More importantly, the unitary modality of these
therapeutics inevitably suffers an inadequate efficacy, due primarily
to the inability to synergistically improve the microbial, immune, and
physicochemical dysfunctions. Therefore, exploration of methods capable
of restoring mucosal barrier functions to resist exogenous damaging
factors is of great significance in disease treatment.
Mucosal surfaces are characteristically colonized by a living and
dynamic microbial ecosystem as the direct exposure to external
environments^[53]14,[54]15. The microbiota plays a fundamental role in
regulating mucosal barrier functions by dialoguing with the epithelium
and mucosal immune system^[55]16. For instance, mucus-dwelling bacteria
can degrade mucin produced by specialized epithelial cells and secrete
metabolites that in turn influence mucus production and epithelial
integrity^[56]17,[57]18. Meanwhile, certain bacteria affect the
functions of both the innate and adaptive immune systems to maintain
immune homeostasis, yet microbiota dysbiosis can trigger several immune
disorders through the activity of macrophages, neutrophils, or T
cells^[58]19,[59]20. Nevertheless, the breakdown of the epithelial
barrier leads to immediate leakage of bacteria and bacterial
derivatives, which contribute to systemic deterioration and
subsequently aggravate the local destruction of the mucosal
barrier^[60]21,[61]22. In addition, inflammatory environment is
conducive to the blooms of certain opportunistic pathogens, which
secrete specific toxins to kill beneficial bacteria and promote the
necrosis of epithelial cells^[62]23. Given these close mutual
interactions, we hence speculate that the restoration of a healthy
microbial structure together with the mitigation of physicochemical and
immune dysfunctions may provide an alternative to recover mucosal
barrier homeostasis. However, to the best of our knowledge, methods
capable to multifacetedly orchestrate mucosal barrier functions have
been rarely reported.
Here, we report the use of in situ formation of a living-synthetic
therapeutic coating (LSTC) to restore the physicochemical, immune, and
microbial homeostasis of the mucosal barrier (Fig. [63]1). With the
help of metal-phenolic complexation and π-π stacking interactions, an
ethyl gallate-Bi^III coating hybridized with living bacteria can be
readily generated on mucosal surfaces. We show that LSTC is flexible to
hybridize diverse bacterial strains and can be steadily formed on the
surface of different types of mucosae. By perfusing Bi^III ions, ethyl
gallate (EG), and Lactobacillus rhamnosus GG (LGG), the resulting LSTC
is able to restore the physicochemical, immune, and microbial functions
of the mucosal barrier, thanks to the combination of the
barrier-repairing ability of Bi^III ions, the immunosuppressive nature
of EG, and the microbiota-regulating behavior of LGG. Consequently,
LSTC recovers the barrier integrity by elevating the expressions of
tight junctions and mucus, alleviates mucosal inflammation by
suppressing inflammasome activation, and maintains normal homeostasis
of the microbiota by inhibiting the colonization of pathogens.
Fig. 1. Schematic illustration.
[64]Fig. 1
[65]Open in a new tab
A Preparation of LSTC by in situ forming an EG-Bi^III coating
hybridized with living LGG on the mucosal surface. B Combination of the
barrier-repairing ability of Bi^III ions, the immunosuppressive feature
of EG, and the microbiota-regulating behavior of LGG enables LSTC to
multifacetedly restore mucosal barrier functions. Specifically, LSTC
improves barrier integrity by increasing the secretions of tight
junctions and mucus, remits mucosal inflammation by suppressing the
production of inflammatory cytokines, and maintains normal homeostasis
of the microbiota by inhibiting the colonization of pathogens. Created
in BioRender.
Vagina is a major mucosal interface connecting the inner and outer
environments in the body^[66]24. Vaginal infections, one of the most
common reasons for women medical consultation, influence about 75% of
adult women worldwide each year^[67]25. Although vaginal infections are
not associated with high mortality rate, they severely increase the
risk of serious consequences, including infertility, cervical cancer,
and acquisition of the human immunodeficiency virus^[68]26–[69]28.
Currently available treatments are antimicrobial therapy, which is
effective but has a high incidence of recurrence and potential risk of
causing antibiotic resistance^[70]29,[71]30. As a proof-of-concept
application, we show that following vaginal delivery, LSTC exhibits
satisfactory efficacies in treating vaginal infections, supported by
the potency to alleviate vaginal pathological injury and reduce vaginal
inflammatory infiltration in two mouse models of aerobic vaginitis and
vaginal candidiasis. We anticipate that LSTC provides a versatile
platform to orchestrate multifaceted barrier functions via synergizing
bacterial agents with conventional therapeutics at the interface of
mucosae.
Results and discussion
Preparation and characterization of LSTC
Previously, metal-polyphenol complexes have been employed to coat
bacteria and improve their intraluminal colonization for enhanced
inflammation treatment^[72]31. While the therapeutic potential of
metal-polyphenol complexes coated on bacterial surface is limited due
to their overall low dose and negligible amount adhered to the mucosal
surface. Therefore, we chose the formation of a metal-polyphenol
coating hybridized with living bacteria on the mucosal surface to play
a therapeutic effect. LGG, which has been chosen as a model probiotic,
because it widely resides the mucosal surfaces of the gastrointestinal,
respiratory, and vaginal tracts and plays a pivotal role in host
health^[73]32–[74]34. To form LSTC, EG extracted from Turkish galls and
Bi^III ions were separately selected considering the ability to
suppress inflammation^[75]35,[76]36 and the activity to promote mucosa
repair^[77]37,[78]38. Note that both EG and Bi^III ions have been
approved by the Food and Drug Administration for use as a food additive
and a nutritional supplement^[79]31,[80]38,[81]39, respectively.
Through the formation of metal-phenolic complexation and π-π stacking
interactions, EG could chelate Bi^III ions to form an adhesive coating,
which was able to hybridize with LGG. To optimize the formation of the
coating, EG and Bi(NO[3])[3]·5H[2]O solutions with different
concentrations were successively added to an agar plate and mildly
stirred for deposition. As shown in Supplementary Fig. [82]1, under the
conditions of 0.6 mg/ml EG and 0.2 mg/ml Bi(NO[3])[3]·5H[2]O, the
resulted coating (termed EG-Bi) showed the highest efficiency for LGG
attachment, accompanied by undetectable adverse effects on bacterial
viability (Supplementary Fig. [83]2). The cytocompatibility of EG-Bi
was verified using a cell counting kit-8 (CCK-8) assay, which displayed
a negligible cytotoxicity against both epithelial and macrophage cell
lines even after incubation for 24 h (Supplementary Fig. [84]3). We
reasoned that the high cytocompatibility of EG-Bi could be attributed
to relatively low dosage of EG and Bi^III ions used for preparation and
rapid chelation during the coating process. Surface morphology of EG-Bi
was characterized by scanning electron microscopy (SEM) and
transmission electron microscopy (TEM), both suggesting a dense
nanoparticulate surface structure (Fig. [85]2A, B). Atomic force
microscopy (AFM) imaging further confirmed the formation of EG-Bi and
highlighted that the thickness of the coating was 96.8 ± 1.1 nm
(Fig. [86]2C). The water contact angle of silicon wafer surface
decreased from 88.5 ± 2.0° to 13.9 ± 1.2° after decorating with EG-Bi
(Fig. [87]2D), suggesting the hydrophilic surface property of the
coating. As the mucosal barrier is generally covered with a gel-forming
mucus layer^[88]40, the plate surface was coated with an artificial
mucus layer containing commercial mucin and agar to assess whether
EG-Bi could form on such a surface. In contrast, obviously increased
fluorescence signaling in the FITC channel was observed in the EG-Bi
group after staining the surface with fluorescein
isothiocyanate-labeled bovine serum albumin (BSA-FITC) (Fig. [89]2E),
verifying the decoration of EG-Bi on the mucus surface. We next
evaluated the flexibility of EG-Bi and found that its thickness
increased with the concentrations of EG and Bi(NO[3])[3]·5H[2]O. When
the concentrations of EG and Bi(NO[3])[3]·5H[2]O were separately
increased to 1.2 and 0.4 mg/ml, three-dimensional laser scanning
confocal microscopy (3D LSCM) images showed a 1.5-fold increment in
EG-Bi thickness (Fig. [90]2F).
Fig. 2. Characterization of LSTC.
[91]Fig. 2
[92]Open in a new tab
SEM (A), TEM (B), and AFM (C) images of the EG-Bi coating. The
experiment was independently repeated at least twice with similar
results. D Water contact angles of the EG-Bi coating tableted on
silicon wafer. E Fluorescence images of the EG-Bi coating labeled with
BSA-FITC. F 3D LSCM images of the EG-Bi coating with a relatively low
(0.6 mg/ml EG and 0.2 mg/ml Bi(NO[3])[3]·5H[2]O) or high (1.2 mg/ml EG
and 0.4 mg/ml Bi(NO[3])[3]·5H[2]O) concentration. SEM (G) and LSCM (H)
images of LSTC containing LGG, EF, LD, or EcN. Red channels indicate
Cy5.5-labeled or mCherry-expressing bacteria. I Counts of adherent LGG,
EF, LD, or EcN on LSTC after a 24-h culture on MRS or LB agar plates at
37 °C (n = 3 independent samples). LSTC without the EG-Bi coating was
used as a control in D, E, G, H, and I. Data are mean ± standard
deviation (SD). Statistical analysis was performed using a two-tailed
Student’s t-test, and p-values were indicated. Source data are provided
as a Source Data file.
Next, we studied the formation of LSTC by examining the hybridization
of living bacteria onto EG-Bi. After incubation EG-Bi with LGG for
5 min, SEM and TEM images visualized the increased amounts of LGG on
the EG-Bi surface compared to those of undecorated mucus surface
(Fig. [93]2G and Supplementary Fig. [94]4). Leveraging Cy5.5-labeled
LGG to form LSTC, the anchored bacteria were visually confirmed by
LSCM, which displayed an obvious fluorescence signaling in the EG-Bi
group (Fig. [95]2H). To quantify the number of hybridized bacteria, 5.0
× 10^7 colony-forming units (CFUs) of LGG was added onto the area of
4.5 cm^2 of EG-Bi for 24-h incubation. The anchored LGG were collected
for bacterial plate counting, verifying that LGG exhibited a 9.8-fold
increase on the EG-Bi surface compared to the undecorated one
(Fig. [96]2I). In addition, we calculated that about 2.7 × 10^7 CFUs of
LGG could be attached to the area of 4.5 cm^2 of EG-Bi. We then
explored the versatility of EG-Bi to bind diverse probiotic strains,
including Gram-positive Enterococcus faecalis (EF) and Lactobacillus
delbrueckii subsp. bulgaricus (LD) as well as Gram-negative Escherichia
coli Nissle 1917 (EcN). Similar to that of LGG, an increased amount of
EF, LD, or EcN was also visualized on the EG-Bi surface (Fig. [97]2G,
H, and Supplementary Fig. [98]4). In comparison to that of naked mucus
surface, bacterial plate counting showed a 20.7-, 30.1-, and 37.2-fold
higher attachment of EF, LD, and EcN on the EG-Bi surface, respectively
(Fig. [99]2I). Taken together, these results verified that LSTC could
be implemented readily to mucus surface in vitro.
Formation of LSTC on mucosal surfaces
Having confirmed the successful preparation of LSTC in vitro, we then
attempted to perform LSTC on mucosal surfaces. First, the validity of
the formation of LSTC at different mucosal interfaces was assessed
using ex vivo models. Cy5.5-labeled LGGs were used to form LSTC, which
was subsequently imaged using in vivo imaging system (IVIS). Vaginal
mucosal interface is unique because it must adapt to complicated
physiological situations, such as acidic pH and hormonal
fluctuation^[100]41. As such, the vaginal tract was sampled from mice
and exposed to the LSTC solution containing 0.6 mg/ml EG, 0.2 mg/ml
Bi(NO[3])[3]·5H[2]O, and 1.0 × 10^6 CFUs/ml Cy5.5-labeled LGG (termed
EG-Bi-LGG). Equivalent Cy5.5-labeled LGG were set as a control.
Clearly, IVIS images presented a higher fluorescence intensity in the
vaginal tissues after decorating with LSTC (Fig. [101]3A, B),
suggesting the feasibility to form LSTC at the mucosal interface within
the vaginal tract. To quantify the number of introduced bacteria in
LSTC, vaginal tissues were homogenized for bacterial plate counting. In
contrast to the control group, EG-Bi-LGG presented a 5.9-fold increment
in the adhesion of LGG to the vagina lining (Fig. [102]3C). In
addition, we measured the concentration of Bi^III ions in the decorated
tissues by inductively coupled plasma mass spectrometry (ICP-MS). As
plotted in Fig. [103]3D, the content of Bi^III ions in the EG-Bi-LGG
group was substantially higher than that in the control group, further
demonstrating the presence of Bi^III ions in the coating. The
gastrointestinal tract is the most widely studied among all mucosal
tissues^[104]42. In agreement with the results from the vaginal tract,
LSTC could efficiently form on the mucosal surface of the intestinal
tract, reflected by a clearly stronger fluorescence signal and a
4.9-fold increase in bacterial number in the decorated colonic tissues
(Fig. [105]3E–G).
Fig. 3. Formation of LSTC on diverse mucosal surfaces.
[106]Fig. 3
[107]Open in a new tab
IVIS image (A) and corresponding quantification of fluorescence
intensity (B) of vaginal tissue after incubation with LGG or LSTC
solution (n = 5 independent samples). C Counts of adherent LGG on
vaginal surface after a 24-h culture on MRS agar plates at 37 °C (n = 5
independent samples). D Content of Bi^III ions on vaginal surface
detected by ICP-MS (n = 4 independent samples). IVIS images (E) and
corresponding quantification of fluorescence intensity (F) of colonic
segments with a length of ~1.5 cm after incubation with LGG or LSTC
solution (n = 5 independent samples). G Counts of adherent LGG on
colonic surface after a 24-h culture on MRS agar plates at 37 °C (n = 5
independent samples). H IVIS images of mice and the sampled vaginal
tracts at 6 h after intravaginal administration with LGG or LSTC (n = 5
independent samples). I Fluorescence intensity derived from the IVIS
images of mice (n = 5 independent samples). J Counts of LGG collected
from the vaginal tracts (n = 5 independent samples). IVIS images of the
sectioned colon 6 h post-administration (K) and corresponding
quantification of fluorescence intensity (n = 5 independent samples)
(L). M Counts of LGG collected from the sectioned colon (n = 5
independent samples). IVIS images (N) and corresponding quantification
of fluorescence intensity (O) of mice at various time points after
intravaginal administration with LGG or LSTC (n = 5 independent
samples). Fluorescence signal indicates bacteria labeled with Cy5.5.
Data are mean ± SD. Statistical analysis was performed using two-tailed
Student’s t-test (B, C, D, F, G, I, J, L, M) or two-way ANOVA with
Fisher’s LSD multiple comparisons test (O), and p-values were
indicated. Source data are provided as a Source Data file.
We further evaluated whether LSTC could be conducted in vivo. Mice were
intravaginally dosed by a two-step procedure and equivalent
Cy5.5-labeled LGG was applied as a control. It was worth mentioning
that the mixing of EG and Bi^III ions as well as the injection, was
conducted rapidly to avoid the formation of a massive complex structure
(Supplementary Fig. [108]5). At 6 h after administration, IVIS images
of mice treated with EG-Bi-LGG exhibited noticeably enhanced
fluorescence intensity in the vagina, which was further confirmed by ex
vivo imaging of the collected vaginal tract (Fig. [109]3H, I). As given
in Fig. [110]3J, quantification of LGG attached to the vaginal tract
exhibited a 4.5-fold increase in the EG-Bi-LGG group. The above data
validated in vivo formation of LSTC at the vagina. Moreover, rectal
administration of the LSTC solution to mice enabled elevated
fluorescence intensity and enriched bacterial distribution in the
sampled colonic tissues of the EG-Bi-LGG group, thoroughly verifying
the formation of LSTC on the inner lining of the intestine
(Fig. [111]3K–M). To investigate the duration of LSTC, mice vaginally
administered with the LSTC solution were monitored using IVIS at a 6-h
interval for 2 days. We found that treatment with LGG alone emerged a
rapid attenuation of fluorescence signal at the vagina within 12 h
(Fig. [112]3N, O). In contrast, the EG-Bi-LGG group remained a higher
degree of fluorescence signal than the LGG group even 2 days
post-administration. The quantification of adherent LGG in the vagina
confirmed that the amount of LGG in the EG-Bi-LGG group was
significantly higher in comparison to that of the control group at
least within 24 h (Supplementary Fig. [113]6A). The stability of LSTC
was further interrogated by evaluating the retention of Bi^III ions in
the vaginal tract. As shown in Supplementary Fig. [114]6B, the content
of Bi^III ions in the vaginal mucosa of the EG-Bi-LGG group exhibited a
gradual decline over time, yet remained at a higher level than the
control group 24 h after administration. Collectively, these data
validated the formation of LSTC on the surface of different types of
mucosae in vivo.
Restoration of the mucosal physicochemical barrier by LSTC
Encouraged by the establishment of LSTC on mucosal surfaces, we then
turned our attention to evaluating the influence of LSTC on the
physicochemical barrier of the mucosa. Tight junctions, such as
occludin and zonula occludens-1 (ZO-1), are intercellular adhesion
complexes in epithelia and play a critical role in maintaining the
integrity of the mucosal physicochemical barrier^[115]43,[116]44. Given
the fact that Bi-containing compounds have a protective effect on
gastric mucosa^[117]37,[118]38,[119]45, we ascertained the impact of
Bi(NO[3])[3]·5H[2]O on the expression of tight junctions. As plotted in
Supplementary Figs. [120]7, [121]8, stimulation with lipopolysaccharide
(LPS) dramatically destroyed the tight junctions on the surface of both
Caco-2 intestinal and VK2 vaginal epithelial cells, as reflected by the
decreased levels of occludin and ZO-1. Incubation with
Bi(NO[3])[3]·5H[2]O significantly enhanced the expressions of occludin
and ZO-1, suggesting the ability of Bi^III ions to recover the
physicochemical barrier of the mucosa. Goblet cells are specialized
epithelial cells responsible for the secretion of mucus, which in turn
coats the intestinal epithelia to play a key role in mucosal
physicochemical barrier^[122]46,[123]47. On the basis of this notion,
LPS-stimulated HT29-MTX-E12 goblet cells were co-cultured with
Bi(NO[3])[3]·5H[2]O to assess mucus secretion. Images of alcian blue
(AB) staining showed an immense decrease in mucus production after LPS
stimulation, which was reversed by treatment with Bi(NO[3])[3]·5H[2]O
(Fig. [124]4A). The data validated that Bi^III ions were able to
effectively repair the destroyed physicochemical barrier of the mucosa.
Furthermore, the barrier integrity was confirmed by measuring
transepithelial electrical resistance (TEER) and transepithelial
permeability. TEER measurement reflects the ionic conductance of the
intercellular pathway by assessing the electrical resistance across a
cellular monolayer^[125]48. VK2 cells were seeded to the transwell
insert and cultured for 4 days to form an epithelial cell monolayer. In
comparison to the unstimulated control, the LPS-stimulated monolayer
displayed a significantly reduced TEER value (Fig. [126]4B), which was
nearly increased to a normal level after incubation with
Bi(NO[3])[3]·5H[2]O. Transepithelial permeability was tested by adding
fluorescein isothiocyanate (FITC)-dextran to the transwell insert. As
shown in Fig. [127]4C, an enhanced fluorescence signal was presented in
the basolateral medium after stimulation with LPS, implying an increase
in permeability across the monolayer. Similarly, treatment with
Bi(NO[3])[3]·5H[2]O effectively reversed the permeability of
FITC-dextran to a level comparable to that of the unstimulated one.
Fig. 4. Restoration of the mucosal physicochemical barrier.
[128]Fig. 4
[129]Open in a new tab
A Images of AB staining of LPS-stimulated HT29-MTX-E12 cells incubated
with PBS or 0.2 mg/ml Bi(NO[3])[3]·5H[2]O for 12 h. B TEER data of
LPS-stimulated epithelial cell monolayer in transwell insert after
incubation with PBS or 3 μg/ml Bi(NO[3])[3]·5H[2]O for 24 h (n = 3
independent samples). C Concentrations of FITC-dextran in the
basolateral medium of transwell measured by a microplate fluorometer
after incubation with PBS or 3 μg/ml Bi(NO[3])[3]·5H[2]O for 4 or 12 h
(n = 3 independent samples). Control group indicates unstimulated cells
in (A–C). D-H Evaluation of vaginal barrier integrity of S.
aureus-infected mice after intravaginal treatment with PBS, LGG, EG-Bi,
or EG-Bi-LGG every day for 7 days. D Images of AF488-WGA (red) staining
of vaginal sections from treated mice. Blue indicates cell nuclei
stained with 4’,6-diamidino-2-phenylindole (DAPI). E Average
fluorescence intensity of AF488-WGA of vaginal sections from D (n = 4
independent samples). F Immunofluorescence images of vaginal occludin
(green) and ZO-1 (red) expressions from treated mice. Blue indicates
cell nuclei stained with DAPI. Average fluorescence intensities of
occludin (G) and ZO-1 (H) of vaginal sections from (F) (n = 3
independent samples). Data are mean ± SD. Statistical analysis was
performed using one-way ANOVA with Fisher’s LSD multiple comparisons
test, and p-values were indicated. Source data are provided as a Source
Data file.
To evaluate the efficacy of LSTC in recovering the physicochemical
barrier in vivo, two mouse models of impaired mucosal barrier,
separately induced by vaginal infection of bacteria and fungi, were
developed. To establish an aerobic vaginitis model, mice were
intraperitoneally treated with 0.2 mg of estradiol benzoate every day
for 3 days and subsequently infected with 4.0 × 10^7 CFUs of S. aureus
every day for 7 days. Mice were intravaginally treated with the LSTC
solution every day for 7 days. Healthy mice and infected mice treated
with phosphate-buffered saline (PBS), LGG, or equivalent EG-Bi were
separately set as controls. Vaginal tissues were collected to evaluate
mucosal barrier functions by assessing the production of mucus and
tight junctions 7 days post treatment. Alexa Fluor 488-labeled wheat
germ agglutinin (AF488-WGA) was used to specifically label mucin, the
major structural component of mucus. Fluorescence images of vaginal
tissues presented a dense mucus layer covering the vaginal epithelium
of healthy mice (Fig. [130]4D, E). Obviously, a dramatic decrease in
mucin was observed after S. aureus infection, confirming the impairment
of the mucosal barrier. Although treatment with EG-Bi could elevate the
mucin level, EG-Bi-LGG resulted in the highest mucin level among all
the groups. In addition, S. aureus infection caused a significant
reduction in the expressions of occludin and ZO-1 of vaginal
epithelium, which were greatly restored by EG-Bi-LGG (Fig. [131]4F–H).
In line with above data, the recovery of mucus and tight junction
productions in vaginal epithelium by EG-Bi-LGG was also demonstrated in
mice infected by C. albicans (Supplementary Figs. [132]9, [133]10).
Note that mice treated with EG-Bi-LGG exhibited a comparable level of
occludin to that of healthy mice, yet a significantly lowered
expression of ZO-1 compared to healthy mice. We speculated that
EG-Bi-LGG preferentially affected the expression of occludin, which
extends into the intercellular spaces to regulate the barrier
permeability^[134]43. Briefly, LSTC was able to effectively restore
physicochemical barrier integrity, which was largely attributed to the
protective effect of Bi^III ions.
Alleviation of mucosal inflammation by LSTC
Excessive inflammation at the mucosal interface can induce the
necroptosis of epithelial cells, contributing to the breakdown of the
mucosal barrier^[135]49,[136]50. We next investigated whether LSTC
could suppress mucosal inflammation to maintain the homeostasis of the
immune barrier. As a natural polyphenol, EG possesses an
anti-inflammatory property that inhibits inducible nitric oxide
synthase (iNOS) expression in macrophages^[137]36,[138]51. To assess
the anti-inflammatory effect of EG, LPS-stimulated J774A.1 cells, a
macrophage cell line, were co-cultured with different concentrations of
EG. As shown in Fig. [139]5A–C, LPS stimulation significantly increased
the production of pro-inflammatory cytokines, including interleukin-1β
(IL-1β), IL-6, and TNF-α, compared to the unstimulated group. Treatment
with EG greatly blunted the levels of pro-inflammatory cytokines,
especially IL-1β and IL-6, in a dose-dependent manner. Meanwhile, LPS
enhanced the levels of late apoptosis and necrosis of epithelial cells,
which were obviously reversed by EG (Fig. [140]5D and Supplementary
Fig. [141]11). These data prompted us to evaluate the in vivo efficacy
of LSTC in suppressing inflammation. Mice intravaginally infected by S.
aureus were administered with the LSTC solution every day for 7 days
and subsequently vaginal tissues were collected for messenger RNA
sequencing (mRNA-Seq). Differential expression gene (DEG) analysis
suggested that there were 134 downregulated and 349 upregulated DEGs in
the vaginal tissue of the infected group compared to those of the
healthy group (Fig. [142]5E). Among the upregulated DEGs in the
infected tissue, 15, 5, and 218 genes were significantly downregulated
after separately treating with LGG, EG-Bi, and EG-Bi-LGG, displaying a
superior ability of EG-Bi-LGG in alleviating S. aureus-induced
inflammatory activation. Pathway analysis using Kyoto Encyclopedia of
Genes and Genomes (KEGG) or Gene Ontology (GO) enrichment evidenced
that in comparison to the infected mice, the downregulated genes in the
EG-Bi-LGG group were enriched in immune-related pathways (Fig. [143]5F,
G). Gene-set enrichment analysis (GSEA) further confirmed the
downregulated immune-related pathways, including inflammatory response,
cytokine-cytokine interaction, Toll-like receptor signaling pathway,
antigen processing and presentation, natural killer cell mediated
cytotoxicity, activation of immune response, lymphocyte mediated
immunity, immunoglobulin mediated immune response, and B cell mediated
immunity (Fig. [144]5H and Supplementary Fig. [145]12). Of note,
EG-Bi-LGG contributed to the decreased expression of numerous genes
related to inflammasome (Supplementary Fig. [146]13). Cell type
enrichment analysis was also conducted to quantify immune cell
distribution within the vagina. As descripted in Fig. [147]5I, S.
aureus infection caused a significant enrichment of innate immune
cells, including granulocyte-monocyte progenitors, neutrophils,
macrophages, and myeloid dendritic cells. This observation was in line
with the notion that granulocyte-monocyte progenitors are capable of
differentiating into neutrophils, macrophages, or dendritic cells in
response to infection, which is the major pathway to produce
pro-inflammatory cytokines^[148]52. In contrast, treatment with
EG-Bi-LGG profoundly decreased the level of these cells compared to
EG-Bi and LGG. Additionally, we found that S. aureus infection elicited
an obvious B cell-associated activation, yet inhibited the T cell
immunity, including both CD8^+ T cells and T-helper 1 (Th1) cells.
Intriguingly, treatment with EG-Bi-LGG preferred to boost the
activation of CD8^+ T cells and Th1 cells, which are the major subsets
to mediate effective anti-infection immunity^[149]53,[150]54. These
results elucidated that EG-Bi-LGG effectively downregulated the
inflammatory responses and triggered an anti-infection immunity within
the vagina.
Fig. 5. Alleviation of mucosal inflammation.
[151]Fig. 5
[152]Open in a new tab
Levels of IL-1β (A), IL-6 (B), and TNF-α (C) in the culture supernatant
of LPS-stimulated J774A.1 cells incubated with different concentrations
of EG (n = 4 independent samples). D Percentages of late apoptotic or
necrotic VK-2 cells (Annexin V^+PI^+) stimulated with LPS after
treatment with PBS or 10 μg/ml EG (n = 6 independent samples). Control
group indicates unstimulated VK-2 cells. E–I S. aureus-infected mice
were intravaginally administered with PBS, LGG, EG-Bi, or EG-Bi-LGG
every day for 7 days and vaginal samples were collected for mRNA-Seq
(n = 3). E Volcano plots showing differentially expressed genes in the
PBS group compared to the healthy group and in the LGG, EG-Bi, or
EG-Bi-LGG group compared to the PBS group, respectively. Red points
indicate genes upregulated in the PBS group compared to the healthy
group. vs, versus. F KEGG pathway enrichment analysis of the
differentially expressed genes that were downregulated in the EG-Bi-LGG
group compared to the PBS group (p < 0.05) and the top 30 enriched
KEGG was shown. G GO enrichment analysis of the downregulated genes in
the EG-Bi-LGG group from the PBS group (p < 0.05). BP and MF indicate
biological process and molecular function, respectively. H GSEA showing
enrichment of genes related to inflammatory responses. I Single-sample
GSEA identifying the difference in immune infiltration in treated mice
(n = 3 independent samples). Data are mean ± SD. p values in A–D
indicate the statistical significance of various groups compared to the
LPS-stimulated groups without EG treatment. p values in I indicate the
statistical significance of various groups compared to the EG-Bi-LGG
group. For E, DEGs were identified using DESeq2 (|log[2] (fold change)|
≥ 1 and FDR ≤ 0.05). For F, KEGG pathway enrichment was analyzed using
a Fisher’s exact test with Benjamini-Hochberg adjustment for multiple
comparisons (adjusted p value ≤ 0.05). For G, GO enrichment was
analyzed using a Fisher’s exact test with Bonferroni, Holm, Sidak and
FDR adjustment for multiple comparisons (adjusted p value ≤ 0.05). For
H, GSEA was performed using R package clusterProfiler. Statistical
analysis was performed using one-way ANOVA with Fisher’s LSD multiple
comparisons test (A–D, I), and p-values were indicated. Source data are
provided as a Source Data file.
Regulation of the microbiota by LSTC
Next, the probiotic effect of LSTC on imbalanced microbial barrier of
the mucosa was evaluated by assessing the homeostasis of the vaginal
microbiota. Given that LGG has been reported to directly antagonize
vaginal pathogen proliferation through peroxide or acid
production^[153]32,[154]55, we tested the potency by co-culturing with
S. aureus and C. albicans, respectively. As shown in Fig. [155]6A, LGG
inhibited the growth of S. aureus in a dose-dependent manner.
Particularly, LGG displayed a potent antagonistic effect on C.
albicans, as reflected by substantially inhibited proliferation even
with the feed ratio decreasing to 1:1 (Fig. [156]6B). In addition, we
further tested the adhesion and growth of vaginal pathogens on LSTC. In
comparison to undecorated mucus surface, an increased amount of S.
aureus or C. albicans on the LSTC surface was observed after a 4-h
incubation (Supplementary Fig. [157]14A, B), suggesting the enhanced
adhesion of vaginal pathogens to LSTC. Worth noting, monitoring the
growth dynamics of S. aureus or C. albicans over a 24-hour period
revealed that LSTC presented the most significant ability in inhibiting
the growth of pathogens (Supplementary Fig. [158]14C, D). To ascertain
the in vivo efficacy of LSTC in inhibiting pathogens and regulating the
microbiota, mice were intravaginally infected with 4 × 10^7 CFUs of
GFP-expressing S. aureus. After administration with the LSTC solution
every day for 7 days, vaginal tissues were sampled to evaluate the
counts of S. aureus. Healthy mice and infected mice treated with PBS,
clindamycin, LGG, or equivalent EG-Bi were separately carried out as
controls. Fluorescence images of the sampled vaginal tissues suggested
that LGG treatment presented a reduction in S. aureus count compared to
infected mice (Fig. [159]6C, D). Of note, among all the treated groups,
the most significant decrease of S. aureus was observed in the
EG-Bi-LGG group. The microbial composition was analyzed using 16S
ribosomal RNA gene sequencing. As expected, infected mice showed
profoundly decreased richness of the vaginal microbiota, as reflected
by a lower abundance-based coverage estimator (ACE) index and Chao1
index compared to healthy mice (Fig. [160]6E, F). In comparison to
clindamycin, LGG, and EG-Bi, EG-Bi-LGG significantly augmented the
richness of the vaginal microbiota. In addition, principal coordinates
analysis (PCoA) at operational taxonomic unit (OTU) and genus levels
indicated that infected mice presented an obvious difference in
bacterial composition compared to healthy mice (Fig. [161]6G). In
contrast to EG-Bi and clindamycin, treatments with LGG and EG-Bi-LGG
displayed a similar bacterial composition to healthy mice, suggesting
the reduced perturbation of the vaginal microbiota induced by S. aureus
infection. Analysis of the relative abundance of bacteria at class
level indicated that the microbial structure of mice treated with LGG
or EG-Bi-LGG was closer to that of healthy mice (Fig. [162]6H).
Specifically, EG-Bi-LGG increased the relative abundances of several
probiotic bacterial genera, such as Lachnospiraceae_NK4A136 group and
Clostridium sensu stricto 1 (Fig. [163]6I, J). Worth noting, LSTC
showed a superior ability to increase the richness and diversity of the
vaginal microbiota compared to clindamycin, further demonstrating its
potential to restore the homeostasis of the vaginal microbiota via
restraining the expansion of pathogens and increasing the richness of
probiotic bacterial genera.
Fig. 6. Regulation of the vaginal microbiota.
[164]Fig. 6
[165]Open in a new tab
In vitro competition between LGG and S. aureus (A) or C. albicans (B)
at various feed ratios. S. aureus expressing GFP or C. albicans were
co-incubated with LGG at 37 °C at different feed ratios of 1:0, 1:1,
1:10, 1:30, and 1:100, respectively (n = 5 independent samples in A;
n = 3 independent samples in B). C-J S. aureus-infected mice were
intravaginally administered with PBS, clindamycin, LGG, EG-Bi, or
EG-Bi-LGG every day for 7 days and vaginal samples were collected for
S. aureus detection and 16S ribosomal RNA gene sequencing. Relative
area of signal in the red channel (C) and immunofluorescence images (D)
of vaginal sections from treated mice (n = 3 independent samples). Red
and blue indicate S. aureus expressing GFP and cell nuclei stained with
DAPI, respectively. ACE index (E) and Chao1 index (F) of the vaginal
microbiota (n = 5 independent samples). G Result of the PCoA of the
vaginal microbiota at OTU and genus levels (n = 5 independent samples).
H Relative abundances of the vaginal microbiota at the class level.
Relative abundances of Lachnospiraceae_NK4A136 group (I) and
Clostridium sensu stricto 1 (J) in the vaginal microbiota at the genus
level (n = 5 independent samples). Data are mean ± SD. Statistical
analysis was performed using one-way ANOVA with Fisher’s LSD multiple
comparisons test, and p-values were indicated. Source data are provided
as a Source Data file.
Efficacy of LSTC in intervening aerobic vaginitis
Aerobic vaginitis is a common vaginal infection defined by a disruption
in dominant Lactobacillus and an increase in aerobic enteric commensals
or pathogens^[166]56,[167]57. Different from other forms of vaginal
infections, aerobic vaginitis presents more extreme inflammation and
epithelial disruption^[168]58. In light of the effectiveness of LSTC in
restoring mucosal barrier functions, we next evaluated its benefits in
treatment of aerobic vaginitis (Fig. [169]7A). Mice were subcutaneously
treated with estradiol benzoate every day for 3 days, followed by
intravaginal infection with 10 μl of 4 × 10^9 CFUs/ml S. aureus every
day for 7 days. To examine the therapeutic value of LSTC, infected mice
were intravaginally administered with the LSTC solution every day for 7
days. Healthy mice and infected mice treated with PBS, LGG, or
equivalent EG-Bi were separately carried out as controls. Clindamycin,
a clinically used gold standard for aerobic vaginitis, was applied as a
benchmark^[170]59. As shown in Fig. [171]7B, S. aureus infection caused
a pronounced loss of body weight, which could be significantly
recovered by clindamycin and EG-Bi-LGG. Vaginal washes were collected
to quantify the number of S. aureus on day 8 after treatment,
demonstrating that treatment with clindamycin and EG-Bi-LGG showed the
most significant reduction in S. aureus number among all the treated
groups (Fig. [172]7C, D). After infection, the vagina appeared red and
swollen, which were substantially relieved after treatment with
EG-Bi-LGG (Supplementary Fig. [173]15). Vaginal tissues of the treated
mice were sampled to assess pathological injury using haematoxylin and
eosin (H&E) staining. Compared to the healthy group, infected mice
presented severe leukocyte infiltration and epithelial desquamation or
keratinization (Fig. [174]7E). In contrast, the EG-Bi-LGG group
exhibited a thinner and denser mucosal multilayer and a fewer leukocyte
infiltration than infected mice with other treatments, implying an
alleviated pathological injury of vaginal mucosal tissue. In addition,
the levels of inflammatory cytokines, including IL-6, TNF-α, and IL-1β,
in serum from EG-Bi-LGG-treated mice were reduced apparently in
contrast to those of the PBS, LGG, and EG-Bi controls (Supplementary
Fig. [175]16). Meanwhile, neutrophil infiltration of vaginal tissue was
identified by myeloperoxidase (MPO), an enzyme mainly expressed in
neutrophils. As depicted in Fig. [176]7F and Supplementary
Fig. [177]17, MPO staining of the sectioned vaginal tissue indicated
that treatment with EG-Bi-LGG greatly reduced the infiltration of
MPO-positive cells compared to those of all other control groups.
Besides, no pathological abnormality was observed after treatment in
major organs, including the heart, liver, spleen, lung, and kidney,
demonstrating satisfactory biosafety of LSTC (Supplementary
Fig. [178]18). Together, LSTC verified its potency to alleviate aerobic
vaginitis-associated pathological injury and inflammatory infiltration.
Fig. 7. Value of LSTC in intervening aerobic vaginitis.
[179]Fig. 7
[180]Open in a new tab
A Experimental design of in vivo assessment using a S. aureus-induced
murine model of aerobic vaginitis. Mice were intraperitoneally treated
with 0.2 mg of estradiol benzoate every day for 3 days and subsequently
were intravaginally infected with 4.0 × 10^9 CFUs of S. aureus every
day for 7 days. The infected mice were then intravaginally administered
with PBS, clindamycin, LGG, EG-Bi, or EG-Bi-LGG every day for 7 days. B
Fluctuation of mouse body weight change (% of initial weight) after
various treatments (n = 5 mice). Counts of S. aureus in vaginal washes
on day 7 post-treatment (C) and corresponding fluorescence images of LB
agar plates for colony counting (D) (n = 5 mice). E Images of H&E
staining of vaginal tissues from treated mice. Black and red arrows
indicate epithelial inflammation and epithelial desquamation or
keratinization, respectively. F Images of MPO staining of vaginal
tissues from treated mice. Brown indicates MPO^+ cells. Data are
mean ± SD. Statistical analysis was performed using one-way ANOVA with
Fisher’s LSD multiple comparisons test, and p-values were indicated.
Source data are provided as a Source Data file. The elements in Fig. 7A
were created in BioRender.
Potential of LSTC in treating vaginal candidiasis
Vaginal candidiasis commonly caused by C. albicans is one of the most
prevalent fungal infections in the vagina^[181]60. Approximately 75% of
women will be infected at least once in their lifetime, with about 8%
suffering recurrent episodes^[182]25. Considering that C. albicans can
grow in yeast, pseudohyphal, and hyphal morphologies, vaginal
candidiasis exhibits a greater resistance to pharmacological treatments
than aerobic vaginitis^[183]32. Next, we investigated the potential of
LSTC in treating fungal infections by establishing a model of vaginal
candidiasis (Fig. [184]8A). After subcutaneous injection of 0.2 mg
estradiol benzoate every other day for three times, 2.0 × 10^7 CFUs of
C. albicans were daily dosed into the vagina for 3 days. To assess the
therapeutic effect of LSTC, infected mice were daily treated with the
LSTC solution for 7 days. Healthy mice and infected mice treated with
PBS, LGG, and equivalent EG-Bi were separately used as controls. Mice
treated with 0.4 mg/kg of clinically used clotrimazole were used as a
benchmark^[185]61. As evidenced in Fig. [186]8B, treatment with PBS,
LGG, or EG-Bi had a negligible effect on the recovery of the decreased
body weight caused by C. albicans infection. In contrast, EG-Bi-LGG
presented an obvious benefit, implying its superior therapeutic
efficacy. In comparison to PBS, LGG, or EG-Bi group, Periodic
acid-Schiff (PAS) staining of vaginal secretions suggested a distinct
decrease in fungal burden in mice treated with EG-Bi-LGG
(Fig. [187]8C), which was in accordance with the relief of red and
swollen vaginal appearance (Supplementary Fig. [188]19). To quantify C.
albicans CFUs in the vagina, vaginal washes were collected for plate
counting, which disclosed the most significantly decreased fungal
burden within the vagina in the clotrimazole and EG-Bi-LGG groups among
all the treated groups (Fig. [189]8D and Supplementary Fig. [190]20).
The levels of inflammatory cytokines were assessed and the results
showed that treatments with clotrimazole and EG-Bi-LGG notably
suppressed the levels of IL-1β, TNF-α, S100A7/A8, and chemoattractant
KC compared to all other groups (Fig. [191]8E, F and Supplementary
Fig. [192]21). Additionally, H&E staining of vaginal tissue suggested
that infection-induced enhancements of vaginal keratinization and
leukocyte infiltration were greatly reduced after treating with
EG-Bi-LGG (Fig. [193]8G, H). MPO staining of the sectioned vaginal
tissue further confirmed the most significant decrease of neutrophil
infiltration in the EG-Bi-LGG group among all treated groups
(Fig. [194]8I and Supplementary Fig. [195]22). In short, these data
supported that LSTC could effectively inhibit the colonization of C.
albicans in the vagina and ameliorate C. albicans-induced mucosal
injury.
Fig. 8. Efficacy of LSTC in treating vaginal candidiasis.
[196]Fig. 8
[197]Open in a new tab
A Experimental design of in vivo assessment using a C. albicans-induced
murine model of vaginal candidiasis. Mice were intraperitoneally
treated with 0.2 mg of estradiol benzoate every other day for three
times and subsequently were intravaginally infected with 2.0 × 10^9
CFUs of C. albicans every day for 3 days. The infected mice were then
intravaginally administered with PBS, clotrimazole, LGG, EG-Bi, or
EG-Bi-LGG every day for 7 days. B Fluctuation of mouse body weight
change (% of initial weight) after various treatments (n = 6 mice). C
Images of PAS staining of vaginal secretions showing C. albicans (n = 5
mice). D Counts of C. albicans in vaginal washes on day 7
post-treatment (n = 6 mice). Levels of IL-1β (E) and TNF-α (F) in serum
(n = 6 mice). H Images of H&E staining of vaginal tissues from treated
mice. Black and red arrows indicate inflammation and epithelial
desquamation or keratinization, respectively. G Quantification of
leukocyte infiltration from (H) (n = 3 independent samples). I Images
of MPO staining of vaginal tissues from treated mice. Brown indicates
MPO^+ cells. Data are mean ± SD. Statistical analysis was performed
using one-way ANOVA with Fisher’s LSD multiple comparisons test, and
p-values were indicated. Source data are provided as a Source Data
file. The elements in Fig. 8A were created in BioRender.
In summary, we present the decoration of mucosal surfaces with LSTC to
multifacetedly restore mucosal barrier functions. Under a simple yet
cytocompatible condition, EG can chelate Bi^III ions to form a coating
that can further hybridize with LGG steadily at mucosal surfaces,
through metal-phenolic complexation and π-π stacking interactions. The
resulting therapeutic coating is applicable to diverse bacterial
species and can be generated on different types of mucosal surfaces.
The combination of the barrier-repairing ability of Bi^III ions, the
immunosuppressive feature of EG, and the microbiota-regulating behavior
of LGG enables LSTC to effectively restore the physicochemical, immune,
and microbial homeostasis of the mucosal barrier. Specifically, LSTC
improves barrier integrity by increasing the productions of tight
junctions and mucus, remits mucosal inflammation by suppressing the
production of inflammatory cytokines, and maintains normal homeostasis
of the microbiota by inhibiting the colonization of pathogens. During
in vivo therapeutic assessment, LSTC potently alleviates vaginal
pathological injury and decreases vaginal inflammatory infiltration in
two murine models of aerobic vaginitis and vaginal candidiasis.
Importantly, LSTC not only demonstrates comparable therapeutic
efficacies to clinical antibiotics, but also outperforms in restoring
microbial homeostasis, implying a low risk of antibiotic resistance and
a prolonged prevention of recurrence. Note that the murine model shows
significant differences in vaginal pH, microbiota composition, and
hormonal regulation compared to humans, future studies on systematic
assessment of biosafety, dosage regiment and treatment frequency, and
batch-to-batch optimization particularly in large animal models are
necessitated before considering for further translation.
Methods
Ethical Statement
ICR mice (female, 6-8 weeks old) were purchased from the SiPeiFu
Biotechnology (Beijing, China) and housed in the SPF environment with
an average temperature of 22 °C and a standard 12 h light/dark cycle.
All animal experiments were carried out according to the institutional
guidelines and approved by Animal Care and Use Committee in Shanghai
Yishang Biotechnology company (IACUC-2023-Mi-214).
Materials
EG (ethyl gallate) was purchased from Shanghai Yuanye Bio-Technology
(China). Bi(NO[3])[3]·5H[2]O (A600233) and lysogeny broth (LB, A507002)
medium were obtained from Sangon Biotech (China). Brain Heart Infusion
Broth (BHI, HB8297), Man Rogosa Sharpe (MRS, HB0384-5), and Yeast
Extract Peptone Dextrose (YPD, HB5193-1) medium were purchased from
Qingdao Hope Biotechnology (China). Fetal bovine serum (FBS, 10099-141)
and Dulbecco’s Modified Eagle Medium (DMEM, 11965092) were purchased
from Gibco. Penicillin-streptomycin (15140-122) was obtained from
Hyclone. BSA-FITC (SF063), clindamycin (C9760), and clotrimazole
(IC0490) were purchased from Solarbio Life Sciences (China). Cyanine
5.5 NHS ester (S276331) was purchased from Aladdin (China).
FITC-dextran (FD4-100MG) and mucin (Type III, M1778) were obtained from
Sigma-Aldrich. LPS (ST1470), CCK-8 Kit (C0038), H&E Staining Kit
(C0105), MPO staining Kit (AF7494), AB staining Kit (C0155S), neutral
balsam (C0173), Antifade Mounting Medium (P0131), and Hoechst 33342
(C1022) were obtained from Beyotime Biotechnology. Annexin V-FITC
Apoptosis Detection Kit (AP101) and IL-1β (EK201B), TNF-α (EK282), and
IL-6 (EK206) ELISA kits were purchased from MultiSciences Biotech
(China). Anti-occludin (sc-133256) was purchased from Santa Cruz
Biotechnology, and Rabbit polyclonal antibody to ZO-1 (AF5145) was
purchased from Affinity Biosciences.
Bacterial and fungal strains and cell lines
EF was obtained from the American Type Culture Collections (USA). S.
aureus was purchased from Hangzhou Forhigh Biotech (China). EcN was
purchased from Ningbo Bionice (China). LD was purchased from Shanghai
Luwei Technology Co., Ltd (China). C. albicans was purchased from
Shanghai Bioresource Collection Center (China). EF, S. aureus, and EcN
were cultured in LB medium at 37 °C. LGG and LD were cultured in MRS
medium at 37 °C. C. albicans was cultured on a YPD agar plate at 37 °C.
Plasmids RN4220 T61-eGFP (erythromycin resistant) and
pBBR1MCS2-Tac-mCherry (kanamycin resistant) were obtained from domestic
suppliers and applied as received. Human vaginal epithelial cell line
(VK2/E6E7) was kindly provided by Lizeng Gao (the Biophysics Chinese
Academy of Sciences, Beijing, China). Mucus-producing intestinal cell
line (HT29-MTX-E12) was kindly provided by Wei Wu (Fudan University,
Shanghai, China). Mouse macrophage cell line (J774A.1), human
epithelial-like cell line (293 T) and human colon carcinoma cell line
(Caco-2) were purchased from iCell Bioscience Inc. (Shanghai, China).
J774A.1, 293 T, Caco-2, VK2/E6E7, and HT29-MTX-E12 cells were cultured
in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin
at 37 °C in a 5% CO[2] incubator.
In vitro preparation of EG-Bi and LSTC
To prepare the EG-Bi coating, 1 mg/ml mucin was dissolved in PBS
containing 1.5% agar to form an artificial mucus layer. Then, 0.6 mg/ml
EG and 0.2 mg/ml Bi(NO[3])[3]·5H[2]O dissolved in ddH[2]O were added to
the surface of the artificial mucus layer and stirred at 300 rpm for
10 min at room temperature (RT). To prepare LSTC, 5.0 × 10^7 CFUs/ml of
LGG, 2.0 × 10^8 CFUs/ml of EF, LD, or EcN were further added onto the
surface of the EG-Bi coating and stirred at 300 rpm for 5 min at RT.
Characterization of EG-Bi
TEM (HITACHI, Japan), SEM (HITACHI, Japan), and AFM (Bruker, USA) were
used to visualize the morphologies of the EG-Bi coating. In brief,
10 μl of ddH[2]O containing 0.6 mg/ml EG and 0.2 mg/ml
Bi(NO[3])[3]·5H[2]O was dropped onto a Formvar/carbon 200-mesh grid and
put for drying in the air at RT. For SEM and AFM observations, grid was
replaced by silicon wafer. For CLSM (TCS SP8, Leica) observation,
0.2 mg/ml BSA-FITC was added to the surface of the resulting EG-Bi
coating and stirred at 300 rpm for 10 min at RT.
Characterization of LSTC
TEM and SEM were used to visualize the morphology of LSTC. In brief,
10 μl of ddH[2]O containing 0.6 mg/ml EG and 0.2 mg/ml
Bi(NO[3])[3]·5H[2]O was dropped onto a Formvar/carbon 200-mesh grid and
put for drying in the air at RT. Then, 5.0 × 10^7 CFUs/ml of LGG, 2.0 ×
10^8 CFUs/ml of EF, LD, or EcN were further added onto the surface of
the EG-Bi coating and the sample was dried completely in air at RT. For
SEM observation, grid was replaced by the silicon wafer. Cy5.5-labeled
LGG, EF, or LD and EcN expressing mCherry were used to prepare LSTC for
CLSM observation. For bacterial plate counting, the resulting LSTC was
homogenized and spread on MRS plates for overnight incubation at 37 °C.
In vitro cytotoxicity of EG-Bi
To assess the toxicity of EG-Bi on bacteria, LGG (1 × 10^5 CFUs per
well, 200 μl) was resuspended in MRS medium within 96-well plate and
incubated with or without the mixture of 0.6 mg/ml EG and 0.2 mg/ml
Bi(NO[3])[3]·5H[2]O at 37 °C with gentle shaking. The absorbance at
600 nm (OD[600]) intensity was recorded for 15 h with a 0.5-h interval
by a microplate reader (BioTek, USA). To assess the cytotoxicity of
EG-Bi on cells, 293 T or J774A.1 cells (1.0 × 10^4 per well, 100 μl)
were seeded into a 96-well plate and cultured overnight at 37 °C. Then,
a mixture of 0.6 mg/ml EG and 0.2 mg/ml Bi(NO[3])[3]·5H[2]O was added
to the plate and incubated at 37 °C for 6, 12, and 24 h, respectively.
Resultant cells were further incubated with CCK-8 solution for 3 h at
37 °C, and the absorbance was measured at 450 nm by a microplate
reader.
Ex vivo preparation of LSTC
To prepare LSTC on ex vivo colon tissue, the freshly sampled colon
tissue from ICR mice (female, 6-8 weeks old) was everted to expose the
mucosal surface. Then, the colon tissue was divided into ~1.5 cm
segments, and the two ends of the everted colon were carefully ligated.
The prepared colon segments were immersed in a ddH[2]O solution
containing 0.6 mg/ml EG and 0.2 mg/ml Bi(NO[3])[3]·5H[2]O and shaken at
300 rpm for 10 min. After washing with PBS for three times, 500 μl of 5
× 10^7 CFUs/ml Cy5.5-labeled LGG were incubated with colon segments
decorated with or without EG-Bi at 300 rpm for 5 min. To prepare LSTC
on ex vivo vaginal tissue, the freshly sampled vaginal tissue from ICR
mice (female, 6-8 weeks old) was sectioned longitudinal and immobilized
on a 6-well plate to ensure an upward orientation of the mucosal layer.
Then, a 500 μl of ddH[2]O solution containing 0.6 mg/ml EG and
0.2 mg/ml Bi(NO[3])[3]·5H[2]O was added on the surface of the mucosal
layer and shaken at 300 rpm for 10 min. After washing with PBS for
three times, 500 μl of 5 × 10^7 CFUs/ml Cy5.5-labeled LGG was incubated
with vaginal tissue decorated with or without EG-Bi at 300 rpm for
20 min. After washing with PBS for three times, the resultant colon
segments or vaginal tissues were imaged using IVIS. To quantify the
hybridized bacteria, homogenates of the colon segments or vaginal
tissues were spread onto MRS plates for overnight incubation at 37 °C.
To quantify the content of Bi^III ions in the coating, homogenates of
the colon segments were centrifuged and the supernatant was detected by
ICP-MS.
In vivo preparation of LSTC
ICR mice (female, 6-8 weeks old) were administered by rectal or vaginal
injection of PBS (50 µl) containing 0.6 mg/ml EG and 0.2 mg/ml
Bi(NO[3])[3]·5H[2]O, followed by PBS (20 µl) containing 5 × 10^7
CFUs/ml Cy5.5-labeled LGG. Mice injected with equivalent Cy5.5-labeled
LGG were used as a control. At 6 h post injection, mice were
anesthetized and imaged using IVIS. Then, mice were euthanized and the
colonic or vaginal tissues were harvested for IVIS imaging. The images
of the mice and tissues were recorded and analyzed by Living Image 4.2.
In addition, equivalent samples were grinded and diluted with 1 ml of
PBS. The dilutions (50 µl) from both tissues were spread onto MRS agar
plate for an overnight incubation at 37 °C.
In vivo stability of LSTC
After in vivo formation of LSTC in the vaginal tract, ICR mice (female,
6-8 weeks old) were anesthetized, and the fluorescence signal of the
mice at the Cy5.5 channel was monitored using IVIS at a 6-h interval
for 2 days. Then, the mice were euthanized, and vaginal tissues were
harvested at 0, 6, 12, 18, or 24 h after administration. The tissues
were homogenized for quantifying the amount of Bi^III ions and the
counts of LGG, respectively.
In vitro microbial competition
To assess the competition between LGG and S. aureus, LGG and S. aureus
(erythromycin resistance, expressing GFP) were separately grown in MRS
and LB medium at 37 °C for 12 h and subsequently diluted to a
concentration of 1 × 10^7 CFUs/ml. S. aureus and LGG were then mixed in
BHI medium (pH 6.0) in a series of ratios (1:1, 1:10, 1:30, 1:100) and
100 µl of each mixture was incubated at 37 °C with shaking. The
relative fluorescence units (RFUs) of GFP expressed by S. aureus were
recorded at 490/530 nm at a 0.5-h interval. To assess the competition
between LGG and C. albicans, LGG and C. albicans were separately grown
in MRS and YPD medium at 37 °C for 12 h and subsequently diluted to a
concentration of 1 × 10^7 CFUs/ml. C. albicans and LGG were mixed in
medium (MRS:YPD = 1:1) in a series of ratios (1:1, 1:10, 1:30, 1:100)
and incubated at 37 °C with shaking. Then, 50 µl of each sample was
diluted and spread onto YPD agar plates at determined time points and
incubated at 37 °C overnight for bacterial counting. To assess the
adhesion and growth of S. aureus and C. albicans at LSTC decorated on
an artificial mucus layer, 3 × 10^5 CFUs of S. aureus or C. albicans
were further added onto the surface of LSTC, followed by stirring at
300 rpm for 5 min at RT. After incubation at 37 °C for various time
points, the intact artificial mucus was collected and homogenized for
bacterial plate counting.
In vitro anti-inflammatory effect of EG
J774A.1 cells (1.0 × 10^5 per well, 500 μl) were seeded into a 48-well
plate and cultured overnight at 37 °C. Then, LPS (1 μg/ml) and
different concentrations of EG (1.25, 2.5, 5, and 10 μg/ml) were added
to the plate and incubated at 37 °C. The cell supernatants were
collected at 4, 8, and 12 h for separately detecting the levels of
TNF-α, IL-6, and IL-1β. These cytokines were detected using ELISA assay
following the manufacturer’s instructions. The results were determined
at 450 nm wavelength within 30 min by a microplate reader. VK2 cells
(2.0 × 10^5 per well, 1 ml) were seeded into a 6-well plate and
cultured overnight at 37 °C. Then, 50 μg/ml LPS and 10 μg/ml EG were
added to the plate and incubated at 37 °C for 24 h. Subsequently, the
resultant cells were harvested and washed twice with precooled PBS.
Then, apoptosis level of VK2 cells was assessed by Annexin V-FITC
Apoptosis Detection Kit according to the manufacturer’s protocol. The
result was detected using FACSVerse flow cytometer (BD Biosciences,
USA), and data analysis was performed using FlowJo (TreeStar).
Permeability and TEER measurements
To form an epithelial cell monolayer, 5 × 10^4 of VK2 cells were seeded
into a 24-well transwell insert (Corning, 0.4 μm pore size) and
cultured for 4 days with a 2-day interval of medium replacement. Then,
3 μg/ml Bi(NO[3])[3]·5H[2]O was added to the apical insert in the
presence of 50 μg/ml LPS and co-incubated for 24 h. For permeability
measurement, 1 mg/ml FITC-dextran (4 kDa) was added to the apical
insert and co-incubated for 4 or 24 h. The medium in basolateral side
was collected and diluted with PBS. The concentration of FITC-dextran
was detected at 490/530 nm by a microplate fluorometer (Infinite M200
PRO, Tecan). For TEER measurement, one electrode was placed in the
apical insert, while a second electrode was placed in the basolateral
side. TEER values were measured using a Millicell-ERS volt-ohmmeter
(EVOM, World Precision Instruments, USA) to monitor the electrical
resistance. TEER values were calculated using the following formula:
[MATH: TEER=R×A
:MATH]
where R is the electrical resistance of the barrier with monolayer and
A is the apical insert area.
In vitro physicochemical barrier function
VK2 cells (5.0 × 10^5) were seeded into glass-bottom dishes and
cultured overnight at 37 °C. Then, cells were stimulated with 50 μg/ml
LPS and concurrently incubated with 3 μg/ml Bi(NO[3])[3]·5H[2]O at
37 °C for 24 h. After washing with PBS, VK2 cells were fixed with 4%
paraformaldehyde (PFA) for 20 min and subsequently blocked with 5% BSA
for 30 min. Then, resultant VK2 cells were stained with anti-mouse
occludin (1:50) and ZO-1 (1:100) at 4 °C overnight, followed by
corresponding secondary antibodies for 1 h at RT. After washing with
PBS, these cells were incubated with 1 μg/ml Hoechst 33342 for 20 min
at RT. The fluorescence intensity was observed and captured by CLSM. To
assess the mucus, VK2 cells were processed AB staining in accordance
with the manufacturer’s instructions.
Efficacy of LSTC in treating aerobic vaginitis and vaginal candidiasis
To establish the murine model of aerobic vaginitis, ICR mice (female,
6-8 weeks old) were subcutaneously injected with 0.1 ml of 2 mg/ml
estradiol benzoate every day for 3 days. Subsequently, mice were
intravaginally administered with 10 μl of 4.0 × 10^9 CFUs/ml S. aureus
every day for 7 days. Then, the mice were randomly divided to five
groups and intravaginally treated with PBS, clindamycin (6.5 mg/kg),
EG-Bi (12 μg of EG and 4 μg of Bi(NO[3])[3]·5H[2]O), LGG (5 × 10^7
CFUs), EG-Bi-LGG (12 μg of EG, 4 μg of Bi(NO[3])[3]·5H[2]O, and 5 ×
10^7 CFUs of LGG) every day for 7 days. To induce the murine model of
vaginal candidiasis, ICR mice (female, 6-8 weeks old) were
subcutaneously injected with 0.1 ml of 2 mg/ml estradiol benzoate every
other day for three times. Subsequently, mice were intravaginally
administered with 10 μl of 2.0 × 10^9 CFUs/ml C. albicans every day for
3 days. Then, the mice were randomly divided to five groups and
intravaginally treated with PBS, clotrimazole (0.4 mg/kg), EG-Bi (12 μg
of EG and 4 μg of Bi(NO[3])[3]·5H[2]O), LGG (5 × 10^7 CFUs), EG-Bi-LGG
(12 μg of EG, 4 μg of Bi(NO[3])[3]·5H[2]O, and 5 × 10^7 CFUs of LGG)
every day for 7 days. Healthy mice without infection were used as a
control in both above models. The body weight of mice was monitored
every day. At the endpoint of the experiment, vaginal washes were
collected for the assessment of S. aureus or C. albicans counts. Serum
was collected for detecting the levels of inflammatory cytokines using
the ELISA kit, and vaginal tissue was sampled for pathological analysis
using H&E staining.
Microbiota 16S ribosomal RNA gene sequencing and analysis
In brief, bacterial DNA was extracted from vaginal washes of S.
aureus-infected female ICR mice after various treatments. Sequencing
was performed by targeting hypervariable V3-V4 regions of the bacterial
16S ribosomal RNA gene with universal primers 27F-1492R (27 F:
AGRGTTTGATYNTGGCTCAG; 1492 R: TASGGHTACCTTGTTASGACTT) and detected on
the Illumina HiSeq platform. The class or genus classification was
obtained on the basis of the sequence composition of the OTU. The alpha
diversity was analyzed with ACE and Shannon index to examine the
richness and diversity of species in individual samples, while beta
diversity was analyzed with PCoA to evaluate the similarity in
bacterial composition. Data analysis was performed on BMKCloud.
Transcriptome sequencing and pathway analysis
Briefly, total RNA was extracted from the vaginal tissue of S.
aureus-infected female ICR mice after various treatments using TRIzol®
Reagent in accordance with manufacturer’s protocol. The RNA quality, in
terms of 260/280 and 260/230 ratios, was measured by 5300 Bioanalyser
(Agilent Technologies), and its quantity was determined using NanoDrop
(ND-2000, Thermo Fisher Scientific). Then, RNA purification, reverse
transcription, library construction, and sequencing were performed at
Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China)
in accordance with the manufacturer’s instructions (Illumina, San
Diego, CA). To identify DEGs between two different samples, the
expression level of each transcript was calculated in accordance with
the transcripts per million (TPM) method. RNA-Seq by
Expectation-Maximization (RSEM) was applied for quantifying transcript
abundances, and DEGs were identified using DESeq2. Genes with |log[2]
(fold change)| ≥ 1 and false discovery rate (FDR) ≤ 0.05 were
considered as DEGs. GO functional enrichment and KEGG pathway analysis
of DEGs were performed using Goatools and KOBAS, respectively. GSEA was
performed using the R package clusterProfiler (version 4.10.0). The
fold changes of genes between case and control group calculated
previously were used to generate the gene list in a descending order.
Immunohistochemical and immunofluorescence analysis
The collected vaginal tissue was fixed in 4% PFA and then embedded in
paraffin. Then, the sample was cut into 4-μm sections. After
deparaffinization and antigen retrieval, the obtained sections were
blocked with 10% goat serum for 1 h. Subsequently, the sections were
incubated with primary antibodies, including occludin (1:200) or ZO-1
(1:200) at 4 °C overnight, followed by corresponding secondary
antibodies conjugated with fluorescence or HRP for 1 h at RT. To
examine mucus, the sections were incubated with AF488-WGA (1:100) at
37 °C for 30 min. For immunohistochemical analysis, samples were
covered by neutral balsam and observed under an upright microscope
(Nikon, Japan). For immunofluorescence analysis, samples were covered
by Antifade Mounting Medium with DAPI and captured by CLSM. ImageJ
software (Media Cybernetics, USA) was used to analyze the positive
signal in the section.
Statistical analysis
All of the data are presented as mean ± SD. The statistical differences
between the two groups were compared using the unpaired Student’s
t-test. One-way ANOVA or two-way ANOVA with Fisher’s LSD post-test was
used to compare multiple comparisons. Statistical analyses were
conducted using GraphPad Prism 8.0. In all cases, statistical
significance was accepted at p < 0.05.
Reporting summary
Further information on research design is available in the [198]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[199]Supplementary information^ (7.4MB, pdf)
[200]Reporting Summary^ (95.5KB, pdf)
[201]Peer Review file^ (8.8MB, pdf)
Source data
[202]Source Data^ (125.5KB, xlsx)
Acknowledgements