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Hydrogen Sulde Protects from Colitis and Restores Intestinal

Microbiota Biolm and Mucus Production
Jean-Paul Motta, PhD,* , Kyle L. Flannigan, PhD, Terence A. Agbor, PhD, Jennifer K. Beatty, PhD,*
Rory W. Blackler, PhD, Matthew L. Workentine, PhD, Gabriela J. Da Silva, PhD, Rui Wang, PhD,k
Andre G. Buret, PhD,* , and John L. Wallace, PhD

Background: Microbiota dysbiosis and impaired barrier function are among the most prominent features of inammatory bowel disease. In the
gastrointestinal tract, hydrogen sulde (H2S) is an important regulator of mucosal homeostasis. We hypothesized that H2S promotes resolution of colonic
inammation through actions on microbiota biolm and the mucus barrier.

Methods: We used mice genetically decient for a key enzyme for H2S production (cystathionine g-lyase) and pharmacologically inhibited that enzyme
during colitis in wild-type mice. We tested the effects of administering an H2S donor (diallyl disulde) to rodents during hapten-induced colitis. Colonic
microbiota biolm was visualized by uorescent in situ hybridization, and mucus granules were quantied with periodic acidalcian blue staining. We
exposed human microbiota biolms and planktonic bacteria to H2S donors ex vivo to determine changes in their growth, viability, and biomass.

Results: Intestinal microbiota formed linear biolms in the colon of healthy rodents. During colitis, microbiota biolms were fragmented and mucus
granule production decreased. Endogenous production of H2S had benecial effects on establishment of microbiota biolms and colonic
mucus production. Therapeutic delivery of H2S into the colon reduced inammation, restored the microbiota biolm, and increased the production
of mucus granules. In ex vivo human microbiota, H2S not only promoted biolm formation but also reduced growth of planktonic bacteria.

Conclusions: Our results suggest that H2S donors could be used therapeutically during colitis, facilitating correction of microbiota biolm dysbiosis
and mucus layer reconstitution.
(Inamm Bowel Dis 2015;21:10061017)
Key Words: colitis, hydrogen sulde, microbiota, biolm, mucus, IBD

he pathogenesis of inammatory bowel disease (IBD) is

incompletely understood. Alterations of the microbiota (dysbiosis) and compromised intestinal barrier function represent critical areas of research in this eld, and there is a need for improved
therapies that will target mucosal inammation and dysbiosis.

Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions of this
article on the journals Web site (
Received for publication November 25, 2014; Accepted January 13, 2015.
From the Departments of *Biological Sciences, and Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada; Department of Medicine,
McMaster University, Hamilton, ON, Canada; Centre of Neurosciences and Cell
Biology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal; and
Department of Biology, Lakehead University, Thunder Bay, ON, Canada.
J.-P. Motta is recipient of CIHR/Canadian Association of Gastroenterology/
CCC, University of Calgary Eyes High and Izaak Walton Killam postdoctoral
J. L. Wallace and A. G. Buret are founders of Antibe Holdings Inc. and Antibe
Therapeutics Inc., companies that are developing novel anti-inammatory drugs.
The remaining authors have no conicts of interest to disclose.
A. G. Buret and J. L. Wallace contributed equally to this study.
Reprints: Andre G. Buret, PhD, Department of Biological Sciences, Faculty of
Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N
4N1, Canada (e-mail:
Copyright 2015 Crohns & Colitis Foundation of America, Inc.
DOI 10.1097/MIB.0000000000000345
Published online 3 March 2015.




In the past decade, hydrogen sulde (H2S) has become

recognized as an important signaling molecule that inuences
many aspects of gastrointestinal (GI) function.1,2 H2S promotes
mucosal defense, accelerates healing of GI ulcers, and promotes
resolution of inammation.24 Garlic (Allium sativum), which is
naturally rich in organosulfurs that release H2S, has traditionally
been used as a medicinal plant for its antibacterial, antifungal, and
antiparasitic properties.5 Although H2S is produced throughout
the intestine, and by certain bacterial species, the effects of H2S
on host mucosal function and on the complex intestinal microbiota in vivo are not completely understood.
Although intestinal bacteria are presumed to be key players
in the pathogenesis of IBD,6 the clinical benets of antibiotics are
uncertain. Promising options have been reported (e.g., rifaximin
in Crohns disease),7 but use of antibiotics is often associated with
signicant adverse effects.8 Most studies examining changes in
microbiota in IBD have focused on single bacterial groups or
species and primarily investigated fecal bacteria.810 Adding to
the invaluable information generated from these reports on fecal
microbiota, studies investigating mucosal microbiota that live in
close contact with the intestinal surface are sorely needed. Indeed,
the bacterial load is greatest in the colon where they live in a symbiotic, but not always harmless, relationship with the host. This
environment offers very high nutrient availability at the cost of
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constant luminal ow, immune pressure, and environmental inuences (e.g., diet, drugs). Under these conditions, bacteria avoid
the planktonic (free-swimming) mode of living and form sessile
biolms.11,12 Biolms are ubiquitous, complex, multispecies colonies encapsulated in a self-secreted polysaccharide matrix.1,13,14
As such, they withstand physical ushing forces by anchoring to
surfaces, and their polysaccharide matrix allows them to compete
with other microbes and to resist exposure to drugs and immune
components.15 Because these biolms represent the phenotype
that lives in close proximity to host tissue, they are key to our
understanding of how gut microbiota can inuence health and
disease. Recent studies indicate that Campylobacter jejuni, an
important cause for postinfectious are-ups in patients with
IBD, was able to promote inammation by increasing the virulence and invasiveness of commensal bacteria, akin to making
them opportunistic planktonic pathogens.1618 These observations
underscore the homeostatic signicance of maintaining gut microbiota in an intact biolm phenotype.
In this study, we tested the hypotheses that H2S (1) modies
gut microbiota by directly reducing planktonic bacteria while promoting the formation of biolms and (2) improves the production
of protective mucus in the gut. We used uorescent in situ hybridization (FISH) to visualize how gut microbiota was organized in
healthy or inamed colonic sections in rodents. We then assessed
the consequences of inhibiting endogenous H2S and the effects of
H2S administration. Other experiments determined the effects of
H2S donors on bacterial survival, growth and biomass, using planktonic enteropathogens, and multispecies planktonic and biolm
populations from human intestinal microbiota. Studies also assessed whether H2S may modulate intestinal mucus production.
Our ndings demonstrate that H2S reduces intestinal
inammation, corrects microbiota dysbiosis, and reverses the
inammation-associated deciency in mucus production. In particular, we show that H2S concomitantly promotes the growth of
microbiota biolms and inhibits the proliferation of planktonic
bacteria and improves the capacity of colonic tissue to produce
mucus during colitis. The results of this study suggest that H2S
donors could be exploited as novel therapeutics for IBD.


H2S Restores Gut Biolms and Mucus Barrier

induced by a single intracolonic instillation of dinitrobenzene

sulfonic acid (DNBS; Sigma-Aldrich, Oakville, Canada) at 50 mg
in 0.5 mL of 50% ethanol for rats and 5 mg in 0.1 mL of 50%
ethanol for mice. Control animals were treated with an equal
volume of phosphate-buffered saline. DNBS was instilled through
a catheter without anesthesia for rats and 3% isourane anesthesia
for mice. After instillation, the animals were kept upside-down for
3 minutes. Groups of 5 to 6 rats were treated intracolonically,
twice daily, with diallyl disulde (DADS, 0.5 mL; 30 mM) or
vehicle (1% carboxymethylcellulose; Sigma-Aldrich). Groups of
5 to 6 mice were treated intraperitonally, once daily with the
CSE inhibitor, b-cyano-L-alanine (BCA, Cayman Chemicals,
Cedarlane, Burlington, Canada; 50 mg/kg). Under isourane
anesthesia (3%), animals were euthanized by cervical dislocation
either 7 or 14 days after DNBS administration. The severity of
colitis was then blindly evaluated, using the criteria outlined in
Table 1, which is slightly modied from that described previously.19 Sections of colonic tissue were processed by routine
techniques for histological examination and for determination of
myeloperoxidase (MPO) activity as a marker of granulocyte inltration into the tissue.20 Body weights were monitored throughout
the study.

Histochemistry and Fluorescence In

Situ Hybridization
Formalin- or Carnoy-xed colonic tissues were parafnembedded and sectioned to 5 mm in thickness. We performed
histological analysis with hematoxylin-eosin stains and periodic
acid Schiffalcian blue (PAB, Newcomer Supply, Middleton,
WI). Mucus granules were informatically enumerated in each
colonic crypt with ImageJ software. For FISH, tissue was hybridized with 10 ng/mL of a universal bacterial 16S uorescent rRNA
probe (EUB338-Cy3, 50 GCTGCCTCCCGTAGGAGT-Cy3, AlphaDNA, Montreal, Canada), control negative probe (NEUB338Cy3, 50 ACTCCTACGGGAGGCAGC-Cy3, AlphaDNA), and
immunostained for DNA by 40 ,6-diamidino-2-phenylindole (DAPI;
Sigma-Aldrich). Our aim was to visualize microbiota biolms.

TABLE 1. Criteria for Scoring Severity of Colitis in




Experiments were performed using male Wistar rats (Charles

River, Montreal, Canada), male C57Bl/6 mice (Charles River), and
male CSE2/2 mice on a C57Bl/6 background (male, 812 wk of
age). The experiments were approved by the respective animal care
committees of the University of Calgary, McMaster University, and
Lakehead University and were conducted in accordance with the
guidelines of the Canadian Council on Animal Care.


Induction and Evaluation of Colitis

All animals were acclimatized to the study conditions
before entering experiments, at 6 to 8 weeks of age. Colitis was

Localized hyperemia, no ulcers
Ulceration without hyperemia or bowel wall thickening
Ulceration with inammation at 1 site
Two or more sites of discrete ulceration and inammation
Major sites of damage extending .1 cm along length of colon
For presence of diarrhea
For bleeding
For mild adhesions
For severe adhesions |



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Motta et al

Thus, images were deliberately over-contrasted, at the expense of

image quality for tissues (see Fig., Supplemental Digital Content 1, We acquired images on Nikon
Eclipse 80i or Leica DMR uorescent microscopes. ImageJ software was used for image postacquisition analysis.

Calgary Biolm Device

The Calgary Biolm Device (CBD; Innovotech, Calgary,
Canada) features microtiter plate lids, on which are distributed 96
plastic pegs that provide a surface for bacteria to adhere, colonize,
and form a biolm.21

Generation of Human-derived Biolm

Mucosal biopsies from the descending colon of 5 healthy
human volunteers were collected during routine colon cancer
screening (Intestinal Inammation Tissue Bank, University of
Calgary). Specic protocols used for this study underwent
ethics approval. We homogenized biopsies in 200 mL of sTSY
broth (tryptic soy base; BD Bioscience, Mississauga, Canada)
supplemented with yeast extract (5 g/mL, BD Bioscience),
L-cysteine (5%, Sigma-Aldrich) and heminmenadione solution
(50 mg of hemin, 1 mg of menadione; Sigma-Aldrich).
Processed biopsy were plated onto CBDs and kept anaerobically at 378C for 72 hours on a gyrotary shaker at 75 rpm
(Model G2; New Brunswick Scientic Company, VWR,
Mississauga, Canada).

Exposure of H2S Donors to Human-derived Biolm

CBD lids containing human-derived biolms were transferred to a challenge plate containing H2S donors dissolved in
sTSY growth medium (sodium hydrosulde, NaHS and DADS,
Sigma-Aldrich; 1100 mM). We incubated anaerobically biolms
for an additional 24 hours at 378C, after which pegs and planktonic bacteria were collected for analysis (cell viability and biomass assays).

Cell Viability Assay

After exposure to H2S donors, we incubated human-derived
biolms or planktonic bacteria with 2,3-Bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT,
50 mM in phosphate-buffered saline; Sigma-Aldrich) for 2 hours
at 378C. Only metabolically active bacteria drive the reduction of
the XTT. The formazan dye produced by this conversion is thus
proportional to the number of viable cells. We collected supernatants into 96-well plates and then read the absorbance at 450 nm
(SpectraMax microplate reader; Molecular Devices Corporation,
Menlo Park, CA). Results were expressed as fold-change of that
of the mean in the vehicle-treated group.

Biomass Assay (Crystal Violet)

After challenge, human-derived biolms were air-dried
and stained with crystal violet solution (1% in water) for
30 minutes. Crystal violet binds to negatively charged molecules, including nucleic acids and polysaccharides, and therefore
serves as an overall indicator of the biolm biomass. We then




resuspended crystal violet in a destaining solution of 95%

ethanol and measured absorbance at 595 nm. Results were
expressed as fold-change of that of the mean in the vehicletreated group.

Minimum Inhibitory Concentration Assay

NaHS (Sigma-Aldrich) was dissolved in growth medium
(lysogeny broth, Oxoid, Fischer Scientic, Nepean, Canada) and
serially diluted across the wells of a microtiter dish starting at
a concentration of 100 mM. Wells were inoculated with a 1:100
dilution of a 0.5 McFarland standard, prepared from the second
subculture of each strain. Wells were overlaid with sterile mineral
oil to prevent evaporation and incubated for 24 hours, with shaking (175 rpm), at 378C. The highest concentration at which no cell
growth (no turbidity in the well) was recovered from any of the 4
replicate wells was recorded as the minimum inhibitory concentration (MIC).

Statistical Analysis
Data representation and statistical analysis were performed
using GraphPad Prism for Macintosh (v5, San Diego, CA).
Statistical signicance was determined by one-way analysis of
variance and Bonferroni multiple comparisons, KruskalWallis
test, Dunns multiple comparisons or unpaired Students t test,
as appropriate. An associated P value of less than 5% was considered signicant. All values in the gures are presented as mean
6 SEM.

Colonic Microbiota Forms a Biolm at the
Surface of Healthy Colonic Tissue
We used FISH to visualize colonic biolm organization in
sections of mouse (Fig. 1A) and rat colon (Fig. 1B). The FISH
probe was specic to bacteria based on competitive probe binding (see Fig., Supplemental Digital Content 1, http://links.lww.
com/IBD/A767), high-resolution morphological analysis
(1,000 magnication; see Fig. A and B, Supplemental Digital
Content 2,, staining with nonspecic DNA dye (DAPI; see Fig. A, Supplemental Digital
Content 2,, and Grams
method (see Fig. B and E, Supplemental Digital Content 2, In healthy control mice and
rats, microbiota formed a linear structure sandwiched between
the sterile mucus layer and the luminal fecal content (Fig. 1A, B
and see Fig. C and D, Supplemental Digital Content 2, http:// Periodic acid Schiffalcian blue
(PAB) was used to stain neutral and acid polysaccharides contained in the mucus, but was also able to stain polysaccharides
surrounding the bacteria (see Fig. D and F, Supplemental Digital
Content 2, All these features
were characteristic of a bacterial biolm structure and thus provided evidence that intestinal bacteria form biolms at the surface of healthy colonic tissue.

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H2S Restores Gut Biolms and Mucus Barrier

staining), host-cell debris (lamentous DNA) and immune cells

(not shown). These biolms were in close contact with host tissue,
associated with extensive bacterial translocation into the lamina
propria (Fig. 1C, D).
Mucus granules in goblet cells are fundamental for
intestinal barrier function as they are required for renewal of the
mucus layer. We used PAB to stain acidic and neutral polysaccharides, and we counted each positive granule per intestinal
crypt (automated particle counting, see Fig. A and B, Supplemental Digital Content 3, Seven
days after induction of colitis in mice and rats, inammation was
accompanied by ulcers, crypt loss, and extensive mucus granule
depletion (Fig. 1E, F and see Fig. C, Supplemental Digital
Content 3, However, number
of mucus granules per intestinal crypt remained unchanged
in adjacent healthy tissue (see Fig. D, Supplemental Digital
Content 3, Together, these ndings demonstrate the development of altered, or dysbiotic biolms
and mucus granule depletion during colitis in rodents.

Endogenous H2S Contributes to the

Development of Colonic Microbiota Biolm
and Mucus Layer

FIGURE 1. Colonic microbiota biolm is fragmented and mucus

granule production is decreased in DNBS-induced colitis. C57Bl/6
mice and Wistar rats were given DNBS intracolonically to induce
colitis and were euthanized 7 days later (n 5 per group). FISH was
performed on colonic sections of mice (A and C) and rats (B and D).
Representative images of healthy control animals (A and B) or
animals with colitis (C and D) are presented. Host nuclei were
colored in red (40 ,6-diamidino-2-phenylindole, DAPI), and FISH
positive cells in green (EUB338-Cy3 probe). Scale bars represent
50 mm, dashed lines represent the limit of mucosa, and asterisks
represent translocated bacteria into the lamina propria (AD).
Average number of colonic mucus granule per microscopic eld
(200) in mice (E) and rats (F) healthy controls and with colitis are
reported (n $ 4 different elds were analyzed per animal). **P ,
0.01, ***P , 0.001 (Students t test).

Colonic Microbiota Biolm Is Fragmented and

Mucus Granule Production Is Decreased in
Hapten-induced Colitis
At day 7 post-DNBS administration, biolm organization
was severely altered both in mice (Fig. 1C) and rats (Fig. 1D).
Microbiota staining with FISH was heterogeneous, ranging from
isolated cells to clusters of different sizes. Bacterial aggregates
were biolms surrounded by polysaccharides (positive PAB

Colonic tissue from CSE-decient mice produced signicantly less H2S but no macroscopically visible changes in colonic
appearance or function as compared with wild-type littermates
(Fig. 2A).22 Consistent with a previous report in rats treated with
a CSE inhibitor,4 we observed that CSE-decient mice had mild
granulocyte inltration in the colon (see Fig. A, Supplemental Digital
Content 4, accompanied by thinning of the inner mucus layer (Fig. 2A, B and see Fig. B and C,
Supplemental Digital Content 4,
Although the microbiota formed a normal linear biolm, some bacterial aggregates were found in close contact with the epithelium (see
Fig. C, Supplemental Digital Content 4,
A770) and inltrating the colonic crypts (see Fig. D, Supplemental
Digital Content 4,
We then investigated if inhibition of endogenous H2S synthesis would have an impact on gut microbiota biolm establishment and on mucus granule production during colitis in mice. The
mice that were treated with an inhibitor of CSE (BCA) lost weight
similarly to those treated with vehicle (Fig. 2C) but developed
more severe colitis (signicantly greater colonic macroscopic damage score, Fig. 2D) and a greater increase of colonic MPO activity
(Fig. 2E). The microbiota in healthy mice (vehicle-treated) formed
a linear biolm, separated from the host cells by a sterile mucus
layer (Fig. 3A). In mice with colitis, the microbiota biolm was
fragmented, and bacteria were in close contact with host tissue
(Fig. 3B). Inammation was accompanied by sporadic accumulation of immune cells in the lumen (see Fig. B, Supplemental
Digital Content 5, As opposed
to mice with colitis treated with vehicle, the microbiota formed 2
different phenotypes in mice with colitis that treated with BCA.
In some areas, the microbiota biolm was still present (Fig. 3C), |



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Motta et al

FIGURE 2. Endogenous H2S contributes to mucus layer thickness in the colon, and its inhibition aggravates DNBS-induced colitis in mice. Sections of
colon tissue from CSE-decient (CSE2/2) mice, and their wild-type littermates (WT) were frozen and Carnoys-xed (n 6 per group). Representative
pictures of periodic acid Schiffalcian blue staining (A) and average inner mucus layer thickness (B) are shown. Scale bars represent 50 mm, and
dashed lines represents area of mucus layer considered for quantication (A and B). C57Bl/6 mice were given DNBS intracolonically to induce colitis
and were euthanized after 7 days (n 56 per group). Healthy mice and mice with colitis were treated with BCA or vehicle (CF). Change in body
weight is shown (C), and severity of disease (blindly evaluated) is reported as the macroscopic damage score (D) and colon myeloperoxidase (MPO)
activity (E). The average number of colonic mucus granules per microscopic eld (200) in groups of control mice and mice with colitis is reported (F,
$4 different elds were analyzed per animal). *P , 0.05, **P , 0.01, ***P , 0.001 (Students t test for data in B, analysis of variance and Bonferroni
multiple comparison test for data in DF).

but in others, it formed small aggregates with visible translocation into the lamina propria (Fig. 3D). However, in both of these
2 phenotypes, there were signicant numbers of immune cells in
the microbiota biolm (Fig. 3C) and in the colonic lumen (Fig.
3D). These cells appeared to be mainly granulocytes and macrophages based on cell and nucleus morphology (hematoxylineosin staining; see Fig., Supplemental Digital Content 5,




Quantication of mucus granules in healthy mice treated

with BCA revealed no change compared with that of healthy mice
treated with vehicle (Fig. 2F). However, mice with colitis that
were treated with BCA had a greater reduction in mucus granule
number than mice with colitis treated with vehicle (Fig. 2F).
These observations indicate that endogenous H2S produced
through CSE promotes the formation of colonic microbiota biolms and plays a role in enhancing mucus barrier function.

Inamm Bowel Dis  Volume 21, Number 5, May 2015

FIGURE 3. Inhibition of endogenous H2S during DNBS-induced colitis

in mice aggravates inammation and alters colonic microbiota biolm.
C57Bl/6 mice were given DNBS intracolonically to induce colitis and
were euthanized 7 days later (n 56 per group). FISH was performed
on colonic sections. Representative images of healthy mice (A), mice
with colitis treated with vehicle (B) or with an inhibitor of H2S synthesis (b-cyanolalanine, BCA, C and D) are presented. Host nuclei were
colored in red (40 ,6-diamidino-2-phenylindole, DAPI), and FISH positive
cells in green (EUB338-Cy3 probe). Scale bars represent 25 mm (A and
C), 50 mm (D), dashed lines represent the limit of mucosa. Asterisk
represents translocated bacteria in lamina propria, and pound signs
represent host cells in lumen.

Therapeutic Delivery of H2S Promotes

Resolution of Colitis and Restores Microbiota
Biolm and Mucus Granule Production
As endogenous H2S appears to be critical for microbiota
biolm development and host mucus production, we examined
the effects of therapeutic delivery of H2S into the colon of rats
with colitis. Rats treated with DADS or vehicle exhibited similar
recoveries of weight loss after induction of colitis (Fig. 4A).
However, rats with colitis treated with DADS exhibited less
severe colonic damage (Fig. 4B) and had lower tissue MPO
activity (Fig. 4C) than rats with colitis treated with vehicle. Likewise, histologically, there was less severe disease in rats with
colitis treated with DADS than in those treated with vehicle
(Fig. 4D, E). Microbiota staining in healthy rats (Fig. 5A), rats
with colitis treated with vehicle (Fig. 5B, C) or treated with
DADS (Fig. 5D) revealed that DADS treatment restored linear
biolm organization, and signs of bacterial translocation were no
longer apparent (Fig. 5A, D). We also observed a sterile mucus
layer that separated microbiota biolm from host tissue, similar to
what was observed in healthy rats (Fig. 5A, D). These ndings

H2S Restores Gut Biolms and Mucus Barrier

demonstrate that luminal administration of H2S was effective

in reducing colonic inammation and restoring microbiota
biolm structure.
We evaluated if delivery of H2S into the colonic lumen for
7 days (Fig. 6A, C, E, G) or 14 days (Fig. 6B, D, F, H) would
restore mucus granule production during colitis. Periodic acid
Schiffalcian blue staining was performed on colonic tissue from
healthy rats treated with vehicle (Fig. 6A, B), healthy rats treated
with DADS (Fig. 6C, D), and rats with colitis treated with vehicle
(Fig. 6E, F) or with DADS (Fig. 6G, H). When rats with colitis
were treated with DADS, either for 7 days (Fig. 4F) or 14 days
(Fig. 4G), mucus granule number per intestinal crypt was significantly greater than in the group of rats with colitis treated with
vehicle. Remarkably, healthy rats treated with DADS for 14 days
(but not for 7 d), had signicantly higher number of mucus granule number than healthy rats treated with vehicle (Figs. 4G and
6B, D). Nevertheless, mucus granule number was signicantly
higher at 7 days in rats with colitis treated with DADS than in
healthy rats treated with DADS (Fig. 4F). The number of colonic
crypts was also increased in rats with colitis treated with DADS
for 7 days, which is consistent with an enhancement of resolution
of tissue damage (see Fig. C, Supplemental Digital Content 3, In rats with colitis that received
vehicle for 14 days, mucus granule number had returned to basal
levels (Figs. 4G and 6B, F). Treatment with DADS for 14 days
signicantly reduced MPO activity (by 58%; P , 0.05) and
reduced macroscopic damage score (by 52%; P , 0.01). Indeed,
14 days after induction of colitis, the histological appearance of
the colon of healthy rats (see Fig. A and C, Supplemental Digital
Content 6,, rats with colitis treated with vehicle (see Fig. A and B, Supplemental Digital Content
6, or with treated with DADS
(see Fig. C and D, Supplemental Digital Content 6, http://links., revealed that intestinal inammation had
resolved more efciently in rats treated with DADS (see Fig. D,
Supplemental Digital Content 6,
than in rats treated with vehicle (see Fig. B, Supplemental Digital
Content 6,

H2S Promotes the Growth of Human-derived

Gut Biolm
To determine whether H2S was able to directly modify microbiota biolms, we exposed human colon-derived multispecies biolms to various concentrations (1100 mM) of H2S donors (NaHS
and DADS). We then evaluated the viability and number of bacteria
(metabolic activity assay or XTT assay, Fig. 7A, C), and the overall
quantity of proteins/polysaccharides/DNA/RNA (biolm biomass
assay or crystal violet assay, Fig. 7B, D) in each of these humanderived biolms. Compared with the vehicle-treated biolms, those
exposed to low concentrations of the H2S donors (1 and 10 mM)
had a higher metabolic activity, suggesting an increased number of
bacteria within the biolm (Fig. 7A, C). Indeed, biolms exposed to
these concentrations of NaHS or DADS had an increased biomass,
implying an increased quantity of cells and proteins (Fig. 7B, D). |



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FIGURE 4. Treatment with an H2S donor reduces the severity of DNBS-induced colitis in rats. Wistar rats were given DNBS intracolonically to
induce colitis. Healthy rats and rats with colitis were euthanized after daily treatment with vehicle or DADS (30 mM in 0.5 mL) for 7 or 14 days (n
45 per group). Effects of DADS on body weight (A; day 7), macroscopic damage score (B; day 7), and colon myeloperoxidase (MPO) activity
(C; day 7) are shown for rats with colitis. Representative images of hematoxylin-eosinstained colonic sections are shown for rats with colitis
treated with vehicle (D; day 7) or with DADS (E; day 7). Scale bars represent 200 mm and dashed lines represents the muscularis mucosae (D and E).
The average number of colonic mucus granules per microscopic eld (200) at day 7 (F) and day 14 (G) are reported in groups of healthy rats and
rats with colitis ($4 different elds were analyzed per animal). *P , 0.05, **P , 0.01, ***P , 0.001 (Students t test for data in B and C, and
nonparametric KruskalWallis test and Dunns multiple comparison tests for data in F and G).

Metabolic activity and biomass of biolms exposed to a higher

concentration (100 mM) of the donors were unmodied.

H2S Kills Planktonic Bacteria

The planktonic phenotype is a free-oating mode of
bacterial growth. This phenotype can be found in vivo
along with the biolm phenotype. Planktonic bacteria can




originate from preformed biolms and can facilitate biolm

dissemination, most of the time to undesired locations.13,23 We
rst examined the effect of H2S on monoculture viability of
planktonic bacteria under aerobic conditions. Exposure to
NaHS for 24 hours led to growth inhibition of 4 different
strains of pathogenic bacteria able to colonize the GI tract: Salmonella typhimurium (MIC 125 mM), Escherichia coli

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FIGURE 5. Treatment with an H2S donor restores microbiota biolm

during DNBS-induced colitis in rats. Wistar rats were given DNBS intracolonically to induce colitis. Healthy rats and rats with colitis were
euthanized after daily treatment with vehicle or DADS (30 mM in 0.5 mL)
for 7 days (n 45 per group). FISH was performed on colonic sections.
Representative images of healthy rats (A), rats with colitis treated with
vehicle (B and C) or with DADS (D) are presented. Host nuclei were
colored in red (40 ,6-diamidino-2-phenylindole, DAPI) and FISH positive
cells in green (EUB338-Cy3 probe). Scale bars represent 50 mm (AD),
dashed lines represent the limit of mucosa, and asterisks represent
translocated bacteria in lamina propria.

(MIC 125 mM), Staphylococcus aureus (MIC 62.5 mM),

and Pseudomonas aeruginosa (MIC 62.5 mM) (Fig. 8A). We
then chose another model to reproduce the biological complexity
of intestinal microbiota. We used human multispecies planktonic
bacteria collected from biolms preformed on CBDs. These
multispecies cultures were exposed under anaerobic conditions
to various concentrations of H2S donors for 24 hours (NaHS and
DADS, 1100 mM).
Growth of human planktonic bacteria in stationary phase
(OD650 1.1) was not affected by the H2S donors (Fig. 8B).
However, concentrations of 50 mM of NaHS and 50 to 100 mM
of DADS were effective in reducing growth of planktonic bacteria in exponential phase (OD650 0.4, Fig. 8C). Correspondingly, concentrations of 50 mM (DADS) and 100 mM (DADS
and NaHS) inhibited the metabolic activity of these planktonic
bacteria in exponential phase (Fig. 8D). The doseresponse
curves of the H2S donors did not strictly parallel one another,
which may be a result of different rates of H2S release from
these donors. H2S is immediately generated from NaHS, unlike
that from DADS. However, these results demonstrated that H2S
donors not only had antimicrobial activity against human

H2S Restores Gut Biolms and Mucus Barrier

FIGURE 6. Treatment with an H2S donor increased mucus granule

number in colonic crypts during DNBS-induced colitis in rat. Wistar
rats were given DNBS intracolonically to induce colitis. Healthy rats
and rats with colitis were euthanized after daily treatment with vehicle
or DADS (30 mM in 0.5 mL) for 7 days (A, C, E, and G) or 14 days (B, D, F,
H). Colonic sections were formalin-xed and stained with periodic acid
Schiffalcian blue. Representative images of healthy rats (A and B),
healthy rats treated with DADS (C and D), rats with colitis treated with
vehicle (E and F) or rats with colitis treated with DADS (G and H) are
presented. Scale bars represent 100 mM.

planktonic bacteria in monoculture but also in multispecies culture evading from biolms.

Maintenance of long-term remission is a key goal for the
treatment of patients suffering from IBD, but current therapies
often fail to achieve this goal. An ideal therapeutic approach may
need to combine both the modulation of inammation and
correction of microbial dysbiosis.8,24 Indeed, the combination of
azathioprine and antibiotics has shown some benets but only for
treating patients at high risk of a surgery-requiring recurrence.25
The formation of bacterial biolms has been mostly regarded as
a detrimental phenotype, both in industry (e.g., pipe corrosion, |



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Motta et al

FIGURE 7. H2S promotes human colonic multispecies microbiota biolms. Human biolms from colon biopsies from healthy volunteers (n 5)
were derived after 72 hours of anaerobic incubation using the CBD. Biolms (n $ 40 per group) were exposed anaerobically to H2S donors for 24
hours: sodium hydrosulde (NaHS, A and C) or DADS (B and D). Effects of different concentrations of NaHS or DADS (1100 mM) on biolm
metabolic activity [2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay, A and C] and biolm biomass
(crystal violet assay, B and D) are presented. *P , 0.05, **P , 0.01, ***P , 0.001, one-way analysis of variance and Bonferroni multiple

ship hull drag) and in medicine (e.g., those forming on surgical

implants, dental plaque).13 Biolms provide an efcient strategy
for bacteria to resist drugs, environmental insults, the host
immune system, and luminal ow in the GI tract. In contrast to
their detrimental effects outlined above, microbiota living as biolms promote gut homeostasis.11,12 Naturally formed intestinal
biolms could benet the host by digesting substrates inaccessible
to host enzymes, modulating immunity, and conferring resistance
against transient enteropathogens.8,15,26
Monospecies biolms generated in vitro have been the
subject of intense research and have provided profound insights
into biolm formation processes.14,27 To characterize complex gut
biolms, we used a classical histological procedure to preserve
tissue integrity, and specic techniques for microbiota detection
(i.e., FISH). Our method using full intestinal tissue preparations,
to minimize experimental artifacts in evaluation of the mucus
layer and loss of microbiota materials incurred from histology
washing, has facilitated novel observations. Together, our ndings conrm that intestinal bacteria form continuous biolms




lining the mucus surface that coats the colonic mucosa.12,28 Previous reports have presented gut microbiota biolms as a recurrent
pathogenic phenotype in IBD.12 Our ndings, consistent with previous observations,28 show that in fact this does not seem to be the
case, and that biolm development is indeed what occurs in the
healthy gut. Our in vivo results further demonstrate that during
colitis, the microbiota biolm is fragmented and adheres closely
to the epithelial surface, possibly representing a novel marker of
microbiota dysbiosis.
Recent data suggest that H2S donors derived from garlic have
antimicrobial effects in vitro on planktonic Gram-negative29,30 and
Gram-positive bacteria.31 Consistent with those observations, this
study demonstrates that H2S can kill several species of planktonic
bacteria found in humans. As microbiota biolm structure was
restored in animals treated with H2S donors during colitis, we investigated whether H2S was able to promote ex vivo human colonic
biolm growth. This was indeed the case, and the exact mechanisms
involved will require further investigation. Recent observations indicate that postinfectious dysbiosis may be associated with disruptions

Inamm Bowel Dis  Volume 21, Number 5, May 2015

H2S Restores Gut Biolms and Mucus Barrier

FIGURE 8. H2S has antimicrobial activity against human planktonic bacteria. Monospecies cultures of 4 different strains of pathogenic bacteria in
liquid media were exposed aerobically to various concentrations of sodium hydrosulde (NaHS, n 4 replicates per species). MIC of NaHS was
reported in each strain, by evaluating survival of bacteria at optical density at 650 nm (OD650nm, A). Multispecies planktonic bacteria were collected
from human biolms formed on CBD (n 5 patients) after 72 hours of anaerobic incubation. Stationary (OD650nm 1.1, B) or exponential phase
(OD650n3m 0.4, C and D) planktonic cultures were exposed anaerobically to various concentrations of NaHS or DADS (1100 mM, n $ 16
different cultures per group) for 24 hours. Effects of NaHS or DADS on growth (C) and on metabolic activity of bacteria [2,3-Bis(2-methoxy-4-nitro5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay, D] are presented. *P , 0.05, **P , 0.01, ***P , 0.001 versus vehicle; one-way
analysis of variance and Bonferroni multiple comparison test.

of microbiota biolms and a concurrent increased release of pathogenic planktonic bacteria from these biolms, which damage tight
junctions and increased epithelial permeability.15,3234 These observations give further signicance to the present ndings, which illustrate that the benets of H2S in colitis are associated with
a promotion of the biolm phenotype, concurrent with a direct antibacterial effects in planktonic populations. Whether these benets
may synergize with the benecial effects of H2S as metabolic fuel
for the colonic epithelium is currently unknown.35,36
The observed benecial effects of DADS in promoting
resolution of colitis in this study is consistent with previous
observations that a range of H2S-releasing agents could accelerate
healing of GI ulcers and reduce tissue inammation.3,4,22,3739 The
mechanisms underlying the anti-inammatory effects of H2S in
experimental colitis include inhibition of leukocyte recruitment,40
upregulation of cyclooxygenase-2 (COX-2) at the ulcer margins,3
stimulation of angiogenesis,41 suppression of expression of proinammatory cytokines, and elevation of expression of interleukin-

10, an anti-inammatory cytokine.4,3739 Promotion of resolution of

colitis by H2S may also include mechanisms such as induction of
neutrophil apoptosis,42 a shift of macrophages to a proresolution
phenotype43 and inhibition of MPO activity.44 The present ndings
are consistent with these, but we described a different and additional mechanism of action of H2S during colitis, namely a direct
action on gut microbiota. We postulate that H2S helps to restore
alterations of healthy gut microbiota biolms during colitis, through
induction of biolm phenotype and growth inhibition of planktonic
Endogenous H2S synthesis is markedly upregulated in
experimental colitis and plays a major role in driving resolution
of inammation and tissue injury.4,22 This increase in H2S production occurs mainly through upregulation of 2 enzymatic pathways: CSE and 3-mercaptopyruvate sulfurtransferase and occurs
selectively at sites of ulceration.22 There is also signicant downregulation of oxidation of H2S at those sites, which together with
increased synthesis, would result in higher local concentrations of |



Inamm Bowel Dis  Volume 21, Number 5, May 2015

Motta et al

H2S in ulcerated tissue.22 Inhibition of H2S synthesis, such as

through suppression of CSE activity with BCA, results in exacerbation of colitis and inhibition of tissue repair.4 In this study,
BCA administration to mice with colitis resulted in signicant
exacerbation of colitis (elevated colonic damage scores and tissue
MPO activity), similarly to what was reported in rats.4 In previous
studies, we observed that BCA reduced colonic H2S synthesis by
41% (P , 0.05), whereas a reduction of colonic H2S synthesis of
61% (P , 0.05) was observed when tissue from CSE-decient
mice was compared with that from wild-type controls.22
Although H2S has sometimes been suggested to be toxic
in the GI tract,45 or to promote mutations in vitro,46 it has
become increasingly clear in recent years that this gasotransmitter performs a number of important physiological
functions,1,47 including the ability to support mitochondrial respiration in circumstances of hypoxia and anoxia.35,48 Colonocytes have been reported to be the best adapted cells for using
H2S as an energy source for driving adenosine triphosphate
(ATP) production, and in vitro, intestinal epithelial cells could
withstand exposure to H2S at a concentration as high as 50
mM.49 Studies in germ-free mice suggest that bacterial production of H2S contributes signicantly to tissue levels in the
host.50,51 H2S released from donors has a very short half-life,
and it is likely that most is converted rapidly to sulfane sulfur or
polysuldes, from which H2S can subsequently be liberated.
Indeed, some effects attributed to H2S may in fact be produced
by polysuldes.52 In a recent study, we observed that the H2S
donor used in this study (DADS) did not produce GI damage or
inammation when administered orally twice daily for 5 days at
doses of up to 60 mmol/kg.53 Indeed, in these studies, the H2S
donor signicantly protected the GI tract from injury induced
by a nonsteroidal anti-inammatory drug.53
A leaky mucus layer that can permit bacterial invasion has
been reported in humans with ulcerative colitis, as well as in murine
models of colitis.54 Despite their extreme density in the colon, we
showed that microbiota biolm bacteria lined the surface of the
mucus layer and did not penetrate host tissues. We also demonstrated
that during DNBS-induced colitis, the number of mucus granules in
goblet cell populations was reduced in association with signs of
bacterial translocation. These observations likely underscore the critical barrier function of the mucus layer in protecting intestinal tissues
against bacterial invasion. Consistent with this hypothesis, a recent
study reported that antibiotics cause a thinning of the mucus layer by
directly reducing mucus granule numbers, an effect that predisposed
mice to further enteric infection with Citrobacter rodentium.55 As
H2S appeared to promote mucus production in this study, future
research investigating whether and how H2S donors might counter
the mucolytic effects of traditional antibiotics seems warranted.
In summary, this study demonstrates that endogenous H2S
has benecial properties in a model of experimental colitis, in part
through reduction of neutrophil inltration, increased production
of mucus granules, and maintenance of mucus layer thickness.
Cystathionine g-lyase is an important source of endogenous
H2S synthesis in this setting. Therapeutic delivery of H2S during




colitis restored production of mucus granules and corrected microbiota biolm dysbiosis. Mechanistic studies showed that the
benets of H2S on microbiota biolms were due, at least in part,
to a direct promotion of the biolm phenotype while inhibiting the
growth of planktonic bacteria. Further research using this model
system will help to identify critical pathways through which microbiota biolms and mucus interactions promote intestinal
homeostasis, and how H2S may by exploited therapeutically to
help restore and maintain intestinal homeostasis.

The authors thank Dr. Paul Beck for providing human
biopsies through the Inammation Tissue Bank at the University
of Calgary. The authors acknowledge nancial support for this work
from Crohns and Colitis Canada, Canadian Institutes of Health
Research and National Sciences and Engineering Research Council.
Authors contributions: J.-P. Motta, K. L. Flannigan, T. A.
Agbor, M. L. Workentine, and R. Wang conducted animal
studies; J.-P. Motta and J. K. Beatty performed human-derived
biolm experiments; J.-P. Motta, R. W. Blackler, M. L. Workentine, and G. J. Da Silva participated in antibacterial in vitro
assay. J.-P. Motta performed histochemistry and wrote the
manuscript. A. G. Buret and J. L. Wallace supervised the research
and wrote the manuscript. All authors were involved in design of
experiments, interpretation of results, and critical revision of the
manuscript. All authors approved the nal manuscript.

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