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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
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 (www.ibdjournal.org).
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
k
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
fellowships.
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: aburet@ucalgary.ca).
Copyright 2015 Crohns & Colitis Foundation of America, Inc.
DOI 10.1097/MIB.0000000000000345
Published online 3 March 2015.
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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.
Animals
Score
0
1
2
3
4
5
+1
+1
+1
+1
Appearance
Normal
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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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.
RESULTS
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, http://links.lww.com/IBD/A768), staining with nonspecic DNA dye (DAPI; see Fig. A, Supplemental Digital
Content 2, http://links.lww.com/IBD/A768), and Grams
method (see Fig. B and E, Supplemental Digital Content 2,
http://links.lww.com/IBD/A768). 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://
links.lww.com/IBD/A768). 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, http://links.lww.com/IBD/A768). 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.
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, http://links.lww.com/IBD/A770) accompanied by thinning of the inner mucus layer (Fig. 2A, B and see Fig. B and C,
Supplemental Digital Content 4, http://links.lww.com/IBD/A770).
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, http://links.lww.com/IBD/
A770) and inltrating the colonic crypts (see Fig. D, Supplemental
Digital Content 4, http://links.lww.com/IBD/A770).
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, http://links.lww.com/IBD/A771). 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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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,
http://links.lww.com/IBD/A771).
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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).
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planktonic bacteria in monoculture but also in multispecies culture evading from biolms.
DISCUSSION
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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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
comparisons.
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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
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-
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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.
ACKNOWLEDGMENTS
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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