Humans are colonized by multitudes of commensal organisms representing members of five of the six kingdoms of life; however, our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms. Imbalances in the composition of the bacterial microbiota, known as dysbiosis, are postulated to be a major factor in human disorders such as inflammatory bowel disease. We report here that the prominent human symbiont Bacteroides fragilis protects animals from experimental colitis induced by Helicobacter hepaticus, a commensal bacterium with pathogenic potential. This beneficial activity requires a single microbial molecule (polysaccharide A, PSA). In animals harbouring B. fragilis not expressing PSA, H. hepaticus colonization leads to disease and pro-inflammatory cytokine production in colonic tissues. Purified PSA administered to animals is required to suppress pro-inflammatory interleukin-17 production by intestinal immune cells and also inhibits in vitro reactions in cell cultures. Furthermore, PSA protects from inflammatory disease through a functional requirement for interleukin-10-producing CD4+ T cells. These results show that molecules of the bacterial microbiota can mediate the critical balance between health and disease. Harnessing the immunomodulatory capacity of symbiosis factors such as PSA might potentially provide therapeutics for human inflammatory disorders on the basis of entirely novel biological principles

Kasper, Dennis L.

Mazmanian, Sarkis K.

Round, June L.

English

Caltech Authors - Main

 
 
 
 
Mutualism by a microbial symbiosis factor prevents intestinal 
inflammatory disease 
 
Sarkis K. Mazmanian1,*, June L. Round1,*, Dennis L. Kasper2,3 
 
1Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA 
2Channing Laboratory, Department of Medicine, Brigham & Women's Hospital, Harvard Medical 
School, Boston, MA 02115, USA  
3Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 
02115, USA 
 
 
 
 
 
 
Supplemental Information 
 
 
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07008
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Supplemental Fig. 1 
 
 
 
 
 
 
 
 
 
 
 
 
S. Fig. 1| Cultures of colons from various groups of animals demonstrate the specific 
increase in pro-inflammatory cytokines in diseased colons (but not in small intestines). This 
increase is significantly reduced in animals co-colonized with PSA-producing B. fragilis. 
Conversely, animals colonized with B. fragilis ΔPSA express greatly increased pro-inflammatory 
cytokine levels over those in control animals (C57BL/6). ELISA results for the pro-inflammatory 
cytokines IL-12p40 (left) and IL-1β (right) are shown. Results are from one trial of 2 independent 
experiments. Error bars indicate SD values from studies of colons recovered from 4 animals per 
group. 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 2 
 
 
 
S. Fig. 2| Expression of the TNFα by CD4+ T cells is reduced by wild-type B. fragilis 
colonization during experimental colitis. CD4+ cells were purified from pooled splenocytes 
from each group (4 mice per group) and restimulated in vitro with PMA and ionomycin in the 
presence of  brefeldin A for 4 hours. Cells were stained for intracellular TNFα. Cells within the 
lymphocyte gate were included in the analysis, and numbers indicate the percentage of cells 
producing TNFα. Purified cells were > 90% CD4+. Animals colonized with PSA-producing B. 
fragilis during protection displayed lower TNFα levels than diseased animals. 
 
 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 3 
a
CM 321 4
b
MDBA E
 
 
S. Fig. 3| Experimental animals remain colonized with H. hepaticus and B. fragilis 
throughout the course of disease. (a) Ethidium bromide–stained gel electrophoresis of H. 
hepaticus–specific Q-PCR shows that co-colonization with B. fragilis does not induce clearance 
of bacteria after 8 weeks. M: Marker. 1: Rag2-/- animals with CD4+CD45Rbhigh T cell transfer 
colonized with H. hepaticus alone. 2: Rag2-/- animals with CD4+CD45Rbhigh T cell transfer 
colonized with H. hepaticus and B. fragilis 9343 (wt). 3: Rag2-/- animals with CD4+CD45Rbhigh T 
cell transfer colonized with H. hepaticus and B. fragilis ΔPSA. 4: C57BL/6 mice colonized with 
H. hepaticus alone. Note: H. hepaticus readily colonized animals but did not induce disease (Fig. 
1). Primers for H. hepaticus 16S rDNA: (HB-15) 5’-GAAACTGTTACTCTG-3’ and (HB-17) 5’-
TCAAGCTCCCCGAAGGG-3’. (b) Ethidium bromide–stained gel electrophoresis of B. fragilis–
specific Q-PCR showing stable bacterial colonization after 8 weeks. A: Rag2-/- animals with 
CD4+CD45Rbhigh T cell transfer colonized with H. hepaticus and B. fragilis 9343 (wt). B: Rag2-/- 
animals with CD4+CD45Rbhigh T cell transfer colonized with H. hepaticus and B. fragilis ΔPSA. 
C: Rag2-/- animals with CD4+CD45Rbhigh T cell transfer colonized with H. hepaticus alone. D: 
C57BL/6 mice colonized with H. hepaticus alone. E: B. fragilis genomic DNA (positive control). 
M: Marker. Primers for B. fragilis ssr3 (finB) gene: (ssr3-F) 5’-TATTTGCGAGAAGGTGAT-3’ 
and (ssr3-r) 5’-TAAACGCTTTGCTGCTAT-3’. 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 4 
 
 
 
 
 
 
 
 
 
 
S. Fig. 4| Quantitation of H. hepaticus colonization demonstrates that the organism is 
present in equal numbers regardless of PSA-mediated protection. Fecal samples were 
collected from each experimental group, and total DNA was extracted (Qiagen DNAeasy tissue 
kit). Equal amounts of DNA (50 ng) were used in Q-PCR (Bio-rad) with H. hepaticus–specific 
primers. Q-PCR for H. hepaticus colonization was assessed according to Young et al., 2004 1 as 
log10 number of copies of a known gene (cytolethal distending toxin). Animals contained 
equivalent levels of H. hepaticus at the end of the experiment. 
 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 5 
 
 
 
 
 
 
 
 
 
 
 
S. Fig. 5| PSA from B. fragilis induces expression of IL-10 in vitro. Results are shown for IL-
10 ELISA of supernatants of primary BMDC–T cell co-cultures incubated for 48 hours with H. 
hepaticus alone or with H. hepaticus andB. fragilis (wild-type or ΔPSA) at a multiplicity of 
infection of 5.. Error bars show SD values for samples run in duplicate and represent 3 
independent experiments. 
 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 6 
 
 
 
 
 
 
 
 
 
 
 
 
S. Fig. 6| PSA reduces expression of IL-1β in an IL-10-dependent manner. Infection of 
BMDC–T cell co-cultures with increasing concentrations of live H. hepaticus (multiplicity of 
infection: 0.1, 1.0, and 10, as depicted by triangles) results in release of the cytokine IL-
1β. Treatment of infected cells with PSA reduces IL-1β levels, as shown in the middle three bars. 
Neutralization of IL-10 signaling by addition of an IL-10 receptor antibody (αIL-10R) alleviates 
suppression of in vitro inflammatory responses, resulting in increased levels of IL-1β (left three 
bars). Error bars show SD values for experiments run in triplicate. 
 
 
 
 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
S. Fig. 7| PSA does not prevent weight loss in TNBS-treated IL-10-/- animals. Groups of 4 
C57BL/6 mice were treated with PSA (or PBS) and then subjected to rectal administration of 
TNBS or vehicle (control). Mean body weights (shown as percentages of initial weight) are 
shown for each group; SD values indicate that, in the absence of IL-10, PSA cannot restore 
TNBS-induced weight loss. ANOVA demonstrates that weight loss in both TNBS-treated groups 
is statistically different from that in control animals.  
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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Supplemental Fig. 8 
 
 
 
S. Fig. 8| PSA does not prevent pathology in TNBS-treated IL-10-/- animals. Groups of 4 
C57BL/6 mice were treated with PSA (or PBS) and then subjected to rectal administration of 
TNBS or vehicle (control). Histologic analysis  of H&E-stained sections from a representative 
animal from each group is shown. Thickening of the colon and epithelial hyperplasia are noted in 
both TNBS-treated groups of IL-10-/- animals, regardless of PSA treatment. Thus, in the absence 
of IL-10, PSA does not reduce intestinal injury in TNBS-treated IL-10-/- mice. Results represent 2 
independent experiments. 
doi: 10.1038/nature07008                                                                                                                                                 SUPPLEMENTARY INFORMATION
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METHODS 
Bacterial strains and animals. B. fragilis NCTC9343 and H. hepaticus ATCC51149 were 
obtained from the American Type Culture Collection. Conventionally reared SPF mice of strains 
C57BL/6NTac, C57BL/6NTac IL-10-/-, and B6.129S6-Rag2tm1Fwa N12 (Rag2-/-) were purchased 
from Taconic Farms (Germantown, NY) and screened negative for B. fragilis and H. hepaticus. 
Swiss-Webster germ-free (SWGF) mice were purchased from Taconic Farms. Upon delivery in 
sterile shipping containers, the mice were transferred to sterile isolators (Class Biologically 
Clean, Madison, WI) in our animal facility. Animals were screened weekly for bacterial, viral, 
and fungal contamination as previously described2. All animals were cared for under established 
protocols and the IACUC guidelines of Harvard Medical School and the California Institute of 
Technology. 
 
Flow cytometry, fluorescence-activated cell sorting (FACS), and staining. Lymphocytes were 
isolated from mouse spleens that were mechanically disrupted into single-cell preparations. Red 
blood cells were lysed, and splenocytes (1x106) were incubated with various combinations of 
antibodies (BD Pharmingen, San Diego, CA) at 2 μg/mL for 30 min at 4°C. Cells were then 
washed and either fixed or used directly. For intracellular cytokine flow cytometry, samples were 
analyzed on a model FC500 cytometer (Beckman Coulter, Fullerton, CA) or a FacsCalibur 
(Becton Dickson), and data were analyzed with RXP Analysis Software (Beckman Coulter) or 
FlowJO.  FACS was performed on a BD FACSAria, and cell purity was always >99%. 
 
In vitro cytokine assays. For colon organ cultures, procedures were followed as previously 
reported3. For co-culture, CD4+ T cells were purified from splenic lymphocytes (prepared as 
described above) with a CD4+ T Cell Subset Kit (R&D Systems, Minneapolis, MN) used as 
instructed by the manufacturer. Cell purity was always >95%. BMDCs were purified from femurs 
of mice after extraction and washing in PBS. Cells were cultured for 8 days in C-RPMI-10 in the 
presence of GM-CSF (20 ng/mL; Biosource, Camarillo, CA). Medium was replaced after 4 days, 
and adherent cells were cultured for an additional 4 days, at which point nonadherent cells were 
recovered, washed, and used directly. Cells were >95% CD11c+ at the time of use. Purified CD4+ 
T cells (1x106) were mixed with purified CD11c+ BMDCs (1x106) in a 48-well plate and were 
incubated at 37°C in an atmosphere containing 5% CO2. Various stimuli were used, as described 
in Results. ELISA was performed with pre-coated plate kits (BD Pharmingen) according to the 
manufacturer’s guidelines. In some assays, H. hepaticus, with or without wild-type B. fragilis or 
B. fragilis ΔPSA, was added at various concentrations.  
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 Induction of experimental colitis. As assessed by PCR, Rag2-/- and control C57Bl/6 mice were 
negative for H. hepaticus colonization at the time of delivery. Splenic lymphocytes were 
harvested from wild-type donor mice, and CD4+CD45Rbhigh cells were purified from lymphocyte 
populations by FACS as described above. Cells were washed with PBS, and 3x105 cells were 
injected intraperitoneally in a volume of 0.2 mL into recipient H. hepaticus–colonized Rag2-/- 
animals. For colonization experiments, both H. hepaticus (1x108 organisms) and B. fragilis 
(1x108 organisms) were introduced at the time of cell transfer. Throughout PSA treatment studies, 
animals received 50 µg of PSA by gavage 3 times per week. Animals were weighed throughout 
the experiment until sacrifice at 8 weeks. 
 
Induction of intestinal inflammation-TNBS colitis. The backs of wild-type (C57BL/6) male 
mice were shaved, and pre-sensitization solution (150 µL; acetone with olive oil in a 4:1 ratio 
mixed with 5% TNBS in a 4:1 ratio) was slowly applied. Seven days after sensitization, mice 
were anesthetized with isofluorene and TNBS solution (100 µL; 1:1 5% TNBS with absolute 
ethanol) administered rectally through a 3.5F catheter (Instech Solomon; SIL-C35). Mice were 
analyzed 4-6 days after TNBS administration.   
 
Histologic tissue analysis. Mouse tissues in Bouin's fixative (VWR, West Chester, PA) were 
embedded in paraffin, sectioned (6-µm slices), mounted onto slides, and stained with hematoxylin 
and eosin. Sections were evaluated in blinded fashion by a single pathologist (Dr. R. T. Bronson, 
Harvard Medical School). 
 
Quantitative real-time PCR. RNA was extracted with Trizol per the manufacturer’s instructions 
(Invitrogen). RNA (1 µg) was reverse transcribed into cDNA with an iScript cDNA synthesis kit 
(Bio-Rad). cDNA was diluted by addition of 60 μL of water, and a 2-μL volume of this solution 
was used for Q-PCR.  Q-PCR was performed using IQ SYBR Green supermix (Bio-Rad) and 
primers were used at 0.2 µm. Q-PCR was performed on a Bio-Rad iCycler IQ5. Sequences of Q-
PCR primers were as follows 5’-3’: IL-23 (p19) F: AGC TAT GAA TCT ACT AAG AGA GGG 
ACA R: GTC CTA GTA GGG AGG TGT GAA GTT G. IL-17A F: TTA AGG TTC TCT CCT 
CTG AA R: TAG GGA GCT AAA TTA TCC AA. TNFα F: ACG GCA TGG ATC TCA AAG 
AC R: GTG GGT GAG GAG CAC GTA GT. IL-10 F: CTG GAC AAC ATA CTG CTA ACC G 
R: GGG CAT CAC TTC TAC CAG GTA A RORyT  F: CCG CTG AGA GGG CTT CAC  R: 
TGC AGG AGT AGG CCA CAT TAC A IL-21 F: ATC CTG AAC TTC TAT CAG CTC CAC 
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R: GCA TTT AGC TAT GTG CTT CTG TTT C IL-27 F:CTG TTG CTG CTA CCC TTG CTT 
R: CAC TCC TGG CAA TCG AGA TTC. 
 
 
 
1. Young, V. B. et al. In vitro and in vivo characterization of Helicobacter hepaticus 
cytolethal distending toxin mutants. Infect Immun 72, 2521-7 (2004). 
2. Mazmanian, S. K., Liu, C. H., Tzianabos, A. O. & Kasper, D. L. An immunomodulatory 
molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 
107-18 (2005). 
3. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. 
Recognition of commensal microflora by toll-like receptors is required for intestinal 
homeostasis. Cell 118, 229-41 (2004). 
 
 
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A microbial symbiosis factor prevents intestinal inflammatory disease

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A microbial symbiosis factor prevents intestinal inflammatory disease

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