SUPPLEMENTAL MATERIAL
The Jour nal of Exper imental Medicine
Fuhrer et al., http://www.jem.org/cgi/content/full/jem.20101098/DC1
Figure S1. Sialyltransferase gene expression in mouse mammary gland. The levels of mRNA transcripts encoding the six ␣2,3 sialyltransferases
(St3gal1 to St3gal6) and the two ␣2,6 sialyltransferases (St6gal1 and St6gal2) were normalized to GAPDH expression (mRNA levels set to 1) at 1 wk postpartum, 2 wk postpartum, and 3 d post-weaning. Data represent the mean ± SEM of 3 mice per time point (n = 3). Similar results were obtained in two
independent experiments.
Figure S2. Determination of sialic acid composition in mouse milk. N-acetylneuraminic acid (NeuAc) was identified in mouse milk after neuraminidase treatment, whereas N-glycolylneuraminic acid (NeuGc) was not detectable. Milk samples before and after neuraminidase treatment were separated
by pulsed amperometry-HPAEC and compared with the following authentic standards: monosaccharides NeuAc and NeuGc and oligosaccharides 2FL
(␣1,2 fucosylated lactose), 6SL, and 3SL. Similar results were obtained in three independent experiments.
[ID]FGS1/
[ID]FGS2/
[ID]FGS3/
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Figure S3. T cell markers and IgA in St3gal4ⴚ/ⴚ and St6gal1ⴚ/ⴚ mice. (A) The ratios of TCR␣␤/␥␦, CD4/CD8, and CD8␣␣/␣␤ CD3+ T cells were
measured in 6-wk-old WT, St3gal4⫺/⫺, St6gal1⫺/⫺, and correspondingly cross-fostered (CF) mice. Values are given as mean ± SEM of four to seven measurements from two independent experiments. (B) IgA levels were measured in feces by ELISA, as described in Materials and methods. Data show means ±
SEM of five mice out of one of two independent experiments.
Figure S4. DSS-induced colitis in St6gal1ⴚ/ⴚ mice. Body weight was determined daily in WT mice (black circle, solid line) and St6gal1⫺/⫺ mice (gray
triangle, dashed line). Values represent the mean ± SEM of 6 mice per time point (n = 6). Similar results were obtained in three independent experiments.
[ID]FGS4/
[ID]FGS5/
[ID]FGS6/
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Figure S5. Leukocyte typing on day 0 and 7 of DSS treatment. (A) Representative dot-plot analysis of infiltrating leukocytes in WT and St3gal4⫺/⫺
mice. Leukocytes were gated as CD45+ cells and further subdivided according to the markers CD4, CD8, CD19, Ly6G, F4/80, Ly6C, and Mac-1. The percentage of CD45+ cells are indicated in the gates and quadrants. The corresponding quantitative analysis of 3–6 mice as obtained from two independent experiments is shown in Fig. 5 C. (B) Th1/Th17 cell ratio in the colon of WT (filled square) and St3gal4⫺/⫺ (filled triangle) mice at day 7 of DSS treatment.
Data show the results of two independent experiments with four to five animals per group. *, P < 0.005.
[ID]FGS7/
[ID]FGS8/
[ID]TBLS1/
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Figure S6. Identification of Ruminococcaceae species. PCR was performed from DNA bands eluted out of temporal temperature gradient gel electrophoresis using the standard HDA-1 and HDA-2 primers (see Materials and methods). The sequences retrieved from the PCR products were aligned
using ClustalW2 (www.ebi.ac.uk/ Tools/clustalw2/index.html). The consensus sequence (underlined with asterisks) was searched in the ribosomal database
project using the seqmatch tool (rdp.cme.msu.edu/seqmatch) and identified as belonging to the family of Ruminococcaceae.
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Figure S7. Microbiota analysis in St6gal1ⴚ/ⴚ mice. Temporal temperature gradient gel electrophoresis profiles of 16S rDNA amplification products
from cecum of 6-wk-old WT and St6gal1⫺/⫺ mice fed with either normal (WT) or 6SL-deficient (St6gal1⫺/⫺) milk. The marker (M) shows amplification
products from the species Lactobacillus plantarum, Lactococcus lactis, Corynebacterium variabile, Brevibacterium linens, and Arthrobacter protophormiae
from top to bottom. The arrowheads mark the position corresponding to the Ruminococcaceae bands described in Fig. 6 A. The data represent the results
of one out of two independent experiments, with six animals per group.
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Figure S8. Definition of a primer specific for the new Ruminococcaceae species. The Ruminococcaceae sequence (R. TTGE) identified from the
temporal temperature gradient gel electrophoresis analysis (see Fig. 6 A and Fig. S5) was aligned with six 16S rDNA sequences from known Ruminococcaceae species using the ClustalW2 algorithm. A region of partial sequence identity was chosen to design a primer enabling the preferential amplification
of the Ruminococcaceae species identified in our study (red box).
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Mother’s milk influences colitis in mice | Fuhrer et al.
Table S1. List of primers for the amplification of sialyltransferase genes, bacterial populations, and chemokine and inflammatory
marker genes
Names
Primers
Sequences 5ⴕ-3ⴕ
References
St3Gal1
mQ_S3G-I Fwd
mQ_S3G-I Rev
mQ_S3G-II Fwd
mQ_S3G-II Rev
mQ_S3G-III Fwd
mQ_S3G-III Rev
mQ_S3G-IV Fwd
mQ_S3G-IV Rev
mQ_S3G-V Fwd
mQ_S3G-V Rev
mQ_S3G-VI Fwd
mQ_S3G-VI Rev
mQ_S6G-I Fwd
mQ_S6G-I Rev
mQ_S6G-II Fwd
mQ_S6G-II Rev
mCXCL-1 Fwd
mCXCL-1 Rev
mCOX-2 Fwd
mCOX-2 Rev
mCCL-2 Fwd
mCCL-2 Rev
mCCL-5 Fwd
mCCL-5 Rev
515F
805R
Bac303R
Bac32F
Eco1457F
Eco1652R
Ccocc1F
Ccocc1R
Clep866mF
Clept1240mR
F_Lacto 05
AR_Lacto 04
Ruminococcaceae
HDA-2
CACCTGCAGACACTGCATCAG
TCAGAGCGTTGTGGACTGTCA
CCGAACAACTCACCATTTCATG
GCGCTGGCAATCCACATTA
GGCTACGCTTCAGCCATGA
CCCAAAAGGTGGCACAAACT
TCCAGGGTGAGGCAGAGAGA
GTCCCAAAGGGCAGCTCATA
GCAGCCTCCACAGGGACTT
AATAGGACGAAGAGGTGCACAGA
CCCAAGGATCAGAAACCCAAA
CATGGTGGCATTCCCGTAGT
GCGCAAGACAGATGTGTGCTA
TCTGTTCCCTCATTGAGATGCTT
CTATGACAGCGACATGCTGTCA
CACCATGCTTGTTGAAGGACAT
CTGGGATTCACCTCAAGAACATC
CAGGGTCAAGGCAAGCCTC
TGAGCAACTATTCCAAACCAGC
GCACGTAGTCTTCGATCACTATC
TTAAAAACCTGGATCGGAACCAA
GCATTAGCTTCAGATTTACGGGT
TGCTGCTTGCCTACCTC
ACACTTGGCGGTTCCTTC
GTGCCAGCMGCCGCGGTAA
GACTACCAGGGTATCTAAT
CCAATGTGGGGGACCTTC
AACGCTAGCTACAGGCTT
CATTGACGTTACCCGCAGAAGAAGC
CTCTACGAGACTCAAGCTTGC
CGGTACCTGACTAAGAAGC
AGTTTYATTCTTGCGAACG
TTAACACAATAAGTWATCCACCTGG
ACCTTCCTCCGTTTTGTCAAC
AGCAGTAGGGAATCTTCC
CGCCACTGGTGTTCYTCCATATA
CTAGGTGAAGATACTGACGGTAACCTG
GTATTACCGCGGCTGCTGGCAC
This study
St3Gal2
St3Gal3
St3Gal4
St3Gal5
St3Gal6
St6Gal1
St6Gal2
CXCL-1
COX-2
CCL2
CCL5
Total bacteria
Bacteroidetes
Enterobacteriaceae
Lachnospiraceae
Clostridial cluster IV
Lactobacillaceae
Ruminococcaceae species
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Laubli et al., 2009
Frank et al., 2007
Dahlgren, 1987
Dahlgren, 1987; Frank et al., 2007
Bartosch et al., 2004
Rinttilä et al., 2004
Ramirez-Farias et al., 2009
Furet et al., 2009
This study
Walter et al., 2000
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Furet, J.-P., O. Firmesse, M. Gourmelon, C. Bridonneau, J. Tap, S. Mondot, J. Doré, and G. Corthier. 2009. Comparative assessment of human and farm
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Läubli, H., K.S. Spanaus, and L. Borsig. 2009. Selectin-mediated activation of endothelial cells induces expression of CCL5 and promotes metastasis through
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Ramirez-Farias, C., K. Slezak, Z. Fuller, A. Duncan, G. Holtrop, and P. Louis. 2009. Effect of inulin on the human gut microbiota: stimulation of
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Rinttilä, T., A. Kassinen, E. Malinen, L. Krogius, and A. Palva. 2004. Development of an extensive set of 16S rDNA-targeted primers for quantification of
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Walter, J., G.W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D.M. Loach, K. Munro, and T. Alatossava. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297–303.
doi:10.1128/AEM.66.1.297-303.2000
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