www.sciencetranslationalmedicine.org/cgi/content/full/4/158/158ra144/DC1

Supplementary Materials for

Food-Grade Bacteria Expressing Elafin Protect Against Inflammation and Restore Colon Homeostasis

Jean-Paul Motta, Luis G. Bermúdez-Humarán, Céline Deraison, Laurence Martin, Corinne Rolland, Perrine Rousset, Jérôme Boue, Gilles Dietrich, Kevin Chapman, Pascale Kharrat, Jean-Pierre Vinel, Laurent Alric, Emmanuel Mas, Jean-Michel

Sallenave, Philippe Langella, Nathalie Vergnolle*

*To whom correspondence should be addressed. E-mail: nathalie.vergnolle@inserm.fr

Published 31 October 2012, Sci. Transl. Med. 4, 158ra144 (2012) DOI: 10.1126/scitranslmed.3004212

The PDF file includes:

Materials and Methods Fig. S1. Effects of Elafin-expressing, IL-10–expressing, and wild-type L. lactis on inflammation. Fig. S2. Presence of L. lactis expressing Elafin in feces and tissue. Table S1. Characteristics and outcomes of IBD patients. Table S2. Primers used for quantitative RT-PCR studies.

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Supplementary Material

Supplementary Material and Methods

Induction of IL-10 production in Lactococcus lactis

Recombinant L. lactis strain expressing IL-10 were constructed as previously described (9)

cultured at 30°C without shaking in M17 medium (Oxoid, Dardilly, France) supplemented

with glucose (0.5%) and chloramphenicol (10µg/mL). Exponential growth cultures

(OD600=0.4-0.6) of L. lactis were treated one hour with nisin (1 ng/mL, Sigma, Saint-Quentin

Fallavier, France) to induce recombinant protein expression. Bacteria were harvested after 1

hour and suspended in corresponding volume of sterile PBS to get 5.109 colony forming units

(cfu) for oral dosing in mice.

Bacterial translocation

Colonic inflammation was induced by treatment with Dextran Sulfate Sodium (DSS),

dissolved in drinking water (5% weight / volume). The animals were free to drink the DSS

solution for 7 days (no differences were reported for the volume consumed between water and

DSS). For the whole period of DSS exposure, mice were daily orally treated for 7 days with

5.109 cfu (in 100 µL PBS) of recombinant L. lactis expressing Elafin. Control animals had

water in their drinking bottles and were daily orally treated for 7 days recombinant L. lactis

expressing Elafin. Stools and samples from the spleen, the liver, the lungs and the arterial

blood were collected in sterile conditions. Tissues were weighted, homogenized in sterile

PBS, and 1/2 dilution were plated. Viable counts for recombinant L.lactis were obtained from

M17 agar + Chloramphenicol (10µg/ml, Difco,) that were incubated aerobically at 37°C for

24H (organs) and 3 to 6H (feces).

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Supplementary Figures

Figure S1: Effects of Elafin-expressing, IL-10–expressing, and wild-type L. lactis on

inflammation. Macroscopic damage score (a) and colonic wall thickness (b) were measured

in colons of mice that had water alone or water + DSS (5%) in their drinking bottles for 7-

days, and that were treated daily orally, with PBS, wild-type, IL-10-expressing or Elafin-

expressing L. lactis (5.109 cfu) (n=10 in each group). Significant differences were noted * for

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Figure S2: Presence of L. lactis expressing Elafin in feces and tissue.

Histograms represent percentage of M17+Chloramphenicol plates positive for the detection of

viable colonies in feces, spleen, liver, lungs and blood. n=8 animals in each group of mice

treated with 5% of DSS and n=9 mice in the group of mice that had water.

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Table S1. Characteristics and outcomes of IBD patients.

Values are number of patients with characteristic unless otherwise indicated.

CTR, Healthy Controls; CD, Crohn’s Disease; UC, Ulcerative Colitis, F, female; M, male,

yrs, years, 5-ASA, 5-aminosalicylic acid).

* including 5 primo-diagnosed patients with Crohn’s Disease; ** severity of disease was

graduated according historical data of patients and histopathological results of biopsy.

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Table S2. Primers used for quantitative RT-PCR studies.

Primers used for the study were from previously published data (PMID of the original study)

or designed in our lab using Primer Express software (Invitrogen). Couple of primers were

validated after following steps: a BLAST for sequences at NCBI website

(http://blast.ncbi.nlm.nih.gov/Blast.cgi), calculation of melting curves Tm calling and

standard curve slopes (≈ 3).