Mucin type 3

Akkermansia muciniphila (ATCC BAA-835) was provided from the DSMZ institute (German Collection of Microorganisms and Cell Cultures GmbH). A. muciniphila strains were cultured in brain heart infusion (BHI) agar medium (Quelab, Canada) supplemented with 0.5% mucin-type III (Sigma-Aldrich, St. Louis, Missouri, USA), hemin (5 μg/ml), menadione (1 μg/ml)^{45 (link)} and 0.05% L-cysteine^{46 (link)} under anaerobic conditions (80% N₂, 10% H₂, and 10% CO₂) at 37 °C for 3–7 days as described previously^{12 (link)}.
Polymerase chain reaction (PCR) test was performed based on 16 s ribosomal ribonucleic acid (rRNA) sequence recognition to confirm the A. muciniphila, in addition to macroscopic and microscopic (Gram staining) assays (Supplementary Table 1).
Cell-free supernatant was obtained from the inoculation of A. muciniphila in 100 mL BHI broth with the above-mentioned supplementations incubated in the same condition reaching optical density (OD) of 1.5 in 600 nm, centrifuged in 8000 × g for 5 min; the pH was adjusted in 7.4 and then purified by passing through 2.22 nm filters and kept at −70 °C until usage.

Cell-free culture supernatants from the proximal and distal colon, both in resting conditions (basal) and after immune challenges (LPS and IL-15), were subsequently used to explore their effect on a stable fecal population via the fecal slurry model. For this purpose, we used a basal media composed of 2 g/l peptone water [Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA] 2 g/l yeast extract (BD), 0.1 g/l NaCl, 0.04 g/l K₂HPO₄, 0.04 g/l KH₂PO₄, 0.01 g/l MgSO₄, 0.01 g/l CaCl₂.⋅2H₂O, 2 g/l NaHCO₃, 2.5 g/l l-Cysteine-HCl, 0.5 g/l bile salts, 2 ml/l Tween-80, 1 g/l arabinogalactan, 2 g/l pectin, 1 g/l xylan, 4 g/l starch, 0.4 g/l glucose, and 0.4 g/l mucin type III (all purchased to Sigma-Aldrich). The mixture was homogenized and autoclaved for 15 min at 121°C, and the following components were added to the cooled media after sterilization by filtration (0.20 μm): 0.05 g/l bovine hemin (Sigma-Aldrich) and 10 μg/l vitamin K (Sigma-Aldrich). Before use, the basal media was maintained overnight at 37°C in anaerobiosis (10% v/v H₂, 10%CO₂, and 80% N₂) in an anaerobic chamber Mac 500 (Don Whitley Scientific, West Yorkshire, UK).