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Page 4 of 12 Sane et al. Microbiome Res Rep 2023;2:18 https://dx.doi.org/10.20517/mrr.2023.12

Deparaffinization was made by immersions in xylene and ethanol. The slides were subjected to hematoxylin
and eosin staining. For each block, a minimum of five slides was obtained to minimise sampling error. Each
slide was examined under a light microscope. Histological scoring was carried out by two blinded and
independent observers from the following criteria: the thickness of articular cartilage, continuity of surface
cartilage, and infiltration of mononuclear cells.

Bacterial enumeration in organs
Organs (distal ileum, caecum and colon fractions, spleen, Peyer’s patches) suspended in 9 mL pre-reduced
Ringer solution (Solabia, Pantin, France) supplemented with cysteine HCl (0.03 %) (VWR, Fontenay-sous-
Bois, France) were weighted and kept frozen until total DNA extraction. After thawing, the total DNA was
extracted using the Nucleospin Tissue kit (Macherey Nagel, Hoerdt, France). DNA content was determined
at 260-nm wavelength using Biophotometer Plus (Eppendorf, Montesson, France). ASF bacteria and
[16]
selected bacteria possibly involved in OA development [12,15] were enumerated by qPCR as described in the
supplementary material [Supplementary Table 1]. Plasma lipopolysaccharide was measured using Charles
River Endosafe kit following the manufacturer’s instructions.

Isolation of Antigen-Presenting Cells (APC)
Femurs and tibias were flushed into 20 mL 50 mM PBS. Spleen fraction was dilacerated into 50 mM PBS.
Cell suspensions were pass-through a 70 µm sieve and washed 3 times with a solution of 50 mM PBS, 2 mM
EDTA, and 2 % BSA. Antigens presenting cells (APC) were isolated from total cells using isolation columns
with Anti-MHC Class II (OX6) MicroBeads (Miltenyi Biotec, Paris, France). Both APC(+) and APC(-) were
counted using Malassez cells following Trypan blue staining to exclude dead cells.

Uptake of labelled Lpps in sham and OA induced rats.
Tetramethylrodamine (TRITC)-labelled Lps and medium extract (MAE, see supplementary methods for
labelling) were administrated to a subgroup of sham and MIA-induced rats by adding at the end of the
survey 1.5 or 15.0 mg/L of Lpps or MAE with 10 µL of the cell-permeable fluorescent dye
5’-carboxyfluorescein succinimidyl ester (CFSE) in the bottles, 12 h before sacrifice. The remaining volumes
were recorded in the morning and the animals were sacrificed within 30 min. Spleen and BM cells were
collected and APCs were isolated as described above. Twenty µL cell suspensions were fixed with 4 % PFA
and Fluor Save ™ (Merck Millipore) on glass slide. Labeled cells were viewed on an Eclipse E600 (Nikon)
confocal microscope.

Statistical analysis
PCAmix, combining a principal component analysis (PCA) with a multiple correspondence analysis
(MCA), was performed using qualitative (diet, OA) and quantitative (bacteria located in the various organs,
APC counts, weight) variables (XLstat, 2019). Partial least squares regression model (PLS-R) was used for
multivariate analyses with bacteria and APC as the dependent variables. The data were visualized using a
99% confidence ellipse. ANOVA, ANCOVA and Kruskal-Wallis with post-hoc tests were used for multiple
2
group univariate analyses, along with chi-squared (χ ) and Fisher tests as indicated in the text. Bivariate
correlations were determined using Spearman’s Rho.

RESULTS
Lpps administration prevents OA development
Osteoarthritis (OA), as defined by macroscopic and histological scoring [Figure 1A and 1B], did not
develop in 8 rats out of 10 receiving 0.3 mg/L (16.4 ± 3.3 µg/kg) 5 days per week for 6 weeks. In contrast, all
rats belonging to the water drinking group developed OA [Figure 1C].