Page 83 - Read Online

Sane et al. Microbiome Res Rep 2023;2:18 https://dx.doi.org/10.20517/mrr.2023.12 Page 7 of 12

Lpps intake harbored more L. johnsonii and less P. goldsteinii in the caecum than the untreated rats.
Mucispirillum schaedleri was also promoted in the colon of protected rats, whereas Akkermansia sp.
colonization was reduced. Additionally, no difference was seen in plasma LPS.

Uptake of Lpps by BM cells
The possible uptake of broth milk proteins retained during Lpps extraction (less than 10 % contamination)
was checked by administrating TRITC-labeled milk aggregate extract (MAE) as a control to OA rats. In
addition, two dosings were used for tracing the lipoprotein core, with only a few amino acid sequences
being accessible for labeling. The highest doses of TRITC-labeled Lpps ingested by sham or OA water-
drinking rats ranged between 0.83-1.13 mg/kg. The rats receiving 1.5 mg·Lpps/L were dosed with around
140 µg/kg, which is still 7 times higher than the mean dose received by Lpps-treated rats over the survey
(21 µg/kg).

No signal of Lpps or MAE was detected in spleen cells. No signal of Lpps was neither observed in APC (+)
from OA rats receiving the lowest dose, which contrasted with the detection of a red signal in BM from the
right tibia of MAE-treated OA rat.

Intake of the highest Lpps dose allows for the detection of the sole red signal in 0.3 % APC(+) cells in the left
femur from a single OA rat [Figure 3A]. Most of the APCs exhibited red and green signals that indicate
uptake of intestinal bacteria loaded with Lpps.

TRITC-labeled MAE administrated at a high dose led to the detection of the red signal in APC(+) cells in
BM from the left (0.2 %) and right (0.3 %) femurs [Figure 3B].

In sham animals, spleen cells were devoid of a single red signal. Cells harboring red fluorescence alone were
detected in BM APC(-) cells from at least three bone samples (right tibia and femur, and left tibia or femur),
suggesting that BCs uptake at high dosing occurs more readily in healthy animals, likely through a different
route [Figure 3C].

DISCUSSION
The aim of this study was to explore the potential of bifidobacterial Lpps to prevent OA progression. There
are obvious limitations with animal models, only mimicking parts or stages of the disease, with no model
completely reproducing human OA complexity. Moreover, the reduction in the microbiome richness
oversimplifies the gut-joint axis. Still, rats developed OA following MIA injection. MIA, an inhibitor of
glyceraldehyde-3-phosphate, disrupts cellular glycolysis, which in turn leads to eventual cell death, mainly
[17]
chondrocyte cell death with cartilage degeneration and subsequent subchondral bone alterations .
Administration of Lpps began in the later stages of the disease (after days 10-14) characterized by
progressive cartilage degradation and remodelling of subchondral bone, replacing the first inflammatory
episodes. Labelled Lpps were not shown to easily reach the spleen nor the bone marrow close to the
subchondral bone in OA rats. At the chronic dosing, it is therefore unlikely that Lpps prevented in situ the
progression of cartilage degeneration. Moreover, a preliminary investigation of cytokine expression (IL-1,
IL-6, and IL-10) in bone marrow APC+ from OA and healthy Lpps-treated rats displayed no significant
difference, which further supported the assumption that the subchondral environment was not targeted by
Lpps (data not shown).

Actually, Lpps were shown to repress bacteria related to OA, i.e., P. goldsteinii and Akkermansia within the
digest tract. PCA also pointed to P. goldsteinii as a contributor to the disease. The involvement of both