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

Conclusion: In rats deprived of bifidobacteria, intake of B.longum Lpps prevented OA development and modulated
the intestinal microbiome with a possible impact on the bacterial end-products. The link between Lpps and the gut
microbial metabolome warrants further investigation.

Keywords: Lipoproteins, bifidobacteria, osteoarthritis, gut bacteria

INTRODUCTION
[1]
Bacterial lipoproteins are secreted membrane-anchored proteins characterized by a lipobox motif . This
lipobox motif directs post-translational modifications at the conserved cysteine through the consecutive
action of three enzymes: diacylglyceryltransferase (Lgt), pro-lipoprotein signal peptidase (LspA) and
N-acyltransferase (Lnt), which results in di- or triacylated forms. Lipoproteins (Lpps) are abundant in all
bacteria including bifidobacteria, but only a few are released from the cell wall during bacterial growth .
[2,3]
Once released, they exhibit a range of biological activities, from immunoregulatory to anti-viral
properties . In vivo, intake of bifidobacterial lipoproteins-containing extracts induces changes in the
[4,5]
microbiota balance with the promotion of the gut bifidobacteria and a decrease in Bacteroides and
clostridia . In addition to the gut microbiome modulation, administration of the aggregated lipoprotein
[6-8]
forms to mice harboring the microbiome from an arthritis donor abrogate the altered antigen presentation
[3]
genes expression in splenic CD11c+ cells . The results emphasized the inflammatory potential of the
arthritis microbiome towards the otherwise healthy host and the beneficial effect of Lpps to restore balance.
Above all, it opens up new avenues for possible protection against microbiome-related osteoarthritis
symptoms.

The present study aims at defining whether Lpps can prevent osteoarthritis (OA) progression. OA is the
most common form of arthritis and a major cause of disability worldwide. OA affects the whole joint,
[9]
leading to cartilage degradation, synovial inflammation and subchondral bone remodelling . Indeed, a link
between dysbiosis and OA progression is observed in the context of obesity. The data in animal models
indicate that cartilage damage and degeneration in diarthrodial joints are related to microbial shifts in the
gut that can be alleviated by the administration of prebiotics or probiotics [10-12] . Moreover, the germ-free
status partially protects mice from OA .
[13]

To address the question of gut-joint axis and the possible protection by Lpps, we induced OA in Altered
Schaedler Flora (ASF) associated rats kept in specific pathogen-free (SPF) conditions. Eight weeks after
injection of monoiodoacetate (MIA), the intestinal colonization and translocation to Peyer’s patches of
selected bacteria was sought by using qPCR quantification in Lpps-treated and untreated rats. An untreated
sub-group of MIA and sham rats were given labelled Lpps prior to sacrifice and antigen-presenting cells
(APC) were collected from bone marrow and spleen. Our results provide evidence that Lpps intake conveys
protection against OA development and induces shifts to gut bacterial colonization.

MATERIAL AND METHODS
Preparation and characterization of Lpps
Bifidobacterium longum subsp longum CBi0703 strain (Bl CBi0703) was grown in an anaerobic chamber for
48 h hours at 37 °C in a pre-reduced broth (2 L) containing lactose (70 g/L) and partially digested whey
proteins as previously described . Bifidobacteria were enumerated at 0 h (7.4 log cfu/mL), 24 h (8.9 log
[3]
cfu/mL) and 48 h (8.3 log cfu/mL) . Monitoring of broth acidification showed a drastic pH drop to 4.5
[6]
within 24 h. The supernatant was collected by centrifugation (11.000 x g for 20 min) and filtrated using a 10
kDa cut-off membrane (Millipore) to remove acetic and lactic acids. The 10 kDa retentate was then filtrated