A8.2 Oral administration of bovine milk-derived extracellular vesicles diminishes cartilage pathology in two arthritis models

Abstract

Development of rheumatoid arthritis (RA) is associated with environmental factors and several studies show a connetion with diet. Recently, exosomes are identified in both bovine and human breast milk. Exosomes are small (30–200 nm) membrane-derived extracellular vesicles that carry immunoregulatory microRNAs, proteins and lipids, and mediate intercellular communication. In this study, we investigated the effect of oral intake of bovine milk-derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra deficient mice and on collagen-induced arthritis (CIA) in DBA1/J mice. BMEVs were isolated from skimmed milk by ultracentrifugation and characterised by nanoparticle tracking analysis, electron microscopy, anti-CD63 staining, and PCR. BMEVs were labelled with PKH-67 to determined the uptake by cells using FACS and confocal microscopy. TGFb levels were measured with a CAGA-fLuc reporter construct. Naïve T cells were cultured for 5 days with an inflammatory cocktail in the presence of milk exosomes, to induce Th17 differentiation as assessed by RORgT and IL-17 mRNA expression. In IL-1Ra-/- mice, BMEVs were administered daily by oral gavage starting at week 5 till 15 after birth and arthritis was scored macroscopically. In CIA, mice received BMEVs via their drinking water starting 1 week before immunisation till day 40. Arthritis was scored macroscopically and on histology. To determine the effect on immunity, serum IgG levels were measured by ELISA, T-cell specific gene expression (T-bet, RORgT, GATA-3) in LPS stimulated splenocytes by Luminex and RT-qPCR. The BMEV particle size was around 120 nm and expressed the exosome markers tetraspanin CD63, miRNA’s (miR-let-7a, -16, -30a, -92a, -223), and milk specific beta-casein and beta-lactoglobulin mRNA. Acidification, up to gastric acid level (pH 2) left the BMEVs intact, and did not alter their inhibitory effect on NFkB-activation. Active TGFb was detected on BMEVs and incubation of naïve T cells with BMEVs induced significant Th17 differentiation to a similar extent as TGFb. However, BMEVs treatment of mice showed a delayed onset of arthritis in both the IL-1Ra-/- and CIA model. Diminished cartilage pathology and bone marrow inflammation was observed in both models. BMEVs also reduced the circulation levels of MCP1 and IL-6 levels and their production by splenic cells. In BMEV treated CIA, circulating anti-collagen type II IgGa level was reduced and this was accompanied by reduced splenic Th1 and Th17 numbers. Bovine milk-derived extracellular vesicles are bioactive, probably can withstand the harsh conditions of the gut, and oral delivery ameliorates disease in two RA models.

New insights into dairy management and the prevention and treatment of osteoporosis: The shift from single nutrient to dairy matrix effects—A review

“…It is presumed that bovine milk exosomes augment the expression of the osteoblast‐specific transcription factor osterix, which promotes osteoblast proliferation and differentiation. The symptoms of arthritic mice were alleviated by the infusion of milk exosomes (25 mg/kg), which also reduced serum levels of monocyte chemotactic protein 1 and IL‐6 (Arntz et al., 2015). Whereas, the functions of the many active components in milk exosomes remain unclear and the study of the molecular mechanisms of their targeted signaling pathways is still in its infancy, translational applications of milk exosomes should focus on improving extraction methods, clarifying therapeutically effective doses, and enhancing targeting and safety.…”