Background/Purpose: Development of rheumatoid arthritis (RA) is associated with different genetic and environmental factors. We postulate that cow milk could be such an environmental trigger and a recent prospective study of Lu et al. (Arthritis Care Res 2014;66(6):802-9) suggests that frequent milk consumption is associated with reduced OA progression in women. Human breast milk contains many components to promote development of neonatal immune competence, such as cytokines, antibodies and immune cells. More recently, exosomes were identified in both bovine and human breast milk. Exosomes are small vesicles recently rediscovered as an important part of intercellular communication. Milk exosomes are known to carry immunoregulatory microRNAs and cytokines, thereby enhancing the antimicrobial defense in newborns. It is however unknown whether exosomes are present in commercial milk and whether they are bioactive. 

Methods: By differential ultracentrifugation, followed by ExoQuick isolation, we isolated exosomes from commercial milk. NanoSight analysis was performed to estimate vesicles size (Ø120nm) and concentration (approx.10¹⁰ exosomes per ml). The expression of milk-derived mRNA and miRNA was confirmed by RT-qPCR. Exosomes were acidified at a gastrointestinal pH2 to test their stability. Cellular uptake of PKH-67 labeled exosomes was analyzed by confocal microscopy and flow cytometry. TGFβ 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. RORγT and IL-17 expression levels were determined by RT-qPCR. 

Results: We found clear levels of messenger- and miRNAs (e.g. miR-let-7a, 124a) in cow milk exosomes. Sucrose gradient followed by electron microscopy revealed an exosome-like morphology. We showed cellular uptake of exosomes in vitro by murine macrophages, splenic antigen presenting cells and non-phagocytic fibroblasts. To test the stability of these vesicles, we used a luciferase reporter assay to measure in vitro NFκB-activation. Acidification, up to gastric acid level kept the exosomes intact, and did not alter the inhibitory effect they had on NFκB-activation. Active TGFβ was detected using CAGA-fluc reporter cells and blocked by addition of anti-TGFβ1,2,3 antibodies. More importantly, incubation of naïve T cells with milk exosomes in the presence of an inflammatory cytokine cocktail induced significant Th17 differentiation. 

Conclusion: We clearly showed that commercial milk contains stable exosomes, which are resistant to acidification. These vesicles can facilitate Th17 differentiation and could therefore play an important role in autoimmune diseases, such as rheumatoid arthritis. To our knowledge, this is the first study to show that commercial milk contains immunoregulatory exosomes with active TGFβ. Our data suggest that bovine milk-derived exosomes, carrying immunoregulatory cargo, could remain intact in the gastrointestinal tract and therefore reach the circulation. This warrants further research to determine their biological effect in both healthy individuals and patients with autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bioactive-tgf-%ce%b2-is-present-on-bovine-milk-derived-exosomes-consequences-for-patients/