Toll-like Receptors and Cytokine Modulation by Goat Milk Extracellular Vesicles in a Model of Intestinal Inflammation.

Extracellular vesicles (EVs) are nanometric spherical structures, enclosed in a lipid bilayer membrane and secreted by multiple cell types under specific physiologic and pathologic conditions. Their complex cargo modulates immune cells within an inflammatory microenvironment. Milk is one of the most promising sources of EVs in terms of massive recovery, and milk extracellular vesicles (mEVs) have immunomodulatory and anti-inflammatory effects. The aim of this study was to characterize goat mEVs' immunomodulating activities on Toll-like receptors (TLRs) and related immune genes, including cytokines, using a porcine intestinal epithelial cell line (IPEC-J2) after the establishment of a pro-inflammatory environment. IPEC-J2 was exposed for 2 h to pro-inflammatory stimuli as a model of inflammatory bowel disease (IBD), namely LPS for Crohn's disease (CD) and H2O2 for ulcerative colitis (UC); then, cells were treated with goat mEVs for 48 h. RT-qPCR and ELISA data showed that cell exposure to LPS or H2O2 caused a pro-inflammatory response, with increased gene expression of CXCL8, TNFA, NOS2 and the release of pro-inflammatory cytokines. In the LPS model, the treatment with mEVs after LPS determined the down-regulation of NOS2, MMP9, TLR5, TGFB1, IFNB, IL18 and IL12A gene expressions, as well as lower release of IL-18 in culture supernatants. At the same time, we observed the increased expression of TLR1, TLR2, TLR8 and EBI3. On the contrary, the treatment with mEVs after H2O2 exposure, the model of UC, determined the increased expression of MMP9 alongside the decrease in TGFB1, TLR8 and DEFB1, with a lower release of IL-1Ra in culture supernatants. Overall, our data showed that a 48 h treatment with mEVs after a pro-inflammatory stimulus significantly modulated the expression of several TLRs and cytokines in swine intestinal cells, in association with a decreased inflammation. These results further highlight the immunomodulatory potential of these nanosized structures and suggest their potential application in vivo.

Cow Milk Extracellular Vesicle Effects on an In Vitro Model of Intestinal Inflammation.

Extracellular vesicles (EVs) are lipid bilayer nano-dimensional spherical structures and act mainly as signaling mediators between cells, in particular modulating immunity and inflammation. Milk-derived EVs (mEVs) can have immunomodulatory and anti-inflammatory effects, and milk is one of the most promising food sources of EVs. In this context, this study aimed to evaluate bovine mEVs anti-inflammatory and immunomodulating effects on an in vitro co-culture (Caco-2 and THP-1) model of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release through ELISA. After establishing a pro-inflammatory environment due to IFN-γ and LPS stimuli, CXCL8, IL1B, TNFA, IL12A, IL23A, TGFB1, NOS2, and MMP9 were significantly up-regulated in inflamed Caco-2 compared to the basal co-culture. Moreover, IL-17, IL-1ß, IL-6, TNF-α release was increased in supernatants of THP-1. The mEV administration partially restored initial conditions with an effective anti-inflammatory activity. Indeed, a decrease in gene expression and protein production of most of the tested cytokines was detected, together with a significant gene expression decrease in MMP9 and the up-regulation of MUC2 and TJP1. These results showed a fundamental capability of mEVs to modulate inflammation and their potential beneficial effect on the intestinal mucosa.