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-1β, 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.

Gut represents a major immunological defense barrier with mucosal immune system and intestinal epithelial cells (IECs). In all intestinal diseases, in particular inflammatory bowel disease (IBD), both the absorption and the local immune system are compromised and alternative effective therapies are sought after. Extracellular Vesicles (EVs) have the capability to regulate immune cells within the inflammatory microenvironment, by dampening inflammation and restoring intestinal barrier integrity. Recently, the immune-modulatory role of EVs has also been confirmed for milk EVs (mEVs), notable for their easy production, high sample volumes, cost-effective scalable production and non-toxic and non-immunogenic behavior. In this context, the aim of this study was to evaluate goat mEV anti-inflammatory and immuno-modulating effects on an in vitro model (IPEC-J2) of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release dosage with ELISA test. After the establishment of a pro-inflammatory environment due to LPS stimuli, IL6, CXCL8, IL12p35, IL12p40, IFNB, IL18, TLR7 and NOS2 resulted significantly up-regulated in stimulated IPEC-J2 cells compared to those of the basal culture. After 48 h of mEV treatment in inflamed IPEC-J2 a partial restoration of initial conditions was detected, with the IL18 and IL12p40 significant down-regulation, and IL12p35, EBI3, TLR7, BD1 and BD3 up-regulation. IL-18 reduced protein production was also detected in supernatants. Moreover, a decrease of MMP9 and NOS2 together with a strong up-regulation of MUC2 indicated a recovery of cellular homeostasis and, therefore, potential beneficial effects on the intestinal mucosa. Nevertheless, 48 h post-treatment, an increased gene expression and protein release of IL-8 was observed. This paper is one of the firsts to assess the effect of goat mEVs and the first one, in particular, of doing this on an in vitro model of gut inflammation. The obtained results show a potential capability of goat mEVs to modulate inflammation and to play beneficial effects on the intestinal mucosa.

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.

Extracellular Vesicles (EVs) are nano-sized double-lipid-membrane-bound structures, acting mainly as signalling mediators between distant cells and, in particular, modulating the immune response and inflammation of targeted cells. Milk and colostrum contain high amounts of EVs that could be exploited as alternative natural systems in antimicrobial fighting. The aim of this study is to evaluate cow colostrum-derived EVs (colosEVs) for their antimicrobial, anti-inflammatory and immunomodulating effects in vitro to assess their suitability as natural antimicrobial agents as a strategy to cope with the drug resistance problem. ColosEVs were evaluated on a model of neonatal calf diarrhoea caused by Escherichia coli infection, a livestock disease where antibiotic therapy often has poor results. Colostrum from Piedmontese cows was collected within 24 h of calving and colosEVs were immediately isolated. IPEC-J2 cell line was pre-treated with colosEVs for 48 h and then infected with EPEC/NTEC field strains for 2 h. Bacterial adherence and IPEC-J2 gene expression analysis (RT-qPCR) of CXCL8, DEFB1, DEFB4A, TLR4, TLR5, NFKB1, MYD88, CGAS, RIGI and STING were evaluated. The colosEVs pre-treatment significantly reduced the ability of EPEC/NTEC strains to adhere to cell surfaces (p = 0.006), suggesting a role of ColosEVs in modulating host–pathogen interactions. Moreover, our results showed a significant decrease in TLR5 (p < 0.05), CGAS (p < 0.05) and STING (p < 0.01) gene expression in cells that were pre-treated with ColosEVs and then infected, thus highlighting a potential antimicrobial activity of ColosEVs. This is the first preliminarily study investigating ColosEV immunomodulatory and anti-inflammatory effects on an in vitro model of neonatal calf diarrhoea, showing its potential as a therapeutic and prophylactic tool.

African swine fever viruses (ASFV), currently a serious threat to the global pig industry, primarily target porcine macrophages. Macrophages are characterized by their remarkable plasticity, being able to modify their phenotype and functions in response to diverse stimuli. Since IL-10 and TGF-β polarize macrophages toward an anti-inflammatory phenotype, we analyzed their impact on porcine monocyte-derived macrophages’ (moMΦ) susceptibility to infection and their responses to two genotype I ASFV strains, virulent 26544/OG10 and attenuated NH/P68. At a low multiplicity of infection (MOI), NH/P68, but not 26544/OG10, presented a higher ability to infect moM(IL-10) compared to moMΦ and moM(TGF-β), but no differences were appreciated at a higher MOI. Both strains replicated efficiently in all moMΦ subsets, with no differences at later times post-infection. Both strains downregulated CD14 and CD16 expression on moMΦ, irrespective of the activation status. ASFV’s modulation of CD163 and MHC II DR expression and cytokine responses to NH/P68 or 26544/OG10 ASFV were not affected by either IL-10 or TGF-β pre-treatment. Our results revealed little impact of these anti-inflammatory cytokines on moMΦ interaction with ASFV, which likely reflects the ability of the virus to effectively modulate macrophage responses.

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.