Antimicrobial and Immunomodulatory Potential of Cow Colostrum Extracellular Vesicles (ColosEVs) in an Intestinal In Vitro Model

Abstract

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.

Keywords: Extracellular Vesicles; antimicrobial; colibacillosis; coloEVs; colostrum.

Figure 1

Morphological characterization of isolated colosEVs: Transmission electron microscopy low (A) and high (B) magnification micrographs showing single and clustered colosEVs indicated by red arrows. Scale bar: A. 100 nm; B. 200 nm; (C) Western blot images obtained using Ab against Tsg101 (Tumor Susceptibility Gene 101 protein) and CD81 (Cluster of Differentiation 81) that are both mEV antigens and calnexin as negative cellular debris control of two colosEV samples (1 and 2); (D) nanoparticle tracking analysis graph indicating colosEV size distribution.

Figure 2

Viability of IPEC-J2 after colosEVs exposure at 48 h. The different concentrations of colosEVs did not determine a significant difference in terms of cell viability after 24 h (see Figure S1), whereas 150 µg colosEVs determined a significant reduction in IPECJ2 vitality at 48 h (p < 0.0001, indicated by *** in the graph). Data are expressed as optical density (OD) ± SD. Differences were evaluated through the Kruskal–Wallis test and applying the post hoc Dunn’s multiple comparison test.

Figure 3

Effect of 48 h +1.5 µg colosEVs on E. Coli-inflamed IPEC-J2 cells. IPEC-J2-tested conditions were: inflamed (E. coli, pink), inflamed 1.5 µg colosEVs (E. coli + 1.5 µg colosEVs, light yellow), untreated (control, dark yellow). Significant differences are reported with respect to infected cells with E. coli. Differences were evaluated through the Kruskal–Wallis test and applying the post hoc Dunn’s multiple comparison test. The asterisks indicate the statistical significance: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 4

Adhesion of E. coli strains on 1.5 µg colosEVs pre-treated cells compared with untreated ones. Data are expressed as log10 CFU of adherent, E. coli/5 × 10⁵ cells and mean value of 3 experiments ± 1 standard deviation. Differences were evaluated through the Student’s t-test. The asterisks indicate the statistical significance: ** p < 0.01.