Monocytes were stimulated with different TLR ligands with or without bovine IL-10 for 24 hours at 37C and 5% CO2

Monocytes were stimulated with different TLR ligands with or without bovine IL-10 for 24 hours at 37C and 5% CO2. response to TLR activation. Bovine IL-10 present in dairy products may thus potentially contribute to the prevention of necrotizing enterocolitis and allergy, enhance mucosal tolerance induction and decrease intestinal inflammation and may therefore be applicable in infant foods and in immunomodulatory diets. Introduction Dietary components are capable of modulating intestinal immune responses [1], [2]. Dairy products, including cow’s milk, are widely consumed in Western societies and contain a wide range of immunoprotective factors such as immunoglobulins, lactoferrin, anti-microbial enzymes and cytokines. Bovine IL-10 was found to have an amino acid sequence identity of 76.8% with human IL-10, indicating that bovine IL-10 may exert functional effects on human immune cells Epoxomicin [3], [4], [5], [6]. Therefore, bovine IL-10 present in dairy and dairy related products could potentially have Epoxomicin immunomodulatory activity in the human consumer. Functional cross species activity of cytokines has been reported for chicken IFN- and turkey IL-2 [5], [6], and both Epoxomicin porcine IL-2 and human IL-2 were reported to enhance proliferation of human, bovine, porcine and murine cells in the lumen of the intestine of mice can induce IL-10 production by cells of the Peyers patch and prevent allergic sensitization to food [33]. Next to this, in a neonatal rat model, decreased necrotising enterocolitis (NEC) correlated with increased in situ IL-10 production [34]. These findings show the potential significance of the presence of IL-10 in the intestine. In this report, we investigated whether bovine IL-10 could exert functional activity on human monocytes and dendritic cells. Bioactive bovine IL-10 could potentially be used for the prevention of inflammatory diseases as NEC and allergy in infant nutrion, or in immunomodulating diets for patients suffering from intestinal inflammatory disorders. We show that bovine IL-10 is usually recognized by the human IL-10 receptor and dose-dependently inhibits cytokine production and surface marker expression during LPS induced DC maturation. Materials and Methods IL-10 sequence analysis IL-10 sequences were obtained from the online databases of NCBI ( and UniProt ( Existing signal peptide data or signalP 3.0 ( were used to identify IL-10 signal peptides, which were removed before performing the sequence alignment. Sequence alignment was done in BioEdit (version using ClustalW Multiple Alignment with default settings. Subsequently, the amino acid sequence identity was calculated using the sequence identity option in BioEdit. Accession numbers of the IL-10 sequences are: Human, UniProt, “type”:”entrez-protein”,”attrs”:”text”:”P22301″,”term_id”:”124292″,”term_text”:”P22301″P22301; Bovine, UniProt, “type”:”entrez-protein”,”attrs”:”text”:”P43480″,”term_id”:”172046854″,”term_text”:”P43480″P43480, Epstein-Barr computer virus (EBV), UniProt, “type”:”entrez-protein”,”attrs”:”text”:”P03180″,”term_id”:”114886″,”term_text”:”P03180″P03180; Rat, NCBI, “type”:”entrez-protein”,”attrs”:”text”:”EDM09836″,”term_id”:”149058679″,”term_text”:”EDM09836″EDM09836; Sheep, NCBI, “type”:”entrez-protein”,”attrs”:”text”:”CAA82546″,”term_id”:”732895″,”term_text”:”CAA82546″CAA82546; Mouse, NCBI, “type”:”entrez-protein”,”attrs”:”text”:”AAI37845″,”term_id”:”187952737″,”term_text”:”AAI37845″AAI37845; Pig, NCBI, “type”:”entrez-protein”,”attrs”:”text”:”CAL29498″,”term_id”:”198033675″,”term_text”:”CAL29498″CAL29498 and Papiine herpesvirus 1 (PapHerp), NCBI, “type”:”entrez-protein”,”attrs”:”text”:”AAF23949″,”term_id”:”6690016″,”term_text”:”AAF23949″AAF23949. Three-dimensional modeling of bovine IL-10 The dimeric structure of human IL-10 (PDB entry: 1j7v, resolution 2.9 ?) was used as a template to model the dimeric bovine IL-10 protein using the program MODELLER (version 9v8 [35], [36]), which incorporates the CVFF pressure field [37]. Stereochemical quality of the homology models was assessed using the program PROCHECK [38]. Protein folding quality was verified using the program PROSAII [39], which independently evaluates the compatibility of each residue to its environment. PBMC isolation Peripheral blood mononuclear cells (PBMCs) were diluted 11 in IMDM (Gibco-BRL, Paisley, Scotland) and isolated by gradient centrifugation on Ficoll-Plaque PLUS (Amersham Biosciences, Uppsala, Sweden) for 5 minutes at 200 g and subsequently TMPRSS2 for 15 min. at 500 g (without brake at 20 C). The PBMCs were harvested from the Ficoll layer, gently resuspended in IMDM and washed two or three occasions in IMDM. Monocyte isolation and stimulation Monocytes from freshly isolated PBMCs were labeled with MicroBeads conjugated to mouse IgG2a monoclonal anti human CD14 antibodies (130-050-201, Myltenyi Biotec, Germany), and isolated using the quadroMACS (Myltenyi Biotec) according to the manufacturers descriptions. Briefly, cells were incubated with MicroBeads for 15 minutes at 4C, washed with MACS buffer, centrifuged and resuspended in MACS buffer. The MACS columns were placed in the quadroMACS and rinsed. Subsequently, the cell suspension was added, rinsed and the columns removed from the quadroMACS; labeled cells were collected in a new tube by rinsing with MACS buffer and the supplied Epoxomicin plunger. Purity of the CD14+ cell populace was between 90 and 95%, as determined by flow cytometric analysis (FACS Canto II.