Modulation of Human Immune Responses by Bovine Interleukin-10

Cytokines can be functionally active across species barriers. Bovine IL-10 has an amino acid sequence identity with human IL-10 of 76.8%. Therefore, the aim of this study was to evaluate whether bovine IL-10 has immunomodulatory activities on human monocytes and dendritic cells. Peripheral blood monocytes were isolated from healthy donors, and used directly or allowed to differentiate to dendritic cells under the influence of IL-4 and GM-CSF. Recombinant bovine IL-10 inhibited TLR induced activation of monocytes, and dose-dependently inhibited LPS-induced activation of monocyte-derived DCs comparable to human IL-10. By using blocking antibodies to either bovine IL-10 or the human IL-10 receptor it was demonstrated that inhibition of monocyte activation by bovine IL-10 was dependent on binding of bovine IL-10 to the human IL-10R. These data demonstrate that bovine IL-10 potently inhibits the activation of human myeloid cells in 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

Summary of bovine IL-10 amino acid substitutions in human IL-10R binding sites.

<p>Data are obtained from a ClustalW Multiple alignment as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018188#pone-0018188-g001" target="_blank">Figure 1A</a>. Of the amino acid substitutions and the substitution burying >5 Ų in the human IL-10R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018188#pone.0018188-Reineke1" target="_blank">[9]</a> the number of homologous amino acid substitutions is indicated.</p

Bovine IL-10 can regulate TLR ligand induced cytokine production by monocytes by binding the IL-10R.

<p>Figures 2B-2D are box plots, showing the median (black horizontal bar), 50% data range (box) and 99% data range (error bars). On the x-axes is the addition indicated (+) of TLR stimuli (panel A), or 10 ng/ml LPS (B–D), 10 ng/ml IL-10 (A-D), bovine IL-10 or human IL-10R blocking antibodies (anti bIL-10 or IL-10R) or isotype control (C–D). <b>A</b>: Freshly isolated monocytes were stimulated for 24 hours with different ligands (lipopolysaccharide (LPS); Flagellin (Flag); peptidoglycan (PGN)) with or without recombinant bovine IL-10. IL-1β (p<0.001) and TNF-α (p<0.001) were significantly inhibited by the three bacterial ligands tested. Data (pg/ml) is shown for three different donors. <b>B:</b> Human monocytes were stimulated with LPS and either human (white box) or bovine (hatched box) IL-10 was added to compare the inhibitory capacity of bovine IL-10 with human IL-10. <b>C:</b> To confirm that the response is specifically inhibited by bovine IL-10 a blocking antibody and an isotope control were pre-incubated with bovine IL-10 and LPS and subsequently added to the monocytes. IL-1β and TNF-α production (pg/ml) is shown of three different donors. <b>D:</b> In order to proof that the bioactivity of bovine IL-10 is mediated through the IL-10R, monocytes were pre-incubated with IL-10R blocking antibodies and subsequently stimulated with LPS and bovine or human IL-10. Data is shown of 3 different donors.</p

Comparison of human and bovine IL-10 in the human IL-10R.

<p><b>A:</b> ClustalW sequence alignment of IL-10 from different species. IL-10 sequences were retrieved from the NCBI and Uniprot databases and analyzed for signal peptides (SignalP 3.0) which were removed from the sequences before performing the alignment (18 amino acids for human and bovine IL-10). At the top left the overall sequence identity is shown. In grey (Ib) and black (Ia) background shading the IL-10 receptor 1 binding sites are indicated as published by Josephson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018188#pone.0018188-Josephson1" target="_blank">[8]</a>. The underlined residues indicate >5 Ų surface area in IL-10RA site I. At the bottom of each row the consensus (*) sequence is shown; “.” and “:” indicated homologous amino acids. <b>B:</b> Bovine IL-10 was modeled using human IL-10 in the human IL-10/IL-10R complex as template. The human IL-10R is shown in green and amino acid substitution between human and bovine IL-10 are depicted in cyan. Cysteine residues are colored yellow, the amino acid colored red (indicated with an arrow for one of the two IL-10 chains) is His 44, which is and amino acid substitution in close contact with the human Il-10R. <b>C:</b> The same model as in B, but displayed using spheres.</p

Dose-dependent inhibition during LPS-induced DC maturation is comparable for human and bovine IL-10.

<p>Data shown are from 3 different donors tested, error bars indicate standard error. <b>A:</b> Dose dependent inhibition of CD83 (p = 0.753) and CD86 (p = 0.936) by human and bovine IL-10. Data were divided by the isotype control and expressed relative to the positive control of only LPS, which was set at 100%. <b>B:</b> Dose dependent inhibition of TNF-α (p = 0.916) and IL-12p70 (p = 0.962) production by human and bovine IL-10, shown in pg/ml.</p

Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans.

Quantitative knowledge of the turnover of different leukocyte populations is a key to our understanding of immune function in health and disease. Much progress has been made thanks to the introduction of stable isotope labeling, the state-of-the-art technique for in vivo quantification of cellular life spans. Yet, even leukocyte life span estimates on the basis of stable isotope labeling can vary up to 10-fold among laboratories. We investigated whether these differences could be the result of variances in the length of the labeling period among studies. To this end, we performed deuterated water-labeling experiments in mice, in which only the length of label administration was varied. The resulting life span estimates were indeed dependent on the length of the labeling period when the data were analyzed using a commonly used single-exponential model. We show that multiexponential models provide the necessary tool to obtain life span estimates that are independent of the length of the labeling period. Use of a multiexponential model enabled us to reduce the gap between human T-cell life span estimates from 2 previously published labeling studies. This provides an important step toward unambiguous understanding of leukocyte turnover in health and disease