Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14

Abstract

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Figure 1

Detection and characterization of sCD14 in human milk. (A) Milk samples (1:25 to 1:200 dilutions) taken after the first week postpartum and normal human serum (NHS) were tested for sCD14 by Western blotting. Shown is the result of one sample representative of 10 donors. (B) Analysis of m-sCD14 in milk samples taken at day 2 or day 10 postpartum from the same mother. Samples from two donors are shown. NHS, normal human serum. (C) m-sCD14 levels determined by ELISA in multiple samples taken from 10 donors at different times postpartum. Values correspond to the mean of triplicate determinations (SD ≤ 6%). The m-sCD14 molecular pattern of each sample was determined by Western blotting and is indicated by the symbols. (D) NH₂-terminal sequence (dashed line) and mass spectrometric analysis followed by amino acid sequencing (solid line) of 48-kD m-sCD14 tryptic peptides showing homology with the predicted sequence from monocyte CD14 cDNA. Thick solid line underlines a peptide analyzed only by mass spectrometry. X, not determined.

Figure 2

48-kD sCD14 production by mammary epithelial cells and absence from commercial infant milk formulas and cows' milk. (A) Detection of sCD14 polypeptides by Western blotting in culture supernatants (Sup.) and total lysates (TL) from serum-free medium–cultured mammary epithelial cell line MCF-7. Parallel analysis of m-sCD14 in milk and normal human serum (NHS) is shown. Mr, relative molecular mass. (B) Infant milk formulas (IF, 1:6 and 1:25 dilutions), cows' milk (bottled milk [BM]), bovine colostrum (BC), and bovine plasma (BP) were tested for sCD14 by Western blotting. Shown is a representative result of 5 IF, 8 BM, 2 BC, and 10 BP.

Figure 3

m-sCD14 mediates LPS activation of CD14-negative cells. (A and B) IL-6 and IL-8 production by the astrocytoma cell line U373 (A) and the myeloid cell line U937 (B), respectively, stimulated with E. coli LPS (100 ng/ml) in medium supplemented with FCS, human AB serum (AB), or human milk (HM). The anti-CD14 mAb MY4 and MEM-18 but not their isotype-matched controls (20 μg/ml) blocked the LPS-induced cell activation. (C) IL-6 production by LPS stimulated U373 cells in the presence of different amounts of purified milk–derived sCD14. All cytokines were tested by ELISA. Results in A and C are means ± SD of triplicate cultures of one experiment representative of four; results in B are the mean ± SD of three independent experiments.

Figure 3

m-sCD14 mediates LPS activation of CD14-negative cells. (A and B) IL-6 and IL-8 production by the astrocytoma cell line U373 (A) and the myeloid cell line U937 (B), respectively, stimulated with E. coli LPS (100 ng/ml) in medium supplemented with FCS, human AB serum (AB), or human milk (HM). The anti-CD14 mAb MY4 and MEM-18 but not their isotype-matched controls (20 μg/ml) blocked the LPS-induced cell activation. (C) IL-6 production by LPS stimulated U373 cells in the presence of different amounts of purified milk–derived sCD14. All cytokines were tested by ELISA. Results in A and C are means ± SD of triplicate cultures of one experiment representative of four; results in B are the mean ± SD of three independent experiments.

Figure 4

IECs can be activated by LPS or whole bacteria by an m-sCD14–dependent mechanism. (A–D) IL-8, TNF-α, and ENA-78 production was tested by ELISA in supernatants of HT29 and SW620 cell lines cultured in medium supplemented with human milk (HM) and stimulated with E. coli LPS or varying numbers of whole E. coli (C and D; 2.5 × 10⁶ E. coli). The anti-CD14 mAb MY4 (IgG2b) but not its isotype-matched control blocked LPS and bacterial stimulation. Representative results with HT29 cells are shown.