Introduction

The mammary glands of mammals are specialized organs whose function is to produce milk, the primary source of nutrition for newborns. Breastfeeding is recognized as one of the most valuable contributors to infant health [1]. Human breast milk protects infants not only against infections but also against chronic diseases. Furthermore, human breast milk contains certain growth factors that help the infant intestine to develop, become able to absorb milk and prepare for food intake. When maternal breast milk is unavailable, the alternative is infant formula. Compared with infants fed on formula, infants fed on breast milk have a lower incidence of digestive problems and are more likely to be protected against gastrointestinal and respiratory infections. Despite the fact that breastfeeding is known to be the best method for nourishing infants, how exactly breastfeeding works to provide the best nutrition and protect infants against disease is not fully understood.

Many immune-related substances are present in human breast milk, and their effects upon the recipient infants are widely recognized [2, 3]. For instance, human breast milk contains large quantities of secretory (s)IgA. These antibodies can bind to pathogens and prevent their attachment to an infant's cells. Furthermore, human breast milk contains measurable levels of leukocytes. In addition to these immunologic components, breast milk contains several nonspecific factors, such as lysozyme, lactoferrin and oligosaccharides, which have antimicrobial effects. Lysozyme inhibits the growth of many bacterial species by disrupting the proteoglycan layer of the bacterial cell wall. Lactoferrin, known as a multifunctional protein in human breast milk, also limits bacterial growth by removing essential iron and by stimulating cytokine production, and enhancing mucosal immunity, natural killer (NK) cell activity and macrophage cytotoxicity. Substantial amounts of oligosaccharides in the mammary gland were found in human breast milk, and these block attachment of microbes to an infant's mucosa, preventing infections. Nucleotides in human breast milk have been shown to enhance the immune function in infants [4]. However, several additional immune regulatory components in milk may explain why breastfeeding can reduce infant mortality.

MicroRNAs (miRNAs) are small regulatory RNA molecules that modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes [5–7]. Specific miRNAs that play important roles in a wide range of physiologic and pathologic processes in mammals may be involved in the control of immunologic reactions [18]. Furthermore, human breast milk miRNAs may induce B-cell differentiation, because the milk is rich in miR-181 and miR-155, both known to induce B-cell differentiation [19, 20], but it is not rich in miR-150, which suppresses B-cell differentiation [21, 22]. Our miRNA microarray detected many different types of miRNAs in human breast milk (Figure 1c); however, the functions of many of these are still unknown. Because immune-related miRNAs are well studied and their functions are clarified in the present report, we focused on their presence in human breast milk in which we believe miRNAs should have many more functions, especially in immunologic conditions such as allergy, including atopy and asthma [23, 24].

The transfer of miRNA among cells means that miRNA is not only a regulatory molecule within the cell, but also, like cytokines, is a regulatory molecule for cell-cell communication. Our study clearly suggests that miRNA is a transferable genetic material from mother to infant. It is estimated that approximately 1.3 × 107 copies/liter/day of miR-181a are received by a breastfed infant. Further studies are needed to examine the potential clinical use of immune-related miRNAs in breast milk and the mechanisms by which these miRNAs act as a tool for molecular communication between mother and infant (Figure 5). As shown in Figure 1c, although there was more variation between individuals, the expression pattern of miRNA in human breast milk samples from the same mother did not differ much with time after birth. These observations suggest that human breast milk reflects a mother's constitution and her living environment such as food intake and climate. The present study provides insight into how breast milk may protect infants from various infections; proposing that the miRNAs there act as immune-regulatory agents.

Figure 5
figure 5

Exosome-like vesicles from human breast milk display the typical size (30--300 nm) and ultrastructure of electron-dense and electron-lucent microvesicles (arrows). miRNAs in the vesicles within breast milk are received by infants and facilitate many aspects of the infant's development. Scale bar, 100 nm.

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Materials and methods

Ethics statement

All the women gave their signed informed consent to participate. The study was approved by Dr. Tadao Ishii, Manager of Morinaga Co., Ltd and the company's ethics committee (the Ethical Committee of Functional Food Creation).

Sample collection

Human breast milk samples were collected from eight women enrolled in a breastfeeding study at Morinaga Milk Industry Co., Ltd. Human breast milk samples were collected when the infant were aged between 4 days and 11 months (see Table 1). The milk was collected and then the collected samples totaling 50-100 ml were put into in storage bags. All samples were stored at - 80°C until analyzed.

Table 1 Time of sample collection
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Sample preparation

We chose the samples given <6 months after birth and samples given between 6 and 12 months after birth. Cells and large debris within the breast milk were removed by centrifugation at 2,000 × g for 10 min twice; the supernatant was then centrifuged at 12,000 × g for 30 min to remove cellular debris. The clear supernatants were used for the analysis.

Total RNA extraction

Total RNA from breast milk was extracted using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA). Breast milk was thawed on ice, diluted with two volumes of mirVana Lysis/Binding Solution, mixed thoroughly by vortex for 30 s and incubated for 5 min. Then 1/10 volumes of miRNA homogenate additive was added, mixed thoroughly by vortex for 30 s and incubated on ice for 10 min. An equal volume of acid/phenol/chloroform (Ambion) was then added to each aliquot. The resulting solutions were mixed by vortex for 1 min and spun for 10 min at 10,000 × g. The resulting aqueous volume was mixed thoroughly with 1.25 volumes of 100% molecular-grade ethanol and passed through a mirVana column in sequential 700 μl aliquots. The column was washed according to the manufacturer's protocol, and RNA was eluted in nuclease-free water at 95°C. RNA extraction from the serum was performed using the same method.

Microarray analysis

To detect the expression of miRNAs in human breast milk, 70 ng of total RNA was labeled and hybridized using a Human microRNA Microarray Kit (Agilent Technologies) according to the manufacturer's protocol (Protocol for Use with Agilent Microrna Microarrays Version 1.5). Hybridization signals were detected by a DNA microarray scanner (Agilent Technologies), and the scanned images were analyzed using Agilent Feature Extraction software.

qRT-PCR

qRT-PCR of miRNA expression was performed using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol. To normalize the sample-to-sample variation in the RNA isolation step, synthetic Caenorhabtidis elegans miRNA cel-miR-39 (synthetic RNA oligonucleotides synthesized by Qiagen, Valencia, CA, USA) was added as a mixture of 25 fmol of each oligonucleotide in a total volume of 1 ml to each denatured sample (that is, after combining the breast milk and serum samples with Lysis Solution (mirVana miRNA isolation kit; Ambion). All experiments were repeated three times.

RNase and freeze-thawing treatment

To confirm that the miRNA is resistant to RNase digestion, human breast milk was treated with 10 U/ml RNase A and 400 U/ml RNase T1 (Ambion) for 60 min at 37°C. After these treatments, the RNA was extracted from the milk as described above.

To investigate the stability of miRNAs in human breast milk, the milk was subjected to three freeze-thaw cycles of at -20°C or treated for 3 h in a low pH solution (pH 1). miRNA levels were assessed by TaqMan qRT-PCR.

Isolation of CD63 positive exosome

Exosomes in human breast milk were specifically isolated by magnetic beads, using anti-CD63 antibody (BD, Erembodgem, Belgium). Human breast milk (0.3 ml) was incubated with anti-CD63 antibody (BD) or mouse IgG1 (Sigma-Aldrich, St Louis, MO, USA) coupled to magnetic microbeads (50 μl). These were mixed and incubated for 16 h at room temperature. The magnetic immune complexes were washed four times with 500 μl of PBS, then the RNA was extracted as described above.

Transmission electron microscopy

Clear supernatants from human breast milk were centrifuged at 100,000 × g for two hours, then washed in phosphate-buffered saline (PBS) and pelleted by ultracentrifugation (100,000 × g). The pellet was diluted in PBS. Resuspended exosomes were fixed in 1% glutaraldehyde in PBS (pH 7.4). The samples were stained for 10 min with 1% uranyl acetate. Excess fluid was removed with a piece of Whatman filter paper. All transmission electron micrographs were obtained using JEM1220 electron microscopy at 120 kv.