Immunomodulatory Properties of Human Breast Milk: MicroRNA Contents and Potential Epigenetic Effects

Abstract

Infants who are exclusively breastfed in the first six months of age receive adequate nutrients, achieving optimal immune protection and growth. In addition to the known nutritional components of human breast milk (HBM), i.e., water, carbohydrates, fats and proteins, it is also a rich source of microRNAs, which impact epigenetic mechanisms. This comprehensive work presents an up-to-date overview of the immunomodulatory constituents of HBM, highlighting its content of circulating microRNAs. The epigenetic effects of HBM are discussed, especially those regulated by miRNAs. HBM contains more than 1400 microRNAs. The majority of these microRNAs originate from the lactating gland and are based on the remodeling of cells in the gland during breastfeeding. These miRNAs can affect epigenetic patterns by several mechanisms, including DNA methylation, histone modifications and RNA regulation, which could ultimately result in alterations in gene expressions. Therefore, the unique microRNA profile of HBM, including exosomal microRNAs, is implicated in the regulation of the genes responsible for a variety of immunological and physiological functions, such as FTO, INS, IGF1, NRF2, GLUT1 and FOXP3 genes. Hence, studying the HBM miRNA composition is important for improving the nutritional approaches for pregnancy and infant's early life and preventing diseases that could occur in the future. Interestingly, the composition of miRNAs in HBM is affected by multiple factors, including diet, environmental and genetic factors.

Keywords: DNA methylation; RNA regulation; breastfeeding; epigenetics; histone modification; lactation; miRNA.

Figure 1

A cross-section scheme of the mammary gland, breast lobe components and process of lactation. Lactation is the process of producing milk from mammary glands in response to hormonal changes, which is secreted in response to an infant sucking. Each mammary gland is composed of a group of alveoli clusters called a lobe, while the alveoli contain balloon-like cavities called alveolus’, which are responsible for milk secretion and storage upon prolactin induction. Alveolus’ are comprised of milk-secreting cuboidal cells called lactocytes surrounded by contractile myoepithelial cells, which in turn respond to oxytocin and push the milk out of the alveoli into the ducts. They also push blood nutrients, immune cells and other molecules across lactocytes into the milk through both the transcellular and paracellular pathways [20,60,61,62]. Created with BioRender.com, accessed on 22 April 2022.

Figure 2

Schematic presentation of HBM components (A) and factors affecting its production and composition (B). Generally, HBM has four main components, water, carbohydrates, fats and protein. In addition, HBM contains a variety of nutrients, vitamins, minerals, prebiotics, probiotics, hormones, immune cells and substances, nucleotides and nucleic acids and other rare elements. This unique mixture of beneficial components varies due to many factors, mainly related to the mother’s body and health conditions, as well as gestation period [82,83,84,85,86]. Created with BioRender.com, accessed on 22 April 2022.

Figure 3

Structural basis for norovirus inhibition by HBM oligosaccharides 2′FL and 3FL. (A) Crystal structure of norovirus GII.10 P domain in complex with 2′FL (PDB code: 5hzb). (B) Crystal structure of the same domain in complex with 3FL (PDB code: 5hza).

Figure 3

Structural basis for norovirus inhibition by HBM oligosaccharides 2′FL and 3FL. (A) Crystal structure of norovirus GII.10 P domain in complex with 2′FL (PDB code: 5hzb). (B) Crystal structure of the same domain in complex with 3FL (PDB code: 5hza).

Figure 4

A scheme of miRNA biogenesis and processing pathways. The process starts when RNA polymerase II transcribes the targeted miRNA from DNA sequences into a primary miRNA (pri-miRNA). The RNase enzymes DROSHA and its partner DGCR8 (DiGeorge critical region 8) play a crucial role as a heterotrimeric microprocessor complex by cleavage of pri-miRNA from different sites. The resulting ~70 nt miRNA, called precursor miRNA (pre-miRNA), has a characteristic stem-loop structure and undergoes extensive processing before crossing from nucleus to cytoplasm. The transportation of pre-mRNAs is controlled by exportin-5 (XPO5) in the presence of guanosine triphosphate (GTP)-binding ras-related nuclear protein (RAN). The cytoplasmic pre-miRNAs released through the Ran-GTP/XPO5 complex are triggered by GTP hydrolysis into GDP, which occurs by RAN. In the cytoplasm, the trans-activation response (TAR) RNA-binding protein (TRBP) forms a complex by interacting with the endoribonuclease Dicer, assisting it in finding and cleavage of pre-miRNAs into miRNA duplexes. The duplexes are unwound by binding to Argonaute proteins (AGO), resulting in mature miRNA incorporated into the multiprotein RNA-induced silencing complex (RISC). The miRNAs guide the RISC to bind to complementary regions within targeted mRNA, mediating gene regulation through several post-transcriptional routes, mainly via endonuclease mRNA cleavage or degradation, translation inhibition and deadenylation of mRNA [55,181,182,183,184]. Created with BioRender.com, accessed on 22 April 2022.

Figure 5

HBM-derived miRNAs and their physiological functions in breastfed infants. Compared to other human biofluids, HBM is an abundant source of miRNAs that present as free molecules or packaged into a type of extracellular vesicle called exosomes. Although the majority of these miRNAs originate from the mammary epithelium, there is a small contribution from the miRNAs that transport from the maternal circulation. Evidence indicates that diet-derived miRNAs from plant and animal sources are presented in human circulation and thus can be transported to HBM. In the infant’s GIT, HBM-derived miRNAs cross intestinal epithelial cells to blood circulation and reach various human organs and tissues. Interactions of miRNAs with their complementary regions within targeted mRNAs affect gene expression and ultimately result in regulating essential physiological functions required for infant growth and development [199,205,206,207]. Created with BioRender.com, accessed on 22 April 2022.

Figure 6

Immunomodulatory actions of HBM-derived miRNAs in both innate and acquired immunity. HBM-derived miRNAs are emerging as key controllers of signaling, differentiation and functions of immune cells, especially T cells. Many miRNAs target cytokine genes in monocytes, T helper type 1 (Th1) and Th2 cells regulating the expression of these cytokines and their circulating levels. Other miRNAs present in HBM have also shown a variety of immunomodulatory actions towards immune cells. For instance, miR-10a is a key regulator of regulatory T cell (T_regs) specialization and stability. Furthermore, miRNAs not only have the potential to regulate B cell development and functions, but some of them regulate the production of immunoglobulin by plasma cells (e.g., miR-155). Other miRNAs exquisitely regulate receptor editing during B cell maturation (e.g., miR-17∼92 cluster), clonal deletion (e.g., miR-148a), antibody class switching to IgG and secretion of IgE in B cells (e.g., miR-146a). Moreover, HBM-derived miRNAs affect other than-immune system components that participate in innate and adaptive immunity. For example, miR-146 regulates the megakaryocytopoiesis process, which produces platelets and red blood cells (RBCs). miR-27b affects the functions and reactivity pathways of platelets that release inflammatory and bioactive molecules and has some immune functions such as engulfing microbes. Further, miR-142 may affect the survival and functions of RBCs that act as modulators of innate immunity, especially by binding and scavenging specific molecules that mediate inflammatory responses (such as mitochondrial DNA and chemokines) in circulation [51,129,604,605,606,607,608,609,610,611,612,613,614,615]. Created with BioRender.com, accessed on 22 April 2022.

Figure 7

A schematic diagram of the epigenetic mechanisms that can be modulated by miRNAs. Without changing the DNA sequence, miRNAs affect gene expression post-transcriptionally, resulting in altered protein levels of target mRNAs. This effect, known as epigenetic regulation, can occur through three major epigenetic mechanisms, including DNA methylation, histone modifications and RNA regulation. (A) Rearrangement of the core histone proteins (H2A, H2B, H3 and H4) involved in chromatin reorganization and regulation of transcription, which come together to form one nucleosome, called histone modifications. These covalent modifications are driven by post-translational addition or removal of acetyl, methyl, phosphate, ubiquityl and sumoyl that attach to the tails of histone proteins. miRNAs could regulate histone modifications by targeting histone-modifying enzymes such as deacetylase and demethylases. (B) RNA regulation is a less well-known epigenetic mechanism and occurs through several models, including miRNA regulation of gene expression upon interaction with targeted mRNAs, as shown in Figure 5. Moreover, it has been shown that RNA regulation is involved in epigenetics by modulating chromatin structure. (C) The most widely discovered epigenetic mechanism is DNA methylation. It predominantly occurs on cytosine-phosphate-guanine (CpG) dinucleotides called CpG islands (CGIs). DNA methylation patterns start with DNA methyltransferase (DNMTs) enzymes transferring a methyl group from the methyl donor S-adenyl methionine (SAM), which is derived from ATP and methionine, to the fifth carbon of cytosine (on a CGI) to produce 5-methylcytosine (5-mC). DNA methylation patterns involve four processes: (1) adding a methyl group to unmethylated DNA by DNMT3A and DNMT3B (de novo methylation); (2) preserving DNA methylation by DNMT1 during cellular DNA replication (maintenance methylation); (3) inhibition of maintenance methylation (passive demethylation); (4) oxidation and deamination of 5-mC to obtain an unmodified cytosine by the ten–eleven translocation (TET) enzymes 1/2/3. miRNAs can directly target DNMTs (e.g., DNMT3A and DNMT3B) and methyl-CpG binding proteins (e.g., MeCP2 and MBD2), resulting in DNA methylations [674,675,676,677,678,679,680,681]. Created with BioRender.com, accessed on 22 April 2022.

Figure 8

The role of lactation-specific exosomal miRNAs in targeting DNA methyltransferases (DNMTs) in the recipient milk. Exosomes are released by (A) mammary gland epithelial cells (MEC) and taken up by a variety of cells, including intestinal epithelial cells (IEC), vascular endothelial cells (VEC), systemic circulation and other body cells [700]. The majority of HBM miRNAs come from MECs, resulting in distinct fractionated milk miRNA profiles [185]. (B) The bilayer membrane is critical for MEX resistance to the gastrointestinal tract’s harsh conditions, where miRNA-148a-3p is the main miRNA of MEX. Other important constituents of MEX are transforming growth factor-β (TGF-β) and Tetraspanins such as CD63, CD81, CD9 and CD83 [701,702]. (C) HBM exosome (MEX) boosts IEC proliferation, goblet cell proliferation and activity and increases the activity and viability of intestinal stem cells by upregulating the stem cell marker leucine-rich-repeat-containing G-protein coupled receptor 5 (Lgr5) [703]. MEX promotes mucus formation, increases mucin 2 (MUC2) synthesis and decreases nuclear factor κB signaling, tumor necrosis factor-α (TNF-α), toll-like receptor 4 (TLR4), myeloperoxidase (MPO) and interleukin 6 (IL-6) to mediate anti-inflammatory activities. MEX also helps to maintain the antimicrobial barrier by upregulating the antibacterial lectin regenerating islet-derived 3y (RegIIIγ) and inducing the production of tight junction proteins. MEX also interacts directly with bacteria in the gut microbiome [702]. (D) Endocytosis by VEC [704] supports the idea that milk-derived exosomes and their miRNA cargo could reach the milk recipient’s systemic circulation and peripheral tissues [700,705,706]. (E) Milk exosomes can cross IEC intercellular gaps, which are linked to increased intestinal permeability, especially during the postnatal period. After entering systemic circulation, milk exosomes may reduce DNA methylation of peripheral target cells, where miRNAs induce DNA promoter demethylation of important CpG islands implicated in the activation of gene expression of key transcription factors such as nuclear factor erythroid 2-related factor 2 (NRF2), sterol regulatory element-binding protein-1 (SREBP1), forkhead box P3 (FOXP3) and nuclear receptor subfamily 4 group a member 3 (NR4A3) [707,708]; metabolic regulators such as insulin gene (INS), insulin-like growth factor-1 (IGF1), caveolin 1 (CAV1), glucose transporter 1 (GLUT1) and lactase gene (LCT) [709,710,711,712,713,714]; as well as the RNA m6A demethylase (fat mass- and obesity-associated gene (FTO)), which promotes FTO-dependent mRNA transcription and mRNA splice variant synthesis, such as the adipogenic short version of runt-related transcription factor 1 (RNX1T1), by removing m6A marks on mRNAs. Moreover, Ghrelin and dopamine receptor 3 (DRD3) mRNAs are targeted by FTO-mediated upregulation. The resultant hyperphagia encourages milk consumption to meet newborn growth needs [700,715]. (F) Anti-inflammatory actions of miRNA-148a and miRNA-22 and DNMT1 on nuclear factor κB signaling. MiRNA-148a increases the expression of FOXP3, a negative regulator of nuclear factor B, via suppressing DNA methyltransferase 1 (DNMT1). MiRNA-148a targets calcium/calmodulin-dependent protein IIα (CaMKIIα), which phosphorylates CARD-containing MAGUK protein 1 (CARMA1) implicated in IκB kinase α (IKKα) and IκB kinase β (IKKβ) activation. MiRNA-148a, in particular, targets IKKα and IKKβ directly, thereby boosting the inhibitory impact of IκB on NF-κB. Furthermore, miRNA-148a targets the interleukin 6 (IL-6) signal transducer gp130. Nuclear receptor co-activator 1 (NCOA1) and cystein-rich protein 61 (CYR61), which activates NF-kB, are targets of miRNA-22, which is substantially abundant in preterm MEX. IL-6 expression is suppressed by miRNA-30b via targeting RIP140. As a result, miRNAs generated from MEX and DNMT1 inhibition provide anti-inflammatory signaling [701,702,716,717,718].

Figure 9

The interaction between DNMT3b (A) and DNMT1 (B) with other proteins. The edges indicate both functional and physical protein associations. Settings included a minimum interaction score of 0.4. Max number of interactions was 10 in the first shell and 0 in the second shell. Active interaction sources included curated databases and experimentally determined data. Dnmt3L, Dnmt3a and Dnmt3b interact in vitro and in vivo with histone deacetylase HDAC1 [721]. In cancer cells, EZH2 was found to interact with DNMT1, DNMT3A and DNMT3B [722], resulting in hypermethylation of genes, causing more silencing of target genes [723]. However, the exact association of EZH2 with DNMTs remains controversial. Endogenous DNMT1 is sumoylated on many lysine residues (645–1113) in the BAH domains by UBE2I. This improves DNMT1’s catalytic action on genomic DNA [724]. AHCY was discovered as a partner of DNMT1 during the cell cycle of HeLa cells in proteomic analysis. Methyltransferase studies revealed that AHCY increases DNMT1 activity in vitro, while AHCY overexpression in HEK293 cells causes a widespread increase in DNA methylation in vivo [725]. AHCYL2 is homologous to IRBIT and regulates ion-transporting proteins. It is a potential regulator of NBCe1-B in mammalian cells [726]. However, its function remains unclear. The methylation of AHCYL2 gene was shown to be associated with tumors. AHCY, denosylhomocysteinase; AHCYL2, AHCY like 2; MAT, methionine adenosyltransferase; EZH2, enhancer of zeste homolog 2; HDAC1, histone deacetylase 1; HDAC2, histone deacetylase 2; UBE2I, ubiquitin-conjugating enzyme 2I; DNMT, DNA methyltransferase; UHRF1, ubiquitin-like with PHD and ring finger domains 1; USP7, ubiquitin-specific protease 7; PCNA, proliferating cell nuclear antigen; RB1, RB transcriptional corepressor 1. Pink and cyan lines indicate interactions experimentally determined and from curated databases, respectively. Illustrations created using STRING database.