Presence and Profile of Innate Lymphoid Cells in Human Breast Milk

Abstract

This in vivo study examines innate lymphoid cells in breast milk.

Human milk contains 32 soluble factors and 5 cell types. The list of both proinflammatory and anti-inflammatory milk cytokines is growing.¹ Studies in the 1960s found cells (neutrophils, macrophages, lymphocytes, stem cells, and epithelial cells) in fresh, unpasteurized milk. Newborns ingest 10⁸ maternal cells/d, with 80% being macrophages, originating from maternal peripheral blood monocytes. These milk components protect the breast from infection while modulating the developing neonatal immune system.² Immunomodulation of an infant’s infection by mother’s milk has been known since 2011, but the precise mechanisms remain incompletely understood. Milk is a dynamic, living fluid and changes with the varying demands of the infant.¹

Innate lymphoid cells (ILCs), a new class of lineage-negative lymphoid cells, are key to intestinal microbiome and the adaptive immunity of the infant.³ Innate lymphoid cells have been classically divided into 3 subgroups of ILC1s, ILC2s, and ILC3s, based on their cytokine secretion and transcription factor profiles. Innate lymphoid cell nomenclature is analogous to that of T helper cells. Innate lymphoid cells are central in inflammation, immunity, and tissue homeostasis. Despite this, to our knowledge, no report of milk ILCs exists. This brief report describes the presence, profile, and potential significance of human milk ILCs.

Methods

We collected 30 mL of human milk from 4 lactating mothers and centrifuged the samples at 2000 rpm for 10 minutes. Next, we performed flow cytometry and cell sorting for ILCs by staining cells with CD127 (interleukin [IL]-7 receptor) and a lineage cocktail (anti-CD3, anti-CD4, anti-CD14, anti-CD16, anti-CD19, anti-CD8, anti-CD15, anti-CD20, anti-CD33, anti-CD34, and anti-CD203) plus CD127. We collected CD127⁺ and lineage-negative cells. In addition, to maximize the yield of cells from the specimen, we used the cytospin technique. All cytospin preparations were incubated with antibodies (anti-human interferon-γ, anti-human IL-5, and anti-human IL-22 (Biolegend USA) for 2 hours in the dark at room temperature. Slides were then washed in phosphate-buffered saline and counterstained using 4′,6-diamidino-2-phenylindole nuclear staining, mounted, and subjected to microscopic examination. Cytokine expression in all sections was quantified using imageJ software (National Institutes of Health). This study was approved by the institutional review board at Augusta University, Medical College of Georgia. The participants provided written informed consent; there was no financial compensation.

Results

All 3 classes of ILCs were present in milk (Figure 1). With use of live sorting, different ILC subsets were characterized by surface markers and specific transcription factors (Figure 1A) and were quantified. In all samples (Figure 1B), the ILC1 count was 3 to 10 times the other 3 ILC subsets (P < .005) and the ILC3 count was 3 times the ILC2 count (P = .049). Cytokine data agreed with surface marker analysis (Figure 1B). Using imageJ, the interferon-γ level by ILC1 was 7 times the IL-22 and 5 times the IL-5 levels produced by ILC3s and ILC2s, respectively (Figure 2) (P = .04).

Figure 1. All 3 Classes of Innate Lymphoid Cells (ILCs) Are Present in Human Milk.

A, Scatterplots and histograms show the gating strategy and the presence of all classes of ILCs. B, Bar graphs demonstrate the quantification as mean values of ILCs with highest frequencies for ILC1, followed by ILC3s and lowest for ILC2s; error bars indicate SD. GATA indicates GATA binding protein 3; RORγt, retinoid orphan receptor γt; and T-bet, T box protein expressed in T cells.

Figure 2. Immunofluorescence Staining of Sorted Innate Lymphoid Cell (ILC) Cytospin Preparations.

Cytokine production (A) and quantified bar graph using imageJ software (B) are shown. Yellow arrowheads indicate the cells that coexpress CD127 and cytokine. DAPI indicates 4',6-diamidino-2-phenylindole; IFN, interferon; and IL, interleukin.

Discussion

The results of this study indicate the presence of ILCs in fresh human milk, with high ILC1s followed by ILC3s and ILC2s. The oral transfer of maternal cells through milk clearly occurs, and these cells survive the gastric pH to live in the intestine of the neonate for 6 days.⁴ Although documented, how mammary glands sense and respond to the changes in the infant microbiome is unclear. Innate lymphoid cells of human milk may shape the infant oral and intestinal microbiomes by modulating neonatal immunity. How maternal ILCs modulate the infant ILC populations also remains largely unknown. The immature immune system of the newborn must rapidly respond to the transition from a sterile intrauterine environment to a microbe-laden external world and differentiate what is to be tolerated from microbes that need elimination through vigorous host responses. Furthermore, lactating mothers must guard against microbial infection of the breasts; the milk leukocytes provide such defense.⁵ Milk ILCs may impart innate immunity in newborns. The next step is to investigate how they shape neonatal immunity and microbiome.