TGF-β2, EGF, and FGF21 Growth Factors Present in Breast Milk Promote Mesenteric Lymph Node Lymphocytes Maturation in Suckling Rats

Abstract

Breast milk, due to its large number of nutrients and bioactive factors, contributes to optimal development and immune maturation in early life. In this study, we aimed to assess the influence of some growth factors present in breast milk, such as transforming growth factor-β2 (TGF-β2), epidermal growth factor (EGF), and fibroblast growth factor 21 (FGF21), on the immune response development. Newborn Wistar rats were supplemented daily with TGF-β2, EGF, or FGF21, throughout the suckling period. At day 14 and 21 of life, lymphocytes from mesenteric lymph nodes (MLNs) were isolated, immunophenotyped, and cultured to evaluate their ability to proliferate and release cytokines. The main results demonstrated that supplementation with TGF-β2, EGF, or FGF21 modified the lymphocyte composition in MLNs. At day 14, all supplementations were able to induce a lower percentage of natural killer (NK) cells with the immature phenotype (CD8+), and they reduced the CD8αα/CD8αβ ratio at day 21. Moreover, the cytokine pattern was modified by the three treatments, with a down regulation of interleukin (IL)-13 secretion. These results showed the contribution of these growth factors in the lymphocytes MLNs immune maturation during the neonatal period.

Modulation of the Systemic Immune Response in Suckling Rats by Breast Milk TGF-β2, EGF and FGF21 Supplementation

“…Some authors researched the effects of TGF-β2, EGF and FGF21 -growth factors present in breast milk- on GALT, but scarce evidence exists regarding the effects of these bioactive factors on systemic immunity in newborn rats [ 37 ]. Previously, some research performed by our group demonstrated the effect of the supplementation with TGF-β2, EGF and FGF21 on the promotion of intestinal lymphocyte maturation in newborn suckling rats [ 25 ]. The present study was designed to determine whether daily supplementation with these three GF could promote systemic immune development during the postnatal period.…”

“…Cells were incubated for 48 h at 37 °C and 5% CO 2 . Four hours before the end time of the incubation, 5-bromo-2’-deoxyuridine (BrdU, 20 μL/well, Merck) was added in order to measure its incorporation as an indicator of DNA synthesis in a colorimetric immunoassay [ 25 , 27 ].…”

“…Lymphocytes from spleens were immunophenotyped by immunofluorescence staining and flow cytometry analysis. The spleen lymphocytes (5 × 10 5 cells/mL) were stained with anti-rat mAb conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin–chlorophyll-a protein (PercP), allophycocyanin (APC) or APC-cyanine (Cy)7, as in previous studies [ 25 , 27 ]. The mAb used in this study were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD45RA and anti-CD25, all from BD Biosciences, anti-CD103 (αE-integrin) and anti-CD62L from Biolegend (San Diego, CA, USA) and anti-Foxp3 from eBioscience.…”

“…In a previous study, we demonstrated that supplementation with EGF, TGF-β2 and FGF21 was able to promote intestinal lymphocyte maturation in newborn suckling rats [ 25 ]. We hypothesized that they could also possess additional developmental roles at the systemic immune level.…”

Lactobacillus fermentum CECT5716 Supplementation in Rats during Pregnancy and Lactation Impacts Maternal and Offspring Lipid Profile, Immune System and Microbiota

“…Cells were isolated from the MLNs of dams by passing the tissue through a 40 µm mesh cell strainer (Thermo Fisher Scientific, Barcelona, Spain). Then, the cell suspension was centrifuged (538× g, 10 min, 4 • C) and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, Madrid, Spain) enriched with 10% fetal bovine serum (FBS; Sigma-Aldrich), 100 IU/mL streptomycin-penicillin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich) and 0•05 mM 2-β-mercaptoethanol (Merck Millipore), as in previous studies [56]. CountessTM Automated Cell Counter (InvitrogenTM, Thermo Fisher Scientific) was used for cell counting and the assessment of viability.…”