Staphylococcus epidermidis' Overload During Suckling Impacts the Immune Development in Rats

Abstract

Mastitis is an inflammation of the mammary gland occurring in 3-33% of the breastfeeding mothers. The majority of mastitis cases have an infectious etiology. More than 75% of infectious mastitis are caused by Staphylococcus epidermidis and Staphylococcus aureus and involves breast milk microbiota alteration, which, may have an impact in lactating infant. The aim of this study was to analyze in rats during the suckling period and later in life the impact of a high and a low overload of Staphylococcus epidermidis, similarly as it occurs during the clinical and the subclinical mastitis, respectively. From days 2 to 21 of life, suckling rats were daily supplemented with low (Ls group) or high (Hs group) dose of S. epidermidis. Body weight and fecal humidity were periodically recorded. On days 21 and 42 of life, morphometry, hematological variables, intestinal gene expression, immunoglobulin (Ig) and cytokine profile and spleen cells' phenotype were measured. Although no differences were found in body weight, Ls and Hs groups showed higher body length and lower fecal humidity. Both doses induced small changes in lymphocytes subpopulations, reduced the plasma levels of Ig and delayed the Th1/Th2 balance causing a bias toward the Th2 response. No changes were found in cytokine concentration. The low dose affected the Tc cells intestinal homing pattern whereas the high dose had an impact on the hematological variables causing leukocytosis and lymphocytosis and also influenced the intestinal barrier maturation. In conclusion, both interventions with Staphylococcus epidermidis overload during suckling, affects the immune system development in short and long term.

Keywords: Staphylococcus epidermidis; immune system; immunoglobulins; intestinal gene expression; mastitis; suckling rat.

FIGURE 1

Body weight (g) of suckling rats during the study (from days 2 to 42 of life). Results are expressed as mean ± SEM (n = 24 animals/group). REF, reference group; Ls, group supplemented with low dose of S. epidermidis and Hs, group supplemented with high dose of S. epidermidis.

FIGURE 2

Fecal humidity during the study (from days 7 to 42 of life) (A) and cecal content pH on day 21 (the end of the supplementation) and day 42 (3 weeks after the end of the supplementation) (B). Results are expressed as mean ± SEM (n = 12–24 animals/group). Statistical differences: ^αp < 0.05 Ls vs. REF, ^βp < 0.05 Hs vs. REF and ^γp < 0.05 Hs vs. Ls. REF, reference group; Ls, group supplemented with low dose of S. epidermidis and Hs, group supplemented with high dose of S. epidermidis.

FIGURE 3

Immunoglobulin concentration in different compartments at the end of the supplementation (day 21 of life) and 3 weeks after (day 42 of life; the end of the study). Intestinal IgA (A), and plasma levels of Ig isotypes (B–H), Th1/Th2 ratio (I) refers to the relationship between Th1 associated Ig (IgG2b + IgG2c) and Th2 associated Ig (IgG1 + IgG2a). Results are expressed as mean ± SEM (n = 12 animals/group). Statistical differences: *p < 0.05 vs. REF, ^#p < 0.05 Hs vs. Ls and ^δ day 42 vs. day 21. REF, reference group; Ls, group supplemented with low dose of S. epidermidis and Hs, group supplemented with high dose of S. epidermidis.

FIGURE 4

αE integrin/CD62L molecular pattern in the total lymphocytes of the spleen of REF (A), Ls (C), Hs (E) animals at day 21 and REF (B), Ls (D), Hs (F) animals at day 42. Representative histograms for each group. Results are expressed as mean ± S.E.M (n = 12 animals/group). REF, reference group; Ls, group supplemented with low dose of S. epidermidis and Hs, group supplemented with high dose of S. epidermidis.

FIGURE 5

Intestinal gene expression of mucin (MUC) 2 (A) and MUC 3 (B), claudin (Cldn) 2, Cldn4 (C,D, respectively), occludin (Ocldn) (E), FcRn (F), Gpr43 (G), and Toll like receptors (TLR) 2 (H), TLR4 (I), TLR5 (J), TLR9 (K) at days 21 and 42. Relative gene expression was calculated with respect to REF animals, which corresponded to 100% of transcription. Results are expressed as mean ± S.E.M (n = 9 animals/group). Statistical significance: *p < 0.05 vs. REF, ^# vs. Ls. REF and ^δday 42 vs. day 21: reference group; Ls, group supplemented with low dose of S. epidermidis and Hs, group supplemented with high dose of S. epidermidis.