Lactobacillus reuteri DSM 17938 changes the frequency of Foxp3+ regulatory T cells in the intestine and mesenteric lymph node in experimental necrotizing enterocolitis

Abstract

Necrotizing enterocolitis (NEC) is an inflammatory disease of the intestine in premature infants. Lactobacillus reuteri DSM 17938 improves survival and reduces the incidence and severity of NEC in a rodent model. Foxp3(+) regulatory T cells (Tregs) maintain intestinal homeostasis by controlling inflammation and inducing tolerance. To determine whether there are insufficient numbers of Tregs to control inflammation in NEC and to determine if LR17938 increases the frequency of Tregs, we studied selected groups of newborn Sprague-Dawley rats according to feeding plan: dam±LR17938, formula±LR17938, and NEC±LR17938. NEC was induced by gavage feeding with special formula and exposure to hypoxic conditions. Lymphocytes isolated from ileum, mesenteric lymph nodes (MLN), spleen and thymus were labeled for T cell surface markers (CD3, CD4, CD8) and intracellular Foxp3; and labeled cells were analyzed by flow cytometry. The percentage of CD3(+) T cells and Foxp3(+) Tregs in the ileum significantly decreased in pups with NEC, compared to normal controls. Feeding LR17938 to neonatal rats with NEC increased the % of Foxp3(+) T cells in the ileum while decreasing the percentage of cells in the MLN. Administration of LR17938 to dam-fed rats significantly increased Foxp3(+)Tregs in the ileum as early as day of life (DOL)1 but did not produce an increase in Tregs in formula-fed rats on DOL1. These results suggest that factors in breast milk may enhance the early immunomodulatory effects of LR17938. An anti-inflammatory effect of LR17938 in NEC was associated with the modulation of immune responses and induction and what appears to be migration of Foxp3(+) Tregs to the diseased gut. Probiotic-facilitated development of Tregs might play an important role in the prevention of NEC.

Figure 1. Phenotypic composition of T cell subsets in the ileum and mesenteric lymph nodes (MLN) of normal rats during day of life (DOL) 0 to DOL 4.

The frequencies of T cell subsets including CD3⁺, CD4⁺, CD8⁺, CD4⁺CD8⁺, CD4⁺Foxp3⁺, and double positive CD4⁺CD8⁺Foxp3⁺ cells, and the comparisons between DOL 0 and DOL 4 were shown by representative flow cytometry plots in ileum (1A–a) and mesenteric lymph node (1B–a) of newborn rats. The bars reflect the percentage of cells, shown as means ± SE, N = 9 pups at each DOL. Comparisons were made between on DOLs 1, 2, 3, or 4 compared with DOL0 in the ileum (1A–b) or MLN (1B–b) of newborn rats, respectively. *p<0.05, **p<0.01, and ***p<0.001.

Figure 2. Effect of breast milk or formula feeding with or without Lactobacillus reuteri DSM 17938 on the frequency of Foxp3⁺ T cells in the ileum and mesenteric lymph node (MLN) of rats during DOL 0 and DOL 4.

Newborn rats were fed immediately after birth for 4 days. (a) and (c): Changes of single CD4⁺Foxp3⁺ Tregs (%) in the ileum (a) and MLN (c); (b) and (d): changes of double CD4⁺CD8⁺Foxp3⁺ T cells in the ileum (b) and MLN (d). The percentage (%) of cells is represented as the mean ± SE of N = 9 observations (pups) in each DOL of each feeding group (total N = 45 for each feeding group).* Dam-fed +17938 vs. Dam-fed, **p<0.01, ***p<0.001. # Formula vs. Dam-fed, # p<0.05, ## p<0.01. &: Formula-fed +17938 vs. Formula-fed, & p<0.05, && p<0.01.

Figure 3. Effect of Lactobacillus reuteri DSM 17938 on survival, CD3⁺ T cells and Foxp3⁺ T cells in the ileum of rats with NEC.

(a) Survival (%) of different groups of rat pups (dam-fed, NEC, and NEC+17938). (b) Changes of % CD3⁺ T cells of lymphocytes in different groups. (c) Immunohistochemical staining of CD3⁺ cells in rat pup ileum, 200 × magnification. (d) Number of CD3⁺ T cells per 5 villi in IHC. (e) Percentage of CD4⁺Foxp3⁺Tregs in each group on day 4 of NEC. (f) Percentage of double positive CD4⁺CD8⁺Foxp3⁺ T cells. The numbers represent percent of T cells (means ± SE). Each dot represents one animal in each group in the figures b, e, and f. Dam-fed: N = 15, NEC: N = 13, NEC+17938: N = 20.

Figure 4. Effect of Lactobacillus reuteriDSM 17938 on CD3⁺, CD4⁺ and Foxp3⁺ T cells in the mesenteric lymph nodes of rats with NEC.

(a): changes of CD3⁺ T cells of lymphocytes, (b) % of CD4⁺ T cells/CD3⁺ T cells, (c) % of CD4⁺Foxp3⁺, and (d) double positive CD4⁺CD8⁺Foxp3⁺ cells in the mesenteric lymph nodes in different groups. The numbers represent percent of T cells (mean ± SE). Each dot represents one animal in each group in the figures. Dam-fed: N = 15, NEC: N = 13, NEC+17938: N = 20.