Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 14;14(1):73-84.
doi: 10.3920/BM2022.0095. Epub 2023 Feb 23.

Probiotic-educated Tregs are more potent than naïve Tregs for immune tolerance in stressed new-born mice

Y Liu  1 , T K Hoang  1 , E S Park  1 , J Freeborn  1 , B Okeugo  1 , D Q Tran  1 , J M Rhoads  1
Affiliations

Probiotic-educated Tregs are more potent than naïve Tregs for immune tolerance in stressed new-born mice

Y Liu et al. Benef Microbes. .

Abstract

When new-born mice are subjected to acute maternal separation stress, cow-milk based formula feeding, and brief recurrent hypoxia with cold stress, they develop gut inflammation similar to the phenotype of neonatal necrotizing enterocolitis, characterised by an increase in gut mucosal effector T (Teffs) and reduced Foxp3+ regulatory T (Tregs) cells. The imbalance can be prevented by probiotic Limosilactobacillus reuteri DSM 17938 (LR 17938). We hypothesised that LR 17938 could potentiate a tolerogenic function of Tregs. To analyse whether LR 17938 can educate Tregs to improve their tolerogenic potency during neonatal stress, we isolated T cells (Tregs and Teffs) from 'donor' mice fed with either LR 17938 (107 cfu) or control media. The cells were adoptively transferred (AT) by intraperitoneal injection (5 × 105 cells/mouse) to new-born (d5) recipient mice. Mice were then separated from their dams, fed formula by gavage, and exposed to hypoxia and cold stress (NeoStress) for 4 days. We analysed the percentage of Tregs in CD4+T helper cells in the intestine (INT) and mesenteric lymph nodes (MLN) of recipient mice. We found that: (1) the percentage of Tregs in the INT and MLN following NeoStress were significantly reduced compared to dam-fed unstressed mice; (2) AT of either naïve Tregs or LR-educated Tregs to mice with Neostress increased the percentage of Tregs in the INT and MLN compared to the percentage in NeoStress mice without Treg treatment; however, LR-educated Tregs increased the Tregs significantly more than naïve Tregs; and (3) AT of LR-educated Tregs reduced pro-inflammatory CD44+Foxp3-NonTregs and inflammatory CX3CR1+ dendritic cells in the intestinal mucosa of NeoStress mice. In conclusion, adoptive transfer of Tregs promotes the generation of and/or migration of endogenous Tregs in the intestinal mucosa of recipient mice. Importantly, probiotic-educated Tregs are more potent than naïve Tregs to enhance immune tolerance following neonatal stress.

Keywords: Limosilactobacillus reuteri; adoptive transfer; immune tolerance; intestinal mucosa; maternal separation; stress.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Experimental design for adoptive transfer. Tregs or Teffs were isolated from Thy1.2 mice fed either probiotic LR 17938 (TregsLR+, TeffsLR+) or control media (TregsLR−, TeffsLR−) and then were injected intraperitoneal to Thy1.1 newborn mouse (5×105 cells/mouse). Thy1.1 newborn mouse was subjected to the NEC procedures, defined as NeoStress. Another set of newborn mice stayed with their dam after adoptive transfer Tregs or Teffs as Dam-fed group.
Figure 2.
Figure 2.
Intestinal histology representative photomicrographs and the incidence of NEC in each experimental group: intestinal histological representative images (40x) and the inserted images (200x) are shown. The numbers of mice in each group are indicated in the Figure table.
Figure 3.
Figure 3.
The percentage of Foxp3+Tregs in the intestinal mucosa of newborn mice affected by adoptive transfer T cells. a: gating strategy to define percentage of Foxp3+Tregs among CD4+T cell populations, as well as CD90.1+Tregs (endogenous) and CD90.2+Tregs (exogenously from donor) in Foxp3+Treg population. b and c: % of Foxp3+Tregs in the intestine (b) and in the mesenteric lymph nodes (c) of newborn mice divided into Neostress and Dam-fed (No NeoStress) groups. d: % of CD90.2+Tregs in the intestine, mesenteric lymph node, and spleen of newborn mice after adoptive transfer Tregs to NeoStress newborn mice. The multiple group comparisons and the numbers of mice are indicated in the Figure.
Figure 3.
Figure 3.
The percentage of Foxp3+Tregs in the intestinal mucosa of newborn mice affected by adoptive transfer T cells. a: gating strategy to define percentage of Foxp3+Tregs among CD4+T cell populations, as well as CD90.1+Tregs (endogenous) and CD90.2+Tregs (exogenously from donor) in Foxp3+Treg population. b and c: % of Foxp3+Tregs in the intestine (b) and in the mesenteric lymph nodes (c) of newborn mice divided into Neostress and Dam-fed (No NeoStress) groups. d: % of CD90.2+Tregs in the intestine, mesenteric lymph node, and spleen of newborn mice after adoptive transfer Tregs to NeoStress newborn mice. The multiple group comparisons and the numbers of mice are indicated in the Figure.
Figure 3.
Figure 3.
The percentage of Foxp3+Tregs in the intestinal mucosa of newborn mice affected by adoptive transfer T cells. a: gating strategy to define percentage of Foxp3+Tregs among CD4+T cell populations, as well as CD90.1+Tregs (endogenous) and CD90.2+Tregs (exogenously from donor) in Foxp3+Treg population. b and c: % of Foxp3+Tregs in the intestine (b) and in the mesenteric lymph nodes (c) of newborn mice divided into Neostress and Dam-fed (No NeoStress) groups. d: % of CD90.2+Tregs in the intestine, mesenteric lymph node, and spleen of newborn mice after adoptive transfer Tregs to NeoStress newborn mice. The multiple group comparisons and the numbers of mice are indicated in the Figure.
Figure 4.
Figure 4.
The percentage of CD44+Foxp3−nonTregs in the intestine of newborn mice affected by adoptive transfer T cells. a: gating strategy to define percentage of CD44+Foxp3-nonTregs among CD4+T cell population. b: % of CD44+Foxp3−nonTregs in the intestine of newborn mice divided into NeoStress and Dam-fed (No NeoStress) groups. The multiple group comparisons and the numbers of mice were indicated in the Figure.
Figure 5.
Figure 5.
Effect of adoptively transferring Tregs to newborn mice on DC maturation and inflammatory markers in the neonatal intestine. a: gating strategy to define percentage of CX3CR1, CD40 and CD86 in CD11c+cells. b: % of CX3CR1+CD11c+ cells, % of CD40+CD11c+cells, and % of CD86+CD11c+cells in the intestine of newborn mice compared the groups of NeoStress mice. c: % of CD40+ or CD86+ CD11c+ cells in the intestine of NeoStress mice compared to Dam-fed mice. The multiple group comparisons and the numbers of mice are indicated in the Figure.
Figure 5.
Figure 5.
Effect of adoptively transferring Tregs to newborn mice on DC maturation and inflammatory markers in the neonatal intestine. a: gating strategy to define percentage of CX3CR1, CD40 and CD86 in CD11c+cells. b: % of CX3CR1+CD11c+ cells, % of CD40+CD11c+cells, and % of CD86+CD11c+cells in the intestine of newborn mice compared the groups of NeoStress mice. c: % of CD40+ or CD86+ CD11c+ cells in the intestine of NeoStress mice compared to Dam-fed mice. The multiple group comparisons and the numbers of mice are indicated in the Figure.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Athalye-Jape G, Rao S and Patole S, 2016. Lactobacillus reuteri DSM 17938 as a Probiotic for Preterm Neonates: A Strain-Specific Systematic Review. JPEN J Parenter Enteral Nutr 40: 783–794. 10.1177/0148607115588113 - DOI - PubMed
    1. Bianchi L, Laghi L, Correani V, Schifano E, Landi C, Uccelletti D and Mattei B, 2020. A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production. Biomolecules 10. 10.3390/biom10010157 - DOI - PMC - PubMed
    1. Bradley JE, Ramirez G and Hagood JS, 2009. Roles and regulation of Thy-1, a context-dependent modulator of cell phenotype. Biofactors 35: 258–265. 10.1002/biof.41 - DOI - PMC - PubMed
    1. Caplan MS, Russell T, Xiao Y, Amer M, Kaup S and Jilling T, 2001. Effect of polyunsaturated fatty acid (PUFA) supplementation on intestinal inflammation and necrotizing enterocolitis (NEC) in a neonatal rat model. Pediatr Res 49: 647–652. 10.1203/00006450-200105000-00007 - DOI - PubMed
    1. Cerny V, Petraskova P, Novotna O, Borakova K, Prokesova L, Kolarova L and Hrdy J, 2020. Value of cord blood Treg population properties and function-associated characteristics for predicting allergy development in childhood. Cent Eur J Immunol 45: 393–402. 10.5114/ceji.2020.103413 - DOI - PMC - PubMed

Substances