Appropriate development of the liver Treg compartment is modulated by the microbiota and requires TGF-β and MyD88

Abstract

Neither the early postnatal development of the liver Treg compartment nor the factors that regulate its development has been characterized. We compared the early developmental patterns of Treg cell accumulation in murine liver, thymus, and spleen. A FoxP3(EGFP) reporter mouse was employed to identify Treg cells. Mononuclear cells were isolated from organs postnatally, stained for CD4, and examined by flow cytometry to enumerate FoxP3(+)CD4(hi) cells. To assess roles for TGF-β1, MyD88, and TLR2, gene-specific knockout pups were generated from heterozygous breeders. To test the role of commensal bacteria, pregnant dams were administered antibiotics during gestation and after parturition. The pattern of appearance of Treg cells differed in liver, spleen, and thymus. Notably, at 1-2 weeks, the frequency of CD4(hi) FoxP3(+) T cells in liver exceeded that in spleen by 1.5- to 2-fold. The relative increase in liver Treg frequency was transient and was dependent upon TGF-β1 and MyD88, but not TLR2, and was abrogated by antibiotic treatment. A relative increase in liver Treg frequency occurs approximately 1-2 weeks after parturition that appears to be driven by colonization of the intestine with commensal bacteria and is mediated by a pathway that requires TGF-β1 and MyD88, but not TLR2.

Figure 1

GFP expression is a reliable marker of FoxP3 expression in liver CD4⁺ cells from FoxP3^EGFP transgenic reporter mice. NPC were isolated from liver of adult BALB/c-background FoxP3^EGFP mice and CD4⁺ cells were sorted by GFP expression. Sorted cell populations were then analyzed by intracellular staining for FoxP3 expression.

Figure 2

Postnatal development of FoxP3⁺CD4⁺ cells in thymus, spleen, and liver. NPC were isolated from thymus, spleen, and liver of BALB/c-background FoxP3^EGFP transgenic reporter mice of the indicated ages. (a) Individual GFP expression profiles of CD4⁺ T cells are shown. (b) Composite data from several mice are shown. Frequency indicates the percentage of CD4⁺ T cells that coexpress eGFP as a reporter of FoxP3 expression. N = 3 to 8 mice per time point. (c) Composite data from several mice are shown. Density indicates the number of CD4⁺eGFP⁺ cells per wet weight of the organ. N = 3 to 8 mice per time point.

Figure 3

The increased frequency of FoxP3⁺CD4⁺ cells in postnatal liver reverses in the adult. (a) Liver and spleen FoxP3⁺CD4⁺ frequency data from BALB/c-background FoxP3^EGFP mice prior to weaning are displayed. Statistical analysis was by 2-way ANOVA. (b) FoxP3⁺CD4⁺ frequency data from preweaned mice (age 1-2 weeks) and adult mice (age 6 weeks; age 13.5 weeks) are shown. For 1-2 weeks, data for 6- to 8-day-old mice were combined with data from 11- to 12-day-old mice. N = 7 to 12 mice per time point. Statistical analyses used the nonparametric Mann-Whitney test.

Figure 4

The increased frequency of FoxP3⁺CD4⁺ cells in postnatal liver depends on TGF-β1. NPC were isolated from thymus, spleen, and liver of BALB/c-background FoxP3^EGFP.Tgfb1 ^+/− mice and littermate FoxP3^EGFP.Tgfb1 ^−/− mice at the indicated ages. (a) Frequency data for FoxP3⁺CD4⁺ cells are shown. (b) Density data for FoxP3⁺CD4⁺ cells are shown. (c) Data for spleen and liver are shown for Tgfb1 ^+/− mice and Tgfb1 ^−/− mice. N = 4 to 9 mice per group at each time point. Statistical analyses were by 2-way ANOVA.

Figure 5

The increased frequency of FoxP3⁺CD4⁺ cells in postnatal liver is blocked by treatment with antibiotics and depends on MyD88. (a) Pregnant C57Bl/6 females were treated continuously with a cocktail of oral antibiotics from two weeks of gestation until pups were 8-9 days of age, at which time spleen and liver NPC were isolated and FoxP3⁺CD4⁺ cells were measured (n = 13 mice). Control mice are untreated C57Bl/6 background pups of the same age (n = 4 mice). Statistical analyses used the nonparametric Mann-Whitney test. (b) Liver and spleen FoxP3⁺CD4⁺ cell frequency data are shown for 8- to 9-day-old littermate Myd88 ^+/+ mice, Myd88 ^+/− mice, and Myd88 ^−/− mice. N = 8 to 13 mice per time point. Myd88 mice were on a mixed BALB/c × C57Bl/6 background, but littermates were used, minimizing background effects. Statistical analyses used the nonparametric Mann-Whitney test.

Figure 6

The increased frequency of FoxP3⁺CD4⁺ cells in postnatal liver is independent of TLR2. Liver and spleen FoxP3⁺CD4⁺ cell frequency data are shown for 8- to 9-day-old littermate C57Bl/6-background Tlr2 ^+/+ mice and Tlr2 ^−/− mice. N = 4 to 5 mice per genotype. Statistical analyses used the nonparametric Mann-Whitney test.