Resistance to lipopolysaccharide-induced preterm delivery mediated by regulatory T cell function in mice

Abstract

Intrauterine or intraperitoneal administration of lipopolysaccharide (LPS) into normal mice at midgestation induces preterm delivery (PTD) within 24 h through a mechanism dependent on Toll-like receptor signaling and expression of inflammatory cytokines. The exact participants in the cellular network involved in PTD are not known. Although the activities of innate immune cells are thought to be important, the extent to which this process depends on T and B cells has yet to be examined. Mice deficient in T and B cells due to genetic deficiency in the recombination activating gene 1 (Rag1(-/-)) were given LPS intraperitoneally on Day 15 of gestation and found to be susceptible to LPS-induced PTD. This was found to involve many of the inflammatory mediators reported as important in normal mice. Moreover, at a low dose (3 microg), pregnant Rag1(-/-) mice were found to be more susceptible to PTD than a cohort of normal mice on the same genetic background. This increased susceptibility was partially reversed by transfer, on Day 10 of gestation, of whole lymphocytes or purified CD4(+) T cells. Transfer of purified CD4(+) T cells to Rag1(-/-) mice resulted in a uterine draining node population of FOXP3(+) cells, suggesting that these cells may contribute to resistance to LPS-induced PTD. Overall, the data suggest that, although T and B lymphocytes are not critical positive regulators of LPS-induced PTD, CD4(+) T cells play a protective and regulatory role, and thus could be a target for preventive or therapeutic manipulation.

FIG. 1.

LPS induces premature delivery in Rag1^−/− mice in a dose-dependent manner. Rag1^−/− females were mated to same-strain males, and, on Day 15 of gestation, they were given either PBS (dose = 0) or varying doses of LPS intraperitoneally. After 24 h, the mice were euthanized and observed for delivery of pups. Y axis, proportion of pregnant females that delivered at least one pup prematurely; x axis: dose of LPS used. n, number of mice tested.

FIG. 2.

LPS-induced gene up-regulation in the uterus of pregnant Rag1^−/− mice. Rag1^−/− females were mated to same-strain males, and, on Day 15 of gestation, they were given either PBS or 3 μg of LPS. At 6 h after injection, the uteri were dissected away from associated placenta and used for RNA isolation. Isolated RNA was assayed for gene expression by quantitative RT-PCR. Y axis, relative expression; x axis, specific genes. Black symbols, uterine RNA from LPS-treated mice; grey symbols, uterine RNA from PBS-treated mice. Each symbol represents tissue from an individual mouse. *Significant increase compared with PBS treatment by the Mann-Whitney U-test. Lines represent the medians for each group.

FIG. 3.

LPS induces antigen-presenting cell activation in the uterus in Rag1^−/− mice. Rag1^−/− females were mated to same-strain males, and, on Day 15 of gestation, they were given either PBS or 3 μg of LPS. After 6 h, the mice were euthanized and uteri from 3–4 mice from each treatment group were used to isolate lymphocytes, pooled and examined by flow cytometry. A) Increase in the presence of CD11C⁺ cells that are MHC class II high. Upper plot: uteri of mice given PBS (control). Lower plot: uteri of mice given LPS. Y axis, MHC class II; x axis, CD11C. B) Increase in the expression of MHC class II on a macrophage population. Left plots: delineation of CD11B⁺CD14⁺ cells (macrophages). Upper plot, PBS-treated mice; lower plot, LPS-treated mice. The plot on the right shows expression of MHC class II in the gated cells. Dotted line, cells from PBS-treated mice; solid line, cells from LPS-treated mice.

FIG. 4.

Rag1^−/− mice are more sensitive to LPS-induced premature delivery, and this is partially reversed by injection of normal whole lymphocytes or CD4⁺ T cells. A) Pregnant Rag1^−/− mice (white bars) or normal C57BL/6 mice (black bars) were injected with 3 μg of LPS and observed for delivery of pups 24 h later. B) Rag1^−/−mice were pretreated on Day 10 of gestation with either whole spleen and lymphocytes or purified CD4⁺ T cells from normal mice. On Day 15 of gestation, they were injected with LPS and observed for delivery of pups 24 h later (Day 16 of gestation). Y axis, proportion of mothers that delivered at least one pup prematurely; x axis, pretreatment and number of mice in the groups tested. P values listed are a comparison between: untreated Rag1^−/− mice and normal B6/J mice; untreated Rag1^−/− mice and Rag1^−/− mice pretreated with whole lymphocytes; or untreated Rag1^−/− mice and Rag1^−/− mice pretreated with CD4⁺ T cells alone (chi-square analysis). Significance was also obtained for each comparison if Fischer exact testing was used (P < 0.0001, P = 0.03, and P = 0.04, respectively).

FIG. 5.

Presence of CD4⁺ T cells in the uterus of Rag1^−/− mice given normal CD4⁺ cells. Pregnant female Rag1^−/− mice were injected with ∼2 million B6 CD4⁺ T cells on Day 10 of gestation and then injected with 3 μg LPS on Gestational Day 15. Shown are representative examples from mice that either did not (left, upper and lower panels) or did (right, upper and lower panels) subsequently deliver 24 h later. Upper plots: the percentage of lymphocytes that were CD4⁺. Lower plots: the percentage of CD4⁺ cells that were TCR positive.

FIG. 6.

CD4⁺TCR⁺ FOXP3⁺ cells in the uterine draining nodes of pregnant mice given LPS. Normal mice (upper panels) were mated and given 3 μg LPS on Day 15 of gestation. After 24 h, the mice were euthanized and found to have not undergone PTD. Nodes draining the uterus were isolated and stained extracellularly for CD4 and T cell receptor, followed by intracellular staining for the molecule FOXP3. Rag1^−/− mice (lower panels) were given CD4⁺ T cells on Day 10 of gestation, and then 3 μg of LPS on Day 15. After 24 h, their tissues were isolated for study. Left and right panels: examples of the data obtained. Left panels show percentage of CD4⁺ T cells in uterine draining node lymphocytes. Right panels show percentage of CD4⁺ T cells that were found to be positive for FOXP3.