Toll-Like Receptor-4 Antagonist (+)-Naloxone Confers Sexually Dimorphic Protection From Inflammation-Induced Fetal Programming in Mice

Abstract

Inflammation elicited by infection or noninfectious insults during gestation induces proinflammatory cytokines that can shift the trajectory of development to alter offspring phenotype, promote adiposity, and increase susceptibility to metabolic disease in later life. In this study, we use mice to investigate the utility of a small molecule Toll-like receptor (TLR)4 antagonist (+)-naloxone, the nonopioid isomer of the opioid receptor antagonist (-)-naloxone, for mitigating altered fetal metabolic programming induced by a modest systemic inflammatory challenge in late gestation. In adult progeny exposed to lipopolysaccharide (LPS) challenge in utero, male but not female offspring exhibited elevated adipose tissue, reduced muscle mass, and elevated plasma leptin at 20 weeks of age. Effects were largely reversed by coadministration of (+)-naloxone following LPS. When given alone without LPS, (+)-naloxone elicited accelerated postweaning growth and elevated muscle and fat mass in adult male but not female offspring. LPS induced expression of inflammatory cytokines Il1a, Il1b, Il6, Tnf, and Il10 in fetal brain, placental, and uterine tissues, and (+)-naloxone suppressed LPS-induced cytokine expression. Fetal sex-specific regulation of cytokine expression was evident, with higher Il1a, Il1b, Il6, and Il10 induced by LPS in tissues associated with male fetuses, and greater suppression by (+)-naloxone of Il6 in females. These data demonstrate that modulating TLR4 signaling with (+)-naloxone provides protection from inflammatory diversion of fetal developmental programming in utero, associated with attenuation of gestational tissue cytokine expression in a fetal sex-specific manner. The results suggest that pharmacologic interventions targeting TLR4 warrant evaluation for attenuating developmental programming effects of fetal exposure to maternal inflammatory mediators.

Figure 1.

Effect of (+)-naloxone with and without LPS challenge on gestation length, litter size, and early postnatal growth. Pregnant B6 mice were administered LPS or PBS IP on gd 16.5, followed by (+)-naloxone or PBS IP on gd 16.5, 17.0, 17.5, and 18.0, then allowed to progress to birth. (A) Number of dams with viable pups, (B) gestation length, (C) number of viable pups born per dam, (D) pup weight at 12 to 24 h, (E) pup weight at 8 d, (F) pup weight at 3 wk, (G) percentage of dams with viable pups at 3 wk, and (H) percentage of pups born surviving at 3 wk are shown. Data are (A, G, and H) percentage analyzed by χ² test or (B–F) mean ± SEM analyzed by two-way ANOVA. Number of pregnant dams per group is shown in parentheses. ^a,bDifferent letters indicate differences between groups (P < 0.05). *P < 0.05, effect of LPS; ^#P < 0.05, effect of (+)-naloxone.

Figure 2.

Effect of (+)-naloxone and LPS challenge on postnatal growth trajectory in progeny. Pregnant B6 mice were administered LPS or PBS IP on gd 16.5, followed by (+)-naloxone or PBS IP on gd 16.5, 17.0, 17.5, and 18.0, then allowed to deliver. (A) Growth trajectory of male progeny and (B) female progeny at 4 wk and every subsequent 2 wk until 20 wk of age. n = 14 to 24 male progeny and n = 8 to 21 female progeny per group. Data are the estimated marginal means ± SEM, analyzed by a mixed model linear repeated measures ANOVA and post hoc Sidak t test. *P < 0.05 compared with PBS control.

Figure 3.

Effect of (+)-naloxone exposure in utero with and without LPS challenge on adipocytokines in adult offspring. (A and C) Leptin and (B and D) adiponectin were quantified by microbead assay in serum, and (E and F) the leptin/adiponectin ratio was calculated for (A, C, and E) male and (B, D, and F) female offspring of B6 dams administered LPS or PBS IP on gd 16.5, followed by (+)-naloxone or PBS IP on gd 16.5, 17.0, 17.5, and 18.0, at 20 wk of age. Data are mean ± SEM, analyzed by ANOVA and post hoc Sidak t test. The number of offspring per group is shown in parentheses. ^a,b,cDifferent letters indicate differences between groups (P < 0.05).

Figure 4.

Effect of (+)-naloxone on proinflammatory cytokine expression induced by LPS challenge in placenta and fetal membranes. Pregnant B6 mice were administered LPS or PBS IP on gd 16.5, followed by (+)-naloxone or PBS IP, and 4 h later placenta and fetal membranes were recovered from two implantation sites. Relative expression of (A, F, and K) Il1a, (B, G, and L), Il1b, (C, H, and M) Il6, (D, I, and N) Tnf, and (E, J, and O) Il10 mRNAs were determined in (A–E) placenta, (F–J) fetal membranes, and (K–O) fetal brain by qPCR and normalized to Actb. Data are mean ± SEM relative gene expression of n = 14 to 16 tissues from n = 8 dams per group and were analyzed by one-way ANOVA and a post hoc Sidak t test. ^a,b,cDifferent letters indicate differences between groups (P < 0.05).

Figure 5.

Effect of (+)-naloxone on proinflammatory cytokine expression induced by LPS challenge in uterine decidua and myometrium. Pregnant B6 mice were administered LPS or PBS on gd 16.5, followed by (+)-naloxone or PBS IP, and 4 h later uterine decidua and myometrium were recovered from two implantation sites. Relative expression of (A and F) Il1a, (B and G) Il1b, (C and H) Il6, (D and I) Tnf, and (E and J) Il10 mRNAs were determined in (A–E) decidua and (F–J) myometrium by qPCR and normalized to Actb. Data are mean ± SEM relative gene expression of n = 14 to 16 tissues from n = 8 dams per group and were analyzed by one-way ANOVA and a post hoc Sidak t test. ^a,b,cDifferent letters indicate differences between groups (P < 0.05).