Prenatal Dexamethasone and Postnatal High-Fat Diet Decrease Interferon Gamma Production through an Age-Dependent Histone Modification in Male Sprague-Dawley Rats

Abstract

Overexposure to prenatal glucocorticoid (GC) disturbs hypothalamic-pituitary-adrenocortical axis-associated neuroendocrine metabolism and susceptibility to metabolic syndrome. A high-fat (HF) diet is a major environmental factor that can cause metabolic syndrome. We aimed to investigate whether prenatal GC plus a postnatal HF diet could alter immune programming in rat offspring. Pregnant Sprague-Dawley rats were given intraperitoneal injections of dexamethasone or saline at 14-21 days of gestation. Male offspring were then divided into four groups: vehicle, prenatal dexamethasone exposure, postnatal HF diet (VHF), and prenatal dexamethasone exposure plus a postnatal HF diet (DHF). The rats were sacrificed and adaptive immune function was evaluated. Compared to the vehicle, the DHF group had lower interferon gamma (IFN-γ) production by splenocytes at postnatal day 120. Decreases in H3K9 acetylation and H3K36me3 levels at the IFN-γ promoter correlated with decreased IFN-γ production. The impaired IFN-γ production and aberrant site-specific histone modification at the IFN-γ promoter by prenatal dexamethasone treatment plus a postnatal HF diet resulted in resilience at postnatal day 180. Prenatal dexamethasone and a postnatal HF diet decreased IFN-γ production through a site-specific and an age-dependent histone modification. These findings suggest a mechanism by which prenatal exposure to GC and a postnatal environment exert effects on fetal immunity programming.

Keywords: IFN-γ; age-dependent; high-fat diet; histone modification; prenatal glucocorticoid.

Figure 1

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of innate cytokine mRNA expressions in the rat spleens at the adolescent stage. Representative RT-PCR analysis of (A) IL-6; (B) IL-8; and (C) TNF-α mRNA levels of the spleens of the four groups at post-natal day 120 (D120) (PPIB as internal control gene) (n = 5 or 6 for each group). Abbreviations: VEH; vehicle: DEX; prenatal dexamethasone exposure: VHF; postnatal high-fat diet exposure: DHF; prenatal dexamethasone plus postnatal high-fat diet exposure.

Figure 2

Effects of prenatal dexamethasone/postnatal high-fat diet treatment on pro-inflammatory mediator production in rat splenocytes. Isolated splenocytes from the VEH, DEX, VHF, and DHF groups at D120 were suspended at 2 × 10⁶/mL in 24-well plates then treated with 100 ng/mL of LPS. The culture supernatants were collected at 24 h and then levels of (A) MCP-1; (B) IP-10; (C) IL-1β; and (D) TNF-α were detected using a Multiplex Assay system or ELISA, respectively (n = 5 or 6 for each group). * p < 0.05.

Figure 3

Effects of prenatal dexamethasone/postnatal high-fat diet treatment on rat splenocyte proliferation and IL-2 productions at D120. (A) Isolated splenocytes were treated with ConA, and splenocyte proliferation was assayed using BrdU assays as described in the Materials and Methods section. The proliferation index was presented as the ratio of optical density of ConA stimulation/optical density of phosphate buffered saline stimulation. The data shown are mean ± SE from five or six replicate experiments (* p < 0.05 compared with the VEH group); (B) For IL-2 production, splenocytes were suspended at 2 × 10⁶/mL in 24-well plates and then treated with 5 μg/mL of ConA. The culture supernatants were collected at 72 h and then IL-2 was detected using a Multiplex Assay system (Millipore).

Figure 4

Effects of prenatal dexamethasone/postnatal high-fat diet treatment on Th1/Th2 cytokine production in rat splenocytes at D120. Isolated splenocytes from the VEH, DEX, VHF, and DHF groups at D120 were suspended at 2 × 10⁶/mL in 24-well plates and then treated with 5 μg/mL of ConA. The culture supernatants were collected at 72 h and then levels of (A) IFN-γ; (B) IL-4; and (C) IL-13 were detected (n = 5 or 6 for each group). * p < 0.05.

Figure 5

Effects of prenatal dexamethasone/postnatal high-fat diet treatment on Th3 cytokine production in rat splenocytes at D120. Isolated splenocytes from the VEH, DEX, VHF and DHF groups at postnatal day 120 were treated with 5 μg/mL of ConA. The culture supernatants were collected at 72 h and then levels of (A) IL-10; (B) TGF-β1; (C) TGF-β2; and (D) TGF-β3 were detected (n = 5 or 6 for each group). * p < 0.05.

Figure 6

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of Th1-related transcription factor T-bet mRNA expression in rat spleens at D120. T-bet mRNA levels in rat spleen tissues are relative to the VEH group at post-natal day 120 (PPIB as internal control gene) (n = 5 or 6 for each group).

Figure 7

Histone H3 lysine acetylation and methylation levels at the IFN-γ promoter of the spleens with/without the indicated treatment at D120. Bar graphs showing the total H3 lysine, H3K9 and H3K14 acetylation, H3K4me1, H3K4me3, and H3K36me3 levels of the indicated treatment group relative to the vehicle group at the IFN-γ promoter. ChIP assays were performed as described in the Materials and Methods section. The results are expressed as fold difference over the vehicle (mean ± SEM; * p < 0.05; n = 3 to 5 for each group).

Figure 8

Effects of prenatal dexamethasone/postnatal high-fat diet treatment on IFN-γ production by rat splenocytes at D180. Isolated splenocytes from the VEH, DEX, VHF, and DHF groups at D180 were suspended at 2 × 10⁶/mL in 24-well plates and then treated with 5 μg/mL of ConA. The culture supernatants were collected at 72 h and then the level of IFN-γ was detected (* p < 0.05; n = 3 to 5 for each group).

Figure 9

Histone H3 lysine acetylation and methylation levels at the IFN-γ promoter of spleens with/without the indicated treatment at D180. Bar graphs show the total H3 lysine, H3K9 and H3K14 acetylation, H3K4me1, H3K4me3, and H3K36me3 levels of the indicated treatment groups relative to the vehicle at the IFN-γ promoter. ChIP assays were performed as described in the Materials and Methods section. The results were expressed as fold difference over the vehicle (mean ± SEM; n = 3 for each group).