Intraamniotic lipopolysaccharide exposure changes cell populations and structure of the ovine fetal thymus

Abstract

Rationale: Chorioamnionitis induces preterm delivery and acute involution of the fetal thymus which is associated with postnatal inflammatory disorders. We studied the immune response, cell composition, and architecture of the fetal thymus following intraamniotic lipopolysaccharide (LPS) exposure.

Methods: Time-mated ewes received an intraamniotic injection of LPS 5, 12, or 24 hours or 2, 4, 8, or 15 days before delivery at 125 days gestational age (term = 150 days).

Results: The LPS exposure resulted in decreased blood lymphocytes within 5 hours and decreased thymic corticomedullary ratio within 24 hours. Thymic interleukin 6 (IL6) and IL17 messenger RNA (mRNA) increased 5-fold 24 hours post-LPS exposure. Increased toll-like receptor 4 (TLR4) mRNA and nuclear factor κB positive cells at 24 hours after LPS delivery demonstrated acute thymic activation. Both TLR4 and IL1 mRNA increased by 5-fold and the number of Foxp3-positive cells (Foxp3+ cells) decreased 15 days after exposure.

Conclusion: Intraamniotic LPS exposure caused a proinflammatory response, involution, and a persistent depletion of thymic Foxp3+ cells indicating disturbance of the fetal immune homeostasis.

Keywords: fetal inflammatory response syndrome; immune modulation; prematurity; thymic involution.

Figure 1.

Corticomedullary (CM) ratio.The CM ratio of the thymus was analyzed in hematoxylin and eosin (H&E) sections. Representative images of controls (A) and 8-day lipopolysaccharide (LPS)-exposed animals (B). The CM ratio decreased after 24 hours and 8 days of LPS exposure compared to controls (C). *P < .05 versus controls.

Figure 2.

Ki67 and cleaved caspase 3 expression in the fetal thymus.The number of cleaved caspase 3 expressing cells increased in the cortex of the thymus 12 hours after the exposure to LPS compared to controls (A). Representative images of cleaved caspase 3 expression in the thymus for controls (B) and 12-hour LPS-exposed animals (C). The percentage of Ki67-positive cells decreased in the cortex of the thymus 4 days after the exposure to LPS compared to controls (D). Representative images for Ki67 expression in the thymus are shown for controls (E) and 4-day LPS-exposed animals (F). *P < .05 versus controls.

Figure 3.

Increased CD3-positive cells in the thymus. The percentage of CD3-positive cells in the medulla of the thymus was evaluated by immunohistochemistry. Representative images of controls (A), 5 hours LPS exposure (B), and 8 days LPS exposure (C). The percentage of CD3-positive cells increased in the medulla of the thymus 5 hours, 2 days, and 8 days after LPS exposure (D). *P < .05 versus controls.

Figure 4.

Decreased Foxp3-positive cells after LPS exposure. Representative images of Foxp3 expression in the thymus for controls (A), 4 days LPS exposure (B), and 15 days LPS exposure (C). The number of Foxp3-positive cells decreased in the medulla 4 and 15 days after LPS exposure compared to controls (D). *P < .05 versus controls.

Figure 5.

Cytokine messenger RNA (mRNA) profile. Interleukin 1 (IL1) mRNA levels (A) increased from 24 hours up to 15 days after the exposure to LPS. The mRNA levels of IL6 (B) increased significantly 12 hours, 24 hours, 2 days, and 15 days after the LPS exposure compared to controls. The IL17 mRNA levels (C) were increased 6-fold at time points 12 hours, 24 hours, 4 days, and 8 days. The interferon γ (IFNγ) mRNA (D) increased up to 5-fold 4, 8, and 15 days after LPS exposure whereas IL10 mRNA levels decreased 5 hours and 2 days after the exposure. *P < .05 versus controls.

Figure 6.

Messenger RNA (mRNA) levels of toll-like receptors (TLR)2 and 4 in the fetal thymus.The TLR2 (A) mRNA increased 12 hours after lipopolysaccharide (LPS) exposure compared to controls. The TLR4 (B) mRNA was increased 2 hours, 8 days, and 15 days after LPS exposure in the fetal thymus. *P < .05 versus controls.

Figure 7.

Increased number of NF)-κB positive cells increased after LPS exposure.Representative images of NF-κB expression in the thymus are shown for controls (A) and 8-day LPS-exposed animals (B).The number of NF-κB positive cells in the medulla of the thymus increased 24 hours and 8 days after the exposure to intraamniotic lipopolysaccharide (LPS; C). *P < .05 versus controls.