Thymic changes after chorioamnionitis induced by intraamniotic lipopolysaccharide in fetal sheep

Abstract

Objective: Regulatory T lymphocytes mediate homeostasis of the immune system and differentiate under the control of the transcription factor FoxP3 in the fetal thymus. We asked whether fetal inflammation caused by chorioamnionitis would modulate thymus development.

Study design: Fetal sheep were exposed to an intraamniotic injection of 10 mg lipopolysaccharide at 5 hours, 1 day, 2 days, or 5 days before delivery at 123 gestation days. Cord blood lymphocytes, plasma cortisol, and thymus weight were measured. Glucocorticoid receptor-, activated caspase-3-, Ki-67-, proliferating cell nuclear antigen-, nuclear factor-kappaB-, and FoxP3-positive cells were immunohistochemically evaluated in thymus.

Results: Intraamniotic lipopolysaccharide exposure decreased the number of circulating lymphocytes by 40% after 1 day. Thymus-to-body weight ratios were reduced in all lipopolysaccharide groups by a maximum of 40% at 5 days. Lipopolysaccharide exposure modestly increased plasma cortisol concentration, increased nuclear factor-kappaB immunostaining in fetal thymus and reduced the number of FoxP3-positive cells by 40% at 1 day.

Conclusion: Intraamniotic exposure to lipopolysaccharide induced thymic changes and influenced thymic FoxP3 expression.

Figure 1

Study design. Four to seven animals per group received LPS 10 mg or NaCl 0.9% (control); 5h, 1d, 2d or 5d after treatment the lambs were delivered and evaluated.

Figure 2

Quantification of blood lymphocytes (A) and thymus weight (B). A: Number of lymphocytes in complete white blood cell counts from cord blood samples at delivery (*p<0.05 vs. control). B: Thymus/body ratio was lower than control after intraamniotic LPS injection at 5h, 1d, 2d or 5d (*p<0.05 vs. control).

Figure 2

Quantification of blood lymphocytes (A) and thymus weight (B). A: Number of lymphocytes in complete white blood cell counts from cord blood samples at delivery (*p<0.05 vs. control). B: Thymus/body ratio was lower than control after intraamniotic LPS injection at 5h, 1d, 2d or 5d (*p<0.05 vs. control).

Figure 3

A: Quantification of cortisol in plasma. B: quantification of glucocorticoid receptor immunostaining in the fetal thymus cortex (*p < 0.05 versus control).

Figure 3

A: Quantification of cortisol in plasma. B: quantification of glucocorticoid receptor immunostaining in the fetal thymus cortex (*p < 0.05 versus control).

Figure 4

Quantification of apoptose in fetal thymus with staining for caspase-3. Evaluation of caspase-3 expression in thymus in control group (A) and in LPS group (1d) (B) by immunohistochemistry. Representative sections for each time point are shown for caspase-3 (A+B). Magnification 200x. C. Quantification of caspase-3-positive thymocytes in thymus sections by immunohistochemistry.

Figure 5

Quantification of cell proliferation in fetal thymus with staining for Ki-67 (A-C) and PCNA (D). Evaluation of Ki-67 expression in thymus in control group (A) and in LPS group (B) by immunohistochemistry. Representative sections for each time point are shown. Magnification 200x. C. Quantification of Ki-67 (C) and caspase-3-positive (D) thymocytes in thymus sections by immunohistochemistry.

Figure 6

Detection of NF-κB-positive thymocytes in cortex of thymus in control group (A) and in LPS group (B) by immunohistochemistry. Representative sections for each time point are shown. Magnification 200x. C. Quantification of NF-κB-positive thymocytes in thymus sections identified by immunohistochemistry. (*p<0.05 vs. controls).

Figure 7

Evaluation of FoxP3 expression in cortex of thymus in control group (A) and in LPS group (B) by immunohistochemistry. Representative sections for each time point are shown. Magnification 200x. C. Quantification of FoxP3-positive thymocytes in thymus sections by immunohistochemistry (*p<0.05 vs. controls).