We investigated the responses of the fetal thymus to chorioamnionitis and antenatal corticosteroids, fetal exposures which are common prior to very preterm delivery 
. We found that intra-amniotic exposure to LPS activated the fetal thymus as shown with increased TLR4
mRNA levels and CD3 expression, decreased Foxp3-positive cells and altered thymic structure.
In organ cultures of the fetal thymus, blocking of Shh signaling accelerated T-cell differentiation 
while additional Shh protein arrested T-cell development 
. Cortical epithelial cells of the thymus also produced BMP4 which controls early T-cell development 
. Inhibition of the BMP4 signaling cascade was required for further differentiation of T-cells at several checkpoints during development 
and BMP4 expression decreased in response to LPS-induced chorioamnionitis indicating increased differentiation of thymic T-cells. This increased differentiation was reflected in an increase in CD3 expression, which is expressed on mature T-cells 
. Taken together these results indicate that exposure to intra-amniotic LPS resulted in differentiation of T-cells with an accumulation of mature T-cells in the medulla and depletion of early progenitor T-cells in the cortex, which was consistent with the changed thymic structure.
Although the involution response of the fetal thymus has been described in several human and animal studies 
, the mechanistic changes behind this response remain unclear. Kunzmann et al. 
showed an acute thymic involution with changes in Foxp3-positive cells in an ovine model of chorioamnionitis up to 5 days after exposure to LPS. Here, we further characterized this process by demonstrating that the effects of LPS on the thymic population and structure were detected 14 days after the LPS exposure and were not due to changes in proliferation or apoptosis. A persistent increase in medulla area due to the accumulation of mature, differentiated T-cells may explain the change in thymic structure.
Based on the anti-inflammatory properties of antenatal corticosteroids, a reduced inflammatory response after exposure to LPS was expected 
. Corticosteroids can exert anti-inflammatory effects by upregulation of the IκB family, which are cytoplasmic inhibitors of NF-κB, and by direct antagonism between the glucocorticoid receptor and NF-κB, resulting in blocked transcription of responsive genes. However, in our study betamethasone administration after the inflammatory stimulus did not reverse the LPS-induced increase in TLR4
and CD3 in the fetal thymus. LPS has a half-life of 1.7 days in the amniotic fluid and was still detectable 15 days after intra-amniotic injection 
. Because of the slow clearance, LPS may induce a persistent inflammatory response which is in line with measurements of pulmonary inflammation in these animals 
Surprisingly, betamethasone administration 7 days before LPS exposure attenuated activation with no signs of inflammation in the fetal thymus. Thymic structure changed slightly due to the pro-apoptotic properties of antenatal corticosteroids 
. Previous reports demonstrated only inhibitory effects of corticosteroids on the immune system for a maximum of 48 hours 
. Our results indicate that the antenatal corticosteroids used clinically can potentially desensitize the fetal immune system and attenuate a response to LPS. Paradoxically, these ‘longer term’ inhibitory effects of corticosteroids on the fetal immune system did not occur in the animals that were exposed to LPS and then betamethasone 7 days later as the immune system remained activated. Corticosteroids are potent immune-modulatory hormones which can have long term effects on the HPA-axis and subsequently on the function of the immune system 
which may be reflected in the unresponsiveness of the fetal immune system to LPS after corticosteroid pre-treatment. The longer-term effects of the changes in cell composition and activation of the fetal thymus after exposure to antenatal corticosteroids may depend on the timing of the exposure and the developmental stage of the immune system and therefore remain to be further determined.
Although administration of antenatal corticosteroids to pregnant women at risk of preterm birth is one of the most effective and important therapies in perinatal medicine, concerns remain about effects on fetal growth and development of the brain and immune system. Antenatal corticosteroid treatment can change the population and function of cord blood lymphocytes of preterm infants 
and may induce thymic involution 
. Antenatal dexamethasone also was associated with decreased T-cell numbers in the fetal rat thymus and spleen and changes in the CD4/CD8 ratio 
. Dexamethasone treatment of neonatal rats changed the peripheral T-cell repertoire and altered endogenous corticosterone production of thymic epithelial cells during neonatal life 
. These changes may impair the functional maturity of the neonatal immune system and could contribute to the increased incidence and adverse outcome of infections 
Our findings contribute to the current concept that events during fetal life can potentially alter the function of the immune system 
. The clinical associations between chorioamnionitis and adverse outcomes in later life such as BPD 
or asthma 
may be mediated in part by changes in immune responses.
In summary, our results demonstrate that fetal exposure to intra-amniotic LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus. However, insights into the effects of LPS and corticosteroids on molecular pathways such as BMP4 and Shh are limited. Due to the low expression BMP4 and a lack of specific reagents for Shh protein for ovine tissue, we were not able to perform more detailed analysis of these pathways. Further analysis at different time intervals of exposure are necessary to better understand the interactive effects of chorioamnionitis and corticosteroids on the fetal thymus. Although the design of the study does not allow us to evaluate the dynamics of the changes induced by LPS and corticosteroids, this report illustrates the complicated interactions of pro- and anti-inflammatory stimuli on the development of the fetal immune system.