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Early life immune challenge alters innate immune responses to lipopolysaccharide: implications for host defense as adults

Hotchkiss Brain Institute, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada

¹Correspondence: Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. N.W., Calgary T2N 4N1, AB, Canada. E-mail: pittman{at}ucalgary.ca

SPECIFIC AIMS

Fever is the most common manifestation of the innate immune response. Animals prevented from developing a fever have increased morbidity and greater mortality after infection. We show here that animals that have received a single neonatal immune challenge display attenuated febrile responses to infection as adults. In this study, we have investigated the role of: 1) proinflammatory cytokine production and 2) activation of the hypothalamic-pituitary-adrenal (HPA) axis in suppressed adult febrile responses caused by neonatal LPS challenge.

PRINCIPAL FINDINGS

1. Animals treated neonatally with LPS show an attenuated adult febrile response to LPS, which is correlated with a reduction in the plasma levels of proinflammatory cytokines
Animals treated neonatally (Pnd14) with lipopolysaccharide (LPS) display a significantly attenuated febrile response to an adult LPS challenge compared with those that had received saline neonatally (Fig. 1 A). To determine the mechanism underlying the attenuation of this important facet of the host-defense response, we measured the proinflammatory plasma cytokine levels in both neonatal treatment groups 2 h after adult LPS challenge. We found that the basal levels (saline as adults) of cytokines TNF-, IL-1ß, and IL-6 were not significantly different between animals that had received LPS or saline neonatally (Fig. 1B-D ). However, when challenged with LPS as adults, the plasma levels of TNF- and IL-6 were significantly attenuated (P<0.05) in animals that had been challenged neonatally with LPS when compared with those that had received saline neonatally (Fig. 1B, D ). A similar trend was observed with IL-1ß, although it was not statistically significant (Fig. 1C ).

View larger version (6K): [in this window] [in a new window]   Figure 1. A) Body temperature responses (mean ±SE) to an intraperitoneal LPS challenge (indicated by the arrow) of adult rats treated neonatally with either saline () or LPS (•). Animals that had received LPS neonatally display suppressed febrile responses compared with animals that had received saline neonatally (fever index: 2.3±0.73 vs. 4.8±0.42°Cxh, P<0.05, n=7 for each group). B–D) Proinflammatory cytokine responses (mean ±SE) 2 h after intraperitoneal saline or LPS challenge in adult rats. The neonatal LPS challenge did not change the basal level of cytokines when compared with animals treated with saline neonatally (L/S vs. S/S, P>0.05). When treated with LPS as adults, those that had received LPS neonatally had significantly reduced levels of TNF- and IL-6 compared with animals treated neonatally with saline (*P<0.05). S/S, neonatal saline + adult saline (n=4); L/S, neonatal LPS + adult saline (n=4); S/L, neonatal saline + adult LPS (n=9); L/L, neonatal LPS + adult LPS (n=9); N/D, not detectable.

2. Animals treated neonatally with LPS have a reduction in the amount of LPS-induced phosphorylation of inhibitory-B- as adults
The production of cytokines after an adult LPS challenge is mainly under the control of the transcription factor NF-B. To assess the amount of NF-B activated after the adult LPS challenge, we used Western blot analysis to determine the amount of inhibitory-B- (P-IB-), a binding protein of NF-B, which has been phosphorylated (i.e., released from NF-B). The basal levels (saline as adults) of P-IB- were not significantly different (P<0.05) in the spleen or liver in adult animals that had received LPS or saline neonatally. When challenged with LPS as adults, those animals that had received saline neonatally responded with a significant increase (P<0.05) in the amount of P-IB- in both the spleen and liver. However, animals that had been treated neonatally with LPS did not show a significant increase (P>0.05) in the amount of P-IB- in the spleen or the liver after adult LPS challenge. This indicates that a neonatal LPS challenge can result in a reduced amount of phosphorylated IB-, thus reflecting reduced activation of NF-B, in response to adult LPS challenge.

3. Animals treated neonatally with LPS show increased corticosterone response to adult LPS challenge
Corticosteroids have potent anti-inflammatory effects through their ability to modify cytokine expression. In light of this, we hypothesized that LPS-induced corticosterone secretion may be underlying the reduction in the proinflammatory cytokines observed in animals treated neonatally with LPS. Trunk blood samples were taken from adult animals, which had been treated neonatally with either LPS or saline at several time points after the adult LPS challenge. Our results show that the basal levels of free corticosterone were unchanged by the neonatal LPS challenge (P>0.05). After the adult LPS challenge, both groups showed a significant increase in plasma corticosterone (P<0.05). However, the peak corticosterone response was significantly higher in adult animals that had been challenged neonatally with LPS when compared with animals that had received saline neonatally (P<0.05, t=60 min.). By 120 min after adult LPS challenge, the corticosterone levels were no longer statistically different between the two groups, but were still significantly higher than baseline values.

4. Blocking the corticosterone effect by either adrenalectomy or RU-486 restored both the febrile and cytokine responses to adult LPS challenge
To assess whether the increase in plasma corticosterone was causing the attenuated febrile response, all animals were adrenalectomized prior to adult LPS challenge. Animals treated neonatally with either LPS or saline displayed almost identical febrile responses (P>0.05). To confirm that these results were due to the loss of corticosterone and not to an alteration caused by the adrenalectomy, animals were administered RU-486, a glucocorticoid receptor blocker, prior to adult LPS challenge. When the vehicle for RU-486 (i.e., DMSO) was given alone before adult LPS challenge, animals that had received LPS neonatally still displayed an attenuated febrile response (P<0.05). When RU-486 was given prior to adult LPS challenge, the two neonatal treatment groups displayed almost identical febrile responses (P>0.05). These results suggest that corticosterone is directly involved in the suppression of fever after a neonatal immune challenge. Consequently, we hypothesized that the mechanism underlying this phenomenon was the suppression of cytokine production. To test this hypothesis, we assayed the plasma levels of proinflammatory cytokines of animals treated neonatally with either LPS or saline and given RU-486 prior to the adult LPS challenge. In this experiment, the plasma levels of TNF-, IL-1ß, and IL-6 were not significantly different between the two neonatal treatment groups that had received the RU-486 prior to the adult LPS challenge, indicating that the effect of RU-486 on the normalization of the febrile response was due to removal of the amplified corticosterone-induced suppression of cytokine synthesis after adult LPS challenge.

CONCLUSIONS AND SIGNIFICANCE

We have shown that a single immune challenge during the neonatal period results in long-lasting alterations in the innate immune response to infection as adults. These alterations are manifested by a reduction in the febrile response elicited by an adult LPS challenge. Our results show that animals that have received a neonatal LPS challenge also showed an attenuated production of the proinflammatory cytokines TNF- and IL-6 after adult LPS challenge. These cytokines are known to be key mediators in the generation of febrile response after an LPS challenge, thus a reduction in the amount of cytokines produced is likely responsible for the suppressed febrile responses observed in animals treated neonatally with LPS. This led us to hypothesize that the effects of a neonatal LPS challenge may be mediated by a shift in the balance between proinflammatory and anti-inflammatory mediators activated in response to an adult LPS challenge.

One of the most potent anti-inflammatory pathways activated by LPS is the hypothalamic-pituitary-adrenal (HPA) axis, which results in the release of corticosterone. The main actions of corticosterone are mediated through its ability to inhibit the activity of the transcription factor NF-B, leading to reduced transcription of proinflammatory mediators. Our results show that animals treated neonatally with LPS display significantly higher free plasma corticosterone levels after adult LPS challenge compared with those that received saline neonatally. These high corticosterone levels can account for the observed reduction in NF-B activity, indicated by the reduction in the amount of P-IB- in peripheral immune organs. These results support the hypothesis that an increase in the anti-inflammatory HPA response caused by neonatal LPS challenge is the underlying factor in suppressing the febrile response.

To confirm this hypothesis, we used two techniques to eliminate the effects of corticosterone on the inflammatory response: 1) adrenalectomy to remove the source of corticosterone; and 2) pharmacological blockade of the glucocorticoid receptor with RU-486 prior to the adult LPS challenge. Here, when the effects of corticosterone are eliminated, the attenuated febrile and cytokine responses seen in animals treated neonatally with LPS are normalized. This gives a direct causal link between the heightened corticosterone response induced by a neonatal LPS challenge and the attenuated cytokine and febrile responses to an immune challenge as adults.

Our results prove for the first time that a single neonatal immune challenge alters the adult acute phase response through a mechanism involving attenuated febrile and cytokine responses elicited by a heightened corticosterone response (Fig. 2 ). Given the importance of the febrile response in the effective host defense response, this alteration may have potentially deleterious consequences on adult host defense response. That is, the ability of an individual to effectively combat disease may be significantly compromised by these alterations elicited by early life immune challenges. These results may also provide a partial basis for individual differences in the susceptibility to infection.

View larger version (11K): [in this window] [in a new window]   Figure 2. Top: Schematic depiction of a normal response to LPS in animals that had not received a neonatal LPS challenge. LPS elicits an NF-B mediated proinflammatory cytokine response which signals to the brain via COS-2/PGE2 dependent mechanism. In the CNS, PGE2 mediates the production of normal febrile and host-defense response. Bottom: Schematic depiction of the alterations in the adult caused by an early life LPS challenge. LPS elicits an increased corticosterone response, which inhibits the activity of NF-B, resulting in decreased production of proinflammatory cytokines, reduced COX-2/PGE2 induction that results in an attenuated febrile and host defense response. ( Activation, Inhibition).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.04-3569fje;

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