Hypothalamo-pituitary and immune-dependent adrenal regulation during systemic inflammation

Abstract

Inflammation-related dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immune-adrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune-adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPA-axis activation mediated by LPS were found in Mx1(Cre+)-MyD88(fl/fl) mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Keywords: Toll-like receptors; adrenal gland insufficiency; the HPA axis.

Fig. 1.

For the adrenocortical cell-specific and general deletion of MyD88, mice with myd88^flox allele were crossed with mice carrying either Akr1b7^Cre transgene (preferentially expressed in adrenocortical cells) or mice with Mx1^Cre transgene (expressed in the liver and hematopoietic system with minor expression in other tissues). The deletion of MyD88 protein was measured by Western blot in (A) spleen tissue lysates of Cre recombinase positive and negative Mx1^Cre-MyD88^fl/fl mice and in (B) adrenal glands of Cre recombinase positive and negative Akr1b7^Cre-MyD88^fl/fl mice. Densitometric analysis of Western blot results revealed a successful deletion of MyD88 in (C) the splenocytes of the Mx1^Cre-MyD88^fl/fl mice (black bars) and in (D) the adrenal glands of Akr1b7^Cre-MyD88^fl/fl mice (gray bars) compared with respective controls (open bars). **P < 0.01.

Fig. 2.

Systemic but not adrenocortical cell-specific TLR signaling is involved in regulation of the HPA axis during endotoxin challenge. Mice with general inactivation of MyD88 signaling (Mx1^Cre+-MyD88^fl/fl; black bars) and specific adrenocortical cell inactivation (Akr1b7^Cre+-MyD88^fl/fl; gray bars) together with respective Cre-negative littermates (open bars) received a single i.p. injection of LPS (1 mg/kg). Three hours and 24 h after LPS injection, mice were killed and plasma (A and B) corticosterone and (C and D) ACTH levels were determined. The results showed that, 3 h after LPS injection, mice with general deletion of MyD88 (A and C) but not mice with specific adrenocortical cell deletion of MyD88 (B and D) demonstrate an impaired glucocorticoid and ACTH response to LPS injection. ***P < 0.001; **P < 0.01; *P < 0.05. n ≥ 6 animals were used in each group. Results are presented as means ± SEM.

Fig. 3.

Systemic but not local adrenocortical cell-specific TLR signaling is involved in the LPS-mediated neutrophil infiltration into adrenal gland. A real-time PCR analysis of neutrophil gene marker PGRP and neutrophil-specific chemokine CXCL2 revealed that (A) systemic but not (B) adrenocortical cell-specific inactivation of TLR signaling within the adrenal microenvironment resulted in decreased leukocyte infiltration into adrenal glands during LPS-induced systemic inflammation. Furthermore, reduced neutrophil infiltration into adrenal glands of (C) Mx1^Cre+-MyD88^fl/fl mice (black bars) was associated with reduced LPS-mediated up-regulation of granulocyte-specific CXCL2 chemokine in the adrenal glands from these animals. In contrast, in mice with (D) adrenocortical-specific deletion of MyD88 (gray bars) and in control animals (open bars), no difference was found. Results are presented as means ± SEM; *P < 0.05.

Fig. 4.

Immune cells are the main regulators involved in the LPS-mediated systemic and intraadrenal inflammation. Real-time PCR results demonstrating almost complete abrogation of LPS-mediated (A) adrenal and (C) plasma IL1 β elevation and a significant decrease in (E) adrenal and (G) plasma IL6 expression at 3 h following LPS injection in mice with general deletion of MyD88 (Mx1^Cre+-MyD88^fl/fl; black bars). In contrast, LPS-modulated (B and F) adrenal and (D and H) plasma IL1 β and IL6 expression did not differ between mice with specific inactivation of TLR signaling in the adrenocortical cells (gray bars) and the respective Cre-negative littermates (open bars). Results are presented as means ± SEM; ***P < 0.001; *P < 0.05.