Production of Proinflammatory Cytokines by Monocytes in Liver-Transplanted Recipients with De Novo Autoimmune Hepatitis Is Enhanced and Induces TH1-like Regulatory T Cells.

A subset of human regulatory T cells (Tregs) can secrete IFN-γ or IL-17, and thus share features of TH1 or TH17 effector cells and lose suppressive function. The main factors driving this differentiation of Tregs toward a proinflammatory phenotype include IL-12 for TH1-like and IL-6 for TH17-type Tregs. In this study we show that Tregs of patients with de novo autoimmune hepatitis (dAIH) display increased frequencies of proinflammatory IFN-γ and IL-17 cytokines. Irrespective of a fully demethylated FOXP3 locus, Tregs of subjects with dAIH are functionally impaired. In line with the observed Treg phenotype, we detected the presence of two dominant cytokines (IL-12 and IL-6) clustering with CD68(+) monocyte/macrophage cells in livers of subjects with dAIH, and isolated monocytes of subjects with dAIH secrete high levels of proinflammatory IL-12 and IL-6, suggesting that this inflammatory milieu is key for functional impairment of Tregs. Importantly, the blockade of IFN-γ partially restores suppressive function of Tregs of subjects with dAIH, indicating that monocyte/macrophage-derived triggers might play a central role in Treg dysfunction and pathogenesis of dAIH.

Endodermal and mesenchymal cross talk: a crossroad for the maturation of foregut organs.

The developmental stages of each foregut organ are intimately linked to the development of the other foregut organs such that the ultimate function of any one foregut organ, such as the metabolic function of the liver, depends on organizational changes associated with the maturation of multiple foregut organs. These changes include: (i) proliferation of the intrahepatic bile ducts and hepatoblasts within the liver coinciding with parenchymal expansion, (ii) elongation of extrahepatic bile ducts, which allows for proper gallbladder (GB) formation, and (iii) duodenal elongation and rotation, which coincides with all of the above to connect the intrahepatic, extrahepatic, and pancreatic ductal systems with the intestine. It is well established that cross talk between endodermal and mesenchymal components of the foregut occurs, particularly regarding the vascularization of developing organs. Furthermore, genetic mutations in mesenchymal and hepatic compartments of the developing foregut result in similar foregut pathologies: hypoplastic liver, absence of GB, biliary atresia (intrahepatic and/or extrahepatic), and failure of gut elongation and rotation. Finally, these shared pathologies can be linked to deficiencies in genes specific to the septum transversum mesenchyme (Hes1, Hlx, and Foxf1) or liver (Hhex and Hnf6), illustrating the complexity of such cross talk.

Deprivation of maternal care has long-lasting consequences for the hypothalamic-pituitary-adrenal axis of zebra finches.

Early-life stress caused by the deprivation of maternal care has been shown to have long-lasting effects on the hypothalamic-pituitary-adrenal (HPA) axis in offspring of uniparental mammalian species. We asked if deprivation of maternal care in biparental species alters stress responsiveness of offspring, using a biparental avian species--the zebra finch, Taeniopygia guttata. In our experiment, one group of birds was raised by both male and female parents (control), and another was raised by males alone (maternally deprived). During adulthood, offspring of both groups were subjected to two stressors (restraint and isolation), and corticosterone concentrations were measured. Additionally, we measured baseline levels of the two corticosteroid receptors--glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)--in the hippocampus, hypothalamus and cerebellum. Our results suggest that maternally deprived offspring are hyper-responsive to isolation in comparison with controls. Furthermore, mRNA levels of both GR and MR receptors are altered in maternally deprived offspring in comparison with controls. Thus, absence of maternal care has lasting consequences for HPA function in a biparental species where paternal care is available.

Evolution of ligand specificity in vertebrate corticosteroid receptors.

BACKGROUND: Corticosteroid receptors include mineralocorticoid (MR) and glucocorticoid (GR) receptors. Teleost fishes have a single MR and duplicate GRs that show variable sensitivities to mineralocorticoids and glucocorticoids. How these receptors compare functionally to tetrapod MR and GR, and the evolutionary significance of maintaining two GRs, remains unclear. RESULTS: We used up to seven steroids (including aldosterone, cortisol and 11-deoxycorticosterone [DOC]) to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal (Mus musculus) and the midshipman fish (Porichthys notatus), a teleost model for steroid regulation of neural and behavioral plasticity. Variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of MR and GRs from the distantly related daffodil cichlid (Neolamprologus pulcher), another teleost model for neurobehavioral plasticity. Both teleost species had a single MR and duplicate GRs. All MRs were sensitive to DOC, consistent with the hypothesis that DOC was the initial ligand of the ancestral MR. Variation in GR steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences. CONCLUSION: The mineralocorticoid sensitivity of duplicate GRs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two GRs.

Corticosteroid receptor expression in a teleost fish that displays alternative male reproductive tactics.

Corticosteroid signaling mechanisms mediate a wide range of adaptive physiological responses, including those essential to reproduction. Here, we investigated the presence and relative abundance of corticosteroid receptors during the breeding season in the plainfin midshipman fish (Porichthys notatus), a species that has two male reproductive morphs. Only type I "singing" males acoustically court females and aggressively defend a nest site, whereas type II "sneaker" males steal fertilizations from nesting type I males. Cloning and sequencing first identified glucocorticoid (GR) and mineralocorticoid (MR) receptors in midshipman that exhibited high sequence identity with other vertebrate GRs and MRs. Absolute-quantitative real-time PCR then revealed higher levels of GR in the central nervous system (CNS) of type II males than type I males and females, while GR levels in the sound-producing, vocal muscle and the liver were higher in type I males than type II males and females. MR expression was also greater in the CNS of type II males than type I males or females, but the differences were more modest in magnitude. Lastly, plasma levels of cortisol, the main glucocorticoid in teleosts, were 2- to 3-fold greater in type II males compared to type I males. Together, the results suggest a link between corticosteroid regulation and physiological and behavioral variation in a teleost fish that displays male alternative reproductive tactics.

Divergent expression of 11beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase genes between male morphs in the central nervous system, sonic muscle and testis of a vocal fish.

The vocalizing midshipman fish, Porichthys notatus, has two male morphs that exhibit alternative mating tactics. Only territorial males acoustically court females with long duration (minutes to >1h) calls, whereas sneaker males attempt to steal fertilizations. During the breeding season, morph-specific tactics are paralleled by a divergence in relative testis and vocal muscle size, plasma levels of the androgen 11-ketotestosterone (11KT) and the glucocorticoid cortisol, and mRNA expression levels in the central nervous system (CNS) of the steroid-synthesizing enzyme aromatase (estrogen synthase). Here, we tested the hypothesis that the midshipman's two male morphs would further differ in the CNS, as well as in the testis and vocal muscle, in mRNA abundance for the enzymes 11beta-hydroxylase (11betaH) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) that directly regulate both 11KT and cortisol synthesis. Quantitative real-time PCR demonstrated male morph-specific profiles for both enzymes. Territorial males had higher 11betaH and 11betaHSD mRNA levels in testis and vocal muscle. By contrast, sneaker males had the higher CNS expression, especially for 11betaHSD, in the region containing an expansive vocal pacemaker circuit that directly determines the temporal attributes of natural calls. We propose for territorial males that higher enzyme expression in testis underlies its greater plasma 11KT levels, which in vocal muscle provides both gluconeogenic and androgenic support for its long duration calling. We further propose for sneaker males that higher enzyme expression in the vocal CNS contributes to known cortisol-specific effects on its vocal physiology.

Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis.

De novo autoimmune hepatitis (DAIH) is an important cause of late allograft dysfunction following liver transplantation, but its cause and underlying pathogenesis remains unclear. We sought to identify specific innate and adaptive immune mechanisms driving the pro-inflammatory cytokine secreting regulatory T cell (Treg) phenotype in DAIH and determine if modulation of these pathways could resolve the inflammatory milieu observed in the livers of patients with DAIH. Here, we demonstrate toll-like receptors (TLRs) 2- and 4-mediated inflammasome activation in CD14++ monocytes, a finding that is key to maintaining dysfunctional Tregs in patients with DAIH. Furthermore, silencing of TLR 2 and 4 in CD14++ monocytes prevented activation of the inflammasome and significantly decreased IFN-γ production by FOXP3+ Tregs. We also observed significantly increase in expression of tumor necrosis factor α-induced protein 3 (TNFAIP3), a negative regulator of the NLRP3 Inflammasome, in monocytes/macrophages of liver transplant subjects who have normal allograft function and do not have DAIH. TNFAIP3 expression was virtually absent in monocytes/macrophages of patients with DAIH. Our findings suggest that autoimmunity in DAIH is promoted by CD14++ monocytes predominantly through activation of inflammatory signaling pathways.

Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling.

Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed in vitro differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene Hhex and show that Hhex(-/-) mESCs fail to differentiate from definitive endoderm (Sox17(+/)Foxa2(+)) to hepatic endoderm (Alb(+)/Dlk(+)). In addition, hepatic culture elicited a >7-fold increase in Vegfa mRNA expression in Hhex(-/-) cells compared to Hhex(+/+) cells. Furthermore, we identified VEGFR2(+)/ALB(+/)CD34(-) in early Hhex(+/+) hepatic cultures. These cells were absent in Hhex(-/-) cultures. Finally, through manipulation of Hhex and Vegfa expression, gain and loss of expression experiments revealed that Hhex shares an inverse relationship with the activity of the Vegf signaling pathway in supporting hepatic differentiation. In summary, our results suggest that Hhex represses Vegf signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of Vegfr2 activity independent of endothelial cells.