MyTrang Nguyen

The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth

Survival of newborn placental mammals depends on closure of the ductus arteriosus (DA), an arterial connection in the fetus which directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Here we show that morphological changes resulting in closure of the DA in mice are virtually identical to those observed in larger mammals, including humans, and that maintenance of the DA in the open, or patent, state in fetal mice is dependent on prostaglandin synthesis. This requirement is absent in mice lacking the prostaglandin E2 EP4 receptor (EP4(−/−) mice). In EP 4(−/−) mice of the 129 strain, remodelling of the DA fails to occur after birth, resulting in a left-to-right shunt of blood and subsequently in death. This suggests that the neonatal drop in prostaglandin E2 (refs 3,4,5,6,7) that triggers ductal closure is sensed through the EP4 receptor. In contrast, 5% of EP4(−/−) mice of mixed genetic background survive, and selective breeding of these mice leads to a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for ductal closure.

Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.

Genetic evidence supports a critical role for the epithelial sodium channel (ENaC) in both clearance of fetal lung liquid at birth and total body electrolyte homeostasis. Evidence from heterologous expression systems suggests that expression of the alphaENaC subunit is essential for channel function, whereas residual channel function can be measured in the absence of beta or gamma subunits. We generated mice without gammaENaC (gammaENaC -/-) to test the role of this subunit in neonatal lung liquid clearance and total body electrolyte balance. Relative to controls, gammaENaC (-/-) pups showed low urinary [K+] and high urinary [Na+] and died between 24 and 36 h, probably from hyperkalemia (gammaENaC -/- 18.3 mEq/l, control littermates 9.7 mEq/l). Newborn gammaENaC (-/-) mice cleared lung liquid more slowly than control littermates, but lung water at 12 h (wet/dry = 5.5) was nearly normal (wet/dry = 5.3). This study suggests that gammaENaC facilitates neonatal lung liquid clearance and is critical for renal Na+ and K+ transport, and that low level Na+ transport may be sufficient for perinatal lung liquid absorption but insufficient to maintain electrolyte balance by the distal nephron. The gammaENaC (-/-) newborn exhibits a phenotype that resembles the clinical manifestations of human neonatal PHA1.

Identification of specific EP receptors responsible for the hemodynamic effects of PGE2

To identify the E-prostanoid (EP) receptors that mediate the hemodynamic actions of PGE2, we studied acute vascular responses to infusions of PGE2 using lines of mice in which each of four EP receptors (EP1 through EP4) have been disrupted by gene targeting. In mixed groups of males and females, vasodepressor responses after infusions of PGE2 were significantly diminished in the EP2 -/- and EP4 -/- lines but not in the EP1 -/- or EP3 -/- lines. Because the actions of other hormonal systems that regulate blood pressure differ between sexes, we compared the roles of individual EP receptors in males and females. We found that the relative contribution of each EP-receptor subclass was strikingly different in males from that in females. In females, the EP2 and EP4 receptors, which signal by stimulating adenylate cyclase, mediate the major portion of the vasodepressor response to PGE2. In males, the EP2 receptor has a modest effect, but most of the vasodepressor effect is mediated by the phospholipase C-coupled EP1 receptor. Finally, in male mice, the EP3 receptor actively opposes the vasodepressor actions of PGE2. Thus the hemodynamic actions of PGE2 are mediated through complex interactions of several EP-receptor subtypes, and the role of individual EP receptors differs dramatically in males from that in females. These differences may contribute to sexual dimorphism of blood pressure regulation.To identify the E-prostanoid (EP) receptors that mediate the hemodynamic actions of PGE2, we studied acute vascular responses to infusions of PGE2using lines of mice in which each of four EP receptors (EP1 through EP4) have been disrupted by gene targeting. In mixed groups of males and females, vasodepressor responses after infusions of PGE2were significantly diminished in the EP2 -/- and EP4 -/- lines but not in the EP1 -/- or EP3 -/- lines. Because the actions of other hormonal systems that regulate blood pressure differ between sexes, we compared the roles of individual EP receptors in males and females. We found that the relative contribution of each EP-receptor subclass was strikingly different in males from that in females. In females, the EP2 and EP4 receptors, which signal by stimulating adenylate cyclase, mediate the major portion of the vasodepressor response to PGE2. In males, the EP2 receptor has a modest effect, but most of the vasodepressor effect is mediated by the phospholipase C-coupled EP1receptor. Finally, in male mice, the EP3 receptor actively opposes the vasodepressor actions of PGE2. Thus the hemodynamic actions of PGE2 are mediated through complex interactions of several EP-receptor subtypes, and the role of individual EP receptors differs dramatically in males from that in females. These differences may contribute to sexual dimorphism of blood pressure regulation.

NLRP1 dependent pyroptosis leads to acute lung injury and morbidity in mice

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from anthrax and that it initiates caspase-1 activation and release of IL-1β. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over 3 d following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1β by the inflammasome but are dependent on caspase-1 expression. In contrast, muramyl dipeptide-mediated inflammasome formation is not dependent on NLRP1 but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism.

Transcellular biosynthesis contributes to the production of leukotrienes during inflammatory responses in vivo

Leukotrienes are lipid mediators that evoke primarily proinflammatory responses by activating receptors present on virtually all cells. The production of leukotrienes is tightly regulated, and expression of 5-lipoxygenase, the enzyme required for the first step in leukotriene synthesis, is generally restricted to leukocytes. Arachidonic acid released from the cell membrane of activated leukocytes is rapidly converted to LTA(4) by 5-lipoxygenase. LTA(4) is further metabolized to either LTC(4) or LTB(4) by the enzyme LTC(4) synthase or LTA(4) hydrolase, respectively. Unlike 5-lipoxygenase, these enzymes are expressed in most tissues. This observation previously has led to the suggestion that LTA(4) produced by leukocytes may, in some cases, be delivered to other cell types before being converted into LTC(4) or LTB(4). While in vitro studies indicate that this process, termed transcellular biosynthesis, can lead to the production of leukotrienes, it has not been possible to determine the significance of this pathway in vivo. Using a series of bone marrow chimeras generated from 5-lipoxygenase- and LTA(4) hydrolase-deficient mice, we show here that transcellular biosynthesis contributes to the production of leukotrienes in vivo and that leukotrienes produced by this pathway are sufficient to contribute significantly to the physiological changes that characterize an ongoing inflammatory response.

Age-Induced Reprogramming of Mast Cell Degranulation

Mast cell degranulation can initiate an acute inflammatory response and contribute to the progression of chronic diseases. Alteration in the cellular programs that determine the requirement for mast cell degranulation would therefore have the potential to dramatically impact disease severity. Mast cells are exposed to increased levels of PGE2 during inflammation. We show that although PGE2 does not trigger the degranulation of dermal mast cells of young animals, in older mice, PGE2 is a potent mast cell stimulator. Intradermal administration of PGE2 leads to an EP3 receptor-dependent degranulation of mast cells, with the number of degranulated cells approaching levels observed in IgE- and Ag-treated controls. Taken together, these studies suggest that the ability of PGE2 to initiate mast cell degranulation changes in the aging animal. Therefore, elevated PGE2 levels might provide an important pathway by which mast cells are engaged to participate in inflammatory responses in the elderly patient.

Genetic Loss of Murine Pyrin, the Familial Mediterranean Fever Protein, Increases Interleukin-1β Levels

Familial Mediterranean Fever (FMF) is an inherited autoinflammatory disorder characterized by unprovoked episodes of fever and inflammation. The associated gene, MEFV (Mediterranean Fever), is expressed primarily by cells of myeloid lineage and encodes the protein pyrin/TRIM20/Marenostrin. The mechanism by which mutations in pyrin alter protein function to cause episodic inflammation is controversial. To address this question, we have generated a mouse line lacking the Mefv gene by removing a 21 kb fragment containing the entire Mefv locus. While the development of immune cell populations appears normal in these animals, we show enhanced interleukin (IL) 1β release by Mefv −/− macrophages in response to a spectrum of inflammatory stimuli, including stimuli dependent on IL-1β processing by the NLRP1b, NLRP3 and NLRC4 inflammasomes. Caspase-1 activity, however, did not change under identical conditions. These results are consistent with a model in which pyrin acts to limit the release of IL-1β generated by activation and assembly of inflammasomes in response to subclinical immune challenges.

A major role for the EP4 receptor in regulation of renin

Prostaglandins have been implicated as paracrine regulators of renin secretion, but the specific pathways and receptor(s) carrying out these functions have not been fully elucidated. To examine the contributions of prostanoid synthetic pathways and receptors to regulation of renin in the intact animal, we used a panel of mice with targeted disruption of several key genes: cyclooxygenase-2 (COX-2), microsomal PGE synthases 1 and 2 (mPGES1, mPGES2), EP2 and EP4 receptors for PGE(2), and the IP receptor for PGI(2). To activate the macula densa signal for renin stimulation, mice were treated with furosemide over 5 days and renin mRNA levels were determined by real-time RT-PCR. At baseline, there were no differences in renin mRNA levels between wild-type and the various strains of mutant mice. Furosemide caused marked stimulation of renin mRNA expression across all groups of wild-type control mice. This response was completely abrogated in the absence of COX-2, but was unaffected in mice lacking mPGES1 or mPGES2. The absence of G(s)/cAMP-linked EP2 receptors had no effect on stimulation of renin by furosemide and there was only a modest, insignificant reduction in renin responses in mice lacking the IP receptor. By contrast, renin stimulation in EP4(-/-) mice was significantly reduced by ∼70% compared with wild-type controls. These data suggest that stimulation of renin by the macula densa mechanism is mediated by PGE(2) through a pathway requiring COX-2 and the EP4 receptor, but not EP2 or IP receptors. Surprisingly, mPGES1 or mPGES2 are not required, suggesting other alternative mechanisms for generating PGE(2) in response to macula densa stimulation.

Genetic Factors Determine the Contribution of Leukotrienes to Acute Inflammatory Responses

Leukotrienes (LT) are potent lipid mediators synthesized by the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LT have been implicated in a broad spectrum of inflammatory processes. To investigate the influence of genetic factors on the contribution of LT to acute inflammation, we generated congenic 5-lipoxygenase-deficient 129, C57BL/6 (B6), and DBA/1Lac (DBA) mouse lines. Topical application of AA evoked a vigorous inflammatory response in 129 and DBA mice, whereas only a modest response was seen in B6 animals. The response to AA in 129 and DBA strains is LT dependent. In contrast, LT make little contribution to this response in B6 mice. AA-induced inflammation in B6 mice is prostanoid dependent, since this response was substantially reduced by treating B6 mice with a cyclooxygenase inhibitor. These data suggest that prostanoids are essential for AA-induced cutaneous inflammation in B6 mice, whereas LT are the major mediators of this response in 129 and DBA strains. In contrast, the response to AA in the peritoneal cavity is robust in the 129 and B6 strains, but was significantly blunted in DBA mice, showing that strain differences in the response to AA are tissue specific. Variations in these and other experimental models of inflammation appear to correlate directly with the ability of a particular mouse strain and a specific tissue to respond to LT, specifically LTC4. Taken together, these findings indicate that the relative contribution of prostanoids and LT to inflammatory responses is variable not only between strains but also between different tissues within these inbred mouse lines.

Phenotypic Analysis of Mice Lacking the Highly Abundant Purkinje Cell- and Bipolar Neuron-Specific PCP2 Protein

The Purkinje cell protein-2 (Pcp2, also known as L7) gene is abundantly expressed only in Purkinje cells of the cerebellum and bipolar neurons of the retina. The spatio-temporal expression pattern of this gene suggests a role for PCP2 in Purkinje cell development or normal cell physiology. A PCP2-deficient mouse was created by gene targeting to test the hypothesis that it is required for Purkinje cell development or function. Although normally present in abundance, the absence of PCP2 in null animals caused no observable cerebellar abnormalities. Behavioral analysis reveals normal abilities for balance and coordination. Null cerebellum has normal Purkinje cell numbers, morphology, and ultrastructure. Retinal bipolar neurons appear similarly unaffected. Aged null animals (22 months) were also examined and no deficits were detected using the same behavioral and histologic analyses. Although the null animal does not reveal the function of PCP2, it does rule out an essential role for PCP2 in Purkinje cell development, in Purkinje cell survival, and in at least some aspects of cerebellar function.