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1.  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 three days 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, MDP 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.
doi:10.4049/jimmunol.1201065
PMCID: PMC3635067  PMID: 22753929
2.  PGE2 produced by the lung augments the effector phase of allergic inflammation 
Elevated PGE2 is a hallmark of most inflammatory lesions. This lipid mediator can induce the cardinal signs of inflammation, and the beneficial actions of non-steroidal anti-inflammatory drugs are attributed to inhibition of cyclooxygenase COX-1 and COX-2, enzymes essential in the biosynthesis of PGE2 from arachidonic acid. However, both clinical studies and rodent models suggest that, in the asthmatic lung, PGE2 acts to restrain the immune response and limit physiological change secondary to inflammation. To directly address the role of PGE2 in the lung, we examined the development of disease in mice lacking microsomal prostaglandin E synthase 1 (mPGES1), which converts COX-1/COX-2 derived PGH2 to PGE2. We show that mPGES1 determines PGE2 levels in the naïve lung and is required for increases in PGE2 after ovalbumin (OVA) induced allergy. While loss of either COX-1 or COX-2 increases the disease severity, surprisingly mPGES1 −/− mice show reduced inflammation. However, an increase in serum IgE is still observed in the mPGES1 −/− mice, suggesting that loss of PGE2 does not impair induction of a TH2 response. Furthermore, mPGES1 −/− mice expressing a transgenic OVA-specific T cell receptor are also protected, indicating that PGE2 acts primarily after challenge with inhaled antigen. PGE2 produced by the lung plays the critical role in this response, as loss of lung mPGES1 is sufficient to protect against disease. Together this supports a model in which mPGES1-dependent PGE2 produced by populations of cells native to the lung contributes to the effector phase of some allergic responses.
doi:10.4049/jimmunol.1101873
PMCID: PMC3324636  PMID: 22412193
3.  Unique populations of lung mast cells are required for antigen-mediated bronchoconstriction 
Background
Studies in both human and mouse indicate that mediators released by mast cells can lead to bronchoconstriction, and thus these are important effector cells in life threatening anaphylaxis. Much of our understanding of the various functions of mast cells emanates from the study of mice lacking theses cells, particularly mice carrying mutations in the tyrosine kinase gene Kit. Definitive evidence for the role of mast cells in the altered immune response requires the demonstration that this response can be normalized by reconstitution of the mice with cultured bone marrow-derived mast cells (BMMCs). While many mast cell niches can be restored with BMMCs, this has not been demonstrated for mast cells present in the airways of the lung, cells poised to mediate bronchoconstriction during allergic responses.
Objective
To determine if mast cell deficient KitWsh/Wsh reconstituted lines are an appropriate model for the study of the role of these cells in bronchoconstriction associated with allergic responses.
Methods
KitWsh/Wsh mice were reconstituted with either whole bone marrow (WBM) or BMMCs and responses to IgE-mediated mast cell activation determined; including systemic hypothermia, mediator release, and bronchoconstriction in anesthetized, mechanically ventilated animals.
Results
Engraftment of KitWsh/Wsh mice with WBM and BMMCs results in reconstitution of the central airways with mast cells. While the treatment of the two groups of animals resulted in systemic changes when challenged with IgE/Ag in a model of passive anaphylaxis, bronchoconstriction was observed only in KitWsh/Wsh animal which had received a bone marrow transplant.
Conclusions
While BMMCs can populate the lung, they cannot restore IgE/Ag-mediated bronchoconstriction to mast cell-deficient animals. This suggests that the mast cell population which mediates this function may be unique, and to fill this niche in the lung cells must undergo a specific developmental program, one that is no longer available to cultured mast cells.
doi:10.1111/j.1365-2222.2010.03583.x
PMCID: PMC3592578  PMID: 20718780
Mast cell; lung; bronchoconstriction; reconstitution
4.  PGE2 through the EP4 receptor controls smooth muscle gene expression patterns in the ductus arteriosus critical for remodeling at birth 
The ductus arteriosus (DA) is a fetal shunt that directs right ventricular outflow away from pulmonary circulation and into the aorta. Critical roles for prostaglandin E2 (PGE2) and the EP4 receptor (EP4) have been established in maintaining both the patency of the vessel in utero and in its closure at birth. Here we have generated mice in which loss of EP4 expression is limited to either the smooth muscle (SMC) or endothelial cells and demonstrated that SMC, but not endothelial cell expression of EP4 is required for DA closure. The genome wide expression analysis of full term wild type and EP4−/− DA indicates that PGE2/EP4 signaling modulates expression of a number of unique pathways, including those involved in SMC proliferation, cell migration, and vascular tone. Together this supports a mechanism by which maturation and increased contractility of the vessel is coupled to the potent smooth muscle dilatory actions of PGE2.
doi:10.1016/j.prostaglandins.2012.02.001
PMCID: PMC3312001  PMID: 22342504
Ductus arteriosus; Prostaglandins; Smooth Muscle; Remodeling; EP4; conditional gene knockout
5.  ONZIN Deficiency Attenuates Contact Hypersensitivity Responses in Mice 
Immunology and cell biology  2012;90(7):733-742.
ONZIN is abundantly expressed in immune cells of both the myeloid and lymphoid lineage. Expression by lymphoid cells has been reported to further increase after cutaneous exposure of mice to antigens and haptens capable of inducing contact hypersensitivity, suggesting that ONZIN plays a critical role in this response. Here, we report that indeed ONZIN-deficient mice develop attenuated CHS to a number of different haptens. Dampened CHS responses correlated with a significant reduction in pro-inflammatory IL-6 at the challenge site in ONZIN-deficient animals compared to wild type controls. Together the study of these animals indicates that loss of ONZIN impacts the effector phase of the CHS response through the regulation of pro-inflammatory factors.
doi:10.1038/icb.2011.107
PMCID: PMC3336033  PMID: 22249203
Cell trafficking; Contact hypersensitivity; Inflammation; ONZIN
6.  PGE2 Promotes Apoptosis Induced by Cytokine Deprivation through EP3 Receptor and Induces Bim in Mouse Mast Cells 
PLoS ONE  2014;9(7):e102948.
Increased mast cell numbers are observed at sites of allergic inflammation and restoration of normal mast cell numbers is critical to the resolution of these responses. Early studies showed that cytokines protect mast cells from apoptosis, suggesting a simple model in which diminished cytokine levels during resolution leads to cell death. The report that prostaglandins can contribute both to recruitment and to the resolution of inflammation together with the demonstration that mast cells express all four PGE2 receptors raises the question of whether a single PGE2 receptor mediates the ability of PGE2 to regulate mast cell survival and apoptosis. We report here that PGE2 through the EP3 receptor promotes cell death of mast cells initiated by cytokine withdrawal. Furthermore, the ability of PGE2 to limit reconstitution of tissues with cultured mast cells is lost in cell lacking the EP3 receptor. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨm), increased caspase-3 activation, chromatin condensation, and low molecular weight DNA cleavage. PGE2 augmented cell death is dependent on an increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE2 can contribute to the resolution of inflammation in part by augmenting the removal of inflammatory cells in this case, mast cells.
doi:10.1371/journal.pone.0102948
PMCID: PMC4108439  PMID: 25054560
7.  Microsomal Prostaglandin E Synthase-2 Is Not Essential For In Vivo Prostaglandin E2 Biosynthesis 
Prostaglandin E2 (PGE2) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE2 from the cyclooxygenase metabolite PGH2 have been described. Here, we examine the contribution of one of these enzymes to PGE2 production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE2 levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE2 synthase.
doi:10.1016/j.prostaglandins.2008.10.003
PMCID: PMC3182462  PMID: 19010439
Microsomal Prostaglandin E2 Synthase-2; Prostaglandin E2
8.  Development of a Mouse Test for Repetitive, Restricted Behaviors: Relevance to Autism 
Behavioural brain research  2007;188(1):178-194.
Repetitive behavior, a core symptom of autism, encompasses stereotyped responses, restricted interests, and resistance to change. These studies investigated whether different components of the repetitive behavior domain could be modeled in the exploratory hole-board task in mice. Four inbred mouse strains, C57BL/6J, BALB/cByJ, BTBR T+tf/J, and FVB/NJ, and mice with reduced expression of Grin1, leading to NMDA receptor hypofunction (NR1neo/neo mice), were tested for exploration and preference for olfactory stimuli in an activity chamber with a 16-hole floor-board. Reduced exploration and high preference for holes located in the corners of the chamber were observed in BALB/cByJ and BTBR T+tf/J mice. All inbred strains had initial high preference for a familiar olfactory stimulus (clean cage bedding). BTBR T+tf/J was the only strain that did not demonstrate a shift in hole preference towards an appetitive olfactory stimulus (cereal or a chocolate chip), following home cage exposure to the food. The NR1neo/neo mice showed lower hole selectivity and aberrant olfactory stimulus preference, in comparison to wildtype controls. The results indicate that NR1neo/neo mice have repetitive nose poke responses that are less modified by environmental contingencies than responses in wildtype mice. 25-30% of NMDA-receptor hypomorphic mice also show self-injurious responses. Findings from the olfactory studies suggest that resistance to change and restricted interests might be modeled in mice by a failure to alter patterns of hole preference following familiarization with an appetitive stimulus, and by high preference persistently demonstrated for one particular olfactory stimulus. Further work is required to determine the characteristics of optimal mouse social stimuli in the olfactory hole-board test.
doi:10.1016/j.bbr.2007.10.029
PMCID: PMC2349090  PMID: 18068825
autism; exploration; olfaction; restricted interests; social preference; stereotypy
9.  Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma gondii 
mBio  2014;5(1):e01117-13.
ABSTRACT
Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis.
IMPORTANCE
Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.
doi:10.1128/mBio.01117-13
PMCID: PMC3944820  PMID: 24549849
10.  Mice lacking NKCC1 are protected from development of bacteremia and hypothermic sepsis secondary to bacterial pneumonia 
The Journal of Experimental Medicine  2007;204(6):1383-1393.
The contribution of the Na+-K+-Cl− transporter (NKCC1) to fluid in ion transport and fluid secretion in the lung and in other secretory epithelia has been well established. Far less is known concerning the role of this cotransporter in the physiological response of the pulmonary system during acute inflammation. Here we show that mice lacking this transporter are protected against hypothermic sepsis and bacteremia developing as a result of Klebsiella pneumoniae infection in the lung. In contrast, this protection was not observed in NKCC1−/− mice with K. pneumoniae—induced peritonitis. Although overall recruitment of cells to the lungs was not altered, the number of cells present in the airways was increased in the NKCC1−/− animals. Despite this robust inflammatory response, the increase in vascular permeability observed in this acute inflammatory model was attenuated in the NKCC1−/− animals. Our studies suggest that NKCC1 plays a unique and untoward unrecognized role in acute inflammatory responses in the lung and that specific inhibition of this NKCC isoform could be beneficial in treatment of sepsis.
doi:10.1084/jem.20061205
PMCID: PMC2118609  PMID: 17517966
11.  Co-operation between mast cells and neurons is essential for antigen-mediated bronchoconstriction1 
Mast cells are important sentinels guarding the interface between the environment and the body: a breach in the integrity of this interface can lead to the release of a plethora of mediators which engage the foreign agent, recruit leukocytes, and initiate adaptive physiological changes in the organism. While these capabilities make mast cells critical players in immune defense, it also makes them important contributors to the pathogenesis of diseases such as asthma. Mast cell mediators induce dramatic changes in smooth muscle physiology, and the expression of receptors for these factors by smooth muscle suggests that they act directly to initiate constriction. Contrary to this view, we show here that mast cell-mediated bronchoconstriction is observed only in animals with intact innervation of the lung and that serotonin release alone is required for this action. While ablation of sensory neurons does not limit bronchoconstriction, constriction after antigen challenge is absent in mice in which the cholinergic pathways are compromised. Linking mast cell function to the cholinergic system likely provides an important means of modulating the function of these resident immune cells to physiology of the lung, but may also provide a safeguard against life-threatening anaphylaxis during mast cell degranulation.
doi:10.4049/jimmunol.0900039
PMCID: PMC3901060  PMID: 19494266
Rodent; Mast Cells; Allergy; Lung
12.  Transcellular biosynthesis contributes to the production of leukotrienes during inflammatory responses in vivo 
The Journal of Clinical Investigation  2002;109(10):1373-1380.
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 LTA4 by 5-lipoxygenase. LTA4 is further metabolized to either LTC4 or LTB4 by the enzyme LTC4 synthase or LTA4 hydrolase, respectively. Unlike 5-lipoxygenase, these enzymes are expressed in most tissues. This observation previously has led to the suggestion that LTA4 produced by leukocytes may, in some cases, be delivered to other cell types before being converted into LTC4 or LTB4. 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 LTA4 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.
doi:10.1172/JCI14869
PMCID: PMC150978  PMID: 12021253
14.  Failure of spermatogenesis in mouse lines deficient in the Na+-K+-2Cl– cotransporter 
Journal of Clinical Investigation  2000;105(4):441-450.
The Na+-K+-2Cl– cotransporter (NKCC1) carries 1 molecule of Na+ and K+ along with 2 molecules of Cl– across the cell membrane. It is expressed in a broad spectrum of tissues and has been implicated in cell volume regulation and in ion transport by secretory epithelial tissue. However, the specific contribution of NKCC1 to the physiology of the various organ systems is largely undefined. We have generated mouse lines carrying either of 2 mutant alleles of the Slc12a2 gene, which encodes this cotransporter: a null allele and a mutation that results in deletion of 72 amino acids of the cytoplasmic domain. Both NKCC1-deficient mouse lines show behavioral abnormalities characteristic of mice with inner ear defects. Male NKCC1-deficient mice are infertile because of defective spermatogenesis, as shown by the absence of spermatozoa in histological sections of their epididymides and the small number of spermatids in their testes. Consistent with this observation, we show that Slc12a2 is expressed in Sertoli cells, pachytene spermatocytes, and round spermatids isolated from wild-type animals. Our results indicate a critical role for NKCC1-mediated ion transport in spermatogenesis and suggest that the cytoplasmic domain of NKCC1 is essential in the normal functioning of this protein.
PMCID: PMC289162  PMID: 10683373
15.  Adenosine and inosine increase cutaneous vasopermeability by activating A3 receptors on mast cells 
Journal of Clinical Investigation  2000;105(3):361-367.
Adenosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A3 adenosine receptor (A3AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A3AR. Adenosine fails to increase vascular permeability in mast cell–deficient mice, suggesting that this tissue response to adenosine is mast cell–dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcεR1) by antigen, as adenosine is equally effective in mediating these changes in FcεR1 β-chain–deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response.
PMCID: PMC377446  PMID: 10675362
16.  The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB 
Nature immunology  2012;13(9):823-831.
Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3−/− mice. LPS-treated Nlrc3−/− macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3−/− mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a ‘TRAFasome’ complex.
doi:10.1038/ni.2378
PMCID: PMC3721195  PMID: 22863753
17.  Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor 
Journal of Clinical Investigation  1999;103(11):1539-1545.
Prostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E2 (PGE2) receptors — specifically, the EP2 receptor — we show that Ep2–/– females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2–/– ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE2 regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE2 receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE2, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum.
PMCID: PMC408376  PMID: 10359563
18.  Role of the 5-Lipoxygenase–activating Protein (FLAP) in Murine Acute Inflammatory Responses 
The Journal of Experimental Medicine  1997;185(6):1065-1076.
Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO–activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor–induced shock compared to controls. Also, edema associated with Zymosan A–induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO–deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.
PMCID: PMC2196234  PMID: 9091580
19.  Analysis of NLRP3 in the Development of Allergic Airway Disease in Mice1 
The contribution of NLRP3, a member of the nucleotide-binding domain leucine-rich repeat containing (NLR) family, in the development of allergic airway disease is currently controversial. Here, we used multiple allergic asthma models to examine the physiologic role of NLRP3. We found no significant differences in airway eosinophilia, histopathology, mucus production and airway hyperreactivity between wild type and Nlrp3-/- mice in either acute (alum-dependent) or chronic (alum-independent) OVA models. In addition to the OVA model, we also did not detect a role for NLRP3 in the development of allergic airway disease induced by either acute or chronic house dust mite (HDM) antigen exposure. While we did not observe significant phenotypic differences in any of the models tested, we did observe a significant reduction of IL-13 and IL-33 in Nlrp3-/- mice compared to wild type controls in the chronic OVA model without added alum. In all of the allergic airway disease models, the levels of the NLRP3 inflammasome associated cytokines IL-1β and IL-18 in the lung were below the level of detection. In sum, this report surveyed four different allergic asthma models and found a modest and selected role for NLRP3 in the alum-free OVA model. However this difference did not greatly alter the clinical outcome of the disease. This suggests that the role of NLRP3 in allergic asthma has to be re-evaluated.
doi:10.4049/jimmunol.1102488
PMCID: PMC3294123  PMID: 22323538
Cryopyrin; NLR; NALP3; Asthma; Inflammasome; Lung Disease; Flexivent; Ovalbumin; Dust Mite Antigen; DMA
20.  Effects of the selective kainate receptor antagonist ACET on altered sensorimotor gating in a genetic model of reduced NMDA receptor function 
Brain Research  2012;1443:98-105.
The pathophysiology of schizophrenia may involve reduced NMDA receptor function. Accordingly, experimental models of NMDA receptor hypofunction may be useful for testing potential new antipsychotic agents and for characterizing neurobiological abnormalities relevant to schizophrenia. We demonstrated previously that mice under-expressing the NR1 subunit of the NMDA receptor show supersensitive behavioral responses to kainic acid and that a kainate receptor antagonist normalized altered behaviors in the mutant mice (NR1neo/neo). The present work examined effects of another selective kainate receptor antagonist, (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxy-5-phenylthiophene-3-yl-methylpyrimidine-2,4-dione (ACET), on altered behavioral phenotypes in the genetic model of NMDA receptor hypofunction. ACET, at a dose of 15 mg/kg, partially reversed the deficits in prepulse inhibition produced by the mutation. The 15 mg/kg dose of ACET was also effective in reversing behavioral effects of the selective kainate agonist ATPA. However, ACET did not significantly reduce the increased locomotor activity and rearing behavior observed in the NR1neo/neo mice. These findings show that a highly selective kainate receptor antagonist can affect the deficits in sensorimotor gating in the NR1neo/neo mice. The results also provide further support for the idea that selective kainate receptor antagonists could be novel therapeutic candidates for schizophrenia.
Section: Disease-Related Neuroscience
doi:10.1016/j.brainres.2012.01.009
PMCID: PMC3294253  PMID: 22297176
Schizophrenia; glutamate; kainate receptor; Grin1; NMDA receptor; kainate receptor antagonist; acoustic startle; prepulse inhibition; NMDA receptor hypofunction
21.  Genetic Loss of Murine Pyrin, the Familial Mediterranean Fever Protein, Increases Interleukin-1β Levels 
PLoS ONE  2012;7(11):e51105.
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.
doi:10.1371/journal.pone.0051105
PMCID: PMC3511413  PMID: 23226472
22.  Role of TNFR-Related 2 Mediated Immune Responses in Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease 
Molecules and Cells  2011;31(2):99-104.
Previous work has suggested that the LIGHT-TR2 costimulatory pathway plays a role in the acute and chronic stages of dextran sulfate sodium (DSS)-induced colitis [Steinberg et al. (2008); Wang et al. (2005)]. To clarify the role of TNFR-related 2 (TR2) signaling in the maintenance of intestinal homeostasis, we generated a TR2 knock-out (KO) mouse. Using DSS to induce colitis, we compared the colitic symptoms and pathological changes in wild type (WT) and TR2 KO mice, and the production of cytokines by the diseased colons. We also studied the role of TR2 in suppressing innate and adaptive immunity in the DSS model. TR2 deficient mice were characterized by reduced symptoms of intestinal inflammation compared with wildtype mice, and reduced production of cytokines. We therefore generated a monoclonal antibody against mouse TR2 which was specific to TR2 and capable of blocking TR2 signals. With this antibody, we demonstrated that antagonizing TR2 during the development of DSS-induced colitis reduced the symptoms of inflammation. Our findings suggest that TR2 is an important mediator in colitis, and may serve as a therapeutic target in inflammatory bowel disease.
doi:10.1007/s10059-011-0013-y
PMCID: PMC3932679  PMID: 21347711
blocking mAb; colitis; DSS; IBD; KO mouse; TR2
23.  THE IMPACT OF MICROSOMAL PROSTAGLANDIN E SYNTHASE 1 (mPGES1) ON BLOOD PRESSURE IS DETERMINED BY GENETIC BACKGROUND 
Hypertension  2010;55(2):531-538.
Prostaglandin (PG) E2 has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. While microsomal PGE synthase 1 (mPGES1) is the only genetically-verified PGE synthase, results of previous studies examining the consequences of mPGES1-deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1−/− mice on two distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1−/− mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1−/− animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1−/− and WT mice developed mild hypertension of similar magnitude, while 129-mPGES1−/− mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1−/− animals. In WT mice of both strains, the increase in urinary excretion of PGE2 with angiotensin II was attenuated in mPGES1−/− animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1−/− mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.
doi:10.1161/HYPERTENSIONAHA.109.145631
PMCID: PMC2836731  PMID: 20065147
prostanoids; PGE synthase; blood pressure; strain; hypertension
24.  Increased sensitivity to kainic acid in a genetic model of reduced NMDA receptor function 
Brain research  2009;1307:166-176.
The pathophysiology of schizophrenia may involve reduced NMDA receptor function and experimental models of NMDA receptor hypofunction have proven useful for characterizing neurobiological abnormalities potentially relevant to schizophrenia. The present study assessed behavioral responses and induction of Fos after administration of kainic acid to wild type mice (NR1+/+) and mice with genetically reduced NMDA receptor expression (NR1neo/neo). At a dose of 20 mg/kg kainic acid induced lethal seizures in 100% of the NR1neo/neo mice tested but produced no lethal seizures in the wild type mice. The NR1neo/neo mice also exhibited enhanced behavioral responses to kainic acid at a dose of 15 mg/kg but no lethal seizures were produced by this dose. A greater induction of Fos was observed in neocortical and limbic cortical regions of the NR1neo/neo compared to NR1+/+ mice after administration of 15 mg/kg kainic acid. In contrast, there were no differences between the genotypes in kainic acid induced of Fos in the amygdala, hippocampus, lateral septum, and nucleus accumbens. In order to determine if altered behavioral phenotypes of the NR1neo/neo mice could be related to increased sensitivity of kainate receptors to endogenous glutamate, effects of the highly selective kainate antagonist LY382884 were examined. The kainate antagonist reduced the exaggerated acoustic startle responses, deficits in prepulse inhibition of acoustic startle, and motor hyperactivity in the NR1neo/neo mice. These findings suggest that selective kainate receptor antagonists could be novel therapeutic candidates for schizophrenia.
doi:10.1016/j.brainres.2009.10.023
PMCID: PMC2787847  PMID: 19840778
Schizophrenia; glutamate; kainic acid; NMDA receptor; hippocampus; cerebral cortex; kainic acid antagonist
25.  Nuclear factor of activated T cells negatively regulates expression of the tumor necrosis factor receptor-related 2 gene in T cells 
Experimental & Molecular Medicine  2010;42(12):805-810.
Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.
doi:10.3858/emm.2010.42.12.083
PMCID: PMC3015154  PMID: 20948279
gene expression regulation; NFATC transcription factors; promoter regions, genetic; T lymphocytes; tumor necrosis factor ligand superfamily member 14

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