p47phox and reactive oxygen species production modulate expression of microRNA-451 in macrophages.

The production of microRNAs (miRNA) is influenced by various stimuli, including environmental stresses. We hypothesized that reactive oxygen species (ROS)-associated stress could regulate macrophage miRNA synthesis. miRNAs undergo unique steps of maturation processing through either one of two pathways of cytoplasmic processing. Unlike the canonical pathway, the regulation of alternative cytoplasmic processing of miRNA has not been fully elucidated yet. We cultured bone marrow derived macrophages (BMDM) from wild type (WT) and p47(phox-/-) mice and profiled miRNA expression using microarrays. We analyzed 375 miRNAs including four endogenous controls to normalize the data. At resting state, p47(phox-/-) BMDM has the markedly reduced expression of miR-451 compared to WT BMDM, without other significant differences. Unlike majority of miRNAs, miR-451 goes through the unique alternative processing pathway, in which Ago2 plays a key role. In spite of significant reduction of mature miR-451, however, its precursor form, pre-mir-451, was similar in both BMDMs, suggesting that the processing of pre-mir-451 is impaired in p47(phox-/-) BMDM. Moreover, p47(phox-/-) BMDM expressed significantly reduced level of Ago2. In contrast, Ago2 mRNA levels were similar in WT and p47(phox-/-) BMDM, suggesting a post-transcriptional defect of Ago2 production in p47(phox-/-) macrophages, which resulted in impaired processing of pre-miR-451. In order to examine the functional significance of miR-451 in macrophages, we cultured BMDMs from miR-451 knock-out mice. Of interest, miR-451-deficient BMDM exhibited reduced ROS generation upon zymosan stimulation, compared to WT BMDM. Our studies suggest functional crosstalk between ROS and miR-451 in the regulation of macrophage oxidant stress.

Expression of TREM-1 is inhibited by PGD2 and PGJ2 in macrophages.

TREM-1 is a superimmunoglobulin receptor present on neutrophils and monocytes, which plays an important role in the amplification of inflammation. The natural ligands for TREM-1 have not been identified; however, Toll-like receptor ligands are known to induce the expression of TREM-1. Blockade of TREM-1 has shown to improve survival in animal models of sepsis. In the present studies, we investigated the role of lipid mediators in the expression of TREM-1. In a macrophage cell line, we show that the expression of TREM-1 in response to LPS and bacteria Pseudomonas aeruginosa is inhibited by PGD(2) and cyclopentanone prostaglandins PGJ(2) and 15-dPGJ(2). The inhibition of TREM-1 by these prostaglandins is independent of the PGD(2) receptors and PPARγ but occurs by activation of Nrf2 and inhibition of NF-κB. Our data suggest a novel mechanism by which these prostaglandins exhibit anti-inflammatory effects and a new therapeutic approach to inhibition of TREM-1.

A system for monitoring and improving animal visibility and its implications for zoological parks.

With the growing trend in zoos to build complex, naturalistic exhibits comes the potential for exhibits to be so densely vegetated or complex that animals are not easily seen by zoo visitors. This can negatively impact the visitor's visiting experience and the zoo's ability to communicate conservation and education messages. Over the past 9 years, Disney's Animal Kingdom has developed a process for monitoring and improving the visibility of animals on display to the public. This animal visibility process utilizes a data collection system whereby systematic observations are collected each week. The percentage of observations where at least one animal was visible is recorded for each species and compared to an 80% visibility criterion. Species that do not reach this criterion for 4 consecutive weeks are discussed at animal management meetings. If the problems associated with animal visibility cannot be easily solved, the animal-care teams partner with the research team to conduct a second process, called the Visibility Issues Process. This process provides additional information for the animal-care team to utilize in developing a plan to improve visibility. Although the processes described here are specific to the infrastructure at Disney's Animal Kingdom, the basic concepts of (1) a formalized visibility data collection process, (2) a visibility criterion to which managers of species are held accountable, and (3) a process for planning to improve animal visibility without negatively impacting animal welfare are fundamental concepts that can be developed at individual institutions and incorporated into that zoo's existing infrastructure.

Genomic analyses reveal global functional alterations that promote tumor growth and novel tumor suppressor genes in natural killer-cell malignancies.

Natural killer (NK)-cell malignancies are among the most aggressive lymphoid neoplasms with very poor prognosis. We performed array comparative genomic hybridization analysis on a number of NK cell lines and primary tumors to gain better understanding of the pathogenesis and tumor biology of these malignancies. We also obtained transcriptional profiles of genes residing in these regions and compared them with normal and activated NK cells. Only 30-50% of the genes residing in the gained or deleted regions showed corresponding increased or decreased expression. However, many of the upregulated genes in regions of gain are functionally important for the proliferation and growth of the neoplastic population. Genes downregulated in regions of loss included many transcription factors or repressors, tumor suppressors or negative regulators of the cell cycle. The minimal common region of deletion in 6q21 included three known genes (PRDM1, ATG5 and AIM1) showing generally low expression. Mutations resulting in truncated PRDM1 and changes in conserved amino-acid sequences of AIM1 were detected. Highly methylated CpG islands 5' of PRDM1 and AIM1 correlated with low expression of the transcripts. Reversal of methylation by Decitabine induced expression of PRDM1 and cell death. In conclusion, we have shown a general tumor-promoting effect of genetic alterations and have identified PRDM1 as the most likely target gene in del6q21. ATG5, an essential gene for autophagy and AIM1, a gene implicated in melanoma, may also participate in the functional abnormalities.

The nuclear factor kappa-B pathway in airway epithelium regulates neutrophil recruitment and host defence following Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa pneumonia usually results from a deficit of the innate immune system. To investigate whether inflammatory signalling by airway epithelial cells provides a pivotal line of defence against P. aeruginosa infection, we utilized two separate lines of inducible transgenic mice that express a constitutive activator of the nuclear factor kappa-B (NF-kappaB) pathway (IKTA) or a dominant inhibitor of NF-kappaB (DNTA) in airway epithelial cells. Compared with control mice, IKTA mice showed an enhanced host response to P. aeruginosa infection with greater neutrophil influx into the lungs, increased expression of Glu-Leu-Arg-positive (ELR(+)) CXC chemokines macrophage inflammatory protein-2 and keratinocyte chemoattractant (KC), superior bacterial clearance and improved survival at 24 h after infection. Neutrophil depletion abrogated the improvement in host defence identified in IKTA mice. In contrast, DNTA mice showed impaired responses to P. aeruginosa infection with higher bacterial colony counts in the lungs, decreased neutrophilic lung inflammation and lower levels of KC in lung lavage fluid. DNTA mice given recombinant KC at the time of P. aeruginosa infection demonstrated improved neutrophil recruitment to the lungs and enhanced bacterial clearance. Our data indicate that the NF-kappaB pathway in airway epithelial cells plays an essential role in defence against P. aeruginosa through generation of CXC chemokines and recruitment of neutrophils.

Functional genomics of silencing TREM-1 on TLR4 signaling in macrophages.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.

Conditional regulation of cyclooxygenase-2 in tracheobronchial epithelial cells modulates pulmonary immunity.

Cyclooxygenase-2 (COX-2) gene expression in the lung is induced in pathological conditions such as asthma and pneumonia; however, the exact impact of COX-2 gene expression in the airway in regulating inflammatory and immunological response in the lung is not understood. To define a physiological role of inducible COX-2 in airway epithelial cells, we developed a novel line of transgenic mice, referred to as CycloOxygenase-2 TransActivated (COTA) mice, that overexpress a COX-2 transgene in the distribution of the CC-10 promoter in response to doxycycline. In response to doxycycline treatment, COX-2 expression was increased in airway epithelium of COTA mice and whole lung tissue contained a three- to sevenfold increase in prostaglandin E(2) (PGE(2)), prostaglandin D(2) (PGD(2)) thromboxane B(2) (TXB(2)) and 6-Keto prostaglandin F(2alpha) (PGF(2alpha)) compared to wild-type and untreated COTA mice. Interestingly, primary mouse tracheal epithelial cells from COTA mice produced only PGE(2) by doxycycline-induced COX-2 activation, providing an indication of cellular specificity in terms of mediator production. In the ovalbumin model, in which doxycycline was given at the sensitization stage, there was an increase in interleukin (IL)-4 level in lung tissue from COTA mice compared to untreated COTA and wild-type mice. In addition, COTA mice that were treated with doxycycline had impaired clearance of Pseudomonas aeruginosa pneumonia compared to wild-type mice. COX-2 gene expression in airway epithelial cells has an important role in determining immunological response to infectious and allergic agents.

Molecular targets for modulating lung inflammation and injury.

The inflammatory response of the lung and airways is one of the main targets for tile development of new therapies for variety of disorders including the acute respiratory distress syndrome, cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Over the last decade our understanding of the molecular biology of the inflammatory response has advanced considerably and has opened up new avenues for therapeutic intervention. Furthermore, the mechanism of action of many of the existing anti-inflammatory agents has been revealed by this burgeoning information. Here, we discuss the functions and therapeutic potential of molecules that might prove promising as targets for treatment of inflammatory lung diseases. These possible molecular targets include cell surface proteins/receptors [toll like receptors (TLRs), triggering receptors expressed on myeloid cells (TREMs), and syndecans)], transcription factors [NF-kappaB, AP-1, PU.1, and high mobility group box 1 (HMGB1)], and regulatory proteins [macrophage migration inhibitory factor (MIF), granulocyte macrophage colony stimulating factor (GM-CSF), cyclooxygenase 2 (COX-2), heme oxygenase 1 (HO-1)].

High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice.

We examined the effects of dexamethasone treatment on nuclear factor (NF)-kappa B activation and lung inflammation in transgenic reporter mice expressing photinus luciferase under the control of an NF-kappa B-dependent promoter (HLL mice). In vitro studies with bone marrow and peritoneal macrophages derived from these mice showed that treatment with dexamethasone blocked luciferase induction after treatment with Escherichia coli lipopolysaccharide (LPS); however, treatment of mice with intraperitoneal injection of dexamethasone at doses of 0.3 microg/g and 1 microg/g failed to inhibit NF-kappa B-dependent luciferase activity in the lungs. Furthermore, intraperitoneal treatment with 10 microg/g of dexamethasone prior to LPS paradoxically resulted in augmented luciferase activity as compared with that of mice treated with LPS alone. NF-kappa B-dependent luciferase expression in the lungs was detected by bioluminescence imaging and by measurement of luciferase activity in homogenized lung tissue. In these studies, there was an excellent correlation between indirect measurement of luciferase activity by bioluminescence in living mice and direct measurement of luciferase activity in lung tissue. Dexamethasone treatment did not affect LPS-induced neutrophilic influx or the concentration of macrophage inflammatory protein-2 in lung lavage fluid. These findings emphasize the potential error of extrapolating in vitro findings to complex in vivo events such as regulation of inflammation.

Impaired pulmonary NF-kappaB activation in response to lipopolysaccharide in NADPH oxidase-deficient mice.

Reactive oxygen species (ROS) are thought to be involved in intracellular signaling, including activation of the transcription factor NF-kappaB. We investigated the role of NADPH oxidase in the NF-kappaB activation pathway by utilizing knockout mice (p47phox-/-) lacking the p47phox component of NADPH oxidase. Wild-type (WT) controls and p47phox-/- mice were treated with intraperitoneal (i.p.) Escherichia coli lipopolysaccharide (LPS) (5 or 20 microg/g of body weight). LPS-induced NF-kappaB binding activity and accumulation of RelA in nuclear protein extracts of lung tissue were markedly increased in WT compared to p47phox-/- mice 90 min after treatment with 20 but not 5 microg of i.p. LPS per g. In another model of lung inflammation, RelA nuclear translocation was reduced in p47phox-/- mice compared to WT mice following treatment with aerosolized LPS. In contrast to NF-kappaB activation in p47phox-/- mice, LPS-induced production of macrophage inflammatory protein 2 in the lungs and neutrophilic lung inflammation were not diminished in these mice compared to WT mice. We conclude that LPS-induced NF-kappaB activation is deficient in the lungs of p47phox-/- mice compared to WT mice, but this abnormality does not result in overt alteration in the acute inflammatory response.

Inhibition of (cytosine C5)-methyltransferase by oligonucleotides containing flexible (cyclopentane) and conformationally constrained (bicyclo[3.1.0]hexane) abasic sites.

Pseudorotationally locked sugar analogues based on bicyclo[3.1.0]-hexane templates were placed in DNA duplexes as abasic target sites in the M. HhaI recognition sequence. The binding affinity of the enzyme increases when the abasic site is constrained to the South conformation and decreases when it is constrained to the North conformation. A structural understanding of these differences is provided.

Dysregulated cytokine production in human cystic fibrosis bronchial epithelial cells.

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) alpha and IL-1beta stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNFalpha stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNFalpha stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1beta. Finally, when CF cells were grown at 27 degrees C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNFalpha-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.

Hypertension and type 2 diabetes comorbidity in adults in the United States: risk of overall and regional adiposity.

OBJECTIVE: To evaluate the impact of generalized, abdominal, and truncal fat deposits on the risk of hypertension and/or diabetes and to determine whether ethnic differences in these fat patterns are independently associated with increased risk for the hypertension-diabetes comorbidity (HDC). RESEARCH METHODS AND PROCEDURES: Data (n = 7075) from the Third U.S. National Health and Nutrition Examination Survey were used for this investigation. To assess risks of hypertension and/or diabetes that were due to different fat patterns, odds ratios of men and women with various cut-points of adiposities were compared with normal subjects in logistic regression models, adjusting for age, smoking, and alcohol intake. To evaluate the contribution of ethnic differences in obesity to the risks of HDC, we compared blacks and Hispanics with whites. RESULTS: Generalized and abdominal obesities were independently associated with increased risk of hypertension, diabetes and HDC in white, black, and Hispanic men and women. The risk of HDC due to generalized, truncal, and abdominal obesities tended to be higher in whites than blacks and Hispanics. In men, the contribution of black and Hispanic ethnicities to the increased risk of HDC due to the various obesity phenotypes was approximately 73% and approximately 61%, respectively. The corresponding values for black and Hispanic women were approximately 115% and approximately 125%, respectively. CONCLUSIONS: In addition to advocating behavioral lifestyles to curb the epidemic of obesity among at-risk populations in the United States, there is also the need for primary health care practitioners to craft their advice to the degree and type of obesity in these at-risk groups.

Suppression of lung inflammation in rats by prevention of NF-kappaB activation in the liver.

Activation of NF-kappaB and production of NF-kappaB-dependent chemokines are thought to be involved in the pathogenesis of neutrophilic lung inflammation. Calpain-1 inhibitor (CI-1) blocks activation of NF-kappaB by preventing proteolysis of the inhibitory protein IkappaB-alpha by the ubiquitin/proteasome pathway. We hypothesized that inhibition of proteasome function with CI-1 would block NF-kappaB activation in vivo after intraperitoneal (i.p.) treatment with bacterial lipopolysaccharide (LPS), and that NF-kappaB inhibition would be associated with suppression of chemokine gene expression and attenuation of neutrophilic alveolitis. We treated rats with a single i.p. injection of CI-1 (10 mg/kg) two hours prior to i.p. LPS (7 mg/kg). Treatment with Cl-1 prevented degradation of IkappaB-alpha and activation of NF-kappaB in the liver in response to LPS; however, Cl-1 treatment had no detected effect on NF-kappaB activation in lung tissue. CI-1 treatment prior to LPS resulted in 40% lower MIP-2 concentration in lung lavage fluid compared to rats treated with vehicle prior to LPS (502 +/- 112 pg/ml vs. 859 +/-144 pg/ml, P < 0.05). In addition, CI-1 treatment substantially inhibited LPS-induced neutrophilic alveolitis (2.7+ /- 1.2 x 10(5) vs. 43.7 +/- 12.2 x 10(5) lung lavage neutrophils, P < 0.01). These data indicate that NF-kappaB inhibition in the liver can alter lung inflammation induced by systemic LPS treatment and suggest that a liver-lung interaction contributes to the inflammatory response of the lung.