Proinflammatory cytokine interferon-γ and microbiome-derived metabolites dictate epigenetic switch between forkhead box protein 3 isoforms in coeliac disease.

Coeliac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong T helper type 1 (Th1)/Th17 immune response in the small intestine. Regulatory T cells (Treg ) are CD4+ CD25++ forkhead box protein 3 (FoxP3+ ) cells that regulate the immune response. Conversely to its counterpart, FoxP3 full length (FL), the alternatively spliced isoform FoxP3 Δ2, cannot properly down-regulate the Th17-driven immune response. As the active state of CD has been associated with impairments in Treg cell function, we aimed at determining whether imbalances between FoxP3 isoforms may be associated with the disease. Intestinal biopsies from patients with active CD showed increased expression of FOXP3 Δ2 isoform over FL, while both isoforms were expressed similarly in non-coeliac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating alternative splicing. The proinflammatory intestinal microenvironment of active patients has been reported to be enriched in butyrate-producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of interferon (IFN)-γ and butyrate triggers the balance between FoxP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore, we report that lactate increases both isoforms in CD patients. Collectively, these findings highlight the importance of the ratio between FoxP3 isoforms in CD and, for the first time, associate the alternative splicing process mechanistically with microbial-derived metabolites.

Differences in body composition between metabolically healthy obese and metabolically abnormal obese adults.

Potential differences in body composition between metabolically healthy obese (MHO) and metabolically abnormal obese (OA) adults were explored with 395 obese adults from the Pennington Center Longitudinal Study (18-68 years). Adults were classified as OA (≥2 risk factors: blood pressure ≥130/85 mmHg; triglycerides ≥150 mg dl(-1); high-density lipoprotein cholesterol: men <40, women <50 mg dl(-1); fasting glucose ≥100 mg dl(-1); waist circumference: ≥102 cm men, women ≥88 cm) or MHO (<2 risk factors). Whole-body bone mineral density and content, percent body fat, fat mass, lean mass and trunk adipose tissue mass were measured with dual-energy X-ray absorptiometry. Visceral (VAT), subcutaneous (SAT) and total abdominal adipose tissue (TAT) were measured with computed tomography. Gender-specific general linear regression models were used to determine differences in body composition between MHO and OA controlling for age, race, smoking status and menopause status. In men, MHO had lower fat mass (kg and %), trunk adipose tissue, VAT, SAT, TAT and lean mass compared with OA. MHO women had lower fat mass (kg), lean mass, trunk adipose tissue, VAT and TAT when compared with OA women. In conclusion, OA and MHO cardiometabolic profiles are characterized by differences in body composition consistent between genders.

Relationships among changes in C-reactive protein and cardiovascular disease risk factors with lifestyle interventions.

BACKGROUND AND AIMS: Inflammation plays a role in the development of cardiovascular disease (CVD). Elevated levels of the inflammatory marker, C-reactive protein (CRP), are cross-sectionally associated with traditional CVD risk factors and are being considered as an emerging CVD risk factor. In a secondary data analysis, we examined changes in CRP and several CVD risk factors after one-year diet and physical activity interventions to assess whether CRP changed concurrently with other risk factors, or was independent of the traditional risk factors. METHODS AND RESULTS: Data were analyzed from 143 men and 133 women with dyslipidemia who were randomized to one-year interventions of low-fat diet only, physical activity only, diet plus physical activity, or control. Plasma high-sensitivity CRP, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides (TG), fasting and 2-hr blood glucose and insulin, blood pressure (BP), and waist circumference were obtained at baseline and follow-up. Multiple linear regression models were used to predict CRP change based on other risk factor changes, controlling for age, race, alcohol intake, and hormone replacement therapy. Treatment groups were combined for analysis. Baseline mean (SD) CRP levels were 1.3 ± 1.3 (men) and 1.9 ± 1.8 mg/L (women), with mean changes of -0.11 ± 1.3 and -0.17 ± 1.5 mg/L, respectively. Plasma CRP change was negatively associated with TG change in men (p = 0.003) and women (p = 0.05), positively associated with change in systolic BP in men (p = 0.01), but was not associated with changes in the other risk factors. CONCLUSION: Dietary and/or physical activity induced changes in CRP may be largely independent of traditional CVD risk factors in persons with dyslipidemia.

Reactive oxygen and nitrogen metabolites modulate fibronectin-induced fibroblast migration in vitro.

Nitration of proteins by peroxynitrite may alter protein function. We hypothesized that reactive nitrogen species modulate fibronectin-induced fibroblast migration. To test this hypothesis, we evaluated fibroblast migration induced by fibronectin incubated with and without peroxynitrite. Peroxynitrite attenuated fibronectin-induced fibroblast migration in a dose-dependent manner but did not attenuate complement-activated serum-induced fibroblast migration. The reducing agents, deferoxamine and dithiothreitol (DTT), and L-tyrosine reversed the inhibition by peroxynitrite. PAPA-NONOate, a nitric oxide (NO) donor, and superoxide generated by the action of xanthine oxidase on lumazine or xanthine, also showed an inhibitory effect on fibroblast migration. The peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), caused a concentration-dependent inhibition of fibroblast migration. Peroxynitrite reduced fibronectin binding to fibroblasts and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of fibronectin binding to fibroblasts and suggest that peroxynitrite may play a role in regulation of fibroblast migration.

Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro.

Peroxynitrite, formed by nitric oxide (NO) and superoxide, can alter protein function by nitrating amino acids such as tyrosine, cysteine, trytophan, or methionine. Inducible nitric oxide synthase (Type 2 NOS or iNOS) converts arginine to citrulline, releasing NO. We hypothesized that peroxynitrite could function as a negative feedback modulator of NO production by nitration of iNOS. Confluent cultures of the murine lung epithelial cell line, LA-4 were stimulated with cytokines to express iNOS, peroxynitrite was added, and the flasks sealed. After 3 h, NO in the headspace above the culture was sampled. Peroxynitrite caused a concentration-dependent decrease in NO. Similar results were obtained when 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, was added to the flasks. PAPA-NONOate, the NO generator, did not affect the headspace NO. Nitration of the iNOS was confirmed by detection of 3-nitrotyrosine by Western blotting. These data suggest a mechanism for inhibition of NO synthesis at inflammatory sites where iNOS, NO, and superoxide would be expected.

The oxidative stress response.

Oxidative stress resulting from toxic effects of reactive oxygen species (ROS) plays an important role in the pathogenesis of a variety of diseases and important biological processes. Toxic effects of these ROS, including the superoxide and hydroxyl radicals, and hydrogen peroxide can cause cellular damage by oxidizing nucleic acids, proteins, and membrane lipids. While the chemical reactions involved in the generation and detoxification of ROS have been studied in great detail, little is known about the cellular and molecular responses to oxidative stress in mammalian cells. This article discusses some of the major aspects of these molecular responses, including alterations in the gene expression of antioxidant enzymes, stress-response genes, and cytokines. The regulatory mechanisms that control this genetic response are highly complex, involving activation of transcription factors and signal transduction pathways. Further characterization of the mechanisms that regulate these molecular responses is essential for understanding the physiologic function of the responses and for the development of new therapeutic modalities to defend and/or adapt to oxidant injury.

Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and inducer-dependent transcription enhancer.

A 161-base pair fragment (AB1) approximately 10 kilobase pairs upstream of the transcription start site of the mouse heme oxygenase-1 gene functions as a basal level and inducer-dependent enhancer. AB1/chloramphenicol acetyltransferase fusion genes stably transfected into mouse hepatoma (Hepa) cells or L929 fibroblasts were activated 7-8- or 17-22-fold, respectively, after treatment of the cells with either CdCl2 or heme. The AB1 fragment is composed largely of three tandem repeats containing two conserved core elements, A and B. Part of core element A (TCCGGAGCTGTG) resembles the consensus-binding site for transcription factor AP-4, whereas core element B (GCTGAGTCANGG) includes the consensus-binding site (TGAGTCA) for the AP-1 family of transcription factors. Nuclear proteins from Hepa cells did not bind to any of the core A elements, but bound to all three copies of the core B element. AB1 derivatives with one or two mutant AP-1-binding elements exhibited reduced but measurable inducer-dependent enhancer activity, but mutation of all three AP-1-binding sites abolished activation by CdCl2 and heme and also by mercury chloride, zinc chloride, H2O2, sodium arsenate, and 12-O-tetradecanoylphorbol-13-acetate. Pretreatment of stably transfected L929 cells with protein kinase C inhibitors, but not with tyrosine kinase inhibitors or N-acetylcysteine, abrogated 12-O-tetradecanoylphorbol-13-acetate-dependent activation of the AB1/chloramphenicol acetyltransferase fusion gene. Induction by H2O2 was unaffected by the kinase inhibitors, but completely abolished by N-acetylcysteine. Heme-dependent induction was not significantly affected by any of these chemicals.

Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation.

Gram-negative sepsis is the most common cause of the adult respiratory distress syndrome (ARDS). Lipopolysaccharide (LPS) when administered in vivo produces pathophysiologic changes similar to those seen in ARDS. The pathogenesis of these changes is mediated in part by oxidative stress. We demonstrate that LPS induces high mRNA levels of the stress-inducible gene heme oxygenase-1 (HO-1) in the rat lung. Increased HO-1 mRNA levels correlate with increased HO-1 protein and enzyme activity. Immunohistochemical analyses of lung tissues from rats treated with LPS reveal abundant HO-1 expression in inflammatory and bronchoalveolar epithelial cells. We further examined the molecular regulation of HO-1 gene expression after exposure of RAW 264.7 macrophage cells to LPS in vitro. These cells respond to LPS with increased HO-1 mRNA expression and HO-1 gene transcription. Transcriptional activation of the mouse HO-1 gene by LPS is mediated by a 5' distal enhancer fragment located approximately 4 kbp upstream from the transcription site. Electrophoretic mobility shift assays show increased activator protein-1 (AP-1) binding activity in RAW 264.7 cells after LPS treatment. Mutation of the AP-1 binding site in this enhancer fragment completely abolishes HO-1 gene activation while mutation of CCAAT/enhancer-binding protein (C/EBP) binding site exerts negligible effect, suggesting that the AP-1 family of transcription factors plays a critical role in regulating HO-1 gene activation following LPS treatment. Furthermore, upstream phosphorylation events modulate this AP-1-dependent expression of the HO-1 gene after LPS treatment.

Methotrexate stimulates lung fibroblasts and epithelial cells to release eosinophil chemotactic activity.

OBJECTIVE: To determine if methotrexate (MTX) induces human lung fibroblast (HFL-1) and epithelial (BEAS 2B) cell lines to release eosinophil chemotactic activity (ECA). METHODS: HFL-1 and BEAS 2B cell supernatant fluids were evaluated for ECA by a blind well chamber technique. RESULTS: HFL-1 and BEAS-2B cells released ECA in a dose and time dependent manner in response to MTX. Partial characterization revealed that ECA was partly heat labile, trypsin sensitive, and ethylacetate extractable. Thus the culture supernatant fluids were evaluated for known eosinophil chemotactic factors. Although several were released constitutively, granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly increased in response to MTX from both cell types. Consistent with these observations, ECA from both cell types was inhibited by GM-CSF antibodies. CONCLUSION: These data suggest that lung fibroblasts and epithelial cells may modulate eosinophil recruitment into the lung by releasing ECA in response to MTX.

Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5' distal enhancers: role of reactive oxygen intermediates and AP-1.

Heme oxygenase-1 (HO-1) is a stress-response protein, the expression of which is transcriptionally regulated by agents that cause oxidative stress. We have previously shown that lipopolysaccharide (LPS)-induced HO-1 gene transcription in RAW 264.7 macrophage cells is mediated by a distal enhancer called SX2, located 4 kb upstream from the HO-1 transcription initiation site (Am. J. Respir. Cell Mol. Biol. 1995;13:387-398). We have recently identified a second distal enhancer, called AB1, located 6 kb upstream from the SX2 distal enhancer (J. Biol. Chem. 1995;270:11977-11984). Here we report the extension of our studies to investigate whether the AB1 distal enhancer and/or other potential regulatory elements in the entire 5' distal flanking sequences (11-kb region) of the HO-1 gene may also mediate HO-1 gene transcription in response to LPS. Using deletional analysis, we found that the AB1 enhancer also mediates LPS-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer and electrophoretic-mobility-shift assays of nuclear extracts from LPS-treated cells further demonstrated that the transcription factor activator protein-1 (AP-1) is critical for AB1-mediated HO-1 gene activation by LPS. We also found increased expression of AP-1 family members c-fos and c-jun by Northern blot analyses after treatment with LPS. Further, we observed that LPS-treated RAW 264.7 cells produced high levels of reactive oxygen intermediates (ROI) as measured through flow-cytometric analysis of dichlorofluoroscein (DCF)-stained cells. Treatment of cells with the antioxidants N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide (DMSO) not only blunts LPS-induced production of ROI, but also significantly attenuates LPS-induced HO-1 messenger RNA (mRNA) expression and gene transcription. Taken together, these data suggest that LPS regulates HO-1 gene transcription in part by inducing the production of ROI, which initiate signal-transduction pathway(s) leading to the activation of AP-1-dependent HO-1 gene transcription.

AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-1.

We have previously shown marked induction of the stress-inducible gene heme oxygenase-1 (HO-1) in vivo and in vitro after hyperoxia. In RAW 264.7 cells, HO-1 induction is transcriptionally regulated and dependent on cooperation between the HO-1 gene promoter and the 5' distal enhancer element SX2. In our present study, further deletional and mutational analyses demonstrate that signal transducer and activator of transcription (STAT) DNA binding sites located in the promoter of HO-1 and activator protein (AP)-1 DNA binding sites in the distal enhancer element SX2 are necessary for optimal HO-1 gene activation after hyperoxia. Interestingly, a second 5' distal enhancer element, AB1, located 10 kb upstream from the HO-1 promoter, alone is activated after hyperoxia but cannot confer maximal hyperoxia-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer shows that AP-1 is essential for AB1-mediated HO-1 gene transcription after hyperoxia. Electromobility shift assays show increased STAT1, STAT3, STAT5, and AP-1 DNA binding activity in RAW 264.7 cells after hyperoxia. Taken together, our data suggest that the 5' distal enhancer elements of the HO-1 gene in concert with the promoter regulate HO-1 gene induction and highlight the complexity of HO-1 gene transcription in response to hyperoxia.