The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.

The roles of Pellino E3 ubiquitin ligases in immunity

Pellino proteins were initially characterized as a family of E3 ubiquitin ligases that can catalyse the ubiquitylation of interleukin-1 receptor-associated kinase 1 (IRAK1) and regulate innate immune signalling pathways. More recently, physiological and molecular roles for members of the Pellino family have been described in the regulation of innate and adaptive immune responses by ubiquitylation. This Review describes the emerging roles of Pellino proteins in innate and adaptive immunity and discusses the mechanistic basis of these functions.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Osteoprotegerin Expression in the Dental Pulp

Objectives: The inflammatory response to pulpal injury or infection has major clinical significance. Osteoprotegerin (OPG) is a soluble decoy receptor for Receptor Activator of NF kappa B Ligand (RANKL), preventing ligand binding to its receptor (RANK), thus inhibiting clastic cell formation. The aim of the study is to investigate the expression of OPG in human dental pulp and the effects of inflammatory mediators. This study will specifically investigate the effects of Transforming Growth Factor Beta-1 (TGF-β1) and Interleukin 1-Beta (IL-1β) on the expression of OPG on pulp fibroblasts in vitro. Method: Five primary pulp fibroblast populations were obtained by explant culture of healthy pulp tissue. Triplicate cultures were grown to confluence in 12-well plates and stimulated for 48 hours with IL-1β (10ng/ml) or TGF-β1 (10ng/ml). The conditioned media was collected and OPG levels detected by ELISA (R+D Systems, UK). Results: All fibroblast populations produced quantifiable levels of OPG in a time-dependant fashion. IL-1β significantly increased the expression of OPG (p<0.05) in all cultures. In contrast, TGF-β1 had no significant effect on OPG expression levels. In addition, previous work in our laboratory demonstrated both TGF-β1 and IL-1β stimulated OPG expression by periodontal ligament fibroblasts. Conclusion: These data indicate that IL-1β-regulated expression of OPG by pulpal fibroblasts may mediate hard tissue turnover in the inflamed dental pulp.

Substance P production by human dental pulp fibroblasts

The inflammatory response to pulpal injury or infection has major clinical significance. Neurogenic inflammation describes the local release of neuropeptides, notably substance P (SP), from afferent neurones, and may play a role in the pathogenesis of pulpal disease. The fibroblast is the most numerous cell type in the dental pulp and recent work has suggested that it is involved in the inflammatory response. Objectives: The aims of the study were to determine whether pulp fibroblasts could produce SP, and to investigate the expression of the SP receptor, NK-1, by these cells. Methods: Primary pulp fibroblast cell populations were isolated by enzymatic digestion from non-carious teeth extracted for orthodontic reasons. Whole pulp tissue was obtained from freshly extracted sound (n=35) and carious (n=39) teeth. Expression of SP and NK-1 mRNA was determined by RT-PCR. The effects of interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) on SP and NK-1 expression were also determined. The presence of NK-1 on fibroblast cell membranes was established by western blotting. The effects of the cytokines on each parameter were analysed by ANOVA. Radioimmunoassay (RIA) was carried out to quantify SP expression by pulp fibroblasts and in whole pulp tissue. Results: SP was expressed by pulpal fibroblasts both at the mRNA level and the protein level. In addition, NK-1 was detected in fibroblast cultures at the mRNA level and appeared as a double band on western blots of membrane extracts. IL-1β and TGF-β1 significantly stimulated the expression of SP and NK-1. SP levels were significantly greater (p<0.05) in carious compared to sound teeth. Conclusion: Pulp fibroblasts are capable of synthesising and secreting SP, as well as expressing the SP receptor, NK-1. These findings suggest that pulp fibroblasts play a role in neurogenic inflammation in pulpal disease. (Supported by the European Society of Endodontology.)

Multiple Markers of Inflammation in Crevicular Fluid During Experimental Gingivitis

Objective: To evaluate temporal changes in GCF levels of substance P, cathepsin G, interleukin 1 beta (IL-1&beta), neutrophil elastase and alpha1-antitrypsin (&alpha1AT) during development of and recovery from experimental gingivitis. Methods: Healthy human volunteers participated in a split-mouth study: experimental gingivitis was induced using a soft vinyl splint to cover test teeth during brushing over 21 days, after which normal brushing was resumed. Modified gingival index (MGI), gingival bleeding index (BI) and modified Quigley and Hein plaque index (PI) were assessed and 30-second GCF samples taken from 4 paired test and contra-lateral control sites in each subject at days 0, 7, 14, 21, 28 and 42. GCF volume was measured and site-specific quantification of one analyte per GCF sample was performed using radioimmunoassay (substance P), enzyme assay (cathepsin G) or ELISA (IL-1&beta, elastase, &alpha1AT). Site-specific data were analysed using analysis of repeated measurements and paired sample tests. Results: 56 subjects completed the study. All measurements at baseline (day 0) and at control sites throughout the study were low. Clinical indices and GCF volumes at the test sites increased from day 0, peaking at day 21 (difference between test and control for PI, BI, MGI and GCF all p<0.0001) and decreased again to control levels by day 28. Levels of four inflammatory markers showed a similar pattern, with significant differences between test and control apparent at 7 days (substance P p=0.0015; cathepsin G p=0.029; IL-1&beta p=0.026; elastase p=0.0129) and peaking at day 21 (substance P p=0.0023; cathepsin G, IL-1&beta and elastase all p<0.0001). Levels of &alpha1AT showed no apparent pattern over the course of the study. Conclusion: GCF levels of substance P, cathepsin G, IL-1&beta and neutrophil elastase have the potential to act as early markers of experimentally-induced gingival inflammation.

Neuropeptide Receptors in the Dental Pulp

Objectives: The inflammatory response to pulpal injury or infection has major clinical significance. The aim of the study is to investigate the presence and regulation of expression of neuropeptide receptors on human pulp fibroblasts and whole pulp tissue. This study will investigate the expression of Substance P (NK-1) and Neuropeptide Y (NPY-Y1) receptors on pulp fibroblasts, determine the effects of Transforming Growth Factor Beta-1 (TGF-b1) and Interleukin 1-Beta (IL-1b) on the expression of NK-1 and NPY-Y1 receptors on pulp fibroblasts and examine the levels of receptor expression in whole pulp samples. Methods: Primary pulp fibroblast cell lines were obtained from patients undergoing extractions for orthodontic reasons. The cells were grown to confluence and stimulated for 5 days with IL-1b or TGF-b1. Pulp tissue fragments were obtained from freshly extracted sound and carious teeth, snap frozen in liquid nitrogen and cracked open using a vice. The monolayer was removed with cell scrapers and pelleted. The cell membranes of the cultured cells and the whole tissue were isolated using a Mem-PER® Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce, UK). The membrane proteins were separated by SDS-PAGE and Western blotting was used to detect the presence of NK-1 and NPY-Y1. Results: Initial results demonstrated the presence of NK-1 and NPY-Y1 in cultured pulp fibroblasts. Following the 5 day incubation with TGF-b1, the cells appeared not to express NK-1. IL-1b had a slight stimulatory effect on NK-1 expression. The NPY-Y1 expression was not affected by either TGF-b1 or IL-1b. In whole pulp samples, levels of NK-1 were increased in carious teeth compared to caries-free teeth. The NPY-Y1 levels were similar in carious and non-carious teeth. Conclusion: These findings give an insight into how pulp cells react to inflammatory stimuli with regards to neuropeptide receptor expression and their roles in health and disease

Cigarette smokers have exaggerated alveolar barrier disruption in response to lipopolysaccharide inhalation

RATIONALE: Cigarette smoke exposure is associated with an increased risk of the acute respiratory distress syndrome (ARDS); however, the mechanisms underlying this relationship remain largely unknown.

OBJECTIVE: To assess pathways of lung injury and inflammation in smokers and non-smokers with and without lipopolysaccharide (LPS) inhalation using established biomarkers.

METHODS: We measured plasma and bronchoalveolar lavage (BAL) biomarkers of inflammation and lung injury in smokers and non-smokers in two distinct cohorts of healthy volunteers, one unstimulated (n=20) and one undergoing 50 μg LPS inhalation (n=30).

MEASUREMENTS AND MAIN RESULTS: After LPS inhalation, cigarette smokers had increased alveolar-capillary membrane permeability as measured by BAL total protein, compared with non-smokers (median 274 vs 208 μg/mL, p=0.04). Smokers had exaggerated inflammation compared with non-smokers, with increased BAL interleukin-1β (p=0.002), neutrophils (p=0.02), plasma interleukin-8 (p=0.003), and plasma matrix metalloproteinase-8 (p=0.006). Alveolar epithelial injury after LPS was more severe in smokers than non-smokers, with increased plasma (p=0.04) and decreased BAL (p=0.02) surfactant protein D. Finally, smokers had decreased BAL vascular endothelial growth factor (VEGF) (p<0.0001) with increased soluble VEGF receptor-1 (p=0.0001).

CONCLUSIONS: Cigarette smoke exposure may predispose to ARDS through an abnormal response to a 'second hit,' with increased alveolar-capillary membrane permeability, exaggerated inflammation, increased epithelial injury and endothelial dysfunction. LPS inhalation may serve as a useful experimental model for evaluation of the acute pulmonary effects of existing and new tobacco products.

Interleukin 18 promoter polymorphisms are not strongly associated with type 1 diabetes in a UK population

Interleukin 18 (IL18) is a proinflammatory cytokine whose levels are increased in the subclinical stage of insulin-dependent (type I) diabetes mellitus. Previous case-control studies have reported associations between IL18 -607C>A and -137G>C promoter polymorphisms and type I diabetes. We performed case-control and family-based association studies employing Pyrosequencing to assess if these IL18 polymorphisms are also associated with the development of type I diabetes in the Northern Ireland population. The chi2 analysis of genotype and allele frequencies for the IL18 polymorphisms in cases (n=433) vs controls (n=426) revealed no significant differences (P>0.05). Assessment of allele transmission distortion from informative parents to affected offspring also failed to confirm previously reported associations. Stratification of these analyses for age-at-onset and HLA-DR type did not reveal any significance associations. In conclusion, our data do not support the strong positive associations of IL18 promoter polymorphisms with type I diabetes reported in previous smaller studies.

Testing the possible negative association of type 1 diabetes and atopic disease by analysis of the interleukin 4 receptor gene

Variations in the interleukin 4 receptor A (IL4RA) gene have been reported to be associated with atopy, asthma, and allergy, which may occur less frequently in subjects with type 1 diabetes (T1D). Since atopy shows a humoral immune reactivity pattern, and T1D results from a cellular (T lymphocyte) response, we hypothesised that alleles predisposing to atopy could be protective for T1D and transmitted less often than the expected 50% from heterozygous parents to offspring with T1D. We genotyped seven exonic single nucleotide polymorphisms (SNPs) and the -3223 C>T SNP in the putative promoter region of IL4RA in up to 3475 T1D families, including 1244 Finnish T1D families. Only the -3223 C>T SNP showed evidence of negative association (P=0.014). There was some evidence for an interaction between -3233 C>T and the T1D locus IDDM2 in the insulin gene region (P=0.001 in the combined and P=0.02 in the Finnish data set). We, therefore, cannot rule out a genetic effect of IL4RA in T1D, but it is not a major one.

Interaction between interleukin 3 and dystrobrevin-binding protein 1 in schizophrenia

Schizophrenia is a common psychotic mental disorder that is believed to result from the effects of multiple genetic and environmental factors. In this study, we explored gene-gene interactions and main effects in both case-control (657 cases and 411 controls) and family-based (273 families, 1350 subjects) datasets of English or Irish ancestry. Fifty three markers in 8 genes were genotyped in the family sample and 44 markers in 7 genes were genotyped in the case-control sample. The Multifactor Dimensionality Reduction Pedigree Disequilibrium Test (MDR-PDT) was used to examine epistasis in the family dataset and a 3-locus model was identified (permuted p=0.003). The 3-locus model involved the IL3 (rs2069803), RGS4 (rs2661319), and DTNBP1 (rs21319539) genes. We used MDR to analyze the case-control dataset containing the same markers typed in the RGS4, IL3 and DTNBP1 genes and found evidence of a joint effect between IL3 (rs31400) and DTNBP1 (rs760761) (cross-validation consistency 4/5, balanced prediction accuracy=56.84%, p=0.019). While this is not a direct replication, the results obtained from both the family and case-control samples collectively suggest that IL3 and DTNBP1 are likely to interact and jointly contribute to increase risk for schizophrenia. We also observed a significant main effect in DTNBP1, which survived correction for multiple comparisons, and numerous nominally significant effects in several genes. (C) 2008 Elsevier B.V. All rights reserved.

The interleukin 1beta gene promoter polymorphism (-511) acts as a risk factor for psychosis in Alzheimer's dementia

The explanation for why some patients develop psychotic change in Alzheimer's disease (AD) is unclear. "Psychosis-modifier genes" may act in the setting of neurodegeneration to produce AD plus psychosis in a similar way to how genetic modulation during neurodevelopment leads to schizophrenia. Because there is increasing interest in the common disruption of cytokine pathways seen in both AD and schizophrenia, we tested the association between the functional interleukin-1beta -511 promoter polymorphism with delusions and hallucinations in AD. Significant associations between psychotic symptoms and the CC genotype (p = 0.001 - p = 0.043) and C allele (p = 0.014 vs p = 0.048) were found, thus confirming the previously noted increased risk in schizophrenia.