46 research outputs found
HMGB1 : regulation of inflammatory functions and therapeutic blockade
The intracellular protein High Mobility Group Box Protein 1 (HMGB1) has been
identified as a pivotal mediator of inflammation. HMGB1 can be released by various
mechanisms and as an inflammatory mediator it induces both migration of
inflammatory cells and cytokine production. Consequently, HMGB1 has been
demonstrated to contribute to pathology in several inflammatory conditions.
Increasing evidence indicate that HMGB1 post-translational modifications (PTMs)
regulate both secretion and function of HMGB1.
The focus of this thesis work has been to investigate how selected PTMs
regulate HMGB1 function and release and to define the presence of such
modifications on HMGB1 in synovial fluid from patients with juvenile idiopathic
arthritis (JIA). Furthermore, I have set the basis for HMGB1-blockade as a clinical
treatment option by generating and characterizing the first known chimeric,
humanized anti-HMGB1 antibody.
To examine the impact of redox-dependent PTMs on HMGB1 function, we first
generated several cysteine redox isoforms of HMGB1. We found that all cysteines
residues (C23, C45 and C106) required a defined redox state. A disulfide bridge
between C23 and C45 with a concomitant C106 thiol was necessary for HMGB1
mediated cytokine-induction. In this disulfide redox isoform, HMGB1 activates
TLR4. Furthermore, I have studied PTMs and their impact on HMGB1 secretion. We
demonstrated that NLRC4 inflammasome activation induces hyperacetylation of
key lysine stretches known to be associated with HMGB1 secretion, independently
of priming signals. Addition of a priming signal induced reactive oxygen species
(ROS) that stimulated a structural transition of HMGB1 to its cytokine-inducing,
disulfide form. Hyperacetylated HMGB1 correlated significantly with inflammatory
HMGB1 redox isoforms in joint fluid from JIA patients, indicating that HMGB1 is
actively secreted during JIA and possesses inflammatory properties.
In addition, I recorded beneficial effects of mouse monoclonal anti-HMGB1
antibody (m2G7) treatment in experimental arthritis and in acetaminopheninduced liver injury. Importantly, I could demonstrate that a partly humanized
version of the antibody (h2G7) retained its in vitro properties and in vivo
therapeutic effects.
In conclusion, this thesis has significantly increased the understanding of the
regulation of HMGB1 secretion and function during inflammation. The generation
of an anti-HMGB1 chimeric antibody is an important step in development of a
clinical anti-HMGB1 treatment
FAM13A and POM121C are candidate genes for fasting insulin: functional follow-up analysis of a genome-wide association study
Aims/hypothesis: By genome-wide association meta-analysis, 17 genetic loci associated with fasting serum insulin (FSI), a
marker of systemic insulin resistance, have been identified. To define potential culprit genes in these loci, in a cross-sectional
study we analysed white adipose tissue (WAT) expression of 120 genes in these loci in relation to systemic and adipose tissue
variables, and functionally evaluated genes demonstrating genotype-specific expression in WAT (eQTLs).
Methods: Abdominal subcutaneous adipose tissue biopsies were obtained from 114 women. Basal lipolytic activity was measured
as glycerol release from adipose tissue explants. Adipocytes were isolated and insulin-stimulated incorporation of
radiolabelled glucose into lipids was used to quantify adipocyte insulin sensitivity. Small interfering RNA-mediated knockout
in human mesenchymal stem cells was used for functional evaluation of genes.
Results: Adipose expression of 48 of the studied candidate genes associated significantly with FSI, whereas expression of 24, 17
and 2 genes, respectively, associated with adipocyte insulin sensitivity, lipolysis and/or WAT morphology (i.e. fat cell size relative
to total body fat mass). Four genetic loci contained eQTLs. In one chromosome 4 locus (rs3822072), the FSI-increasing allele
associated with lower FAM13A expression and FAM13A expression associated with a beneficial metabolic profile including
decreased WAT lipolysis (regression coefficient, R = â0.50, p = 5.6 Ă 10â7). Knockdown of FAM13A increased lipolysis by ~1.5-
fold and the expression of LIPE (encoding hormone-sensitive lipase, a rate-limiting enzyme in lipolysis). At the chromosome 7
locus (rs1167800), the FSI-increasing allele associated with lower POM121C expression. Consistent with an insulin-sensitising
function, POM121C expression associated with systemic insulin sensitivity (R = â0.22, p = 2.0 Ă 10â2), adipocyte insulin sensitivity
(R = 0.28, p = 3.4 Ă 10â3) and adipose hyperplasia (R = â0.29, p = 2.6 Ă 10â2). POM121C knockdown decreased expression
of all adipocyte-specific markers by 25â50%, suggesting that POM121C is necessary for adipogenesis.
Conclusions/interpretation: Gene expression and adipocyte functional studies support the notion that FAM13A and POM121C
control adipocyte lipolysis and adipogenesis, respectively, and might thereby be involved in genetic control of systemic insulin
sensitivity
Genome-wide association study of diabetogenic adipose morphology in the GENetics of Adipocyte Lipolysis (GENiAL) Cohort
An increased adipocyte size relative to the size of fat depots, also denoted hypertrophic adipose morphology, is a strong risk factor for the future development of insulin resistance and type 2 diabetes. The regulation of adipose morphology is poorly understood. We set out to identify genetic loci associated with adipose morphology and functionally evaluate candidate genes for impact on adipocyte development. We performed a genome-wide association study (GWAS) in the unique GENetics of Adipocyte Lipolysis (GENiAL) cohort comprising 948 participants who have undergone abdominal subcutaneous adipose biopsy with a determination of average adipose volume and morphology. The GWAS identified 31 genetic loci displaying suggestive association with adipose morphology. Functional evaluation of candidate genes by small interfering RNAs (siRNA)-mediated knockdown in adipose-derived precursor cells identified six genes controlling adipocyte renewal and differentiation, and thus of potential importance for adipose hypertrophy. In conclusion, genetic and functional studies implicate a regulatory role for ATL2, ARHGEF10, CYP1B1, TMEM200A, C17orf51, and L3MBTL3 in adipose morphology by their impact on adipogenesis
Genome-wide association study of adipocyte lipolysis in the GENetics of adipocyte lipolysis (GENiAL) cohort.
OBJECTIVES:Lipolysis, hydrolysis of triglycerides to fatty acids in adipocytes, is tightly regulated, poorly understood, and, if perturbed, can lead to metabolic diseases including obesity and type 2 diabetes. The goal of this study was to identify the genetic regulators of lipolysis and elucidate their molecular mechanisms. METHODS:Adipocytes from abdominal subcutaneous adipose tissue biopsies were isolated and were incubated without (spontaneous lipolysis) or with a catecholamine (stimulated lipolysis) to analyze lipolysis. DNA was extracted and genome-wide genotyping and imputation conducted. After quality control, 939 samples with genetic and lipolysis data were available. Genome-wide association studies of spontaneous and stimulated lipolysis were conducted. Subsequent in vitro gene expression analyses were used to identify candidate genes and explore their regulation of adipose tissue biology. RESULTS:One locus on chromosome 19 demonstrated genome-wide significance with spontaneous lipolysis. 60 loci showed suggestive associations with spontaneous or stimulated lipolysis, of which many influenced both traits. In the chromosome 19 locus, only HIF3A was expressed in the adipocytes and displayed genotype-dependent gene expression. HIF3A knockdown in vitro increased lipolysis and the expression of key lipolysis-regulating genes. CONCLUSIONS:In conclusion, we identified a genetic regulator of spontaneous lipolysis and provided evidence of HIF3A as a novel key regulator of lipolysis in subcutaneous adipocytes as the mechanism through which the locus influences adipose tissue biology
Redox modifications of cysteine residues regulate the cytokine activity of HMGB1.
BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is passively released during cell death and secreted by activated cells of many lineages. HMGB1 contains three conserved redox-sensitive cysteine residues: cysteines in position 23 and 45 (C23 and C45) can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. METHODS: Primary human macrophages or murine macrophage-like RAW 264.7 cells were activated in cell cultures by redox-modified or point-mutated (C45A) recombinant HMGB1 preparations or by lipopolysaccharide (E. coli.0111: B4). Cellular phosphorylated NF-ÎșB p65 subunit and subsequent TNF-α release were quantified by commercial enzyme-linked immunosorbent assays. RESULTS: Cell cultures with primary human macrophages and RAW 264.7 cells demonstrated that fully reduced HMGB1 with all three cysteines expressing thiol side chains failed to generate phosphorylated NF-ĐB p65 subunit or TNF-α. Mild oxidation forming a C23-C45 disulfide bond, while leaving C106 with a thiol group, was required for HMGB1 to induce phosphorylated NF-ĐB p65 subunit and TNF-α production. The importance of a C23-C45 disulfide bond was confirmed by mutation of C45 to C45A HMGB1, which abolished the ability for cytokine induction. Further oxidation of the disulfide isoform also inactivated HMGB1. CONCLUSIONS: These results reveal critical post-translational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during inflammation
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Novel role of PKR in inflammasome activation and HMGB1 release
The inflammasome regulates release of caspase activation-dependent cytokines, including IL-1ÎČ, IL-18, and high-mobility group box 1 (HMGB1)1-5. During the course of studying HMGB1 release mechanisms, we discovered an important role of double-stranded RNA dependent protein kinase (PKR) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, rotenone, live E. coli, anthrax lethal toxin, DNA transfection, and S. Typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1beta, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with multiple inflammasome components, including NLR family pyrin domain-containing 3 (NLRP3), NLR family pyrin domain-containing 1 (NLRP1), NLR family CARD domain-containing protein 4 (NLRC4), Absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. These results reveal a critical role of PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation
MD-2 is required for disulfide HMGB1-dependent TLR4 signaling
Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2âdeficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2âHMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4âMD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness
Changes in lung function in European adults born between 1884 and 1996 and implications for the diagnosis of lung disease:a cross-sectional analysis of ten population-based studies
Background: During the past century, socioeconomic and scientific advances have resulted in changes in the health and physique of European populations. Accompanying improvements in lung function, if unrecognised, could result in the misclassification of lung function measurements and misdiagnosis of lung diseases. We therefore investigated changes in population lung function with birth year across the past century, accounting for increasing population height, and examined how such changes might influence the interpretation of lung function measurements.
Methods: In our analyses of cross-sectional data from ten European population-based studies, we included individuals aged 20-94 years who were born between 1884 and 1996, regardless of previous respiratory diagnoses or symptoms. FEV1, forced vital capacity (FVC), height, weight, and smoking behaviour were measured between 1965 and 2016. We used meta-regression to investigate how FEV1 and FVC (adjusting for age, study, height, sex, smoking status, smoking pack-years, and weight) and the FEV1/FVC ratio (adjusting for age, study, sex, and smoking status) changed with birth year. Using estimates from these models, we graphically explored how mean lung function values would be expected to progressively deviate from predicted values. To substantiate our findings, we used linear regression to investigate how the FEV1 and FVC values predicted by 32 reference equations published between 1961 and 2015 changed with estimated birth year.
Findings: Across the ten included studies, we included 243 465 European participants (mean age 51·4 years, 95% CI 51·4-51·5) in our analysis, of whom 136 275 (56·0%) were female and 107 190 (44·0%) were male. After full adjustment, FEV1 increased by 4·8 mL/birth year (95% CI 2·6-7·0; p<0·0001) and FVC increased by 8·8 mL/birth year (5·7-12·0; p<0·0001). Birth year-related increases in the FEV1 and FVC values predicted by published reference equations corroborated these findings. This height-independent increase in FEV1 and FVC across the last century will have caused mean population values to progressively exceed previously predicted values. However, the population mean adjusted FEV1/FVC ratio decreased by 0·11 per 100 birth years (95% CI 0·09-0·14; p<0·0001).
Interpretation: If current diagnostic criteria remain unchanged, the identified shifts in European values will allow the easier fulfilment of diagnostic criteria for lung diseases such as chronic obstructive pulmonary disease, but the systematic underestimation of lung disease severity.
Funding: The European Respiratory Society, AstraZeneca, Chiesi Farmaceutici, GlaxoSmithKline, Menarini, and Sanofi-Genzyme
COPD in never smokers: results from the population-based burden of obstructive lung disease study.
To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.BACKGROUND: Never smokers comprise a substantial proportion of patients with COPD. Their characteristics and possible risk factors in this population are not yet well defined. METHODS: We analyzed data from 14 countries that participated in the international, population-based Burden of Obstructive Lung Disease (BOLD) study. Participants were aged â„ 40 years and completed postbronchodilator spirometry testing plus questionnaires about respiratory symptoms, health status, and exposure to COPD risk factors. A diagnosis of COPD was based on the postbronchodilator FEVâ/FVC ratio, according to current GOLD (Global Initiative for Obstructive Lung Disease) guidelines. In addition to this, the lower limit of normal (LLN) was evaluated as an alternative threshold for the FEVâ/FVC ratio. RESULTS: Among 4,291 never smokers, 6.6% met criteria for mild (GOLD stage I) COPD, and 5.6% met criteria for moderate to very severe (GOLD stage II+) COPD. Although never smokers were less likely to have COPD and had less severe COPD than ever smokers, never smokers nonetheless comprised 23.3% (240/1,031) of those classified with GOLD stage II+ COPD. This proportion was similar, 20.5% (171/832), even when the LLN was used as a threshold for the FEVâ/FVC ratio. Predictors of COPD in never smokers include age, education, occupational exposure, childhood respiratory diseases, and BMI alterations. CONCLUSION: This multicenter international study confirms previous evidence that never smokers comprise a substantial proportion of individuals with COPD. Our data suggest that, in addition to increased age, a prior diagnosis of asthma and, among women, lower education levels are associated with an increased risk for COPD among never smokers.ALTANA
Aventis
AstraZeneca
Boehringer-Ingleheim
Chiesi
GlaxoSmithKline
Merck
Novartis
Pfizer Inc
Schering-Plough
Sunovion Pharmaceuticals Inc
University of Kentucky
Schering Plough
Sepracor
AstraZeneca, Spai