Dibutyltin Disrupts Glucocorticoid Receptor Function and Impairs Glucocorticoid-Induced Suppression of Cytokine Production

BACKGROUND: Organotins are highly toxic and widely distributed environmental chemicals. Dibutyltin (DBT) is used as stabilizer in the production of polyvinyl chloride plastics, and it is also the major metabolite formed from tributyltin (TBT) in vivo. DBT is immunotoxic, however, the responsible targets remain to be defined. Due to the importance of glucocorticoids in immune-modulation, we investigated whether DBT could interfere with glucocorticoid receptor (GR) function. METHODOLOGY: We used HEK-293 cells transiently transfected with human GR as well as rat H4IIE hepatoma cells and native human macrophages and human THP-1 macrophages expressing endogenous receptor to study organotin effects on GR function. Docking of organotins was used to investigate the binding mechanism. PRINCIPAL FINDINGS: We found that nanomolar concentrations of DBT, but not other organotins tested, inhibit ligand binding to GR and its transcriptional activity. Docking analysis indicated that DBT inhibits GR activation allosterically by inserting into a site close to the steroid-binding pocket, which disrupts a key interaction between the A-ring of the glucocorticoid and the GR. DBT inhibited glucocorticoid-induced expression of phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine-aminotransferase (TAT) and abolished the glucocorticoid-mediated transrepression of TNF-alpha-induced NF-kappaB activity. Moreover, DBT abrogated the glucocorticoid-mediated suppression of interleukin-6 (IL-6) and TNF-alpha production in lipopolysaccharide (LPS)-stimulated native human macrophages and human THP-1 macrophages. CONCLUSIONS: DBT inhibits ligand binding to GR and subsequent activation of the receptor. By blocking GR activation, DBT may disturb metabolic functions and modulation of the immune system, providing an explanation for some of the toxic effects of this organotin

Suppression of Ribosomal Function Triggers Innate Immune Signaling through Activation of the NLRP3 Inflammasome

Some inflammatory stimuli trigger activation of the NLRP3 inflammasome by inducing efflux of cellular potassium. Loss of cellular potassium is known to potently suppress protein synthesis, leading us to test whether the inhibition of protein synthesis itself serves as an activating signal for the NLRP3 inflammasome. Murine bone marrow-derived macrophages, either primed by LPS or unprimed, were exposed to a panel of inhibitors of ribosomal function: ricin, cycloheximide, puromycin, pactamycin, and anisomycin. Macrophages were also exposed to nigericin, ATP, monosodium urate (MSU), and poly I:C. Synthesis of pro-IL-ß and release of IL-1ß from cells in response to these agents was detected by immunoblotting and ELISA. Release of intracellular potassium was measured by mass spectrometry. Inhibition of translation by each of the tested translation inhibitors led to processing of IL-1ß, which was released from cells. Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome. Despite the inability of these inhibitors to trigger efflux of intracellular potassium, the addition of high extracellular potassium suppressed activation of the NLRP3 inflammasome. MSU and double-stranded RNA, which are known to activate the NLRP3 inflammasome, also substantially inhibited protein translation, supporting a close association between inhibition of translation and inflammasome activation. These data demonstrate that translational inhibition itself constitutes a heretofore-unrecognized mechanism underlying IL-1ß dependent inflammatory signaling and that other physical, chemical, or pathogen-associated agents that impair translation may lead to IL-1ß-dependent inflammation through activation of the NLRP3 inflammasome. For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome. For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined

YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity

Construction of a large scale integrated map of macrophage pathogen recognition and effector systems

<p>Abstract</p> <p>Background</p> <p>In an effort to better understand the molecular networks that underpin macrophage activation we have been assembling a map of relevant pathways. Manual curation of the published literature was carried out in order to define the components of these pathways and the interactions between them. This information has been assembled into a large integrated directional network and represented graphically using the modified Edinburgh Pathway Notation (mEPN) scheme.</p> <p>Results</p> <p>The diagram includes detailed views of the toll-like receptor (TLR) pathways, other pathogen recognition systems, NF-kappa-B, apoptosis, interferon signalling, MAP-kinase cascades, MHC antigen presentation and proteasome assembly, as well as selected views of the transcriptional networks they regulate. The integrated pathway includes a total of 496 unique proteins, the complexes formed between them and the processes in which they are involved. This produces a network of 2,170 nodes connected by 2,553 edges.</p> <p>Conclusions</p> <p>The pathway diagram is a navigable visual aid for displaying a consensus view of the pathway information available for these systems. It is also a valuable resource for computational modelling and aid in the interpretation of functional genomics data. We envisage that this work will be of value to those interested in macrophage biology and also contribute to the ongoing Systems Biology community effort to develop a standard notation scheme for the graphical representation of biological pathways.</p

Activation of an NLRP3 Inflammasome Restricts Mycobacterium kansasii Infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii

Ageing, Muscle Power and Physical Function: A Systematic Review and Implications for Pragmatic Training Interventions.

BACKGROUND: The physiological impairments most strongly associated with functional performance in older people are logically the most efficient therapeutic targets for exercise training interventions aimed at improving function and maintaining independence in later life. OBJECTIVES: The objectives of this review were to (1) systematically review the relationship between muscle power and functional performance in older people; (2) systematically review the effect of power training (PT) interventions on functional performance in older people; and (3) identify components of successful PT interventions relevant to pragmatic trials by scoping the literature. METHODS: Our approach involved three stages. First, we systematically reviewed evidence on the relationship between muscle power, muscle strength and functional performance and, second, we systematically reviewed PT intervention studies that included both muscle power and at least one index of functional performance as outcome measures. Finally, taking a strong pragmatic perspective, we conducted a scoping review of the PT evidence to identify the successful components of training interventions needed to provide a minimally effective training dose to improve physical function. RESULTS: Evidence from 44 studies revealed a positive association between muscle power and indices of physical function, and that muscle power is a marginally superior predictor of functional performance than muscle strength. Nine studies revealed maximal angular velocity of movement, an important component of muscle power, to be positively associated with functional performance and a better predictor of functional performance than muscle strength. We identified 31 PT studies, characterised by small sample sizes and incomplete reporting of interventions, resulting in less than one-in-five studies judged as having a low risk of bias. Thirteen studies compared traditional resistance training with PT, with ten studies reporting the superiority of PT for either muscle power or functional performance. Further studies demonstrated the efficacy of various methods of resistance and functional task PT on muscle power and functional performance, including low-load PT and low-volume interventions. CONCLUSIONS: Maximal intended movement velocity, low training load, simple training methods, low-volume training and low-frequency training were revealed as components offering potential for the development of a pragmatic intervention. Additionally, the research area is dominated by short-term interventions producing short-term gains with little consideration of the long-term maintenance of functional performance. We believe the area would benefit from larger and higher-quality studies and consideration of optimal long-term strategies to develop and maintain muscle power and physical function over years rather than weeks

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y6 receptor mediated impact on interleukin (IL)-8 production, (2) P2Y11 receptor-mediated affects on IL-12/23 output, and (3) P2X7 receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases

Identifying an indoor air exposure limit for formaldehyde considering both irritation and cancer hazards

Formaldehyde is a well-studied chemical and effects from inhalation exposures have been extensively characterized in numerous controlled studies with human volunteers, including asthmatics and other sensitive individuals, which provide a rich database on exposure concentrations that can reliably produce the symptoms of sensory irritation. Although individuals can differ in their sensitivity to odor and eye irritation, the majority of authoritative reviews of the formaldehyde literature have concluded that an air concentration of 0.3 ppm will provide protection from eye irritation for virtually everyone. A weight of evidence-based formaldehyde exposure limit of 0.1 ppm (100 ppb) is recommended as an indoor air level for all individuals for odor detection and sensory irritation. It has recently been suggested by the International Agency for Research on Cancer (IARC), the National Toxicology Program (NTP), and the US Environmental Protection Agency (US EPA) that formaldehyde is causally associated with nasopharyngeal cancer (NPC) and leukemia. This has led US EPA to conclude that irritation is not the most sensitive toxic endpoint and that carcinogenicity should dictate how to establish exposure limits for formaldehyde. In this review, a number of lines of reasoning and substantial scientific evidence are described and discussed, which leads to a conclusion that neither point of contact nor systemic effects of any type, including NPC or leukemia, are causally associated with exposure to formaldehyde. This conclusion supports the view that the equivocal epidemiology studies that suggest otherwise are almost certainly flawed by identified or yet to be unidentified confounding variables. Thus, this assessment concludes that a formaldehyde indoor air limit of 0.1 ppm should protect even particularly susceptible individuals from both irritation effects and any potential cancer hazard

Image-guided surgery influences perioperative morbidity from endoscopic sinus surgery: a systematic review and meta-analysis

ObjectiveAlthough image-guided surgery (IGS) is considered a valuable tool, its impact on perioperative morbidity for endoscopic sinus surgery (ESS) remains unclear. The evidence from reported literature is systematically reviewed with meta-analysis.Data sourcesMEDLINE (1946 to September 14, 2012, week 2) and EMBASE (1974 to September 14, 2012, week 37).Review methodsMEDLINE and EMBASE were searched using a search strategy for publications on IGS during ESS that reported original data on perioperative morbidity. PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) guidelines were followed. Both comparative cohort studies with non-IGS cases and case series were included. Primary outcome was major and total complications. Secondary outcomes were specific orbital and intracranial injury, major hemorrhage, ability to complete the operation, and revision surgery. The incidence of these events was defined as dichotomous variables and expressed as a risk ratio (RR) in a fixed-effects model.ResultsIn total, 2586 articles fulfilled the search, producing 55 included studies. Fourteen were comparative cohorts of IGS and non-IGS sinus surgical patient populations used for meta-analysis. Among the cohorts, major complications were more common in the non-IGS group (RR = 0.48; 95% confidence interval [CI], 0.28-0.82; P = .007). Total complications were greater in the non-IGS group (RR = 0.66; 95% CI, 0.47-0.94; P = .02). All other outcomes did not reach significance on meta-analysis.ConclusionContrary to current review articles on the topic of IGS use during ESS, there is evidence from published studies that the use of IGS for sinus surgery, within selected populations, is associated with a lower risk of major and total complications compared with non-IGS sinus surgery.Dustin M. Dalgorf, Raymond Sacks, Peter-John Wormald, Yuresh Naidoo, Ben Panizza, Brent Uren, Chris Brown, John Curotta, Kornkiat Snidvongs, and Richard J. Harve