Cigarette smoke attenuates the production of cytokines by human plasmacytoid dendritic cells and enhances the release of IL-8 in response to TLR-9 stimulation

Myeloid and plasmacytoid dendritic cells (mDCs, pDC) are crucial to the immune system, detecting microorganisms and linking the innate and adaptive immunity. pDC are present in small quantities in tissues that are in contact with the external environment; mainly the skin, the inner lining of the nose, lungs, stomach and intestines. They produce large amounts of IFN-α after stimulation and are pivotal for the induction of antiviral responses. Chronic obstructive pulmonary disease (COPD) patients are known to be more susceptible to viral infections. We have demonstrated that exposure of mDC to cigarette smoke extract (CSE) leads to the release of chemokines, however, not much is known about the role of pDC in COPD. In this study, we addressed several key questions with respect to the mechanism of action of CSE on human pDC in an in vitro model. Human pDCs were isolated from normal healthy volunteers and subjected to fresh CSE and the levels of IL-8, TNF-α, IP-10, IL-6, IL-1, IL-12 and IL-10 and IFN-α were studied by both ELISA and real time PCR methods. We observed that CSE augmented the production of IL-8 and suppressed the release of TNF-α, IL-6 and IFN-α. Moreover, CSE suppressed PI3K/Akt signalling in pDC. In conclusion, our data indicate that CSE has both the potential to diminish anti-viral immunity by downregulating the release of IFN-α and other pro-inflammatory cytokines while, at the same time, augmenting the pathogenesis of COPD via an IL-8 induced recruitment of neutrophils.     

Involvement of VDAC,Bax and Ceramides in the Efflux of AIF from Mitochondria during Curcumin-Induced Apoptosis

Background

We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process.

Principal Findings

Curcumin-induced apoptosis could not be blocked by caspase-inhibitors and we could not detect any caspase-3/7 activity. Curcumin predominantly induced mitochondria-mediated ROS formation and stimulated the expression of the redox-sensitive pro-apoptotic factor p53. Inhibition of the pro-apoptotic signaling enzyme glycogen synthase kinase-3β (GSK-3β) blocked curcumin-induced apoptosis. Apoptosis was associated with high molecular weight DNA damage, a possible indicator of apoptosis-inducing factor (AIF) activity. Indeed, curcumin caused nuclear translocation of AIF, which could be blocked by the antioxidant N-acetyl cysteine. We next investigated how AIF is effluxed from mitochondria in more detail. The permeability transition pore complex (PTPC), of which the voltage-dependent anion channel (VDAC) is a component, could be involved since the VDAC-inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) efficiently blocked AIF translocation. However, PTPC is not involved in AIF release since cyclosporine A, a specific inhibitor of the complex did not block apoptosis. Alternatively, the pro-apoptotic protein Bax could have formed mitochondrial channels and interacted with VDAC. Curcumin caused mitochondrial translocation of Bax, which was blocked by DIDS, suggesting a Bax-VDAC interaction. Interestingly, ceramide channels can also release apoptogenic factors from mitochondria and we found that addition of ceramide induced caspase-independent apoptosis. Surprisingly, this process could also be blocked by DIDS, suggesting the concerted action of Bax, VDAC and ceramide in the efflux of AIF from the mitochondrion.

Conclusions

Curcumin-induced fibroblast apoptosis is totally caspase-independent and relies on the mitochondrial formation of ROS and the subsequent nuclear translocation of AIF, which is released from a mitochondrial pore that involves VDAC, Bax and possibly ceramides. The composition of the AIF-releasing channel seems to be much more complex than previously thought.     

High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

Background

High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated.

Methodology/Principal Findings

To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-¹³C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 µg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 µg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8±0.4% vs. 8.1±0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized.

Conclusions/Significance

High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

Metabolism and excretion of anabolic steroids in doping control--new steroids and new insights

The use of anabolic steroids in sports is prohibited by the World Anti-Doping Agency. Until the 1990s, anabolic steroids were solely manufactured by pharmaceutical companies, albeit sometimes on demand from national sports agencies as part of their doping program. Recently the list of prohibited anabolic steroids in sports has grown due to the addition of numerous steroids that have been introduced on the market by non-pharmaceutical companies. Moreover, several designer steroids, specifically developed to circumvent doping control, have also been detected. Because anabolic steroids are most often intensively subjected to phase I metabolism and seldom excreted unchanged, excretion studies need to be performed in order to detect their misuse.

This review attempts to summarise the results of excretion studies of recent additions to the list of prohibited steroids in sports. Additionally an update and insight on new aspects for “older” steroids with respect to doping control is given.     

An assessment of the biotechnological use of hemoglobin modulation in cereals

Non‐symbiotic hemoglobin (nsHb) genes are ubiquitous in plants, but their biological functions have mostly been studied in model plant species rather than in crops. nsHb influences cell signaling and metabolism by modulating the levels of nitric oxide (NO). Class 1 nsHb is upregulated under hypoxia and is involved in various biotic and abiotic stress responses. Ectopic overexpression of nsHb in Arabidopsis thaliana accelerates development, whilst targeted overexpression in seeds can increase seed yield. Such observations suggest that manipulating nsHb could be a valid biotechnological target. We studied the effects of overexpression of class 1 nsHb in the monocotyledonous crop plant barley (Hordeum vulgare cv. Golden Promise). nsHb was shown to be involved in NO metabolism in barley, as ectopic overexpression reduced the amount of NO released during hypoxia. Further, as in Arabidopsis, nsHb overexpression compromised basal resistance toward pathogens in barley. However, unlike Arabidopsis, nsHb ectopic overexpression delayed growth and development in barley, and seed specific overexpression reduced seed yield. Thus, nsHb overexpression in barley does not seem to be an efficient strategy for increasing yield in cereal crops. These findings highlight the necessity for using actual crop plants rather than laboratory model plants when assessing the effects of biotechnological approaches to crop improvement.     

Microcephaly with simplified gyration, epilepsy, and infantile diabetes linked to inappropriate apoptosis of neural progenitors

We describe a syndrome of primary microcephaly with simplified gyral pattern in combination with severe infantile epileptic encephalopathy and early-onset permanent diabetes in two unrelated consanguineous families with at least three affected children. Linkage analysis revealed a region on chromosome 18 with a significant LOD score of 4.3. In this area, two homozygous nonconserved missense mutations in immediate early response 3 interacting protein 1 (IER3IP1) were found in patients from both families. IER3IP1 is highly expressed in the fetal brain cortex and fetal pancreas and is thought to be involved in endoplasmic reticulum stress response. We reported one of these families previously in a paper on Wolcott-Rallison syndrome (WRS). WRS is characterized by increased apoptotic cell death as part of an uncontrolled unfolded protein response. Increased apoptosis has been shown to be a cause of microcephaly in animal models. An autopsy specimen from one patient showed increased apoptosis in the cerebral cortex and pancreas beta cells, implicating premature cell death as the pathogenetic mechanism. Both patient fibroblasts and control fibroblasts treated with siRNA specific for IER3IP1 showed an increased susceptibility to apoptotic cell death under stress conditions in comparison to controls. This directly implicates IER3IP1 in the regulation of cell survival. Identification of IER3IP1 mutations sheds light on the mechanisms of brain development and on the pathogenesis of infantile epilepsy and early-onset permanent diabetes.     

Plant Functional Traits and the Distribution of West African Rain Forest Trees along the Rainfall Gradient

Plant morphological and physiological traits affect the way plants tolerate environmental stresses and therefore play an important role in shaping species distribution patterns in relation to environmental gradients. Despite our growing knowledge on the role of functional traits in structuring plant communities, few studies have tested their importance at large scales in the wet tropics. Here, we describe the distribution patterns of the most important West African rain forest timber species along the regional rainfall gradient and relate them to their functional traits. We found that the distribution patterns of 25 out of the 31 studied species (80%) were significantly related to mean annual rainfall. Shade tolerance and drought resistance were identified as the main strategy axes of variation. Wood density and leaf deciduousness emerged as the best predictor traits of species position along the rainfall gradient, explaining respectively 32 and 15 percent of the variation. Species traits tended to show stronger relationships with estimated optimum annual rainfall for each species than to the extreme rainfall conditions where they occur. The significant role of rainfall in shaping timber species distribution and the strong relationships between species traits and rainfall indicate that changes in climate, especially declining rainfall, could have strong effects on species composition and abundance in these tropical forests.