RIP kinases: key decision makers in cell death and innate immunity

Innate immunity represents the first line of defence against invading pathogens. It consists of an initial inflammatory response that recruits white blood cells to the site of infection in an effort to destroy and eliminate the pathogen. Some pathogens replicate within host cells, and cell death by apoptosis is an important effector mechanism to remove the replication niche for such microbes. However, some microbes have evolved evasive strategies to block apoptosis, and in these cases host cells may employ further countermeasures, including an inflammatory form of cell death know as necroptosis. This review aims to highlight the importance of the RIP kinase family in controlling these various defence strategies. RIP1 is initially discussed as a key component of death receptor signalling and in the context of dictating whether a cell triggers a pathway of pro-inflammatory gene expression or cell death by apoptosis. The molecular and functional interplay of RIP1 and RIP3 is described, especially with respect to mediating necroptosis and as key mediators of inflammation. The function of RIP2, with particular emphasis on its role in NOD signalling, is also explored. Special attention is given to emphasizing the physiological and pathophysiological contexts for these various functions of RIP kinases.

Activated protein C inhibits tumor necrosis factor and macrophage migration inhibitory factor production in monocytes

The precise regulatory mechanisms of amplification and downregulation of the pro- and anti-inflammatory cytokines in the inflammatory response have not been fully delineated. Although activated protein C (APC) and its precursor protein C (PC) have recently been reported to be promising therapeutic agents in the management of meningococcal sepsis, direct evidence for the anti-inflammatory effect remains scarce. We report that APC inhibits in vitro the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor (MIF), two known cytokine mediators of bacterial septic shock, from lipopolysaccharide (LPS)-stimulated human monocytes. The THP-1 monocytic cell line, when stimulated with LPS and concomitant APC, exhibited a marked reduction in the release of TNF and MIF protein in a concentration-dependent manner compared to cells stimulated with LPS alone. This effect was observed only when incubations were performed in serum-free media, but not in the presence of 1-10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations to far beyond physiological levels, suggesting the presence of endogenous serum-derived APC inhibitors. Inhibition of MIF release by APC was found to be independent of TNF, as stimulation of MIF release by LPS was unaltered in the presence of anti-TNF antibodies. Our data confirm that the suggested anti-inflammatory properties of APC are due to direct inhibition of the release of the pro-inflammatory monokine TNF, and imply that the anti-inflammatory action of APC is also mediated via inhibition of MIF release.

Targeting Siglecs with a sialic acid-decorated nanoparticle abrogates inflammation

Sepsis is the most frequent cause of death in hospitalized patients, and severe sepsis is a leading contributory factor to acute respiratory distress syndrome (ARDS). At present, there is no effective treatment for these conditions, and care is primarily supportive. Murine sialic acid-binding immunoglobulin-like lectin-E (Siglec-E) and its human orthologs Siglec-7 and Siglec-9 are immunomodulatory receptors found predominantly on hematopoietic cells. These receptors are important negative regulators of acute inflammatory responses and are potential targets for the treatment of sepsis and ARDS. We describe a Siglec-targeting platform consisting of poly(lactic-co-glycolic acid) nanoparticles decorated with a natural Siglec ligand, di(α2→8) N-acetylneuraminic acid (α2,8 NANA-NP). This nanoparticle induced enhanced oligomerization of the murine Siglec-E receptor on the surface of macrophages, unlike the free α2,8 NANA ligand. Furthermore, treatment of murine macrophages with these nanoparticles blocked the production of lipopolysaccharide-induced inflammatory cytokines in a Siglec-E-dependent manner. The nanoparticles were also therapeutically beneficial in vivo in both systemic and pulmonary murine models replicating inflammatory features of sepsis and ARDS. Moreover, we confirmed the anti-inflammatory effect of these nanoparticles on human monocytes and macrophages in vitro and in a human ex vivo lung perfusion (EVLP) model of lung injury. We also established that interleukin-10 (IL-10) induced Siglec-E expression and α2,8 NANA-NP further augmented the expression of IL-10. Indeed, the effectiveness of the nanoparticle depended on IL-10. Collectively, these results demonstrated a therapeutic effect of targeting Siglec receptors with a nanoparticle-based platform under inflammatory conditions.

Evaluation of La1−xSrxMnO3 (0 ≤ x < 0.4) synthesised via a modified sol–gel method as mediators for magnetic fluid hyperthermia

A range of lanthanum strontium manganates (La1−xSrxMnO3–LSMO) where 0 ≤ x < 0.4 were prepared using a modified peroxide sol–gel synthesis method. The magnetic nanoparticle (MNP) clusters obtained for each of the materials were characterised using scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and infra-red (IR) spectroscopy in order to confirm the crystalline phases, crystallite size and cluster morphology. The magnetic properties of the materials were assessed using the Superconducting quantum interference device (SQUID) to evaluate the magnetic susceptibility, Curie temperature (Tc) and static hysteretic losses. Induction heating experiments also provided an insight into the magnetocaloric effect for each material. The specific absorption rate (SAR) of the materials was evaluated experimentally and via numerical simulations. The magnetic properties and heating data were linked with the crystalline structure to make predictions with respect to the best LSMO composition for mild hyperthermia (41 °C ≤ T ≤ 46 °C). La0.65Sr0.35MnO3, with crystallite diameter of 82.4 nm, (agglomerate size of ∼10 μm), Tc of 89 °C and SAR of 56 W gMn−1 at a concentration 10 mg mL−1 gave the optimal induction heating results (Tmax of 46.7 °C) and was therefore deemed as most suitable for the purposes of mild hyperthermia, vide infra.

Predictive Value of Depressive Symptoms for All-Cause Mortality: Findings From the PRIME Belfast Study Examining the Role of Inflammation and Cardiovascular Risk Markers

OBJECTIVES: To improve understanding about the potential underlying biological mechanisms in the link between depression and all-cause mortality and to investigate the role that inflammatory and other cardiovascular risk factors may play in the relationship between depressive symptoms and mortality.

METHODS: Depression and blood-based biological markers were assessed in the Belfast PRIME prospective cohort study (N = 2389 men, aged 50-59 years) in which participants were followed up for 18 years. Depression was measured using the 10-item Welsh Pure Depression Inventory. Inflammation markers (C-reactive protein [CRP], neopterin, interleukin [IL]-1 receptor antagonist [IL-1Ra], and IL-18) and cardiovascular-specific risk factors (N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, C-terminal pro-endothelin-1 [CT-proET]) were obtained at baseline. We used Cox proportional hazards modeling to examine the association between depression and biological measures in relation to all-cause mortality and explore the mediating effects.

RESULTS: During follow-up, 418 participants died. Higher levels of depressive symptoms were associated with higher levels of CRP, IL-1Ra, and CT-proET. After adjustment for socioeconomic and life-style risk factors, depressive symptoms were significantly associated with all-cause mortality (hazard ratio = 1.10 per scale unit, 95% confidence interval = 1.04-1.16). This association was partly explained by CRP (7.3%) suggesting a minimal mediation effect. IL-1Ra, N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, and CT-proET contributed marginally to the association between depression and subsequent mortality.

CONCLUSIONS: Inflammatory and cardiovascular risk markers are associated with depression and with increased mortality. However, depression and biological measures show additive effects rather than a pattern of meditation of biological factors in the association between depression and mortality.

Drug induced gingival overgrowth: A role for Protease Activated Receptors?

Background: Protease activated receptors (PAR) belong to a subfamily of G protein coupled receptors. They consist of seven transmembrane domains but are not classical receptors as their agonist is a circulating serine proteinase. This proteinase cleaves an N-terminal extracellular domain of the receptor to reveal a new N-terminal tethered ligand which binds intramolecularly, thus converting an extracellular proteolytic event into a transmembrane signal. Therefore, the cleavage and activation of PARs provide a mechanism whereby proteinases can directly influence the inflammatory response. Gingival hyperplasia or gingival enlargement is a side effect of some drugs such as cyclosporine, a potent immunosuppressant. To date, the potential role of PAR in the inflammation associated with the pathogenesis of gingival overgrowth has not been studied. Objectives: The present study was designed to determine whether proteinases derived from extracts of cyclosporine induced hyperplasia were capable of activating PAR in vitro. Methods: Cell lysates were derived from tissue obtained from gingival overgrowth of patients requiring surgical excision. Cell lines over-expressing PARs were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% foetal calf serum (FCS) in 5% CO2. The cells were treated with gingival overgrowth lysates and agonist stimulated calcium release from the cells was recorded using the Fluo-4-Direct™ Calcium Assay Kit from Invitrogen, according to manufacturer's instructions. Results: Calcium release by activated PAR on tumour cells was detected in those treated with gingival hyperplasia lysates. Samples from healthy gingival fibroblasts did not elicit this response. Conclusions: The identification of mediators of the molecular events central to the inflammatory phenotype elicited by gingival hyperplasia is important. To this end, our experiments show that in vitro, enzymes derived from overgrown gingival tissue are capable of activating PAR and thereby provide evidence for the potential role of PAR in sustaining gingival hyperplasia.

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.

Endothelial Function in Hypertension: Victim or Culprit?

Far from simply lining the inner surface of blood vessels, the cellular monolayer that comprises the endothelium is a highly active organ that regulates vascular tone. In health, the endothelium maintains the balance between opposing dilator and constrictor influences, while in disease, it is the common ground on which cardiovascular risk factors act to initiate the atherosclerotic process. As such, it is the site at which cardiovascular disease begins and consequently acts as a barometer of an individual's likely future cardiovascular health. The vascular endothelium is a very active organ responsible for the regulation of vascular tone through the effects of locally synthesized mediators, predominantly nitric oxide (NO), endothelial NO synthase (eNOS), and superoxide. NO is abundantly evident in normally functioning vasculature where it acts as a vasodilator, inhibits inflammation, and has an antiaggregant effect on platelets. Its depletion is both a sign and cause of endothelial dysfunction resulting from reduced activity of eNOS and amplified production of nicotinamide adenine dinucleotide oxidase, which, in turn, results in raised levels of reactive oxygen species. This cascade is the basis for reduced vascular compliance through an imbalanced regulation of tone with a predominance of vasoconstrictive elements. Further, structural changes in the microvasculature are a critical early step in the loss of normal function. This microvascular dysfunction is known to be highly predictive of future macrovascular events and is consequently a very attractive target for intervention in the hypertensive population in order to prevent cardiovascular events.

Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro- Inflammation in Brain, Cognitive Deficits,and Alterations in Apurinic Endonuclease 1

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

Persistent endothelial activation and inflammation after Plasmodium falciparum Infection in Malawian children

Endothelial dysregulation is central to the pathogenesis of acute Plasmodium falciparum infection. It has been assumed that this dysregulation resolves rapidly after treatment, but this return to normality has been neither demonstrated nor quantified. We therefore measured a panel of plasma endothelial markers acutely and in convalescence in Malawian children with uncomplicated or cerebral malaria. Evidence of persistent endothelial activation and inflammation, indicated by increased plasma levels of soluble intracellular adhesion molecule 1, angiopoetin 2, and C-reactive protein, were observed at 1 month follow-up visits. These vascular changes may represent a previously unrecognized contributor to ongoing malaria-associated morbidity and mortality.