Inflammatory signatures for eosinophilic vs. neutrophilic allergic pulmonary inflammation reveal critical regulatory checkpoints.

Contrary to the T-helper (Th)-2 bias and eosinophil-dominated bronchial inflammation encountered in most asthmatic subjects, other patients may exhibit neutrophil-predominant asthma subphenotypes, along with Th-1 and Th-17 cells. However, the etiology of many neutrophil-dominated asthma subphenotypes remains ill-understood, in part due to a lack of appropriate experimental models. To better understand the distinct immune-pathological features of eosinophilic vs. neutrophilic asthma types, we developed an ovalbumin (OVA)-based mouse model of neutrophil-dominated allergic pulmonary inflammation. Consequently, we probed for particular inflammatory signatures and checkpoints underlying the immune pathology in this new model, as well as in a conventional, eosinophil-dominated asthma model. Briefly, mice were OVA sensitized using either aluminum hydroxide (alum) or complete Freund's adjuvants, followed by OVA aerosol challenge. T-cell, granulocyte, and inflammatory mediator profiles were determined, along with alveolar macrophage genomewide transcriptome profiling. In contrast to the Th-2-dominated phenotype provoked by alum, OVA/ complete Freund's adjuvants adjuvant-based sensitization, followed by allergen challenge, elicited a pulmonary inflammation that was poorly controlled by dexamethasone, and in which Th-1 and Th-17 cells additionally participated. Analysis of the overall pulmonary and alveolar macrophage inflammatory mediator profiles revealed remarkable similarities between both models. Nevertheless, we observed pronounced differences in the IL-12/IFN-γ axis and its control by IL-18 and IL-18 binding protein, but also in macrophage arachidonic acid metabolism and expression of T-cell instructive ligands. These differential signatures, superimposed onto a generic inflammatory signature, denote distinctive inflammatory checkpoints potentially involved in orchestrating neutrophil-dominated asthma.

Effects of marginal iron overload on iron homeostasis and immune function in alveolar macrophages isolated from pregnant and normal rats.

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.

Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity.

The transcription factor NFkappaB plays a critical role in normal and pathophysiological immune responses. Therefore, NFkappaB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFkappaB activation by preventing the tumor necrosis factor-induced activation of IkappaB kinase beta via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IkappaB kinase beta inhibition by a plant-derived inhibitor, coinciding with MEK1/ERK-dependent Ser-181 hyperphosphorylation. This prevents IkappaB phosphorylation and degradation, which subsequently blocks NFkappaB translocation, NFkappaB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFkappaB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

Changes in function of iron-loaded alveolar macrophages after in vivo administration of desferrioxamine and/or chloroquine.

Both desferrioxamine (DFO) and chloroquine can significantly reduce hepatic iron in experimental animals with iron overload by chelating iron from the low-molecular-weight pool or decreasing iron uptake by the transferrin-transferrin receptor cycle, respectively. However, no previous studies have investigated whether combination therapy of these two drugs would further decrease the tissue iron overload as well as iron-induced toxicity. Chloroquine administration, 15 mg/kg, 5x/week, to rats during the iron loading regime, 10 mg/kg, 3x/week for 4 weeks, significantly decreased both hepatic (54%) and macrophage iron content (24%). However when administered in combination with desferrioxamine, 10 mg/kg, 3x/week for 2 weeks at the cessation of iron loading, no further reduction of hepatic iron content was noted while the iron content of the macrophages significantly increased, possibly indicating the flux of ferrioxamine through these cells. Further studies are warranted to investigate the speciation of iron within these macrophages. Macrophages isolated from chloroquine-treated iron loaded rats showed a reduction in latent NFkappaB activation and a significant increase in lipopolysaccharide-stimulated nitrite release by comparison to these parameters in iron loaded macrophages. Co-administration of chloroquine and desferrioxamine normalised the latent activity of NFkappaB to that of control macrophages as well as increasing LPS-stimulated NO release towards control values. However, DFO alone did not have any significant effect upon either of these parameters. Such results may have important relevance for the reduced immune function of iron loaded macrophages isolated from thalassaemia patients receiving chelation therapy and their propensity to increased infection.