Vishwa D. Dixit

Gamma secretase–mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme γ-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Ghrelin and immunity : A young player in an old field

There is increasing evidence of the coupling of immune status to the metabolic system. The communication between the state of systemic and cellular energy balance to immune compartment is mediated via a complex array of cytokines, hormones and neuropeptides. Ghrelin, a recently described orexigenic peptide hormone, is predominantly produced by the stomach and functions as a positive regulator of the somatotropic axis and a peripheral signal of negative energy balance. Apart from its well-studied metabolic effects, ghrelin also exerts multiple regulatory effects on several other organ systems including the cardiovascular, central nervous and immune systems. Here, we summarize the growing evidence of ghrelin as a significant player in the regulation of inflammation and the immune function and the potential therapeutic targeting of ghrelin or its receptor, the growth hormone secretagogue receptor (GHS-R), in various inflammatory and cachexic disease states.

CXCL12-induced partitioning of flotillin-1 with lipid rafts plays a role in CXCR4 function

Lipid rafts play an important role in signal integration and in the cellular activation of a number of cytokine and growth factor receptors. It has recently been demonstrated that flotillin proteins are recruited to lipid raft microdomains upon cellular activation and play a role in neural cell regeneration, receptor signaling and lymphocyte activation. However, little is known about the relevance of the flotillin proteins during T cell responses to chemoattractant stimulation. To this end, cytoplasmic and lipid raft fractions from human T cells were analyzed for flotillin protein redistribution prior to and after CXCL12 stimulation. Flotillin‐1, but not flotillin‐2, redistributes to lipid rafts upon CXCR4 ligation. Moreover, in CXCL12‐treated T cells, flotillin‐1 also associates with several raft proteins including LAT, CD48 and CD11a but not Lck. In addition, an increase in CXCR4 association with flotillin‐1 in lipid rafts was observed after chemokine treatment. RNAi technology was also utilized to inhibit the expression of flotillin‐1, resulting in an inhibition of CXCL12‐mediated signaling, function and CXCR4 recruitment into lipid rafts. Together, these data suggest that the increased association of cellular flotillin‐1 with lipid raft microdomains during chemokine exposure may play an important role in chemokine receptor signaling and receptor partitioning with lipid rafts.

Reduction in hypophyseal growth hormone and prolactin expression due to deficiency in ghrelin receptor signaling is associated with Pit-1 suppression: Relevance to the immune system

In mice and in rats, reduced levels of the growth hormone secretagogue receptor (GHS-R1a) results in reduced body weight and lower levels of serum insulin-like growth factor I (IGF-I). However, the mechanism leading to these impairments has not been elucidated. Studies in primary cultures of pituitary cells from very young mice have shown that GHS-R1a agonists, including ghrelin, increase expression of the pituitary-specific transcription factor (Pit-1) that is critical for differentiation of pituitary cells into somatotrophs, lactotrophs, and thyrotrophs. Hence, we hypothesized that ablation of Ghsr would reduce Pit-1 expression and as a consequence reduce growth hormone (GH) production explaining the lower body weight of Ghsr-/- mice. Here, we now show that Pit-1 mRNA levels are significantly lower in the pituitary gland of Ghsr-/- mice compared to wild-type littermates and also with advancing age. This Pit-1 loss is associated with reduced GH mRNA and fewer GH producing cells. To determine whether reduced GH is caused by reduced expression of Pit-1 in Ghsr-/- mice, we also measured prolactin (PRL) expression in the pituitary gland and in the circulation. PRL mRNA was significantly reduced in Ghsr-/- mice compared to wild-type littermates and fewer cells expressed PRL. The reduction in expression of both GH and PRL is consistent with a Pit-1 regulated pathway and demonstrates that the GHS-R has an important role in the pituitary gland as a modulator of Pit-1 expression and provides a possible mechanism to explain the lower plasma IGF-1 and modestly reduced body weight exhibited by Ghsr-/- mice. We also believe that lower systemic and lymphoid hormone expression may also account, in part, for the enhanced thymic involution and reduced thymic output in Ghsr-/- mice.

Controlled meal frequency without caloric restriction alters peripheral blood mononuclear cell cytokine production

BackgroundIntermittent fasting (IF) improves healthy lifespan in animals by a mechanism involving reduced oxidative damage and increased resistance to stress. However, no studies have evaluated the impact of controlled meal frequency on immune responses in human subjects.ObjectiveA study was conducted to establish the effects of controlled diets with different meal frequencies, but similar daily energy intakes, on cytokine production in healthy male and female subjects.DesignIn a crossover study design with an intervening washout period, healthy normal weight middle-age male and female subjects (n = 15) were maintained for 2 months on controlled on-site one meal per day (OMD) or three meals per day (TMD) isocaloric diets. Serum samples and peripheral blood mononuclear cells (PBMCs) culture supernatants from subjects were analyzed for the presence of inflammatory markers using a multiplex assay.ResultsThere were no significant differences in the inflammatory markers in the serum of subjects on the OMD or TMD diets. There was an increase in the capacity of PBMCs to produce cytokines in subjects during the first month on the OMD or TMD diets.Lower levels of TNF-α, IL-17, MCP-1 and MIP-1β were produced by PBMCs from subjects on the OMD versus TMD diet.ConclusionsPBMCs of subjects on controlled diets exhibit hypersensitivities to cellular stimulation suggesting that stress associated with altered eating behavior might affect cytokine production by immune cells upon stimulation. Moreover, stimulated PBMCs derived from healthy individuals on a reduced meal frequency diet respond with a reduced capability to produce cytokines.

Age-associated alterations in CXCL1 chemokine expression by murine B cells

BackgroundThe CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes.ResultsHere, we demonstrate that highly purified murine splenic B cells are capable of expressing both MIP-2 and KC protein and mRNA upon activation with lipopolysaccharide (LPS) but not in response to anti-μ and anti-CD40 in combination with interleukin-4 (IL-4) stimulation. Moreover, these chemokines are expressed at higher levels in B cells derived from young (4 m) compared to old (24–29 m) mice. Upon fractionation into distinct B-cell subsets, we found that the expression of MIP-2 and KC by aged follicular (FO) B cells is significantly decreased when compared to the same cells from younger mice, while only MIP-2 production was found to be diminished in aged marginal zone (MZ) B cells. Interestingly, MIP-2 and KC production by newly formed (NF) B cells did not significantly differ with age. Moreover, the potential relevance of these findings is supported by the poor ability of LPS-activated aged B cells to specifically mediate CXCL1-dependent leukocyte recruitment when compared to younger B cells.ConclusionOverall, the decreased expression of CXCL1 chemokines by aged B cells in response to LPS may have potential implications on the secondary recruitment of leukocytes to sites of microbial infections and inflammation possibly contributing to the increased susceptibility of older subjects to pathogen challenge.

Identification of genes differentially expressed in T cells following stimulation with the chemokines CXCL12 and CXCL10.

BackgroundChemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines.MethodsWe have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis.ResultsOne hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment.ConclusionsTogether, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology.

T cell derived ghrelin is required for regulation of NFκB and T-helper 1 cytokines

IFN-a in healthy humans during a regular sleep-wake cycle and during a 24-h period of continuous wakefulness. Blood was sampled every 1.5 h during night-time and every 3 h during daytime. Sleep strongly decreased the absolute count of CD14dimCD16 monocytes, while the major CD14CD monocyte population remained unaffected. Pre-mDC and PDC counts revealed a circadian rhythm but were not influenced by sleep. Compared with sustained wakefulness, nocturnal sleep strongly increased the number of IL-12 producing pre-mDC throughout the night, whereas IFN-a production showed only a circadian rhythm with peak time during late night and early morning, in paralleled with the peak of PDC blood numbers. Correlation analyses identified hormones (noradrenalin, cortisol, prolactin) as factors modulating APC counts and function during sleep. The profound influence of sleep on APC provides evidence that sleep beyond circadian rhythms critically contribute to the regulation of the adaptive immune processes.

Fasting induced rewiring of human peripheral blood mononuclear cell cytokine secretion

rochrome-labeled Ab by IST and were visualized by CM. Results: Data from 10 normal individuals demonstrate that the b2AR was detected by FC in all lymphocyte and mononuclear cell populations when compared to negative controls with variation of fluorescence between subjects. CM studies demonstrated compartmentalization of the b2AR to both intracellular and membrane bound domains. Samples remain stable for up to 1 week for FC and for one month for CM. Summary: These studies indicate that immunophenotyping of leukocyte populations with b2AR can be readily accomplished with a commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the b2AR. The FC assay has applications for measuring alterations in total b2AR in human leukocyte populations as changes in fluorescence intensity and/or percent positive cells. In addition, CM confirms that both surface and intracellular compartments stain positively for the b2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.