Tight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a ...synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-α blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-α in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-α suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-κB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-α suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.
The nutritional demands of honeybees are met by two plant-produced components: nectar and pollen, the contents of which vary among floral sources. In Israel, there is an extraordinary richness in ...plant species, and one of the dominant insect pollinators is the honeybee (Apis mellifera L.). July to February is characterized as a period of low flower abundance for local species in general and for bee forage plants in particular. In this study, we monitored the amount and number of pollen sources collected by honeybees, and where possible also identified the plant source of pollen, in four geographically distinct sites in Israel. We also assessed honeybee colonies (population level, sealed brood area, and pollen and honey stores) and studied the effect of pollen levels on population growth. Our results show that peak pollen-collection times differ according to site. The number of pollen sources from trapped pollen pellets varied during the year, between sites, and between colonies in the same site, and ranged between 5 and 20 plant species per sampling date per site. The most abundant pollen source in each sample comprised between 22 and 94% of the pollen pellets. There were only a few cases in which pollen was collected from fewer than five or more than nine plants in each colony's sample. Thus, colonies seem to specialize in only a small number of species of the available flora. Overall, in all sites, the daily amount of pollen collected was significantly correlated with sealed brood and pollen store areas.
Abstract Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor α (TNFα), and thus as a putative drug for the ...treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFα production in RAW264.7 macrophages in vitro . We therefore hypothesized that PCERA-1 targets TNFα production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFα, interleukin (IL)-10 and IL-12p40, in vivo , and ex vivo . We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFα and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-γ. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFα production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFα and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases.
The role of CREB in LPS signaling is controversial. The objective of this study was to evaluate the effect of LPS on phosphorylation and transcriptional activation of CREB, in comparison to ...isoproterenol, a beta-adrenergic receptor agonist. We show here that LPS elevates intra-cellular cAMP level in RAW264.7 macrophages, with slower kinetics and lower magnitude than isoproterenol. The two agents stimulated CREB phosphorylation on Ser-133 to a similar extent, but with a different mechanism; rapid and mostly PKA-mediated for isoproterenol; slow and MSK1-mediated for LPS. Interestingly, LPS-stimulated phosphorylation of CREB did not result in transcriptional activation of a CRE-regulated luciferase reporter, in contrast to stimulation by isoproterenol. Furthermore, inhibitors of p38 and MSK1, but not PKA, completely blocked the production of IL-10 and TNFalpha in LPS-stimulated macrophages. Distinctively, the PKA inhibitor H89 blocked the suppressive effect of isoproterenol on TNFalpha production, as well as its stimulatory effect on IL-10 induction, in LPS-stimulated macrophages. Likewise, while over-expression of dominant negative CREB had no effect on LPS-stimulated TNFalpha production, it blocked the suppressive effect of isoproterenol on TNFalpha production in the LPS-stimulated macrophages. Our results thus indicate that PKA-mediated phosphorylation of CREB promotes TNFalpha suppression and IL-10 induction, whereas the same phosphorylation event initiated by LPS and mediated by MSK1 is non-functional for transcriptional modulation.
Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha), and thus as a putative drug for the ...treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFalpha production in RAW264.7 macrophages in vitro. We therefore hypothesized that PCERA-1 targets TNFalpha production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFalpha, interleukin (IL)-10 and IL-12p40, in vivo, and ex vivo. We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFalpha and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-gamma. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFalpha production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFalpha and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases.
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