Mayumi Hidaka

Vitamin D3 derivatives increase soluble CD14 release through ERK1/2 activation and decrease IL-8 production in intestinal epithelial cells

Dysfunction of the innate immune system has been reported to cause intestinal inflammation. Vitamin D3 is known to be an important immune system regulator and exerts anti-inflammatory effects. We investigated in vitro effects of vitamin D3 and its derivatives on the innate immune system in HT-29 cells, a line of human colon adenocarcinoma cells. Among the innate immune-related receptors such as Toll-like receptor (TLR) 1, 2, 4, 6, and CD14 examined by flow cytometry, only CD14 was up-regulated by vitamin D3 derivatives. Release of soluble form CD14 (sCD14) was also increased by vitamin D3 derivatives. The 1α,25-dihydroxy-22-oxavitamin D3 (Oxa-D3) induced-sCD14 release was inhibited by U0126 (a specific inhibitor of extracellular signal-regulated kinase; ERK1/2) but not by SB203580 (a specific inhibitor of p38 MAPK), and ERK1/2 phosphorylation was accelerated by Oxa-D3. These results indicate that Oxa-D3 facilitates the release of sCD14 through ERK1/2 activation. IL-8 production stimulated with LPS was diminished by vitamin D3 derivatives. Recombinant sCD14 also lowered the LPS-stimulated IL-8 production, suggesting neutralization of LPS by sCD14. The anti-inflammatory effect of vitamin D3 derivatives was thus associated with diminution of IL-8 production due to increased release of sCD14.

Inhibition of CXCL10 release by monomeric C3bi and C4b

Cellulose acetate (CA) beads are often used for leucocyte apheresis therapy against inflammatory bowel disease. In order to clarify the mechanism of the anti‐inflammatory effects of CA, global analysis of the molecules generated in blood by the interaction with CA beads was performed in this study. An activated medium was collected from whole blood that had been preincubated with CA beads, and the effects of the CA‐activated medium on leucocyte function were investigated. Fresh blood was stimulated with lipopolysaccharide (LPS) or interferon (IFN)‐β in the presence of the activated medium, and levels of chemokines and cytokines, including CXCL10 (IFN‐inducible protein‐10), and phosphorylated STAT1 (signal transducer and activator of transcription 1), which is known to be essential for CXCL10 production in leucocytes, were measured. IFN‐β‐ or LPS‐induced CXCL10 production, expression of CXCL10 mRNA and phosphorylation of STAT1 were significantly reduced in the presence of the medium pretreated with CA beads compared with the control without the CA bead treatment. The factors inhibiting CXCL10 production were identified as the C3 and C4 fragments by mass spectrometry. The monomeric C3bi and C4b proteins were abundant in the medium pretreated with CA beads. Furthermore, purified C3bi and C4b were found to inhibit IFN‐β‐induced CXCL10 production and STAT1 phosphorylation. Thus, STAT1‐mediated CXCL10 production induced by stimulation with LPS or IFN was potently inhibited by monomeric C3bi and C4b generated by the interaction of blood with CA beads. These mechanisms mediated by monomeric C3bi and C4b may be involved in the anti‐inflammatory effects of CA.

Down-modulation of toll-like receptor 2 expression on granulocytes and suppression of interleukin-8 production due to in vitro treatment with cellulose acetate beads.

The Adacolumn, which is filled with cellulose acetate beads (CA beads), has been used as a medical device for inflammatory diseases. The CA beads selectively adsorb granulocytes and monocytes and remove them from the peripheral blood. The anti‐inflammatory effects of the Adacolumn are possibly caused by removal of these cells but also due to the functional changes in the processed cells. In this study, we investigated the effects of CA beads treatment on modulation of the expression of innate immunity receptors such as the Toll‐like receptor (TLR) family and production of an inflammatory cytokine, interleukin‐8 (IL‐8). Changes in the expressions of TLR1, 2, 4 and 6 in peripheral leukocytes exposed to CA beads were examined by flow cytometry. TLR2 expression on the surface of granulocytes exposed to CA beads was decreased, but the amount of intracellular TLR2 was increased, possibly by internalization. These changes were not observed in monocytes or lymphocytes. Peptidoglycan (PGN) treatment produced similar changes in TLR2 on granulocytes. We also measured the amounts of IL‐8 in cultured blood treated with lipopolysaccharide (LPS) and PGN, which are known TLR agonists. PGN‐induced IL‐8 production was lower in CA beads‐treated leukocytes than that in non‐treated leukocytes, but LPS did not induce these changes. Based on these findings, we conclude that the down‐modulation of TLR2 and suppression of IL‐8 production on granulocytes by CA beads, may play an important role in the anti‐inflammatory effects of the Adacolumn.

Peripheral tissue levels and molecular species compositions of N-acyl-phosphatidylethanolamine and its metabolites in mice lacking N-acyl-phosphatidylethanolamine-specific phospholipase D

N-acylethanolamines (NAEs), a class of lipid mediators, are produced from N-acyl-phosphatidylethanolamine (NAPE) by several pathways, including the direct release by NAPE-specific phospholipase D (NAPE-PLD) or the multistep pathway via sn-glycero-3-phospho-N-acylethanolamine (Gp-NAE). Using liquid chromatography-tandem mass spectrometry, we compared peripheral tissue levels of NAPE, Gp-NAE and NAE in NAPE-PLD-deficient (NAPE-PLD-/-) and wild type (WT) mice. NAPE-PLD was suggested to play a major role in the NAPE degradation in heart, kidney, and liver, but not in jejunum, because the NAPE levels except jejunum were significantly higher in NAPE-PLD-/- mice than in WT mice. The deletion of NAPE-PLD failed to alter the NAE levels of these tissues, suggesting its limited role in the NAE production. The enzyme assays with tissue homogenates confirmed the presence of NAPE-PLD-independent pathways in these peripheral tissues. Gp-NAE species having an acyl moiety with 22 carbons and 6 double bonds was enriched in these peripheral tissues. As for sn-2 acyl species of NAPE, 18:2-acyl-containing NAPE species were predominant over 18:1-containing species in heart, liver, and jejunum. Our results show that both molecular species composition of NAPE, NAE and Gp-NAE and their dependencies on Napepld are different among the peripheral tissues, suggesting that each tissue has distinct metabolic pathways and these NAE-containing lipids play tissue-specific roles.

Preferable existence of polyunsaturated lysophosphatidic acids in human follicular fluid from patients programmed with in vitro fertilization

Lysophosphatidic acid (LPA) exerts diverse physiological effects on various types of animal cells, including reproductive cells, through its binding to six LPA receptors. We previously found that LPA promoted maturation of the nucleus and cytoplasm of mouse and hamster oocytes surrounded by cumulus cells in vitro. Using gas-liquid chromatography, we previously reported detection of several species of LPA by analyzing the fatty acid methyl esters derived from thin layer chromatography-purified LPA in lipid extract from incubated follicular fluids programmed with in vitro fertilization. In this study using liquid chromatography- tandem mass spectrometry, we directly detected high levels of linoleoyl, arachidonoyl, and docosahexaenoyl LPAs in human follicular fluid. This unique molecular species composition of LPA was suggested to be due to a balance between the low LPA-degrading activity and high LPA-producing activity of autotaxin in human follicular fluid. Our results suggest that polyunsaturated LPAs produced by autotaxin in human follicular fluid exert unknown physiological effects on cumulus cells.

Three lysophosphatidic acids with a distinct long chain moiety differently affect cell differentiation of human colon epithelial cells to goblet cells

Aim: The intestinal mucus layer helps maintain intestinal homeostasis. In this study, we investigated the effects of lysophosphatidic acids (LPA) on differentiation of human colon carcinoma cell line, HT‐29, to goblet cells with and without sodium butyrate, a known differentiation factor for intestinal cells. Main methods: Number and average size of cells with goblet‐like morphology in five photographs per dish were measured for assessment of differentiation of HT‐29 cells to goblet cells as well as their relative portion of surface of to whole surface area of the photograph. Key findings: Our results revealed that 18:1 LPA enhanced butyrate‐induced differentiation of HT‐29 cells. Because increased mRNA expression of LPA5 and decreased mRNA expression of LPA6 were observed in HT‐29 cells after treatment with butyrate, we explored the effects of alkyl LPA and 20:4 LPA, which show preferentially higher affinities to LPA5 and LPA6, respectively. As a result, the cell differentiation to goblet cell was increased by alkyl LPA but decreased by 20:4 LPA. Further, alkyl LPA and 18:1 LPA, but not 20:4 LPA, were found to reduce the numbers of cells surviving after incubation in a standard culture medium containing 10% fetal calf serum. Significance: We suggest that the three LPAs positively and negatively affect the differentiation of HT‐29 cells to goblet cells, which may be associated with their reduced survival through the activation of distinct LPA receptor(s).

Addition of high load of lysophosphatidic acid to standard and high-fat chows causes no significant changes of its circulating and peripheral tissue levels but affects body weight and visceral fat mass of mice: Effects of LPA-rich diet on mouse body weight and fat mass

Oral administration of lysophosphatidic acid (LPA), a critical intercellular lipid mediator, exerts wound healing and antiulcer effects on gastrointestinal system. To evaluate effects of food-derived LPA on body homeostasis, we measured LPA levels by liquid chromatography-tandem mass spectrometry in chows, feces, plasma, liver, and visceral fat of mice fed a normal or high-fat chow supplemented with or without LPA-rich soybean phospholipids for 30 days. Reductions in daily body weight gains and visceral fat mass were mainly related to lower chow intake by mice fed the LPA-rich high-fat chow, whereas reduced body weight gains and fat mass were mainly related to decreased intestinal triacylglycerol absorption in mice fed LPA-rich chow. Our results showed no significant increase in plasma, liver, or adipose LPA levels, even if a quite high LPA concentration (2.0%) in chows was ingested daily, suggesting limited effects of food-derived LPA on the lumen side of the digestive tract.