Dietary Fish Oil Increases the Number of CD11b+CD27- NK Cells at the Inflammatory Site and Enhances Key Hallmarks of Resolution of Murine Antigen-Induced Peritonitis.

PURPOSE: To determine the effects of dietary omega-3 polyunsaturated fatty acids (PUFAs) on recruitment of natural killer (NK) cells and resolution responses in antigen-induced peritonitis in mice. METHODS: Mice were fed fish oil-enriched or control diets, immunized twice and challenged intraperitoneally with methylated bovine serum albumin. Prior to and at different time-points following inflammation induction, expression of surface molecules on peritoneal cells was determined by flow cytometry, concentration of soluble mediators in peritoneal fluid by ELISA or Luminex, and of lipid mediators by LC-MS/MS, and number of apoptotic cells in mesenteric lymph nodes by TUNEL staining. RESULTS: Mice fed the fish oil diet had higher number of CD11b+CD27- NK cells as well as a higher proportion of CD107a+ NK cells in their peritoneum 6 h after inflammation induction than mice fed the control diet. They also had higher numbers of CCR5+ NK cells and higher concentrations of CCL5 and CXCL12. Additionally, a higher fraction of apoptotic neutrophils but lower fraction of CD47+ neutrophils were present in the peritoneum of mice fed the fish oil diet 6 h after inflammation induction and the fish oil fed mice had a shorter resolution interval. They also had lower concentrations of pro-inflammatory mediators but higher concentrations of the anti-inflammatory/pro-resolution mediators TGF-ß, IGF-1, and soluble TNF RII, as well as higher ratios of hydroxyeicosapentaenoic acid (HEPE) to hydroxyeicosatetraenoic acid (HETE) than mice fed the control diet. CONCLUSION: The results demonstrate that dietary fish oil increases the number of mature NK cells at the inflamed site in antigen-induced peritonitis and enhances several key hallmarks of resolution of inflammation, casting light on the potential mechanisms involved.

Emu Oil and Saireito in combination reduce tumour development and clinical indicators of disease in a mouse model of colitis-associated colorectal cancer.

BACKGROUND: Emu Oil (EO) previously demonstrated therapeutic potential in a mouse model of colitis-associated CRC (CA-CRC). Saireito, a traditional Japanese medicine, has not been investigated in CA-CRC. AIM: To determine whether EO and Saireito could be therapeutic in an azoxymethane (AOM)/dextran sulphate sodium (DSS) model of CA-CRC. METHODS: Female C57BL/6 mice were assigned to groups (n = 10/group); 1) saline control, 2) saline+Saireito, 3) saline+EO, 4) saline+EO/Saireito, 5) AOM/DSS control, 6) AOM/DSS+Saireito, 7) AOM/DSS+EO and 8) AOM/DSS+EO/Saireito. Mice were intraperitoneally injected with saline or AOM (7.4 mg/kg) on day 0 and underwent three DSS/water cycles (2%w/v DSS for 7 days, 14 days water). Mice were orally-gavaged with either water (80 µL), Saireito (80 µL), EO (80 µL) or EO/Saireito (160 µL; 80 µL EO + 80 µL Saireito) thrice weekly. Daily bodyweight and disease activity index (DAI) were recorded and colonoscopies performed on days 20, 41 and 62. Mice were euthanized on day 63. p < 0.05 was considered statistically significant. RESULTS: AOM/DSS induced significant bodyweight loss throughout the trial (max -36%), which was attenuated by Saireito (max +7%), EO (max +5%) and EO/Saireito (max +14%; p < 0.05). AOM/DSS increased DAI compared to saline controls (p < 0.05), which was reduced by Saireito, EO and EO/Saireito (p < 0.05). All treatments reduced colonoscopically-assessed colitis severity (days 20 and 41; p < 0.05). EO/Saireito further decreased colitis severity compared to Saireito and EO alone (day 20; p < 0.05). Finally, EO and EO/Saireito resulted in fewer colonic tumours compared to AOM/DSS controls (p < 0.05). CONCLUSION: Combined EO and Saireito reduced disease and tumour development in AOM/DSS mice, suggesting therapeutic potential in CA-CRC.

Anti-Inflammatory and Proresolving Effects of the Omega-6 Polyunsaturated Fatty Acid Adrenic Acid.

Polyunsaturated fatty acids (PUFAs) and their metabolites are potent regulators of inflammation. Generally, omega (n)-3 PUFAs are considered proresolving whereas n-6 PUFAs are classified as proinflammatory. In this study, we characterized the inflammatory response in murine peritonitis and unexpectedly found the accumulation of adrenic acid (AdA), a poorly studied n-6 PUFA. Functional studies revealed that AdA potently inhibited the formation of the chemoattractant leukotriene B4 (LTB4), specifically in human neutrophils, and this correlated with a reduction of its precursor arachidonic acid (AA) in free form. AdA exposure in human monocyte-derived macrophages enhanced efferocytosis of apoptotic human neutrophils. In vivo, AdA treatment significantly alleviated arthritis in an LTB4-dependent murine arthritis model. Our findings are, to our knowledge, the first to indicate that the n-6 fatty acid AdA effectively blocks production of LTB4 by neutrophils and could play a role in resolution of inflammation in vivo.

The BLIMP1-EZH2 nexus in a non-Hodgkin lymphoma.

Waldenström's macroglobulinemia (WM) is a non-Hodgkin lymphoma, resulting in antibody-secreting lymphoplasmacytic cells in the bone marrow and pathologies resulting from high levels of monoclonal immunoglobulin M (IgM) in the blood. Despite the key role for BLIMP1 in plasma cell maturation and antibody secretion, its potential effect on WM cell biology has not yet been explored. Here we provide evidence of a crucial role for BLIMP1 in the survival of cells from WM cell line models and further demonstrate that BLIMP1 is necessary for the expression of the histone methyltransferase EZH2 in both WM and multiple myeloma cell lines. The effect of BLIMP1 on EZH2 levels is post-translational, at least partially through the regulation of proteasomal targeting of EZH2. Chromatin immunoprecipitation analysis and transcriptome profiling suggest that the two factors co-operate in regulating genes involved in cancer cell immune evasion. Co-cultures of natural killer cells and cells from a WM cell line further suggest that both factors participate in immune evasion by promoting escape from natural killer cell-mediated cytotoxicity. Together, the interplay of BLIMP1 and EZH2 plays a vital role in promoting the survival of WM cell lines, suggesting a role for the two factors in Waldenström's macroglobulinaemia.

A polysaccharide fraction from Achillea millefolium increases cytokine secretion and reduces activation of Akt, ERK and NF-κB in THP-1 monocytes.

Achillea millefolium has been used in traditional medicine for a number of ailments, including skin inflammation and wounds. A polysaccharide fraction (Am-25-d) isolated from aqueous extract from A. millefolium had an average molecular weight of 270 kDa and a monosaccharide composition of GalA, Gal, Ara, Xyl, Rha in molar ratio of 28:26:23:9:7. THP-1 cells primed with IFN-γ and stimulated with LPS in the presence of Am-25-d secreted more IL-1ß, IL-8, IL-10, IL-12p40, IL-23 and TNF-α than THP-1 cells stimulated in the absence of Am-25-d. However, when added to unstimulated cells Am-25-d did not increase secretion of the cytokines examined. Stimulating THP-1 monocytes in the presence of Am-25-d led to decreased nuclear concentrations of NF-κB and phosphorylation of ERK1/2 and Akt kinases compared with that when the cells were stimulated without Am-25-d. These findings indicate that Am-25-d isolated from A. millefolium has immunoenhancing properties that may be mediated via the Akt pathway.

Murine antigen-induced inflammation--a model for studying induction, resolution and the adaptive phase of inflammation.

Murine zymosan-induced peritonitis is the model most frequently used to study resolution of inflammation. However, the antigen-induced peritonitis model may be better suited for studying resolution of inflammation and the adaptive phase that follows. The objective of this study was to provide an evaluation of the kinetics of cells and mediators during induction, resolution and the adaptive immune phases of a murine antigen-induced inflammation. Female C57BL/6 mice were immunized twice subcutaneously with mBSA and three weeks after the initial immunization they were injected intraperitoneally (i.p.) with mBSA, which induced peritonitis. Peritoneal cells were counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in peritoneal fluid were determined by ELISA. Two neutrophil populations, differing in size and granularity and slightly in expression of surface molecules, were observed in the peritoneal cavity after induction of inflammation. Macrophages disappeared from the peritoneal cavity following i.p. administration of mBSA but appeared again as they differentiated from recruited monocytes and peaked in numbers at 48 h. At that time point, two distinct populations of macrophages were present in the peritoneal cavity; one with high expression of F4/80, also expressing the atypical chemokine receptor D6 as well as CCR7; the other expressing low levels of F4/80 and also expressing CD11c and CD138. Eosinophils appeared in the peritoneum 3h following i.p. administration of mBSA and peaked at 48 h. At that time point they had upregulated their expression of CCR3 but decreased their expression of CD11b. Peritoneal levels of CCL11 peaked at 6h and may have led to recruitment of the eosinophils. NK cells and T cells peaked at 48 h, whereas B cells peaked at 5 days, with the majority being B1 cells. Peritoneal concentrations of pro-inflammatory cytokines (IL-ß and IL-6) and chemokines (CCL2 and CCL3) peaked at 3h, whereas IL-1ra peaked at 6h, sTNF-R at 24h and sIL-6R and TGF-ß at 48 h. The results show kinetic alterations in cell populations and mediators in a murine model that may be an excellent model to study initiation and resolution of inflammation and the following adaptive phase.

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt.

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.

Dietary fish oil reduces the acute inflammatory response and enhances resolution of antigen-induced peritonitis.

Dietary n-3 polyunsaturated fatty acids (PUFA) influence the inductive phase of inflammation but less is known about their effects on the resolution phase. This study examined the effects of dietary fish oil on induction and resolution of antigen-induced inflammation in mice. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and inflammation induced by intraperitoneal injection of mBSA. Prior to and at different time points after mBSA administration, peritoneal cells were analyzed and expression of surface molecules determined by flow cytometry. Concentration of chemokines, cytokines and soluble cytokine receptors was determined by ELISA. Mice fed the fish oil diet had fewer peritoneal neutrophils, shorter resolution interval and lower levels of pro-inflammatory cytokines and chemokines than mice fed the control diet. In mice fed the fish oil diet there was an early peak in peritoneal levels of the immunosuppressive molecules sIL-6R and TGF-ß, that was not seen in mice fed the control diet. In the resolution phase, peritoneal macrophages from mice fed the fish oil diet expressed more of the atypical chemokine receptor D6 and peritoneal TGF-ß levels were higher than that in mice fed the control diet. Furthermore, in the late-resolution phase there were more peritoneal eosinophils and macrophages in mice fed the fish oil diet than in mice fed the control diet. These results demonstrate a suppressive effect of n-3 PUFA on the inductive phase of inflammation and indicate an enhancing effect of n-3 PUFA on resolution of inflammation.

Dietary fish oil increases the proportion of a specific neutrophil subpopulation in blood and total neutrophils in peritoneum of mice following endotoxin-induced inflammation.

Omega-3 polyunsaturated fatty acids may have beneficial effects in inflammation, where neutrophil migration and activation are of importance. The effects of dietary fish oil on neutrophil numbers and subpopulations in healthy mice and mice with endotoxin-induced inflammation were determined. Mice were fed a control diet with or without 2.8% fish oil, and half of them were injected intraperitoneally with endotoxin. Blood, peritoneal lavage, bone marrow and spleen were collected. Expression of cell surface molecules was analyzed by flow cytometry, and chemokine concentrations were determined by enzyme-linked immunosorbent assay. Dietary fish oil did not alter the proportion of total neutrophils in blood but increased the proportion of a specific subpopulation of neutrophils 48 h following endotoxin administration. This subpopulation of neutrophils expressed higher levels of CD11b, Ly6G and major histocompatibility complex-II, suggesting a different role for these neutrophils in the inflammatory response. Dietary fish oil did not affect neutrophil numbers in the peritoneum of healthy mice, but 12 h after endotoxin administration, there were fewer neutrophils in the peritoneum of mice fed the fish oil diet than in mice fed the control diet. However, 48 h after endotoxin administration, mice fed the fish oil diet had more neutrophils in peritoneum than mice fed the control diet. These results indicate that, although dietary fish oil may delay recruitment of neutrophils from blood to the peritoneum early in inflammation, it has the potential to increase the number of peritoneal neutrophils later, which may be of benefit as impaired neutrophil migration and activation have been associated with immunosuppression late in inflammation.

Dietary fish oil decreases the proportion of classical monocytes in blood in healthy mice but increases their proportion upon induction of inflammation.

Fish oil can have beneficial effects in health and disease. In healthy individuals, reduction of the inflammatory status may be of benefit, whereas in patients with systemic inflammation, such as sepsis, it is important to diminish the immunosuppression that is thought to contribute to poor outcome. The objective of this study was to determine the effects of dietary fish oil on monocytes/macrophages in blood, bone marrow, spleen, and peritoneum and chemokine concentrations in blood and peritoneum in healthy mice and mice with endotoxin-induced inflammation. Mice were fed a Western-type diet without fish oil (C) or with 2.8% fish oil (FO) for 6 wk and then either killed (healthy mice) or injected i.p. with endotoxin (LPS) and killed after 3, 8, 12, 24, or 48 h. Blood, bone marrow, spleen, and peritoneal lavage were collected. Expression of cell surface molecules and chemokine receptors was analyzed by flow cytometry and chemokine concentrations measured by ELISA. Healthy mice in the FO group had lower proportions of classical monocytes in blood than healthy mice in the C group. LPS administration increased the proportion of classical monocytes in blood in mice in the FO group but not in those in the C group. Healthy mice in the FO group had lower serum concentrations of CCL2 than mice in the C group, but in inflamed mice, CCL2 concentrations were higher in the FO group than in the C group. These results indicate that dietary fish oil can attenuate the inflammatory status in homeostasis but intensify the immune response upon inflammation.

Dietary fish oil decreases secretion of T helper (Th) 1-type cytokines by a direct effect on murine splenic T cells but enhances secretion of a Th2-type cytokine by an effect on accessory cells.

Dietary fish oil is considered to have anti-inflammatory effects based primarily on its effects on T-cell proliferation and IL-2 secretion. Its effects on the secretion of T helper (Th) 1-type cytokines vary and few studies have examined its effects on the secretion of Th2-type cytokines. In the present study, we examined the effects of dietary fish oil on the secretion of Th1 and Th2-type cytokines by splenocytes and the mechanism by which dietary fish oil affects Th2-type cytokine secretion. Mice were fed diets supplemented with 18 % fish oil (w/w) +2 % maize oil or 20 % maize oil for 6 weeks. Spleen cells, isolated splenic T cells and accessory cells (splenocytes depleted of T cells) were stimulated with anti-CD3/anti-CD28. The secretion of interferon (IFN)-gamma, TNF-alpha, IL-4 and IL-10 was measured by ELISA. Dietary fish oil decreased the secretion of IFN-gamma and TNF-alpha by total splenocytes and isolated T cells. In contrast, dietary fish oil increased the secretion of IL-4 by total splenocytes but had no effect on IL-4 secretion by isolated T cells. When isolated T cells were cultured with CD11b+ cells (mainly macrophages), cells from mice fed the fish oil diet secreted more IL-4 than cells from mice fed the maize oil diet. These results demonstrate that dietary fish oil directs cytokine secretion by splenocytes towards a Th2 phenotype and that the effects of dietary fish oil on the secretion of a Th2-type cytokine are mediated by its effect on CD11b+ accessory cells.

Positive association between DNA strand breaks in peripheral blood mononuclear cells and polyunsaturated fatty acids in red blood cells from women.

Lipid peroxidation of polyunsaturated fatty acids (PUFA) generates reactive products that may cause DNA damage. To examine the possible relationship between DNA damage in peripheral blood mononuclear cells (PBMC) and the concentration of PUFA in red blood cells (RBC), endogenous DNA strand breaks, formamidopyrimidine DNA glycosylase (FPG) sites, and hydrogen peroxide (H2O2) sensitive sites were evaluated by the comet assay in blood samples from 98 Icelandic women. Fatty acid composition of RBC was analyzed by gas chromatography. Endogenous DNA strand breaks in PBMC correlated positively with the concentration of total PUFA, total n-3 PUFA, docosahexaenoic acid, linoleic acid, oleic acid, and palmitic acid in RBC. However, there was no association between FPG sites or H(2)O(2) sensitive sites in DNA in PBMC and the concentration of total PUFA or total saturated fatty acid in RBC. As there was no association between oxidative DNA damage or sensitivity of DNA to oxidative stress and the concentration of PUFA in RBC, the positive association between endogenous DNA strand breaks in PBMC and the concentration of total PUFA in RBC is probably not related to oxidative stress.

The effects of omega-3 polyunsaturated fatty acids on TNF-alpha and IL-10 secretion by murine peritoneal cells in vitro.

Omega-3 polyunsaturated fatty acids (PUFA) affect immune response, partly by affecting cytokine secretion. Omega-3 PUFA decrease tumor necrosis factor (TNF)-alpha secretion by RAW 264.7 macrophages but increase TNF-alpha secretion by primary elicited peritoneal macrophages in vitro. In this study, the effects of omega-3 and omega-6 PUFA on lipopolysaccharide induced TNF-alpha and interleukin (IL)-10 secretion by murine primary resident and elicited peritoneal macrophages and by RAW 264.7 macrophages, were examined in vitro using an enzyme-linked immunosorbent assay. In addition, the effects of dietary omega-3 PUFA on the number of cells secreting these cytokines were examined with enzyme-linked immunospot assay. All cell types secreted more TNF-alpha but similar amounts of IL-10 when incubated with the omega-3 PUFA, eicosapentaenoic acid or docosahexaenoic acid, compared with that when incubated with the omega-6 PUFA, linoleic acid or arachidonic acid. Dietary fish oil did not affect the number of TNF-alpha secreting resident peritoneal macrophages but decreased the number of macrophages secreting IL-10 ex vivo. These results show that dietary omega-3 PUFA and omega-3 PUFA added to cells in vitro increase TNF-alpha secretion by resident peritoneal macrophages, probably by a direct effect on the cells. In contrast, omega-3 PUFA did not affect IL-10 secretion by the cells but decreased the number of cells secreting IL-10 ex vivo, possibly by affecting cell recruitment, maturation or proliferation.

Mucosal tolerance to KLH reduces BSA-induced arthritis in rats--an indication of bystander suppression.

Mucosal tolerance has been shown to reduce disease severity in animal models mimicking human autoimmune diseases. The objective of this study was to examine whether mucosal tolerance against keyhole limpet haemocyanin (KLH) could be used to reduce bovine serum albumin (BSA)-induced arthritis in rats and whether anti-inflammatory drugs or passive cigarette smoke affected tolerance induction. Arthritis was induced by immunizing rats with BSA and then injecting BSA into one knee and saline into the other knee for comparison. Prior to BSA immunization, the rats were treated intranasally with KLH or saline and KLH then injected in the knee joints at the time of BSA injection, or the rats were treated with or without anti-inflammatory drugs or subjected to cigarette smoke prior to and during intranasal treatment with BSA. The rats that received intranasal treatment with KLH had a significantly less inflammation in their left knee joint compared to rats that received intranasal saline treatment. Beclamethasone increased the tolerance effect of BSA, whereas passive cigarette smoke abrogated the mucosal tolerance. This data suggests that bystander suppression can be used to treat arthritis and other autoimmune diseases, even when the autoantigen is not known.

Dietary fish oil increases the number of splenic macrophages secreting TNF-alpha and IL-10 but decreases the secretion of these cytokines by splenic T cells from mice.

Dietary fish oil has immunomodulatory effects that are partly mediated by its effects on cytokine secretion. In this paper, we examine whether dietary fish oil has different effects on cytokine secretion by T cells and macrophages. Female BalbC mice were fed diets supplemented with 18% fish oil + 2% corn oil or 20% corn oil. Concanavalin A (ConA)- and LPS-induced TNF-alpha and IL-10 secretion by splenocytes was examined using ELISA. Dietary fish oil decreased ConA induced-, but increased LPS-induced, TNF-alpha and IL-10 secretion by total murine splenocytes. Dietary fish oil increased the number of splenocytes secreting TNF-alpha and IL-10, following stimulation with LPS, by 123 and 38%, respectively, but did not affect cytokine secretion by each cell, as determined using enzyme-linked immunospot. Spleens from mice fed the fish oil diet had over 2-fold higher proportion of macrophages with high expression of CD11b than spleens from mice fed the corn oil diet. In addition, fish oil increased the proportion of total and CD11b(+) splenocytes that expressed the LPS receptor complex molecules, CD14 and toll-like receptor (TLR)4/myeloid differentiation factor-2 (MD-2), by 85 and 28%, respectively. The increased proportion of macrophages expressing the LPS receptor complex molecules, CD14 and TLR4/MD-2, in spleens from mice fed the fish oil diet may explain the increased number of cells that secreted the cytokines after LPS stimulation.

Dietary fish oil increases tumor necrosis factor secretion but decreases interleukin-10 secretion by murine peritoneal macrophages.

Dietary fish oil has immunomodulatory effects that are mediated in part by its effects on cytokines. Secretion of the inflammatory and the anti-inflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-10 by murine resident peritoneal macrophages was monitored after ex vivo stimulation with lipopolysaccharide. Macrophages were obtained from mice fed a corn oil diet containing 200 g/kg corn oil or a fish oil diet containing 180 g/kg fish oil and 20 g/kg corn oil. Dietary fish oil increased secretion of the proinflammatory cytokine, TNF, but decreased secretion of the anti-inflammatory cytokine, IL-10. The cytokines appeared in the medium after 1.5 h and peaked at 6-12 h. Neutralizing antibodies against TNF and IL-10 had little effect on secretion of the other cytokine, indicating that the effects of fish oil on TNF and IL-10 secretion by these cells are independent of one another. Furthermore, although inhibiting prostaglandin production enhanced TNF secretion by macrophages from mice fed the corn oil diet, it did not affect IL-10 secretion by macrophages in this group. Blocking leukotriene B(4) production also did not affect IL-10 secretion in macrophages from mice fed a nonpurified diet. These results demonstrate that fish oil has an overall pro-inflammatory effect given its effects on secretion of both inflammatory and anti-inflammatory cytokines by resident peritoneal macrophages.