Virgin olive oil and its phenol fraction modulate monocyte/macrophage functionality: a potential therapeutic strategy in the treatment of systemic lupus erythematosus.

Monocytes and macrophages are critical effectors and regulators of inflammation and innate immune response, which appear altered in different autoimmune diseases such as systemic lupus erythematosus (SLE). Recent studies suggested that virgin olive oil (VOO) and particularly its phenol compounds might possess preventive effects on different immune-inflammatory diseases, including SLE. Here, we evaluated the effects of VOO (and sunflower oil) on lipopolysaccharide (LPS)-activated peritoneal macrophages from a model of pristane-induced SLE in BALB/c mice, as well as those of the phenol fraction (PF) from VOO on the immune-inflammatory activity and plasticity in monocytes and monocyte-derived macrophages from healthy volunteers. The release of nitrite and inflammatory cytokines was lower in LPS-treated peritoneal macrophages from pristane-SLE mice fed the VOO diet when compared with the sunflower oil diet. PF from VOO similarly decreased the secretion of nitrite and inflammatory cytokines and expression of inducible nitric oxide, PPARγ and Toll-like receptor 4 in LPS-treated human monocytes. PF from VOO also prevented the deregulation of human monocyte subset distribution by LPS and blocked the genetic signature of M1 macrophages while favouring the phenotype of M2 macrophages upon canonical polarisation of naïve human macrophages. For the first time, our study provides several lines of in vivo and in vitro evidence that VOO and PF from VOO target and counteract inflammatory pathways in the monocyte-macrophage lineage of mice with pristane-induced SLE and of healthy subjects, which is a meaningful foundation for further development and application in preclinical and clinical use of PF from VOO in patients with SLE.

Daidzein and Equol: Ex Vivo and In Silico Approaches Targeting COX-2, iNOS, and the Canonical Inflammasome Signaling Pathway.

BACKGROUND: The inflammasome is a cytosolic multiprotein complex associated with multiple autoimmune diseases. Phytochemical compounds in soy (Glycine max) foods, such as isoflavones, have been reported for their anti-inflammatory properties. AIM: the anti-inflammatory activity of DZ (daidzein) and EQ (equol) were investigated in an ex vivo model of LPS-stimulated murine peritoneal macrophages and by molecular docking correlation. METHODS: Cells were pre-treated with DZ (25, 50, and 100 µM) or EQ (5, 10, and 25 µM), followed by LPS stimulation. The levels of PGE2, NO, TNF-α, IL-6, and IL-1ß were analyzed by ELISA, whereas the expressions of COX-2, iNOS, NLRP3, ASC, caspase 1, and IL-18 were measured by Western blotting. Also, the potential for transcriptional modulation by targeting NF-κB, COX-2, iNOS, NLRP3, ASC, and caspase 1 was investigated by molecular docking. RESULTS: The anti-inflammatory responses observed may be due to the modulation of NF-κB due to the binding of DZ or EQ, which is translated into decreased TNF-α, COX-2, iNOS, NLRP3, and ASC levels. CONCLUSION: This study establishes that DZ and EQ inhibit LPS-induced inflammatory responses in peritoneal murine macrophages via down-regulation of NO and PGE2 generation, as well as the inhibition of the canonical inflammasome pathway, regulating NLRP3, and consequently decreasing IL-1ß and IL-18 activation.

Oleuropein, a secoiridoid derived from olive tree, inhibits the proliferation of human colorectal cancer cell through downregulation of HIF-1α.

Oleuropein (OL) is the most prominent phenolic compound in the fruit of olive tree. Although OL has shown powerful anticancer activity the underlying action mechanism remains largely unknown. The present study evaluated the effects of OL on hydroxityrosol (HT)-29 human colon adenocarcinoma cells in comparison to hydroxytyrosol, its hydrolysis product, and to elucidate the underlying anticancer molecular mechanisms involved. Cell proliferation was determined using SRB assay. Cell cycle and apoptosis were assessed by flow cytometry and changes in MAPK cascade protein expression, HIF-1α, p53, PPARγ, and NFKß signaling pathways by Western blot. Although OL showed less potency than HT, in terms of cell growth inhibition, induced significant changes in cell cycle analysis and caused a significant increase in the apoptotic population. Both compounds produced a remarkable decrease in HIF-1α protein and an upregulation of p53 protein expression. However, no significant changes in IkB-α and MAPK cascade protein expressions were observed. HT produced a significant upregulation in peroxisome proliferator-activated receptor gamma (PPARγ) expression whereas OL failed. PPARγ upregulation may be one of the principal mechanisms of the tumor shrinkage by HT. Our novel findings demonstrate that OL limits the growth and induces apoptosis in HT-29 cells via p53 pathway activation adapting the HIF-1α response to hypoxia.

Mechanisms involved in the antiproliferative and proapoptotic effects of unsaponifiable fraction of extra virgin olive oil on HT-29 cancer cells.

Extra virgin olive oil (EVOO) has demonstrated great oncostatic and antiinflammatory properties. Nowadays, it is clear that unsaponifiable fraction (UF) as well as other minor EVOO components have a key role in these beneficial effects. The present study was designed to evaluate UF effect in HT-29 human colon adenocarcinoma cells. Cell growth and viability assays were determined by sulphorhodamine B test at different time points (24, 48, and 72 h). The proapoptotic effect was evaluated by flow cytometric studies and different protein expression were determined by immunoblotting. UF µg/mL concentrations' range significantly reduced the growth of HT-29 cell line. Moreover UF induced intrinsic apoptotic pathway in HT-29 human colon adenocarcinoma cells through PPARγ and NFκB signaling pathways coming up to COX-2 downregulation and modulating p53 suppressor protein levels. The results suggest that UF of EVOO may exert an important role in the anticancer effect of EVOO and provide a natural resource for the prevention or treatment of human colon cancer.

Dietary supplementation of an ellagic acid-enriched pomegranate extract attenuates chronic colonic inflammation in rats.

Dietary polyphenols present in Punica granatum (pomegranate), such as ellagitannins and ellagic acid (EA) have shown to exert anti-inflammatory and antioxidant properties. This study was designed to evaluate the effects of a dietary EA-enriched pomegranate extract (PE) in a murine chronic model of Cronh's disease (CD). Colonic injury was induced by intracolonic instillation of trinitrobenzensulfonic acid (TNBS). Rats were fed with different diets during 30 days before TNBS instillation and 2 weeks before killing: (i) standard, (ii) PE 250 mg/kg/day, (iii) PE 500 mg/kg/day, (iv) EA 10 mg/kg/day and (v) EA 10 mg/kg/day enriched-PE 250 mg/kg/day. Inflammation response was assessed by histology and MPO activity and TNF-α production. Besides, colonic expressions of iNOS, COX-2, p38, JNK, pERK1/2 MAPKs, IKBα and nuclear p65 NF-κB were studied by western blotting. MPO activity and the TNF-α levels were significantly reduced in dietary fed rats when compared with TNBS group. Similarly, PE and an EA-enriched PE diets drastically decreased COX-2 and iNOS overexpression, reduced MAPKs phosporylation and prevented the nuclear NF-κB translocation. Dietary supplementation of EA contributes in the beneficial effect of PE in this experimental colitis model and may be a novel therapeutic strategy to manage inflammatory bowel disease (IBD).

Activated T follicular helper-like cells are released into blood after oral vaccination and correlate with vaccine specific mucosal B-cell memory.

T follicular helper (Tfh)-like cells with potent B-cell helping ability are mobilized into human circulation after parenteral vaccination and are generally held to reflect ongoing germinal center reactions. However, whether mucosal vaccination induces systemic Tfh responses and how such responses may relate to IgA production are unknown. We investigated the frequencies, phenotype and function of circulating Tfh-like CD4+CXCR5+ T cells (cTfh) in adults receiving an oral inactivated enterotoxigenic Escherichia coli vaccine. Subjects were classified as vaccine responders or weak/non-responders based on their intestine-derived antibody-secreting cell (ASC) IgA responses to major vaccine antigens. Oral immunization induced significantly increased proportions of cTfh cells expressing the cTfh activation marker inducible costimulator (ICOS) in ASC responders, but not in weak/non-responders. Vaccination also enhanced the expression of IL-21, Th17 markers and integrin ß7 by activated cTfh cells, supporting functionality and gut homing potential. cTfh cells promoted total and vaccine specific IgA production from cocultured B cells. Magnitudes of cTfh responses assessed within a week after primary vaccinations correlated with memory intestine-derived vaccine specific IgA responses 1-2 years later. We conclude that activated ICOS+ Tfh-like cells are mobilized into blood after oral vaccination and may be used as biomarkers of vaccine specific mucosal memory in humans.

The phenolic fraction of extra virgin olive oil modulates the activation and the inflammatory response of T cells from patients with systemic lupus erythematosus and healthy donors.

SCOPE: Systemic lupus erythematosus (SLE) is a chronic multiorgan autoimmune disease characterized by immune deregulation, which involves altered T-cell response and imbalance of cytokine production. The phenolic fraction (PE) of extra virgin olive oil (EVOO) possesses anti-inflammatory and immunomodulatory properties and exerts preventive effects in murine models of immune-inflammatory diseases, such as SLE. The present study was designed to determine the in vitro effects of the PE from EVOO on peripheral blood mononuclear cells (PBMC) from inactive patients with SLE and healthy donors. METHODS AND RESULTS: T-cell phenotype was investigated by flow cytometry, cytokine levels were determined by ELISA, and protein expression was detected by Western blot. The PE of EVOO decreased the frequency of CD69+ cells and the secretion of IFN-γ, TNF-α, IL-6, IL-1ß, and IL-10. Moreover, PE increased the expression of I-kappa-B-α and decreased extracellular signal regulated kinase phosphorylation on PBMC from patients with SLE and healthy donors. CONCLUSION: PE modulates cytokine production and attenuates induced T-cell activation, probably through NF-κB signaling pathway, providing the first evidence that PE from EVOO has an anti-inflammatory and immunomodulatory role in SLE patients and it might therefore be considered as a dietary complement in SLE management.

Apigenin supplementation protects the development of dextran sulfate sodium-induced murine experimental colitis by inhibiting canonical and non-canonical inflammasome signaling pathways.

The present study was designed to elucidate the protective effects of dietary apigenin (API) enrichment in a chronic colitis model induced by DSS in mice. Inflammatory mediators and the possible role of canonical and non-canonical NLRP3 inflammasome signaling pathways in the beneficial effects of API under chronic inflammatory conditions were also explored. Six-week-old mice were randomized in four dietary groups: sham and control groups received standard diet (SD), and other two groups were fed with API at 0.1%. After 30days, all groups except sham received 3% DSS in drinking water for 5days followed by a regime of 21days of water. Our results revealed that dietary API supplementation decreased the macroscopic and microscopic damage signs of colitis; also, it was capable to down-regulate mPGES, COX-2 and iNOS enzyme colonic expressions and to decrease serum matrix metalloproteinase (MMP-3) levels. Similarly, API diet reduced IL-1ß and TNF-α proinflammatory cytokine secretions in primary LPS-stimulated splenocytes. Furthermore, we demonstrated that API anti-inflammatory activity was related with an inhibition of both canonical and non-canonical NLRP3 inflammasome pathways by decreasing proinflammatory IL-1ß and IL-18 cytokine levels as a consequence of regulation of cleaved caspase-1 and caspase-11 enzymes. We conclude that API supplement might provide a basis for developing a new dietary strategy for the prevention of chronic ulcerative colitis.

Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf-2 antioxidant pathway and suppression of JAK/STAT, NF-κB and MAPK activation.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a widespread organ involvement. Recent studies have suggested that extra virgin olive oil (EVOO) might possess preventive effects on this immunoinflammation-related disease. However, its role in SLE remained unknown. In this work, we evaluated the effects of EVOO diet in a pristane-induced SLE model in mice. Three-month-old mice received an injection of pristane or saline solution and were fed with different experimental diets: sunflower oil diet or EVOO diet. After 24weeks, mice were sacrificed, spleens were collected and kidneys were removed for immunoinflammatory detections. The kidney expression of microsomal prostaglandin E synthase 1, heme oxygenase 1 (HO-1), nuclear factor E2-related factor 2 (Nrf-2), mitogen-activated protein kinases (MAPKs), Janus kinase/signal transducer and activator of transcription (JAK/STAT) and nuclear transcription factor-kappa B (NF-κB) pathways were studied by western blotting. In addition to macroscopic and histological analyses, serum matrix metalloproteinase 3 (MMP-3) levels and proinflammatory cytokines production in splenocytes were evaluated by enzyme-linked immunoassay. We have demonstrated that EVOO diet significantly reduced renal damage and decreased MMP-3 serum and PGE2 kidney levels as well as the proinflammatory cytokines production in splenocytes. Our data indicate that Nrf-2 and HO-1 protein expressions were up-regulated in those mice fed with EVOO and the activation of JAK/STAT, MAPK and NF-κB pathways were drastically ameliorated. These results support the interest of EVOO as a beneficial functional food exerting a preventive/palliative role in the management of SLE.

Effects of dietary virgin olive oil polyphenols: hydroxytyrosyl acetate and 3, 4-dihydroxyphenylglycol on DSS-induced acute colitis in mice.

Hydroxytyrosol, a polyphenolic compound from extra virgin olive oil (EVOO) has exhibited an improvement in a model of DSS-induced colitis. However, other phenolic compounds present such as hydroxytyrosyl acetate (HTy-Ac) and 3,4-dihydroxyphenylglycol (DHPG) need to be explored to complete the understanding of the overall effects of EVOO on inflammatory colon mucosa. This study was designed to evaluate the effect of both HTy-Ac and DHPG dietary supplementation in the inflammatory response associated to colitis model. Six-week-old mice were randomized in four dietary groups: sham and control groups received standard diet, and other two groups were fed with HTy-Ac and DHPG, respectively, at 0.1%. After 30 days, all groups except sham received 3% DSS in drinking water for 5 days followed by a regime of 5 days of water. Acute inflammation was evaluated by Disease Activity Index (DAI), histology and myeloperoxidase (MPO) activity. Colonic expression of iNOS, COX-2, MAPKs, NF-kB and FOXP3 were determined by western blotting. Only HTy-Ac-supplemented group showed a significant DAI reduction as well as an improvement of histological damage and MPO. COX-2 and iNOS protein expression were also significantly reduced. In addition, this dietary group down-regulated JNK phosphorylation and prevented the DSS-induced nuclear translocation level of p65. However, no significant differences were observed in the FOXP3 expression. These results demonstrated, for the first time, that HTy-Ac exerts an antiinflammatory effect on acute ulcerative colitis. We concluded that HTy-Ac supplement might provide a basis for developing a new dietary strategy for the prevention of ulcerative colitis.

Unsaponifiable fraction from extra virgin olive oil inhibits the inflammatory response in LPS-activated murine macrophages.

Extra virgin olive oil (EVOO) has demonstrated great anti-inflammatory properties. Nowadays, it is clear that its minor components have a key role in these beneficial effects. However, the contribution of the unsaponifiable fraction (UF) to these healthy effects remains unknown. The present study was designed to evaluate UF in LPS stimulated peritoneal macrophages isolated from mice. NO production was analysed by the Griess method and intracellular ROS by fluorescence analysis. In addition, MAPK family activation, IKBα degradation, NFκB-p65, iNOS and COX-2 expression were determined by Western blot. UF exerted anti-inflammatory and anti-oxidant effects inhibiting LPS-induced intracellular ROS and nitrite production. Additionally, UF decreased COX-2 and iNOS protein expression. These effects were related with a down-regulation in NFκB signal signalling pathways and in MAPK phosphorylation. UF of EVOO compounds could play an important role in the anti-inflammatory effect of virgin olive oils and probably provide an attractive complement in management of inflammatory diseases.

Dietary extra virgin olive oil polyphenols supplementation modulates DSS-induced chronic colitis in mice.

We evaluated the protective effect of dietary extra virgin olive oil (EVOO) polyphenol extract (PE) supplementation in the inflammatory response associated to chronic colitis model. Six-week-old mice were randomized in four dietary groups: standard diet (SD), EVOO diet and both enriched with PE (850 ppm) (SD+PE and EVOO+PE). After 30 days, animals that were exposed to dextran sodium sulfate (DSS) (3%) followed by 3 weeks of drinking water developed chronic colitis, which was evaluated by disease activity index (DAI) and histology. Cell proliferation was analyzed by immunohistochemical and changes in monocyte chemotactic protein (MCP)-1 and tumor necrosis factor (TNF)-α mRNA expression by quantitative real-time polymerase chain reaction. Colonic expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, mitogen-activated protein kinases (MAPKs), IκBα inhibitory and peroxisome proliferator-activated receptor gamma (PPARγ) were determined by western blotting. SD-DSS group showed a significant increase of DAI, histological damage and cell proliferation, as well as an up-regulation of TNF-α, MCP-1, COX-2 and iNOS proteins. p38 and JNK MAPKs phosphorylation, IκBα degradation and PPARγ deactivation were also observed. However, in DSS-treated and EVOO+PE-fed mice, DAI and cell proliferation were significantly reduced, as well as MCP-1, TNF-α, COX-2 and iNOS expression levels. In addition, this dietary group, notably down-regulated JNK phosphorylation, prevented IκBα degradation and PPARγ deactivation. These results demonstrated, for the first time, that EVOO-PE supplementation possessed marked protective effects on experimental colitis through PPARγ up-regulation and nuclear transcription factor-kappa B and MAPK signaling pathway inhibition, decreasing the inflammatory cascade. We concluded that PE-enriched EVOO diet could be a beneficial functional food on ulcerative colitis.

Dietary supplementation of resveratrol attenuates chronic colonic inflammation in mice.

Ulcerative colitis is a nonspecific inflammatory disorder characterized by oxidative and nitrosative stress, leucocyte infiltration and upregulation of inflammatory mediators. Resveratrol is a polyphenolic compound found in grapes and wine, with multiple pharmacological actions, mainly anti-inflammatory, antioxidant, antitumour and immunomodulatory activities. The aim of this study was to investigate the effect of dietary resveratrol on chronic dextran sulphate sodium (DSS)-induced colitis. Six-week-old mice were randomized into two dietary groups: one standard diet and the other enriched with resveratrol at 20mg/kg of diet. After 30days, mice were exposed to 3% DSS for 5days developing acute colitis that progressed to severe chronic inflammation after 21days of water. Our results demonstrated that resveratrol group significantly attenuated the clinical signs such as loss of body weight, diarrhea and rectal bleeding improving results from disease activity index and inflammatory score. Moreover, the totality of resveratrol-fed animals survived and finished the treatment while animals fed with standard diet showed a mortality of 40%. Three weeks after DSS removal, the polyphenol caused substantial reductions of the rise of pro-inflammatory cytokines, TNF-alpha and IL-1beta and an increase of the anti-inflammatory cytokine IL-10. Also resveratrol reduced prostaglandin E synthase-1 (PGES-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins expression, via downregulation of p38, a mitogen-activated protein kinases (MAPK) signal pathway. We conclude that resveratrol diet represents a novel approach to the treatment of chronic intestinal inflammation.

Anti-inflammatory intestinal activity of Abarema cochliacarpos (Gomes) Barneby & Grimes in TNBS colitis model.

AIM OF THE STUDY: To assess the anti-inflammatory effect of butanolic fraction of methanolic extract from bark of Abarema cochliacarpos in acute ulcerative colitis model induced by intracolonic administration of trinitrobenzene sulfonic acid (TNBS) in Wistar rats. MATERIALS AND METHODS: Abarema cochliacarpos (100 and 150mg/kg/day) was administered by gavage 48, 24 and 1h prior to the induction of colitis with 10mg/kg of TNBS and, 24h later. RESULTS: Phytochemical studies by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) revealed that catechins were a major component into condensate class of tannins. Treatment with Abarema cochliacarpos decreased significantly macroscopic damage as compared with TNBS (p<0.05). Histological analysis showed that both doses of the extract improved the microscopic structure and preserved some areas of the colonic mucosa structure. In addition, myeloperoxidase activity (MPO), as a marker of neutrophil infiltration, was decreased in a dose-dependent way (p<0.01 and p<0.001 respectively), TNF-alpha level was also diminished with the highest dose of the extract (p<0.001) and, IL-10 level obtained no significant results. In order to elucidate some of the mechanisms, expression of inducible inflammatory enzymes, such as cyclooxygenase (COX)-2 and nitric oxide synthase (iNOS), were studied showing a significant reduction. Finally, the involvement of c-Jun N-terminal kinase (JNK) signalling demonstrated a reduction in the JNK activation with the highest dose (p<0.05 vs TNBS). CONCLUSIONS: We have shown for the first time that the extracts obtained from Abarema cochliacarpos bark possess active substances, which exert marked protective effects in acute experimental colitis, confirming and justifying, at least in part, the popular use of this plant to treat gastrointestinal diseases.

Chronic administration of Abarema cochliacarpos attenuates colonic inflammation in rats

Inflammatory bowel diseases are characterized by a chronic clinical course of relapse and remission associated with self-destructive inflammation of the gastrointestinal tract. Active extracts from plants have emerged as natural potential candidates for its treatment. Abarema cochliacarpos (Gomes) Barneby & Grimes, Fabaceae (Barbatimão), is a native medicinal plant in to Brazil. Previously we have demonstrated in an acute colitis model a marked protective effect of a butanolic extract, so we decided to assess its anti-inflammatory effect in a chronic ulcerative colitis model induced by trinitrobenzensulfonic acid (TNBS). Abarema cochliacarpos (150 mg/day, v.o.) was administered for fourteen consecutive days. This treatment decreased significantly macroscopic damage as compared with TNBS. Histological analysis showed that the extract improved the microscopic structure. Myeloperoxidase activity (MPO) was significantly decreased. Study of cytokines showed that TNF-α was diminished and IL-10 level was increased after Abarema cochliacarpos treatment. In order to elucidate inflammatory mechanisms, expression of cyclooxygenase (COX)-2 and nitric oxide synthase (iNOS) were studied showing a significant downregulation. In addition, there was reduction in the JNK and p-38 activation. Finally, IκB degradation was blocked by Abarema cochliacarpos treatment being consistent with an up-regulation of the NF-kappaB-binding activity. These results reinforce the anti-inflammatory effects described previously suggesting that Abarema cochliacarpos could provide a source for the search for new anti-inflammatory compounds useful in ulcerative colitis treatment.