sphingosine-1-phosphate and Colitis

Baicalein has been shown to have anti-inflammatory and anti-tumor activities. However, the mechanisms underlying its anti-inflammatory effect on colitis remain unclear.. A dextran sodium sulfate (DSS)-induced model of acute colitis was established in BALB/c mice (6-8 weeks old, weighing 18-22 g). Six groups of mice received: (1) water for 10 days (control), n = 6; (2) DSS 4% solution in the drinking water for 7 days, followed by normal water for 3 days, n = 7; (3), (4), and (5) as for group 2 plus baicalein (10, 20, 40 mg/kg) administered once daily starting on day 1, n = 6; and (6) as for (2) plus 5-aminosalicylic acid (50 mg/kg) administered once daily starting on day 1, n = 6. Body weights, stool consistency, and hematochezia were recorded, and the severity of colitis was evaluated using a disease activity index. On day 11, the mice were euthanized, and organs and blood were collected for analysis. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay; CD11b-positive cells were analyzed by immunofluorescence microscopy; expression of retinoic-acid-receptor-related orphan nuclear receptor gamma, sphingosine kinase 1 (SPHK1), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) was detected by immunohistochemistry; and expression of nucleotide-binding oligomerization domain 2 (NOD2), SPHK1, sphingosine 1-phosphate receptor 1 (S1PR1), total STAT3, and p-STAT3 were detected by western blotting analysis. Inter-group differences were compared using Student's t test.. Baicalein treatment dose-dependently reduced DSS-induced weight loss (P < 0.01 or P < 0.05), splenomegaly (P < 0.01), and colonic damage, as reflected by amelioration of diarrhea, rectal bleeding, and colonic ulceration, congestion, edema (shown as colon length, P < 0.05 or P < 0.01), and inflammatory cell infiltration. Baicalein also significantly decreased the levels of inflammatory mediators in the serum (P < 0.01) and colon, and significantly inhibited expression of NOD2 SPHK1, S1PR1, and p-STAT3 in the colon (P < 0.05).. Baicalein treatment ameliorated colitis in mice by inhibiting S1P-STAT3 signaling, suggesting that this flavonoid might be beneficial in the treatment of colitis.

Topics: Animals; Colitis; Dextran Sulfate; Female; Flavanones; Lysophospholipids; Mice; Mice, Inbred BALB C; Nod2 Signaling Adaptor Protein; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; STAT3 Transcription Factor

Triptolide (TP) exhibits effective activity against colon cancer in multiple preclinical models, but the mechanisms underlying the observed effects are not fully understood. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of colon cancer progression. The aim of this study was to investigate the effect of TP on the sphingosine kinase (SPHK)-S1P signaling pathway in colitis-associated colon cancer.. An azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model and the THP-1 cell line were used to evaluate the therapeutic effects and mechanisms of TP in colitis-associated colon cancer (CACC). Various molecular cell biology experiments, including Western blotting, real-time PCR and immunofluorescence, were used to obtain relevant experimental data. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was also established to detect the levels of S1P in tissue and plasma.. In the AOM/DSS mouse model, TP treatment induced a dose-dependent decrease in tumor incidence and inhibited macrophage recruitment and M2 polarization in the tumors. TP also efficiently decreased the S1P levels and SPHK1/S1PR1/S1PR2 expression and significantly inhibited activation of the S1P-mediated phosphorylation of ERK protein in macrophages.. The results indicated that TP might influence the recruitment and polarization of tumor-associated macrophages by suppressing the SPHK-S1P signaling pathway.

Topics: Animals; Azoxymethane; Colitis; Colitis-Associated Neoplasms; Colon; Dextran Sulfate; Disease Models, Animal; Diterpenes; Epoxy Compounds; Female; Humans; Lysophospholipids; Male; Mice, Inbred BALB C; Mice, Inbred ICR; Mice, Nude; Phenanthrenes; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; THP-1 Cells; Tumor-Associated Macrophages

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease, which often affects colon or rectum or both. It is now well recognized that sphingosine kinases-1/sphingosine-1-phosphate (S1P) signaling may have a very significant potential as targets for therapeutic intervention in UC. Compared with the pure dextran sodium sulfate group, administration of PF543 significantly reduced clinical symptoms with less weight loss, diarrhea, and shortening of the colon. The severity of colitis was improved with reduced disease activity index and degree of histological damage in colon. Moreover, treatment with PF543 not only decreased S1P but also inhibited mRNA expression of proinflammatory factors such as interleukin (IL)-1β and IL-6. This suggests that PF543 might exhibit an anti-inflammatory function against colitis through inhibition of expression of proinflammatory factors.

Topics: Animals; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Enzyme Inhibitors; Lysophospholipids; Male; Methanol; Mice; Mice, Inbred C57BL; Organ Size; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Sphingosine; Spleen; Substrate Specificity; Sulfones

Ulcerative colitis (UC) is a chronic gastrointestinal disorder interfering with life quality. A total of 60 male Wistar rats were divided into four equal groups: Control (group I), hesperidin only (group II), UC untreated (group III), and UC treated with hesperidin (group IV). Hesperidin had modulatory effects on UC pathogenesis, which might be through alleviating colonic sphingosine phosphate phosphatase 2 messenger RNA expression and sphingosine kinase-1 levels, thus suppressing the subsequent downstream inflammatory and apoptotic cascades represented by decreased macrophage inflammatory protein-1α and enhancement of B-cell lymphoma 2 immunohistochemistry expression. Also, it improved mitochondrial biogenesis by increasing the peroxisome proliferator-activated receptor-gamma-coactivator 1-α level. It successfully restored redox potential as evidenced by marked alleviations of the nitric oxide and peroxynitrite levels, increasing total antioxidant capacity, and activating the superoxide dismutase enzyme. Also, hesperidin alleviated the UC disease activity index and improved the histopathological picture. These findings may offer a new therapeutic strategy for UC treatment.

Topics: Animals; Apoptosis; Colitis; Dextran Sulfate; Drug Delivery Systems; Hesperidin; Inflammation; Lysophospholipids; Male; Mitochondria; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Sphingosine

Topics: Aldehyde-Lyases; Animals; Carcinogenesis; Cells, Cultured; Colitis; Colon; Colonic Neoplasms; Female; Inflammation; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Signal Transduction; Sphingosine; Tumor Microenvironment

Epithelial cells line the intestinal mucosa and form an important barrier for maintaining host health. This study aimed to explore the mechanism of the Sphingosine-1-phosphate (S1P)/Sphingosine-1-phosphate receptor 2 (S1PR2) pathway in intestinal epithelial cells (IECs) that participate in the intestinal barrier function.. In this study, we constructed a knockout of the S1PR2 gene in mice, and Dextra sulfate sodium (DSS) was used to induce colitis. We isolated IECs from wild type (WT) and S1PR2-/- mice, and the endogenous expression of S1PR2 and Zonula occludens 1 (ZO-1) in IEC were detected by Western blot. Next, the major histocompatibility complex II (MHC-II) expression was analyzed by reverse transcription quantitative real-time (RT-qPCR) and flow cytometry. The in vivo and in vitro intestinal permeability were evaluated by serum fluorescein isothiocyanate (FITC) concentration. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in cell suspension were analyzed by enzyme-linked immuno sorbent assay (ELISA). A carboxyfluorescein diacetate succinimidyl ester (CFSE) assay was used to detect the T-cell proliferation in a co-culture system.. The intestinal mucosal barrier damage in S1PR2-/- mice was more severe than in the WT mice, and there were more CD4+T-cells in the colon tissue of DSS-treated S1PR2-/- mice. Either the mouse colon carcinoma cell line (CT26. WT) or the IECs upregulated MHC-II expression, which then promoted CD4+T-cell proliferation. The S1P/S1PR2 pathway controlled MHC-II expression to regulate CD4+T-cell proliferation via the extracellular signal-regulated kinase (ERK) pathway. In addition, the IFN-γ that was secreted by CD4+T-cells increased DSS-induced damage of intestinal epithelial cell barrier function. ZO-1 expression was increased by S1P in CT26.WT cells, while S1PR2 antagonist JTE-013 expression was downregulated. However, in CT26.WTsi-S1PR2 cells, S1P had no effect on ZO-1 expression.. The S1P/S1PR2 axis in IECs mediated CD4+T-cell activation via the ERK pathway and MHC-II expression to regulate intestinal barrier function.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Communication; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cells, Cultured; Colitis; Epithelial Cells; Female; Intestinal Absorption; Intestinal Mucosa; Lysophospholipids; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Mice, Knockout; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis.  These results, if confirmed by in vitro studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease.

Topics: Animals; Colitis; Disease Models, Animal; Fibrosis; Immunohistochemistry; Intestines; Lysophospholipids; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Reference Standards; Signal Transduction; Smad3 Protein; Sphingosine; Transforming Growth Factor beta

Gut-associated inflammation plays a crucial role in the progression of colon cancer. Here, we identify a novel pathogen-host interaction that promotes gut inflammation and the development of colon cancer. We find that enteropathogenic bacteria-secreted particles (ET-BSPs) stimulate intestinal epithelium to produce IDENs (intestinal mucosa-derived exosome-like nanoparticles) containing elevated levels of sphingosine-1-phosphate, CCL20 and prostaglandin E2 (PGE2). CCL20 and PGE2 are required for the recruitment and proliferation, respectively, of Th17 cells, and these processes also involve the MyD88-mediated pathway. By influencing the recruitment and proliferation of Th17 cells in the intestine, IDENs promote colon cancer. We demonstrate the biological effect of sphingosine-1-phosphate contained in IDENs on tumour growth in spontaneous and transplanted colon cancer mouse models. These findings provide deeper insights into how host-microbe relationships are mediated by particles secreted from both bacterial and host cells.

Topics: Adenocarcinoma; Animals; Azoxymethane; Bacteroides fragilis; Blotting, Western; Carcinogenesis; Carcinogens; Cell Line, Tumor; Cell Proliferation; Chemokine CCL20; Colitis; Colonic Neoplasms; Dextran Sulfate; Dinoprostone; Disease Models, Animal; Enterobacteriaceae; Exosomes; Immunohistochemistry; In Situ Hybridization, Fluorescence; Inflammation; Intestinal Mucosa; Lysophospholipids; Mice; Myeloid Differentiation Factor 88; Nanoparticles; Neoplasm Transplantation; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Th17 Cells

Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.

Topics: Animals; Colitis; Hematopoietic System; Inflammation; Inflammatory Bowel Diseases; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine

Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The prodrug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC, even in Sphk2(-/-) mice, and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

Topics: Animals; Cell Transformation, Neoplastic; Colitis; Colon; Fingolimod Hydrochloride; Gene Deletion; Gene Expression Regulation, Neoplastic; Interleukin-6; Lysophospholipids; Mice; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Sphingosine; STAT3 Transcription Factor; Tumor Microenvironment

Although predominantly expressed on lymphocytic and hematopoietic cells, the role of sphingosine-1-phospate receptor 4 (S1P(4)) in immune homeostasis is still poorly understood. In this report, we used a S1P(4)-deficient murine model to characterize the biological role of S1P(4)-mediated S1P signaling in the immune system. S1p(4)(-/-) animals showed normal peripheral lymphocyte numbers and a regular architecture of secondary lymphoid organs. Interestingly, S1P(4) only marginally affects T-cell function in vivo. In contrast, dendritic cell (DC) migration and cytokine secretion are profoundly affected by S1P(4) deficiency. Lack of S1P(4) expression on DCs significantly reduces T(H)17 differentiation of T(H) cells. Furthermore, in various in vivo models of T(H)1- or T(H)2-dominated immune reactions, S1P(4) deficiency consistently increased the amplitude of T(H)2-dominated immune responses, while those depending on T(H)1-dominated mechanisms were diminished. Finally, S1p(4)(-/-) mice showed decreased pathology in a model of dextran sulfate sodium-induced colitis. In summary, for the first time, we show that S1P(4) signaling is involved in the regulation of DC function and T(H)17 T-cell differentiation. S1P(4)-mediated S1P signaling also modifies the course of various immune diseases in a murine model. We propose that S1P(4) may constitute an interesting target to influence the course of various autoimmune pathologies.

Topics: Animals; Cell Differentiation; Colitis; Dendritic Cells; Dextran Sulfate; Immunoglobulins; Lysophospholipids; Mice; Mice, Knockout; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Th17 Cells; Th2 Cells

Sphingolipid metabolism is driven by inflammatory cytokines. These cascade of events include the activation of sphingosine kinase (SK), and subsequent production of the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). Overall, S1P is one of the crucial components in inflammation, making SK an excellent target for the development of new anti-inflammatory drugs. We have recently shown that SK inhibitors suppress colitis and hypothesize here that the novel SK inhibitor, ABC294640, prevents the development of colon cancer. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model, there was a dose-dependent decrease in tumor incidence with SK inhibitor treatment. The tumor incidence (number of animals with tumors per group) in the vehicle, ABC294640 (20 mg/kg) and ABC294640 (50 mg/kg) groups were 80, 40 and 30%, respectively. Tumor multiplicity (number of tumors per animal) also decreased from 2.1 ± 0.23 tumors per animal in the AOM + DSS + vehicle group to 1.2 ± 0 tumors per animal in the AOM + DSS + ABC294640 (20 mg/kg) and to 0.8 ± 0.4 tumors per animal in the AOM + DSS + ABC294640 (50 mg/kg) group. Importantly, with ABC294640, there were no observed toxic side effects. To explore mechanisms, we isolated cells from the colon (CD45-, representing primarily colon epithelial cells) and (CD45+, representing primarily colon inflammatory cells) then measured known targets of SK that control cell survival. Results are consistent with the hypothesis that the inhibition of SK activity by our novel SK inhibitor modulates key pathways involved in cell survival and may be a viable treatment strategy for the chemoprevention colitis-driven colon cancer.

Topics: Adamantane; Animals; Azoxymethane; Colitis; Colon; Colonic Neoplasms; Dextran Sulfate; Extracellular Signal-Regulated MAP Kinases; Lysophospholipids; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Pyridines; Sphingosine

The bioactive lipid sphingosine-1-phosphate (S1P) is emerging as an important mediator of immune and inflammatory responses. S1P formation is catalyzed by sphingosine kinase (SK), of which the SK1 isoenzyme is activated by tumor necrosis alpha (TNF-alpha). SK1 has been shown to be required for mediating TNF-alpha inflammatory responses in cells, including induction of cyclooxygenase 2 (COX-2). Because TNF-alpha and COX-2 are increased in patients with inflammatory bowel disease (IBD), we investigated the role of SK1 in a murine model of colitis. SK1(-/-) mice treated with dextran sulfate sodium (DSS) had significantly less blood loss, weight loss, colon shortening, colon histological damage, and splenomegaly than did wild-type (WT) mice. In addition, SK1(-/-) mice had no systemic inflammatory response. Moreover, WT but not SK1(-/-) mice treated with dextran sulfate sodium had significant increases in blood S1P levels, colon SK1 message and activity, and colon neutrophilic infiltrate. Unlike WT mice, SK1(-/-) mice failed to show colonic COX-2 induction despite an exaggerated TNF-alpha response; thus implicating for the first time SK1 in TNF-alpha-mediated COX-2 induction in vivo. Inhibition of SK1 may prove to be a valuable therapeutic target by inhibiting systemic and local inflammation in IBD.

Topics: Animals; Body Weight; Colitis; Colitis, Ulcerative; Colon; Cyclooxygenase 2; Dextran Sulfate; Erythrocytes; Gene Expression Regulation; Humans; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Size; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Spleen; Tumor Necrosis Factor-alpha

Anti-lymphocyte antibodies (Abs) that suppress T-cell chemotactic and other responses to sphingosine 1-phosphate (S1P), but not to chemokines, were found in a lymphopenic patient with recurrent infections. Lymphocyte type 1 S1P receptor (S1P(1)) that transduces S1P chemotactic stimulation was recognized by patient Abs in Western blots of T cells, S1P(1) transfectants, and S1P(1)-hemagglutinin purified by monoclonal anti-hemagglutinin Ab absorption. The amino terminus of S1P(1), but not any extracellular loop, prevented anti-S1P(1) Ab suppression of S1P(1) signaling and T-cell chemotaxis to S1P. Human purified anti-S1P(1) Abs decreased mouse blood lymphocyte levels by a mean of 72%, suppressed mouse T-cell chemotaxis to S1P in vivo, and significantly reduced the severity of dextran sodium sulfate-induced colitis in mice. Human Abs to the amino terminus of S1P(1) suppress T-cell trafficking sufficiently to impair host defense and provide therapeutic immunosuppression.

Topics: Aged; Animals; Antigens; Autoantibodies; Bacterial Infections; Colitis; Female; Humans; Immunosuppressive Agents; Lysophospholipids; Mice; Mice, Inbred C57BL; Protein Isoforms; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocytes