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• W2886871503 abstract "Intestinal alkaline SMase (Alk-SMase) cleaves phosphocholine from SM, platelet-activating factor (PAF), and lysophosphatidylcholine. We recently found that colitis-associated colon cancer was 4- to 5-fold enhanced in Alk-SMase KO mice. Here, we further studied the pathogenesis of colitis induced by dextran sulfate sodium (DSS) in WT and KO mice. Compared with WT mice, KO mice demonstrated greater body weight loss, more severe bloody diarrhea, broader inflammatory cell infiltration, and more serious epithelial injury. Higher levels of PAF and lower levels of interleukin (IL)10 were identified in KO mice 2 days after DSS treatment. A greater and progressive increase of lysophosphatidic acid (LPA) was identified. The change was associated with increased autotaxin expression in both small intestine and colon, which was identified by immunohistochemistry study, Western blot, and sandwich ELISA. The upregulation of autotaxin coincided with an early increase of PAF. IL6 and TNFα were increased in both WT and KO mice. At the later stage (day 8), significant decreases in IL6, IL10, and PAF were identified, and the decreases were greater in KO mice. In conclusion, deficiency of Alk-SMase enhances DSS-induced colitis by mechanisms related to increased autotaxin expression and LPA formation. The early increase of PAF might be a trigger for such reactions. Intestinal alkaline SMase (Alk-SMase) cleaves phosphocholine from SM, platelet-activating factor (PAF), and lysophosphatidylcholine. We recently found that colitis-associated colon cancer was 4- to 5-fold enhanced in Alk-SMase KO mice. Here, we further studied the pathogenesis of colitis induced by dextran sulfate sodium (DSS) in WT and KO mice. Compared with WT mice, KO mice demonstrated greater body weight loss, more severe bloody diarrhea, broader inflammatory cell infiltration, and more serious epithelial injury. Higher levels of PAF and lower levels of interleukin (IL)10 were identified in KO mice 2 days after DSS treatment. A greater and progressive increase of lysophosphatidic acid (LPA) was identified. The change was associated with increased autotaxin expression in both small intestine and colon, which was identified by immunohistochemistry study, Western blot, and sandwich ELISA. The upregulation of autotaxin coincided with an early increase of PAF. IL6 and TNFα were increased in both WT and KO mice. At the later stage (day 8), significant decreases in IL6, IL10, and PAF were identified, and the decreases were greater in KO mice. In conclusion, deficiency of Alk-SMase enhances DSS-induced colitis by mechanisms related to increased autotaxin expression and LPA formation. The early increase of PAF might be a trigger for such reactions. Alkaline SMase (Alk-SMase) was originally identified as an enzyme that hydrolyzes SM to ceramide in the intestinal tract (1.Nilsson Å. The presence of sphingomyelin- and ceramide-cleaving enzymes in the small intestinal tract.Biochim. Biophys. Acta. 1969; 176: 339-347Crossref PubMed Scopus (173) Google Scholar). Studies with Alk-SMase KO mice have confirmed the crucial role of the enzyme in SM digestion (2.Zhang Y. Cheng Y. Hansen G.H. Niels-Christiansen L.L. Koentgen F. Ohlsson L. Nilsson A. Duan R.D. Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.J. Lipid Res. 2011; 52: 771-781Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Alk-SMase is an ecto-enzyme that anchors on the mucosal membrane with a short hydrophobic domain, and is released into the lumen in an active form by bile salt and pancreatic trypsin (3.Duan R.D. Alkaline sphingomyelinase: an old enzyme with novel implications.Biochim. Biophys. Acta. 2006; 1761: 281-291Crossref PubMed Scopus (117) Google Scholar). The enzyme shares no structural similarities with acid and neutral SMases, but belongs to the ecto-nucleotide pyrophosphatase phosphodiesterase (NPP) family (4.Duan R.D. Bergman T. Xu N. Wu J. Cheng Y. Duan J. Nelander S. Palmberg C. Nilsson A. Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family.J. Biol. Chem. 2003; 278: 38528-38536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). As a novel member of the family, Alk-SMase is also called NPP7. Further characterizations of purified and recombinant Alk-SMase revealed that it is a phosphocholine-specific enzyme that cleaves the phosphocholine group not only from SM but also from platelet-activating factor (PAF) (5.Wu J. Nilsson A. Jonsson B.A. Stenstad H. Agace W. Cheng Y. Duan R.D. Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity.Biochem. J. 2006; 394: 299-308Crossref PubMed Scopus (40) Google Scholar) and lysophosphatidylcholine (lyso-PC) (4.Duan R.D. Bergman T. Xu N. Wu J. Cheng Y. Duan J. Nelander S. Palmberg C. Nilsson A. Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family.J. Biol. Chem. 2003; 278: 38528-38536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). These effects render the enzyme important implications in carcinogenesis and inflammation, because the metabolism of these substrates is deeply involved in inflammation and cancer (6.Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2440) Google Scholar–8.Tsoupras A.B. Iatrou C. Frangia C. Demopoulos C.A. The implication of platelet activating factor in cancer growth and metastasis: potent beneficial role of PAF-inhibitors and antioxidants.Infect. Disord. Drug Targets. 2009; 9: 390-399Crossref PubMed Scopus (107) Google Scholar). In line with this hypothesis, dietary SM was previously found to inhibit carcinogen-induced colon cancer in animals (9.Dillehay D.L. Webb S.K. Schmelz E.M. Merrill Jr., A.H. Dietary sphingomyelin inhibits 1,2-dimethylhydrazine-induced colon cancer in CF1 mice.J. Nutr. 1994; 124: 615-620Crossref PubMed Scopus (241) Google Scholar) and progressive reduction of Alk-SMase activity was found in patients with ulcerative colitis, sporadic adenomas, colonic carcinomas, and familial adenomatous polyposis (10.Hertervig E. Nilsson A. Bjork J. Hultkrantz R. Duan R.D. Familial adenomatous polyposis is associated with a marked decrease in alkaline sphingomyelinase activity: a key factor to the unrestrained cell proliferation?.Br. J. Cancer. 1999; 81: 232-236Crossref PubMed Scopus (60) Google Scholar, 11.Hertervig E. Nilsson A. Nyberg L. Duan R.D. Alkaline sphingomyelinase activity is decreased in human colorectal carcinoma.Cancer. 1997; 79: 448-453Crossref PubMed Scopus (124) Google Scholar, 12.Sjöqvist U. Hertervig E. Nilsson A. Duan R.D. Ost A. Tribukait B. Lofberg R. Chronic colitis is associated with a reduction of mucosal alkaline sphingomyelinase activity.Inflamm. Bowel Dis. 2002; 8: 258-263Crossref PubMed Scopus (65) Google Scholar, 13.Zhang P. Li B. Gao S. Duan R.D. Dietary sphingomyelin inhibits colonic tumorigenesis with an up-regulation of alkaline sphingomyelinase expression in ICR mice.Anticancer Res. 2008; 28: 3631-3635PubMed Google Scholar). Recently, we found that colitis-associated colon cancer was enhanced in Alk-SMase KO mice (14.Chen Y. Zhang P. Xu S.C. Yang L. Voss U. Ekblad E. Wu Y. Min Y. Hertervig E. Nilsson A. et al.Enhanced colonic tumorigenesis in alkaline sphingomyelinase (NPP7) knockout mice.Mol. Cancer Ther. 2015; 14: 259-267Crossref PubMed Scopus (28) Google Scholar), indicating that the anticancer properties of Alk-SMase are linked to its anti-inflammatory effects. Considering the three major substrates of Alk-SMase, its anti-inflammatory effect may be more closely related to its activity against PAF and lyso-PC, which are two pro-inflammatory phospholipids (7.Moolenaar W.H. Lysophospholipids in the limelight: autotaxin takes center stage.J. Cell Biol. 2002; 158: 197-199Crossref PubMed Scopus (90) Google Scholar, 15.Yost C.C. Weyrich A.S. Zimmerman G.A. The platelet activating factor (PAF) signaling cascade in systemic inflammatory responses.Biochimie. 2010; 92: 692-697Crossref PubMed Scopus (109) Google Scholar). Increased PAF has been found in inflammatory diseases in the intestine and liver (16.Wardle T.D. Hall L. Turnberg L.A. Platelet activating factor: release from colonic mucosa in patients with ulcerative colitis and its effect on colonic secretion.Gut. 1996; 38: 355-361Crossref PubMed Scopus (32) Google Scholar, 17.Yang Y. Nemoto E.M. Harvey S.A. Subbotin V.M. Gandhi C.R. Increased hepatic platelet activating factor (PAF) and PAF receptors in carbon tetrachloride induced liver cirrhosis.Gut. 2004; 53: 877-883Crossref PubMed Scopus (43) Google Scholar). By cleaving phosphocholine from PAF, Alk-SMase inactivates PAF and blocks PAF-induced MAP kinase activation, chemotaxis, and cytokine release (5.Wu J. Nilsson A. Jonsson B.A. Stenstad H. Agace W. Cheng Y. Duan R.D. Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity.Biochem. J. 2006; 394: 299-308Crossref PubMed Scopus (40) Google Scholar); and by cleaving phosphocholine from lyso-PC, Alk-SMase converts lyso-PC to monoacylglycerol, which may decrease formation of lysophosphatidic acid (LPA) by lysophospholipase D (LPD) (3.Duan R.D. Alkaline sphingomyelinase: an old enzyme with novel implications.Biochim. Biophys. Acta. 2006; 1761: 281-291Crossref PubMed Scopus (117) Google Scholar). LPA has emerged as a potent inflammatory factor that activates multiple signal transduction pathways, such as Ras, Rac, and PI3 kinase, to promote inflammation via different receptors (18.Knowlden S. Georas S.N. The autotaxin-LPA axis emerges as a novel regulator of lymphocyte homing and inflammation.J. Immunol. 2014; 192: 851-857Crossref PubMed Scopus (112) Google Scholar). LPA is mainly generated by an enzyme called autotaxin, which was first identified as a nucleotidase and then turned out to be an important LPD for LPA generation (19.Stracke M.L. Krutzsch H.C. Unsworth E.J. Arestad A. Cioce V. Schiffmann E. Liotta L.A. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.J. Biol. Chem. 1992; 267: 2524-2529Abstract Full Text PDF PubMed Google Scholar, 20.Tokumura A. Majima E. Kariya Y. Tominaga K. Kogure K. Yasuda K. Fukuzawa K. Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase.J. Biol. Chem. 2002; 277: 39436-39442Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). Similar to Alk-SMase, autotaxin is a member of the NPP family, and is named NPP2 (21.Stefan C. Jansen S. Bollen M. NPP-type ectophosphodiesterases: unity in diversity.Trends Biochem. Sci. 2005; 30: 542-550Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). The aim of this study was to examine the changes of colitis induced by dextran sulfate sodium (DSS) in WT and Alk-SMase KO mice, with particular attention paid to the changes of PAF, LPA, and autotaxin. Our study demonstrated that deficiency of Alk-SMase (NPP7) enhanced colitis with upregulation of autotaxin (NPP2) and increased formation of LPA and PAF. The Alk-SMase KO mice were generated as reported previously (2.Zhang Y. Cheng Y. Hansen G.H. Niels-Christiansen L.L. Koentgen F. Ohlsson L. Nilsson A. Duan R.D. Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.J. Lipid Res. 2011; 52: 771-781Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The genetic background of the mice was C57BL/6. Deletion by Cre recombinase induces a shift of reading frame, which creates an early stop codon, resulting in blocking the translation of the transcript at early stage. Both WT and Alk-SMase KO mice used in this study were bred from intercross of heterozygous mice and then further bred to get enough animals for the investigation. The genotype and phenotype of the animals were confirmed by PCR and fecal Alk-SMase assay, as reported before. All mice were housed in the animal facilities at Daqing campus, Harbin Medical University and fed commercial standard pellets with free access to water. The mice were euthanized by cervical dislocation under inhaled isoflurane anesthesia. The experimental protocols were approved by the Animal Ethics Committee of Harbin Medical University, China. DSS with a molecular mass of 36–50 kDa was purchased from MP Biomedicals (Stockholm, Sweden). The kits for analysis of PAF, LPA, interleukin (IL)10, IL6, and TNFα were purchased from Cloud-Clone Corp., USA (Wuhan, China). The antibody against autotaxin was purchased from Abcam (Shanghai, China) and the goat against mouse IgG antibody conjugated with horseradish peroxidase was from MXB Biotechnologies, China. The sandwich ELISA kit for autotaxin quantification was purchased from Echelon Biosciences Inc. (Salt Lake City, UT). The kit for mouse occult blood assay was purchased from ACON Biotechnologies Company (Hangzhou, China). The colitis was induced by DSS treatment as previously described (22.Andersson D. Kotarsky K. Wu J. Agace W. Duan R.D. Expression of alkaline sphingomyelinase in yeast cells and anti-inflammatory effects of the expressed enzyme in a rat colitis model.Dig. Dis. Sci. 2009; 54: 1440-1448Crossref PubMed Scopus (26) Google Scholar). At 9 weeks of age, both WT and KO mice were provided with 3% DSS dissolved in sterilized drinking water ad libitum for 8 days. The body weight was measured every day at 9:00 AM and the changes in percentage related to the original body weight on day 0 were calculated. The properties of the stools and the blood stain around the anus were examined. When no visible blood stain was found on the stools, the occult blood tests were performed. The disease activity index (DAI) was scored based on the weight loss, stool consistency, and blood in stool as shown in Table 1, which is essentially according to Maines et al. (23.Maines L.W. Fitzpatrick L.R. French K.J. Zhuang Y. Xia Z. Keller S.N. Upson J.J. Smith C.D. Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase.Dig. Dis. Sci. 2008; 53: 997-1012Crossref PubMed Scopus (150) Google Scholar).TABLE 1Assessment of DAIScoreWeight LossStool ConsistencyBlood in Stool0NoneNormal pelletsNegative11–5%Slightly loose but in shapeHemoccult positive25–10%Loose pelletVisual slightly bleeding310–15%Loose feces and no shapeObvious bleeding but no adhesion around anus4>15%DiarrheaGross bleeding and blood incrustation around anus Open table in a new tab The mice were euthanized on day 8 after DSS treatment. The colon was removed and its length measured from the ileocecal valve to the end of rectum. The segments were cut open and the gross changes of the tissue, including degrees of edema, bleeding, and ulceration, were examined under a dissecting microscope and scored according to Table 2 (23.Maines L.W. Fitzpatrick L.R. French K.J. Zhuang Y. Xia Z. Keller S.N. Upson J.J. Smith C.D. Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase.Dig. Dis. Sci. 2008; 53: 997-1012Crossref PubMed Scopus (150) Google Scholar). The liver, spleen, and thymus were removed and their wet weights determined.TABLE 2Macroscopic change score of the colitisScoreEdemaBleedingUlceration0NoneNoneNone1MildMildMild2ModerateModerateModerate3SevereSevereSevere Open table in a new tab A proximal part (1 cm) of colon was cut and fixed in 4% paraformaldehyde, embedded in paraffin, further cut in 0.5 mm sections, and stained with H&E. The ulcerations, infiltration of inflammatory cells, crypt loss, epithelial cell hyperplasia, goblet cell reduction, and epithelial regeneration were examined in 10 randomly selected fields under microscopy and scored according to Table 3 (23.Maines L.W. Fitzpatrick L.R. French K.J. Zhuang Y. Xia Z. Keller S.N. Upson J.J. Smith C.D. Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase.Dig. Dis. Sci. 2008; 53: 997-1012Crossref PubMed Scopus (150) Google Scholar). For statistical analysis, the histology scores from each observed field were summed up and multiplied with the percentage of the fields with positive findings.TABLE 3Histology score of the colitisChangesScoreDegreeDamage of epithelium0Morphologically normal1Zonal destruction of the epithelial surface2Diffuse epithelial destruction and/or mucosal ulcerations involving submucosa3Severe epithelial destructionInflammatory cell infiltration0Absence of infiltrate or fewer than five cells1Mild infiltration to the lamina propria2Moderate infiltration to the muscularis mucosae3High infiltration to the muscularis mucosae4Severe infiltration involving the submucosaGoblet cell depletion0No depletion1Presence of nonorganized goblet cells2Presence of one to three areas without goblet cells3More than three areas without goblet cells4Complete depletion of goblet cellsCrypt damage0None1One-third of crypt damaged2Two-thirds of crypt damaged3Crypts lost, surface epithelium intact4Crypts lost, surface epithelium lostPercent involvement0011–25%226–50%351–75%476–100% Open table in a new tab To determine the changes of PAF, LPA, and cytokines, both WT and KO mice were treated with DSS and euthanized on days 0, 2, 4, and 8, respectively. The colon was removed, cut open longitudinally, and rinsed with saline solution. The colonic mucosa was scraped and homogenized in lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, and 1 mM PMSF. After centrifugation, the supernatant was collected and the levels of IL6, IL10, TNFα, PAF, and LPA were determined by individual ELISA kits following the manufacturer's instructions. The animals were treated with DSS as above and euthanized on days 0, 2, 4, and 8. A 0.5 cm-long segment was taken from the middle of the small intestine and colon of both WT and KO mice and fixed in 4% paraformaldehyde overnight and embedded in paraffin. The section was deparaffinized, followed by boiling in sodium citrate buffer (pH 6) for 20 min for antigen retrieval. The section was incubated with anti-NPP2 antibody for 15 min and then with goat against mouse IgG antibody conjugated with horseradish peroxidase for 30 min at room temperature. Diaminobenzidine (MXB Biotechnologies, China) was used as the chromogen. The section was then counterstained with hematoxylin and observed under a microscope. To semi-quantify the autotaxin expression in the WT and KO mice, Western blot was performed 4 days after DSS treatment; as at this time point, expression of autotaxin was clearly demonstrated in immunohistochemistry (IHC) studies in both small intestine and colon, and LPA levels were significantly increased. The mucosal tissues from both small intestine and colon were scraped, homogenized, and centrifuged. Proteins of 50 μg of the supernatant were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane electrophoretically. After blocking with fat-free milk, the membrane was incubated with anti-NPP2 antibody (1:400) for 2 h, and then with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (ZSGB-Bio, Beijing, China) for 1 h. The bands were identified by ECL advance reagents and the remitted light was recorded on X-ray film. The densities of the bands were measured by Image J software (National Institutes of Health; https://imagej.nih.gov/ij/). To confirm the Western blot results, a quantitative sandwich ELISA was employed to measure autotaxin levels in the homogenates of the small intestine and colon after DSS treatment for 4 days. According to the instructions of the kit, the mucosal samples were diluted to 2 mg protein per milliliter. After adding the samples, the reaction was incubated for 1 h at room temperature. The anti-autotaxin antibody and the secondary detector were then added followed by incubation for an additional hour. After stopping the reaction and color generation, the absorbance was assayed at 450 nm. Standard samples with different concentrations of autotaxin were assayed simultaneously. The levels of autotaxin in each sample were determined with reference of the standard curve generated. Statistical significance for multiple comparisons was determined by one-way ANOVA followed by Newman-Keuls analysis using Prism 5.0 (GraphPad Software). All data are presented as the mean ± SEM from at least three repeated experiments. P < 0.05 was defined as statistically significant. As shown in Fig. 1A, the body weights of both WT and the Alk-SMase KO mice were stable for the first 3 days and started decreasing on day 4. The reduction was greater in KO than in WT mice. At the end of the experiment, the body weight had decreased by 30% in KO mice and by 23% in WT mice. Although WT and KO mice started showing blood stain on stool or anus on day 5, bloody diarrhea rapidly occurred and became more serious in KO than in WT mice. At day 8, most stools in the WT group were still shaped, while those in KO group showed serious bloody diarrhea. The KO mice were also more sluggish than the WT mice. Thus the DAI in KO mice was significantly higher than in WT mice from day 6 to day 8 (Fig. 1B). Macroscopic signs of inflammation in the colon were found in both groups, but the severity was greater in KO than in WT mice. KO mice exhibited broader mucosal bleeding, more extensive tissue edema, and more severe ulceration, as shown by a pair of representative samples in Fig. 2A. Compared with WT mice, the length of the colon in KO mice was significantly shortened (Fig. 2B). The more pronounced tissue edema, bleeding, and visible ulcerations in KO mice gave a significantly higher macroscopic index (Fig. 2C). The wet weights of thymus and liver were significantly reduced and that of spleen slightly increased (P = 0.134) in KO mice compared with WT mice (Fig. 2D). Histopathological characterization revealed more marked damage of the crypts in KO mice. As shown in Fig. 3A, the height of the mucosa in KO mice was shortened, the surface was more irregular, and the destruction of the crypt architecture was more obvious (arrowed) compared with WT mice. Figure 3B shows detailed pathological changes with higher magnifications. A larger number of inflammatory cells and broader infiltrations, even to serosa, were observed in KO mice (Fig. 3Ba, d); whereas in WT mice, the infiltration was largely spread to muscularis (Fig. 3Bd). Glandular hyperplasia in KO mice was more severe (Fig. 3Bb) than in WT mice (Fig. 3Bc). Diffusion of inflammatory cells to blood vessels was observed in both groups [KO (Fig. 3Bc) and WT (Fig. 3Ba)], with that in KO mice more extensive. Figure 3Bb (WT) shows the tendency of inflammatory cells to form follicles. In addition, goblet cell reduction, nucleus distortion, and colonic erosion were more obvious in KO (Fig. 3Bb) than in WT mice (Fig. 3Bc). These changes led to higher microscopic inflammatory scores in KO than in WT mice (Fig. 3C). In order to find potential mechanisms for the more severe colitis in Alk-SMase KO mice, we examined two inflammatory lysophospholipids, PAF and LPA, which are closely related to the substrates of Alk-SMase (3.Duan R.D. Alkaline sphingomyelinase: an old enzyme with novel implications.Biochim. Biophys. Acta. 2006; 1761: 281-291Crossref PubMed Scopus (117) Google Scholar). As shown in Fig. 4, before DSS treatment (day 0), the levels of PAF and LPA in WT and KO mice were all low. However, after 2 days of DSS treatment, PAF in the KO mice was significantly higher than that in the WT group. The increase of PAF in WT mice occurred later than in KO mice. The increase reached maximal levels on day 4 in both groups and declined on day 8. The levels of PAF on day 8, although low, were still significantly higher (P < 0.05) in KO mice than in WT mice. The LPA levels increased with time in both WT and KO mice, and the levels in KO mice were about 60% (P < 0.05) higher than those in WT mice on day 4 and 36% (P < 0.05) higher on day 8. Because LPA is mainly produced by autotaxin, we further examined the expression of autotaxin. We first studied the expression of autotaxin by IHC methods. The results from small intestine are shown in Fig. 5A, and those from colon in Fig. 5B. In the small intestine, before DSS treatment (day 0), there was a dispersed weak expression (brown stains), which was identified in the lamina propria of the villi and was most likely produced by the immune cells (arrowed) in both WT and KO mice. On day 2, while the positive IHC staining in WT mice was only slightly increased, that in KO mice was significantly intensified. Expression was also found in the endothelial cells of blood vessels or high epithelial venule-like structures (arrowed). On day 4, higher expression was found in both groups and the positive staining was mainly displayed along the middle of the lamina propria in the conduit system (arrowed). As in colon (Fig. 5B), there was no positive staining identified before DSS treatment (day 0) in either WT or KO mice, but it became clearly visible on day 2, with the expression more intensive in KO than in WT mice. The expression was also found in the lamina propria region of the crypt in some types of immune cells (arrowed). The expression of autotaxin on day 4 in the colon was sharply increased for both WT and KO mice, the increase in KO mice being more pronounced. Expression in the endothelial cells of blood vessels in the colon was also identified in both WT and KO mice (Fig. 5C). However, expression of autotaxin was not found on the surface of the mucosa in either the small intestine or the colon. Due to the severe damage of the colonic mucosa, the IHC results on day 8 could not be interpreted (not shown). In addition, Fig. 5A and also show that on day 0, no inflammation signs were identified for either WT or KO mice, indicating that the lack of Alk-SMase alone did not induce colitis. To compare the intensity of autotaxin expression between WT and KO mice, Western blot for autotaxin on day 4 was performed and the densities of the bands were determined. As shown in the right panels of Fig. 6, the autotaxin bands in KO and WT mice treated with DSS were clearly demonstrated in both small intestine and colon, with the expression more obvious in KO than in WT mice. The densities of the bands are shown in the left panels of Fig. 6. The autotaxin expression was approximately 98% higher in the small intestine and 77% higher in the colon in KO mice than in WT mice. To confirm the semi-quantitative results from Western blot, autotaxin was further determined by sandwich ELISA 4 days after DSS treatment of the mice (Fig. 7). In support of the results from Western blot, the levels of autotaxin were six to seven times higher in KO than in WT mice for both small intestine and colon. The changes of the cytokines, IL6, IL10, and TNFα, after DSS treatment in KO and WT mice are shown in Fig. 8. These cytokines were low on days 0 and 2 when inflammation signs did not display. However, at this time point, notably lower IL10 levels were demonstrated in KO mice than in WT mice. DSS treatment induced increases of both IL10 and IL6 on day 4, followed by a significant reduction on day 8 in both WT and KO mice, with the decrease in KO mice more obvious than in WT mice. TNFα showed a time-dependent increase in both WT and KO mice. No difference was found from day 0 to day 4, but the levels on day 8 in KO mice were significantly lower than in WT mice. In the present study, we examined DSS-induced colitis in Alk-SMase KO mice and WT mice. We found that the deficiency of Alk-SMase significantly enhanced the inflammation, as shown by the greater loss of body, thymus, and liver weights, by more severe bloody diarrhea and mucosal injury, and by microscopic findings of crypt distortion and more intensive inflammatory cell infiltration. Earlier studies suggested that Alk-SMase might indeed have anti-inflammatory functions in the gut. Patients with chronic ulcerative colitis had lowered Alk-SMase activity (12.Sjöqvist U. Hertervig E. Nilsson A. Duan R.D. Ost A. Tribukait B. Lofberg R. Chronic colitis is associated with a reduction of mucosal alkaline sphingomyelinase activity.Inflamm. Bowel Dis. 2002; 8: 258-263Crossref PubMed Scopus (65) Google Scholar), and recombinant human Alk-SMase given rectally alleviated DSS-induced acute colitis in rats (22.Andersson D. Kotarsky K. Wu J. Agace W. Duan R.D. Expression of alkaline sphingomyelinase in yeast cells and anti-inflammatory effects of the expressed enzyme in a rat colitis model.Dig. Dis. Sci. 2009; 54: 1440-1448Crossref PubMed Scopus (26) Google Scholar). Furthermore, it was recently reported that upregulation of the ENPP7 gene is a feature of the immunosuppressive response to chronic infection with enterohemorrhagic Escherichia coli in calves (24.Kieckens E. Rybarczyk J. Li R.W. Vanrompay D. Cox E. Potential immunosuppressive effects of Escherichia coli O157:H7 experimental infection on the bovine host.BMC Genomics. 2016; 17: 1049Crossref PubMed Scopus (11) Google Scholar). The present study provides strong direct evidence that Alk-SMase not only plays crucial roles in digestion of dietary SM (2.Zhang Y. Cheng Y. Hansen G.H. Niels-Christiansen L.L. Koentgen F. Ohlsson L. Nilsson A. Duan R.D. Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.J. Lipid Res. 2011; 52: 771-781Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and cholesterol absorption (25." @default.
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• W2886871503 title "Deficiency of alkaline SMase enhances dextran sulfate sodium-induced colitis in mice with upregulation of autotaxin" @default.
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