Production of lipid mediators across different disease stages of dextran sodium sulfate-induced colitis in mice

Abstract

Although several studies have revealed the role of different lipid mediators in colitis, the comprehensive analysis of their production across different phases of colitis remained unclear. Here, we performed the following analysis in the dextran sodium sulfate (DSS)-induced colitis model using LC-MS/MS. Oral administration of 2% DSS in mice for 4 days resulted in severe intestinal inflammation by day 7, which gradually subsided by day 18. Based on the disease scoring index (assigned on the basis of fecal condition and weight loss), we defined the phases of colitis as induction (days 0–4), acute inflammation (days 4–7), recovery (days 7–9), and late recovery (days 9–18). Across all phases, 58 lipid mediators were detected in the inflamed colon tissue. In the induction phase, the production of n-6 fatty acid-derived prostaglandin E2 and thromboxane B2 increased by ∼2-fold. In the acute inflammation phase, the production of n-6 fatty acid-derived leukotrienes increased by >10-fold, while that of n-3 fatty acid-derived hydroxyeicosapentaenoic acids and dihydroxyeicosatetraenoic acids decreased. In the recovery phase, a precursor of protectin D1 (17-hydroxydocosahexaenoic acid) increased over 3-fold. These observations suggested dynamic changes in the production of lipid mediators across different phases of the disease and their potential regulation in healing colitis. Although several studies have revealed the role of different lipid mediators in colitis, the comprehensive analysis of their production across different phases of colitis remained unclear. Here, we performed the following analysis in the dextran sodium sulfate (DSS)-induced colitis model using LC-MS/MS. Oral administration of 2% DSS in mice for 4 days resulted in severe intestinal inflammation by day 7, which gradually subsided by day 18. Based on the disease scoring index (assigned on the basis of fecal condition and weight loss), we defined the phases of colitis as induction (days 0–4), acute inflammation (days 4–7), recovery (days 7–9), and late recovery (days 9–18). Across all phases, 58 lipid mediators were detected in the inflamed colon tissue. In the induction phase, the production of n-6 fatty acid-derived prostaglandin E2 and thromboxane B2 increased by ∼2-fold. In the acute inflammation phase, the production of n-6 fatty acid-derived leukotrienes increased by >10-fold, while that of n-3 fatty acid-derived hydroxyeicosapentaenoic acids and dihydroxyeicosatetraenoic acids decreased. In the recovery phase, a precursor of protectin D1 (17-hydroxydocosahexaenoic acid) increased over 3-fold. These observations suggested dynamic changes in the production of lipid mediators across different phases of the disease and their potential regulation in healing colitis. Inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), is a chronic gastrointestinal disease characterized by periods of remission and relapse of symptoms like diarrhea and loss of body weight (1.Podolsky D.K. Inflammatory bowel disease.N. Engl. J. Med. 2002; 347: 417-429Crossref PubMed Scopus (3148) Google Scholar, 2.Abraham C. Cho J.H. Inflammatory bowel disease.N. Engl. J. Med. 2009; 361: 2066-2078Crossref PubMed Scopus (2107) Google Scholar). In UC, inflammation is limited to the colonic mucosa and results in severe ulceration, edema, and hemorrhage. In CD, inflammation can extend throughout the gastrointestinal tract, most commonly affecting the ileocecal region. Currently, in the United States, the number of IBD patients is estimated to be around 1 to 1.3 million (3.Loftus Jr, E.V. Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences.Gastroenterology. 2004; 126: 1504-1517Abstract Full Text Full Text PDF PubMed Scopus (2371) Google Scholar, 4.Kappelman M.D. Rifas-Shiman S.L. Kleinman K. Ollendorf D. Bousvaros A. Grand R.J. Finkelstein J.A. The prevalence and geographic distribution of Crohn's disease and ulcerative colitis in the United States.Clin. Gastroenterol. Hepatol. 2007; 5: 1424-1429Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar). Although the pathophysiology of IBD has been investigated previously, its etiology remained elusive. In order to investigate the pathophysiology of IBD, chemically induced murine IBD models, such as dextran sodium sulfate (DSS)-induced and trinitrobenzene sulfonic acid (TNBS)-induced colitis models, have been widely employed. DSS exerts direct toxicity toward gut epithelial cells of the basal crypts and affects the integrity of the mucosal barrier. This results in acute colitis, which is characterized by bloody diarrhea, colonic ulceration, and granulocyte (neutrophil) infiltration into the colonic mucosa (5.Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat. Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1151) Google Scholar). On the other hand, TNBS haptenizes colonic autologous or microbiota proteins rendering them immunogenic to the host immune system (5.Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat. Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1151) Google Scholar). Lipid mediators are bioactive substances produced via enzymatic [catalyzed by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450 (CYP)] or nonenzymatic mechanisms for fatty acid oxidation. Over one hundred lipid mediators have been identified, which differentially regulate the promotion and/or resolution of inflammatory responses (6.Stables M.J. Gilroy D.W. Old and new generation lipid mediators in acute inflammation and resolution.Prog. Lipid Res. 2011; 50: 35-51Crossref PubMed Scopus (247) Google Scholar). They are mainly categorized into two groups on the basis of their molecular structures. The first group comprises the lipid mediators produced from arachidonic acid (AA), an omega-6 (n-6) polyunsaturated fatty acid, which possesses the first double bond between the sixth and seventh carbon atom from the methyl end of the fatty acid. The second group comprises the lipid mediators produced from the omega-3 (n-3) fatty acids, such as EPA, DHA, and α-linolenic acid (ALA), which possess a double bond at the third carbon atom from the end of the carbon chain. The n-6 fatty acid-derived lipid mediators have been reported to mediate both the pro- and anti-inflammatory responses in colitis (7.Hata A.N. Breyer R.M. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation.Pharmacol. Ther. 2004; 103: 147-166Crossref PubMed Scopus (676) Google Scholar). The mucosal biopsies from IBD patients revealed an increase in the production of the COX-derived mediators from n-6 fatty acids, such as prostaglandin (PG)E2 and thromboxane (TX)B2 (8.Masoodi M. Pearl D.S. Eiden M. Shute J.K. Brown J.F. Calder P.C. Trebble T.M. Altered colonic mucosal polyunsaturated fatty acid (PUFA) derived lipid mediators in ulcerative colitis: new insight into relationship with disease activity and pathophysiology.PLoS One. 2013; 8: e76532Crossref PubMed Scopus (50) Google Scholar). Lack of the PGE2 receptor gene, namely E-type prostanoid receptor 4 (ep4), aggravated the symptoms of DSS-induced colitis in mice (9.Kabashima K. Saji T. Murata T. Nagamachi M. Matsuoka T. Segi E. Tsuboi K. Sugimoto Y. Kobayashi T. Miyachi Y. et al.The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut.J. Clin. Invest. 2002; 109: 883-893Crossref PubMed Scopus (409) Google Scholar). The administration of PGE2 or an EP4 agonist accelerated mucosal healing in the DSS-induced colitis in rats (10.Sasaki S. Hirata I. Maemura K. Hamamoto N. Murano M. Toshina K. Katsu K. Prostaglandin E2 inhibits lesion formation in dextran sodium sulphate-induced colitis in rats and reduces the levels of mucosal inflammatory cytokines.Scand. J. Immunol. 2000; 51: 23-28Crossref PubMed Scopus (66) Google Scholar, 11.Hatazawa R. Tanaka A. Tanigami M. Amagase K. Kato S. Ashida Y. Takeuchi K. Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors.Am. J. Physiol. Gastrointest. Liver Physiol. 2007; 293: G788-G797Crossref PubMed Scopus (79) Google Scholar). Inhibition of TXA2 synthase or administration of a derivative of lipoxin A4 (LXA4; a LOX-derived metabolite from n-6 fatty acids) ameliorated the symptoms of TNBS-induced colitis in mice and rats (12.Tozaki H. Fujita T. Odoriba T. Terabe A. Suzuki T. Tanaka C. Okabe S. Muranishi S. Yamamoto A. Colon-specific delivery of R68070, a new thromboxane synthase inhibitor, using chitosan capsules: therapeutic effects against 2,4,6-trinitrobenzene sulfonic acid-induced ulcerative colitis in rats.Life Sci. 1999; 64: 1155-1162Crossref PubMed Scopus (64) Google Scholar, 13.Fiorucci S. Wallace J.L. Mencarelli A. Distrutti E. Rizzo G. Farneti S. Morelli A. Tseng J.L. Suramanyam B. Guilford W.J. et al.A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction.Proc. Natl. Acad. Sci. USA. 2004; 101: 15736-15741Crossref PubMed Scopus (142) Google Scholar). Some n-3 fatty acid-derived lipid mediators have been reported to mediate anti-inflammatory responses during colitis (14.Serhan C.N. Pro-resolving lipid mediators are leads for resolution physiology.Nature. 2014; 510: 92-101Crossref PubMed Scopus (1834) Google Scholar). Administration of isomers of the LOX-derived metabolites from n-3 fatty acids, namely protectin D1 or resolvin E1, ameliorated the symptoms of DSS-induced colitis in mice (15.Masterson J.C. McNamee E.N. Fillon S.A. Hosford L. Harris R. Fernando S.D. Jedlicka P. Iwamoto R. Jacobsen E. Protheroe C. et al.Eosinophil-mediated signalling attenuates inflammatory responses in experimental colitis.Gut. 2015; 64: 1236-1247Crossref PubMed Scopus (91) Google Scholar, 16.Ishida T. Yoshida M. Arita M. Nishitani Y. Nishiumi S. Masuda A. Mizuno S. Takagawa T. Morita Y. Kutsumi H. et al.Resolvin E1, an endogenous lipid mediator derived from eicosapentaenoic acid, prevents dextran sulfate sodium-induced colitis.Inflamm. Bowel Dis. 2010; 16: 87-95Crossref PubMed Scopus (133) Google Scholar). As described above, several studies have revealed the role of each lipid mediator in colitis. However, these mediators coordinately and intricately modulate the progression and/or resolution of the inflammatory responses during colitis. Thus, it is essential to understand when and how these mediators are produced and metabolized for improving our understanding regarding the etiology of colitis and developing a novel therapy in combating it. Recently, the simultaneous quantification of a number of lipid mediators has become possible using LC-MS/MS (17.Yamada M. Kita Y. Kohira T. Yoshida K. Hamano F. Tokuoka S.M. Shimizu T. A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2015; 995–996: 74-84Crossref PubMed Scopus (53) Google Scholar). Using this technique, in the present study, we performed a comprehensive analysis of the lipid mediators that were produced during the progression and resolution of the inflammatory responses in the DSS-induced model of murine colitis. Tetranor-PGEM-d6, 6-keto PGF1α-d4, TXB2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, leukotriene (LT)C4-d5, LTB4-d4, 5(S) HETE-d8, 12(S) HETE-d8, 15(S) HETE-d8, PAF C16-d4, and oleoyl ethanolamide-d4 were from Cayman Chemical; DSS salt reagent grade was from MP Biomedicals; and indomethacin was from Sigma. Eight- to twelve-week-old male C57BL/6 mice were used. All experimental procedures in this study were approved by the Institutional Animal Care and Use Committee at the University of Tokyo (P11-576 and P08-258). Mice were provided 2% DSS containing water ad libitum for 4 days. Control mice were given water. Fecal condition and body weight change were checked every day for assessment of colitis severity. Fecal condition was scored as previously reported (Table 1) (5.Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat. Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1151) Google Scholar).TABLE 1Fecal scoreFecal ConditionScoreNormal0Soft but still formed1Very soft2Diarrhea3 Open table in a new tab Colon tissue was fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. The sections (2 μm) were stained with hematoxylin and eosin based on conventional methods. For assessment of colitis severity, histological scoring was performed as shown in Table 2 (18.Siegmund B. Lehr H.A. Fantuzzi G. Dinarello C.A. IL-1 beta-converting enzyme (caspase-1) in intestinal inflammation.Proc. Natl. Acad. Sci. USA. 2001; 98: 13249-13254Crossref PubMed Scopus (359) Google Scholar).TABLE 2Histological scoreScoreCell infiltrationLow frequency in the lamina propria0High frequency in the lamina into the of the mucosal mucosal or mucosal damage and into of the bowel Open table in a new tab The of the mice were and liquid and to by of were with the tissue and at for The was with acid in water containing are shown in Table and a The was with acid, and the on the were with This was by for and in of were into an The is shown in Table analysis of lipid metabolites was performed by using a method for lipid mediators to the were by of Open table in a new tab A B Open table in a new tab was from colon tissue on each disease by using reagent and to using was performed using and as and and and and and and and and The was performed using an for each was to using the are as the was for the of and The were by and tissue The was performed as of of of of of were using by for between two was as The mice that water DSS change in their fecal their body weight gradually increased during the of the The mice that water with DSS 1) on day The in fecal was on day the fecal by day and on day 18. with the the mice weight from day 4 to day However, weight by day and on day 18. is that the of the colon in mice with colitis Y. A. Y. S. Y. K. T. Y. prevents the of dextran sulfate sodium (DSS)-induced experimental Dis. Sci. 2007; PubMed Scopus Google Scholar). DSS the colon of mice from day 4 to day The of the colon to increase from day to day it was with the on day In the revealed infiltration and mucosal and cell on day 7, which on day The for the histological assessment of inflammation increased DSS was to be on day 7, and by day Based on these we defined day as day 4 as day as acute day as and day as late recovery the role of in the progression of murine colitis (days As shown in administration of a inhibitor, indomethacin for 4 days in aggravated the colitis and weight in the performed a comprehensive analysis of the lipid mediators produced in the inflamed colon tissue at each of the disease using LC-MS/MS. Across all the phases of colitis, a of n-6 fatty acid and n-3 fatty acid DHA, and lipid mediators were have the number of n-6 and n-3 fatty acid-derived lipid mediators in and the change in production of each lipid mediator in The number of n-6 fatty acid-derived lipid mediators that increased during the inflammatory was on day on day 7, on day and on day 18. Thus, the number of lipid mediators was the on day 4 and gradually by day recovery On the other hand, the number of n-3 fatty acid-derived lipid mediators that increased during the inflammatory was on day on day 7, on day and on day 18. Thus, the number of lipid mediators was the on day inflammation and the on day These results suggested that the of fatty acid for the production of lipid mediators from n-6 to n-3 as the disease categorized the lipid mediators on the basis of their as shown in and the change in production of each lipid mediator in Across all the phases of the the production of several lipid mediators the LOX-derived lipid The number of LOX-derived lipid mediators produced was on day on day 7, on day and on day 18. Thus, the number of mediators produced on day 4 was it increased on day On the other hand, the number of COX-derived lipid mediators was on day on day 7, on day and on day 18. Thus, the number of mediators produced was the on day 4 and by day recovery These results that the synthase for the production of these lipid mediators from to as the disease of the COX-derived lipid mediators from n-6 fatty PGE2 and increased on day 4 and by day recovery The production of all LOX-derived as and as increased on day and day The production of increased on day inflammation while that of increased on day The production of all n-3 fatty acid DHA, or lipid hydroxyeicosapentaenoic acids such as and dihydroxyeicosatetraenoic acids such as on day inflammation the production of was to that of was to of the The production of as and acids such as increased on day the production of increased that of precursor of protectin increased over 3-fold. In the production of acid increased over on day investigated how production of each lipid mediator in murine colitis on day Oral administration of indomethacin for 4 days in the production of metabolites but other lipid and metabolites in the we the of each enzyme that produced lipid mediators in the inflamed colon tissue The of which fatty acids from cell increased on day The of increased on day The of and to increase on day 4 and day 7, on day on day The levels of and change all the DSS-induced colitis in mice the immune such as infiltration of in patients with IBD in patients with (5.Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat. Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1151) Google Scholar). In the present study, using this disease we revealed the dynamic changes in the production of 58 lipid mediators during the progression of colitis. analysis revealed that the n-6 fatty acid-derived lipid mediators were mainly produced during the induction phase, while the n-3 fatty acid-derived were mainly produced during the recovery potential be for this change in the fatty acid for the of the lipid The first be that the of n-6 fatty acids to of n-3 fatty this the of n-6 fatty acid) the induction of colitis, while that of and fatty acids) The second be that the of which fatty acids from cell changes as the disease of such as and are A study that the of the production of n-6 fatty while the of the production of n-3 fatty acid) M. M. G. acid and arachidonic acid in is by two of and is by and J. PubMed Scopus Google in the we a increase in the of in the induction of colitis. study that continuous of containing a high of n-6 fatty acids increased the of developing in J.K. B. 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These observations suggested that the fatty acid for lipid mediators was for healing colitis. analysis related to the for of lipid mediators that the COX-derived lipid mediators were mainly produced during the induction phase, while the LOX-derived were mainly produced during the recovery the of increased in the induction phase, while of to increase in the acute inflammation studies have reported that the of was to be in the colonic epithelial cells and lamina of patients R. Cho J.H. Clinical and pathophysiology of inflammatory bowel 2002; PubMed Scopus Google Scholar). In several a high of in the M.J. B. M. et eicosanoids are by in and in and murine 2011; PubMed Scopus Google Scholar). et N. A. Arita M. E. S. C.N. of formation and of and Immunol. PubMed Scopus Google suggested the relationship between cell and production of lipid mediator with progression of in Here, we that the intestinal to the of disease epithelial and These changes in and were to the of different as as Although several studies have on the role of in colitis, its role unclear. et M.D. M.D. K. of anti-inflammatory in of inflammatory bowel Clin. Gastroenterol. 50: PubMed Scopus Google reported that the of as anti-inflammatory increased the of developing IBD, while et Y. S. I. M. The gastrointestinal and on disease activity of a in inflammatory bowel J. Gastroenterol. 101: PubMed Scopus Google reported that the of was for the IBD PGE2 is a COX-derived lipid A study that the of the receptor for aggravated the symptoms of TNBS-induced colitis in mice J.H. T. F. M. R. D. The of prostaglandin E2 in experimental inflammatory bowel disease is the Immunol. 2007; PubMed Scopus Google Scholar). of the aggravated the symptoms of DSS-induced colitis in mice (9.Kabashima K. Saji T. Murata T. Nagamachi M. Matsuoka T. Segi E. Tsuboi K. Sugimoto Y. Kobayashi T. Miyachi Y. et al.The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut.J. Clin. Invest. 2002; 109: 883-893Crossref PubMed Scopus (409) Google Scholar). have been several regarding the role of The of or the production of its metabolite ameliorated the symptoms of and DSS-induced colitis in mice C. A. K. A. of inflammation and production in colitis by a novel of leukotriene A4 J. PubMed Scopus Google Scholar, J. F. E. R. I. M. administration of a inhibitor, accelerates healing in a model of chronic colitis.Gut. PubMed Scopus Google Scholar). In other studies reported that the of aggravated the symptoms of DSS-induced colitis in mice M.J. L. B. C. in inflammatory bowel Lipid PubMed Scopus Google Scholar). In the present study, we revealed that aggravated the DSS-induced colitis by changes in production of metabolites as as Thus, a of the of each of lipid the and of its and of activation be for the of IBD in the detected lipid mediators with such as was derived from by the of (17.Yamada M. Kita Y. Kohira T. Yoshida K. Hamano F. Tokuoka S.M. Shimizu T. A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2015; 995–996: 74-84Crossref PubMed Scopus (53) Google and increased during the recovery and was derived from by the of (17.Yamada M. Kita Y. Kohira T. Yoshida K. Hamano F. Tokuoka S.M. Shimizu T. A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2015; 995–996: 74-84Crossref PubMed Scopus (53) Google and to of its during the acute inflammation These mediators roles in the progression of colitis. In this study, we using the DSS-induced colitis model in mice that the production of of lipid mediators with the progression of the These in the pathophysiology of regulation of lipid with their and for their be for therapeutic in combating with arachidonic acid α-linolenic acid Crohn's disease cyclooxygenase cytochrome p450 dihydroxyeicosatetraenoic acid dextran sodium sulfate E-type prostanoid receptor 4 acid hydroxyeicosapentaenoic acid acid inflammatory bowel disease leukotriene lipoxygenase lipoxin A4 prostaglandin trinitrobenzene sulfonic acid thromboxane ulcerative colitis