CD1d基因敲除小鼠拮抗DSS诱导结肠炎
|
摘要
目的以葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的CD1d~(-/-)小鼠实验性结肠炎模型为研究对象,探究CD1d分子在小鼠结肠炎进程中的作用。方法取SPF级C57BL/6小鼠和CD1d~(-/-)(C57BL/6背景)小鼠,每天给予2.5%DSS喂养,连续6 d,每天记录小鼠的体质量变化、粪便性状、活动情况等,评估小鼠的疾病活动指数(DAI),记录小鼠的生存情况。第6天用FITC-dextran灌胃检测肠通透性,测量结肠长度,并用HE染色法观察结肠组织病理学变化,免疫组化法与免疫荧光法检测肠上皮增殖情况,免疫印迹(Western blot)检测小鼠结肠组织细胞炎性因子的表达。结果与C57/BL6小鼠相比,CD1d~(-/-)小鼠生存率高(P<0.05),体质量减轻缓慢、疾病活动指数(DAI)低(P<0.05),结肠长度长、肠通透性低(P<0.01),肠黏膜损伤轻,结肠上皮细胞增殖明显(P<0.05),肠组织IL-1beta及IL-18表达高(P<0.05)。结论 CD1d基因敲除小鼠拮抗DSS诱导的结肠炎,可能是通过促进结肠表达NLRP3炎性小体及其底物IL-1beta及IL-18。
This study designed to observe the effect of CD1d in the progress of mouse colitis by using the dextran sulfate sodium(DSS)-induced colitis model of CD1d~(-/-)(genetic background: C57BL/6)mice. The colitis mouse model was established by immunization of CD1d~(-/-)(genetic background: C57BL/6)mice and C57BL/6 mice.They were given daily 2.5% DSS feeding for 6 days. The changes in body weight, fecal traits, activity conditions were recorded every day. Disease activity index(DAI)of mice was assessed and the survival of the mice was recorded.On the 6 thday, intestinal permeability was measured by FITC-dextran gavage and the length of the colon was measured. The histopathological changes of the colon were observed by HE staining. The intestinal epithelial proliferation was detected by immunohistochemistry and immunofluiorescence. Western blot was used to detect the expression of inflammatory cytokines in colon tissue of mice.Data showed that compared with the WT mice of, the survival of CD1d~(-/-)mice was higher(P<0.05), the weight loss was slower and disease activity index(DAI)was lower(P<0.05), and the colon length was longer and intestinal permeability was lower(P<0.01), intestinal mucosal damage was lighter and colonic epithelial positive cells proliferated significantly(P<0.05). The expression levels of IL-1 beta and IL-18 were highter(P<0.05).These combined data suggest that CD1d knockout mice can antagonize DSS-induced colitis may by promoting colon expression of NLRP3 inflammasome and its substrates IL-1 beta and IL-18.
This study designed to observe the effect of CD1d in the progress of mouse colitis by using the dextran sulfate sodium(DSS)-induced colitis model of CD1d~(-/-)(genetic background: C57BL/6)mice. The colitis mouse model was established by immunization of CD1d~(-/-)(genetic background: C57BL/6)mice and C57BL/6 mice.They were given daily 2.5% DSS feeding for 6 days. The changes in body weight, fecal traits, activity conditions were recorded every day. Disease activity index(DAI)of mice was assessed and the survival of the mice was recorded.On the 6 thday, intestinal permeability was measured by FITC-dextran gavage and the length of the colon was measured. The histopathological changes of the colon were observed by HE staining. The intestinal epithelial proliferation was detected by immunohistochemistry and immunofluiorescence. Western blot was used to detect the expression of inflammatory cytokines in colon tissue of mice.Data showed that compared with the WT mice of, the survival of CD1d~(-/-)mice was higher(P<0.05), the weight loss was slower and disease activity index(DAI)was lower(P<0.05), and the colon length was longer and intestinal permeability was lower(P<0.01), intestinal mucosal damage was lighter and colonic epithelial positive cells proliferated significantly(P<0.05). The expression levels of IL-1 beta and IL-18 were highter(P<0.05).These combined data suggest that CD1d knockout mice can antagonize DSS-induced colitis may by promoting colon expression of NLRP3 inflammasome and its substrates IL-1 beta and IL-18.
引文
[1]Ge Y,Li Y,Gong J,et al.Mesenteric organ lymphatics and inflammatory bowel disease[J].Ann Anat,2018,218:199-204.
[2]Kaser A,Zeissig S,Blumberg RS.Inflammatory bowel disease[J].Annu Rev Immunol,2010,28:573–621.
[3]Rogler G.Inflammatory bowel disease cancer risk detection and surveillance[J].Dig Dis,2012,30(Suppl 2):48-54.
[4]Yashiro M.Ulcerative colitis-associated colorectal cancer[J].Wond J Gastroenterol,2014,20(44):16389-16397.
[5]Zajonc DM,Maricicl,Wu D,et al.Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity[J].J Exp Med,2005,202(11):1517-1526
[6]Skold M,Xiong X,Illarionov PA,et al.Interplay of cytokines and microbial signals in regulation of CD1d expression and NKT cell activation[J].J Immunol,2005,175:3584–3593.
[7]Fuss IJ,Heuer F,Boirivant M,et al.Nonclassical CD1drestricted NKT cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis[J].J Clin Invest,2004,113:1490–1497.
[8]Liao CM,Zimmer MI,Shanmuganad S,et al.dysregulation of CD1d-restricted type II natural killer T cells leads to spontaneous development of colitis in mice[J].Gastroenterology,2012,142:326–334.
[9]Saubermann LJ,Beck P,De Jong YP,et al.Activation of natural killer T cells by alpha-galactosylceramide in the presence of CD1d provides protection against colitis in mice[J].Gastroenterology,2000,119:119–128.
[10]Cooper HS,Murthy SN,Shah RS,et al.Clinicopathologic study of dextran sulfate sodium experimental murine colitis[J].Lab Invest,1993,69(2):238-249.
[11]Wirtz S,Neufert C,Weigmann B,et al.Chemically induced mouse models of intestinal inflammation[J].Nat Protoc,2007,2(3):541–546.
[12]Hamamoto N,Maemura K,Hirata I,et al.Inhibition of dextran sulphate sodium(DSS)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules(endothelial leucocyte adhesion molecule-1(ELAM-1)or intercellular adhesion molecule-1(ICAM-1)[J].Clin Exp Immunol,1999,117(3):462-468.
[13]Furuta GT,Turner JR,Taylor CT,et al.Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia[J].J Exp Med,2001,193(9):1027–1034.
[14]Rakoff Nahoum S,Paglino J,Eslami Varzaneh F,et al.Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis[J].Cell,2004,118(2):229–241.
[15]陈淑珍.内质网应激调控NLRP3炎症小体的研究进展[J].免疫学杂志,2017,33(10):905-910.
[16]Latz E,Xiao TS,Stutz A.Activation and regulation of theinflammasomes[J].Nat Rev Immunol,2013,13(6):397-411.
[17]Sutterwala FS,Ogura Y,Szczepanik M,et al.Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1[J].Immunity,2006,24(3):317–327.
[18]Zaki MH,Boyd KL,Vogel P,et al.The NLRP3inflammasome protects against loss of epithelial integrity and mortality during experimental colitis[J].Immunity,2010,32(3):379-391.
[19]Zaki MH,Lamkanf M,Kanneganti TD.The Nlrp3 in fl ammasome:contributions to intestinal homeostasis[J].Trends Immunol,2011,32(4):171–179.
[20]Hirota SA,Ng J,Lueng A,et al.NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis[J].Inflamm Bowel Dis,2011,17(6):1359-1372.
[21]Dupaul Chicoine J,Yeretssian G,Doiron K,et al.Control of intestinal homeostasis,colitis,and colitis-associated colorectal cancer by the in flammatory caspases[J].Immunity,2010,32(3):367–378.
[2]Kaser A,Zeissig S,Blumberg RS.Inflammatory bowel disease[J].Annu Rev Immunol,2010,28:573–621.
[3]Rogler G.Inflammatory bowel disease cancer risk detection and surveillance[J].Dig Dis,2012,30(Suppl 2):48-54.
[4]Yashiro M.Ulcerative colitis-associated colorectal cancer[J].Wond J Gastroenterol,2014,20(44):16389-16397.
[5]Zajonc DM,Maricicl,Wu D,et al.Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity[J].J Exp Med,2005,202(11):1517-1526
[6]Skold M,Xiong X,Illarionov PA,et al.Interplay of cytokines and microbial signals in regulation of CD1d expression and NKT cell activation[J].J Immunol,2005,175:3584–3593.
[7]Fuss IJ,Heuer F,Boirivant M,et al.Nonclassical CD1drestricted NKT cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis[J].J Clin Invest,2004,113:1490–1497.
[8]Liao CM,Zimmer MI,Shanmuganad S,et al.dysregulation of CD1d-restricted type II natural killer T cells leads to spontaneous development of colitis in mice[J].Gastroenterology,2012,142:326–334.
[9]Saubermann LJ,Beck P,De Jong YP,et al.Activation of natural killer T cells by alpha-galactosylceramide in the presence of CD1d provides protection against colitis in mice[J].Gastroenterology,2000,119:119–128.
[10]Cooper HS,Murthy SN,Shah RS,et al.Clinicopathologic study of dextran sulfate sodium experimental murine colitis[J].Lab Invest,1993,69(2):238-249.
[11]Wirtz S,Neufert C,Weigmann B,et al.Chemically induced mouse models of intestinal inflammation[J].Nat Protoc,2007,2(3):541–546.
[12]Hamamoto N,Maemura K,Hirata I,et al.Inhibition of dextran sulphate sodium(DSS)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules(endothelial leucocyte adhesion molecule-1(ELAM-1)or intercellular adhesion molecule-1(ICAM-1)[J].Clin Exp Immunol,1999,117(3):462-468.
[13]Furuta GT,Turner JR,Taylor CT,et al.Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia[J].J Exp Med,2001,193(9):1027–1034.
[14]Rakoff Nahoum S,Paglino J,Eslami Varzaneh F,et al.Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis[J].Cell,2004,118(2):229–241.
[15]陈淑珍.内质网应激调控NLRP3炎症小体的研究进展[J].免疫学杂志,2017,33(10):905-910.
[16]Latz E,Xiao TS,Stutz A.Activation and regulation of theinflammasomes[J].Nat Rev Immunol,2013,13(6):397-411.
[17]Sutterwala FS,Ogura Y,Szczepanik M,et al.Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1[J].Immunity,2006,24(3):317–327.
[18]Zaki MH,Boyd KL,Vogel P,et al.The NLRP3inflammasome protects against loss of epithelial integrity and mortality during experimental colitis[J].Immunity,2010,32(3):379-391.
[19]Zaki MH,Lamkanf M,Kanneganti TD.The Nlrp3 in fl ammasome:contributions to intestinal homeostasis[J].Trends Immunol,2011,32(4):171–179.
[20]Hirota SA,Ng J,Lueng A,et al.NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis[J].Inflamm Bowel Dis,2011,17(6):1359-1372.
[21]Dupaul Chicoine J,Yeretssian G,Doiron K,et al.Control of intestinal homeostasis,colitis,and colitis-associated colorectal cancer by the in flammatory caspases[J].Immunity,2010,32(3):367–378.