OJGas  Vol.3 No.1 , February 2013
Gut sterilization in experimental colitis leukocyte mediated colon injury, and effects on angiogenesis/lymphangiogenesis

Inappropriate responses to normal commensal bacteria trigger immune activation in both inflammatory bowel disease and experimental colitis. How gut flora contribute to the pathogenesis of inflammatory bowel disease is unclear, but may involve entrapment of leukocytes and remodeling of the vascular system. Here we evaluated how the progression and tissue remodeling in experimental colitis differ in a germ- free model of mouse colitis. Four treatment groups were used: control, antibiotic-treated (ABX), dextran sulfate colitis (DSS) and DSS pre- and co-treated with antibiotics (DSS + ABX). In days 0 - 3 of the study, germ-free mice received antibiotics (vancomycin, neomycin, and metronidazole). During the next 11 days, antibiotics were continued and DSS (3%) added to “colitis” groups. Disease activity, weight, stool form and blood were monitored daily. Mice were sacrificed and tissue samples harvested. Histopathological scores in controls (0.00) and in ABX (1.0+/0.81) were significantly (p < 0.001) lower than DSS (12+/0). Extents of injury, inflammation and crypt damage were all improved in DSS + ABX. The Disease Activity Index score (day 11) was significantly worse in the DSS group compared to the DSS + ABX group. Stool blood and form scores were also significantly improved among these groups. Importantly, myeloper- oxidase was significantly reduced in DSS + ABX, indicating that neutrophil infiltration was blocked. Colitis was associated with an increase in blood and lymphatic vessels; both of these events were also significantly reduced by gut sterilization. Our experiment shows that clinical and histopathological severity of colitis was significantly worse in the DSS colitis group compared to the DSS + ABX group, supporting the hypothesis that development of IBD is likely to be less severe with appropriate antibiotic treatment. In particular, gut sterilization effectively reduces leuko- cyte-dependent (PMN) injury to improve outcomes and may be an important target for therapy.

Cite this paper
Patel, M. , Olinde, J. , Tatum, A. , Ganta, C. , Cromer, W. , Sheth, A. , Jennings, M. , Mathis, J. , Testerman, T. , Jordan, P. , Manas, K. , Monceaux, C. and Alexander, J. (2013) Gut sterilization in experimental colitis leukocyte mediated colon injury, and effects on angiogenesis/lymphangiogenesis. Open Journal of Gastroenterology, 3, 12-24. doi: 10.4236/ojgas.2013.31003.
[1]   Jantchou, P., Monnet, E. and Carbonnel, F. (2006) Envi ronmetal risk factors in Crohn’s disease and ulcerative colitis (excluding tobacco and appendicectomy). Gastro entérologie Clinique et Biologique, 30, 859-867. doi:10.1016/S0399-8320(06)73333-4

[2]   Veluswamy, H., Suryawala, K., Sheth, A., Wells, S., Salvatierra, E., Cromer, W., Chaitanya, G.V., Painter, A., Patel, M., Manas, K., Zwank, E., Boktor, M., Baig, K., Datti, B., Mathis, M.J., Minagar, A., Jordan, P.A. and Alexander, J.S. (2010) African-American inflammatory bowel disease in a Southern US health center. BMC Gastroenterology, 10, 104. doi:10.1186/1471-230X-10-104

[3]   Buhner, S., Buning, C., Genschel, J., Kling, K., Hermann, D., Dignass, A., Kuechler, I., Krueger, S., Schmidt, H.H. and Lochs, H. (2006) Genetic basis for increased intestinal permeability in families with Crohn’s disease: Role of CARD15 3020insC mutation? Gut, 55, 342-347. doi:10.1136/gut.2005.065557

[4]   Sartor, R.B. and Muehlbauer, M. (2007) Microbial host interactions in IBD: Implications for pathogenesis and therapy. Current Gastroenterology Reports, 9, 497-507. doi:10.1007/s11894-007-0066-4

[5]   Reuter, B.K. and Pizarro, T.T. (2009) Mechanisms of tight junction dysregulation in the SAMP1/YitFc model of Crohn’s disease-like ileitis. Annals of the New York Academy of Sciences, 1165, 301-307. doi:10.1111/j.1749-6632.2009.04035.x

[6]   Cromer, W., Jennings, M.H., Odaka, Y., Mathis, J.M. and Alexander, J.S. (2010) Murine rVEGF164b, an inhibitory VEGF reduces VEGF-A-dependent endothelial proliferation and barrier dysfunction. Microcirculation, 17, 536 547.

[7]   Podolsky, D.K. (1997) Lessons from genetic models of inflammatory bowel disease. Acta Gastroenterologica Belgica, 60, 163-165.

[8]   Laukoetter, M.G., Nava, P. and Nusrat, A. (2008) Role of the intestinal barrier in inflammatory bowel disease. World Journal of Gastroenterology, 14, 401-407. doi:10.3748/wjg.14.401

[9]   Soriano, A., Salas, A., Salas, A., Sans, M., Gironella, M., Elena, M., Anderson, D.C., Pique, J.M. and Panes, J. (2000) VCAM-1, but not ICAM-1 or MAdCAM-1, immunoblockade ameli rates DSS-induced colitis in mice. Laboratory Investigation, 80, 1541-1551. doi:10.1038/labinvest.3780164

[10]   Oshima, T., Laroux, F.S., Coe, L.L., Morise, Z., Kawachi, S., Bauer, P., Grisham, M.B., Specian, R.D., Carter, P., Jennings, S., Granger, D.N., Joh, T. and Alexander, J.S. (2001) Interferon gamma and interleukin-10 reciprocally regulate endothelial junction integrity and barrier function. Microvascular Research, 61, 130-143. doi:10.1006/mvre.2001.2322

[11]   Sydora, B.C., Martin, S.M., Lupicki, M., Dieleman, L.A., Doyle, J., Walker, J.W. and Fedorak, R.N. (2006) Bacterial antigens alone can influence intestinal barrier integrity, but live bacteria are required for initiation of intestinal inflammation and injury. Inflammatory Bowel Disease, 12, 429-436. doi:10.1097/00054725-200606000-00001

[12]   Tellez, H.S. (2010) Digestive physiology and the role of microorganisms. Journal of Applied Poultry Research, 15, 136-144.

[13]   Madsen, K.L., Doyle, J.S., Tavernini, M.M., Jewell, L.D., Rennie, R.P. and Fedorak, R.N. (2000) Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice. Gastroenterology, 118, 1094-1105. doi:10.1016/S0016-5085(00)70362-3

[14]   Wold, J.S. and Turnipseed, S.A. (1981) Toxicology of vancom-ycin in laboratory animals. Reviews of Infection Disease, 3, S224-S229. doi:10.1093/clinids/3.Supplement_2.S224

[15]   Dieleman, L.A., Pena, A.S., Meuwissen, S.G. and Van Rees, E.P. (1997) Role of animal models for the pathogenesis and treatment of inflammatory bowel disease. Scandanavian Journal of Gastroenterology, 223, 99-104.

[16]   Kim, H.S. and Berstad, A. (1992) Experimental colitis in animal models. Scandanavian Journal of Gastroenterology, 27, 529-537. doi:10.3109/00365529209000116

[17]   Sasaki, M., Bharwani, S., Jordan, P., Elrod, J.W., Grisham, M.B., Jackson, T.H., Lefer, D.J. and Alexander, J.S. (2003) Increased disease activity in eNOS-deficient mice in experimental colitis. Free Radical Biology and Medicine, 35, 1679-1687. doi:10.1016/j.freeradbiomed.2003.09.016

[18]   Sasaki, M., Bharwani, S., Jordan, P., Joh, T., Manas, K., Warren, A., Harada, H., Carter, P., Elrod, J.W., Wolcott, M., Grisham, M.B. and Alexander, J.S. (2003) The3-hy droxy-3-methylglutaryl-CoA reductase inhibitor pravastatin reduces disease activity and inflammation in dextran-sulfate induced colitis. Journal of Pharmacology and Experimental Therapeutics, 305, 78-85. doi:10.1124/jpet.102.044099

[19]   Sasaki, M., Mathis, J.M., Jennings, M.H., Jordan, P., Wang, Y., Ando, T., Joh, T. and Alexander, J.S. (2005) Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector. Journal of Inflammation, 2, 13. doi:10.1186/1476-9255-2-13

[20]   Cooper, H.S., Murthy, S.N., Shah, R.S. and Sedergran, D.J. (1993) Clinicopathologic study of dextran sulfate sodium exper mental murine colitis. Laboratory Investigation, 69, 238-249.

[21]   Grisham, M.B., Granger, D.N. and Lefer, D.J. (1998) Modulation of leukocyte-endothelial interactions by reactive metabolites of oxygen and nitrogen: Relevance to ischemic heart disease. Free Radical Biology and Medicine, 25, 404-433. doi:10.1016/S0891-5849(98)00094-X

[22]   Hausmann, M., Obermeier, F., Paper, D.H., Balan, K., Dunger, N., Menzel, K., Falk, W., Schoelmerich, J., Herfarth, H. and Rogler, G. (2007) In vivo treatment with the herbal phenyletanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis. Clinical & Experimental Immunology, 148, 373-381. doi:10.1111/j.1365-2249.2007.03350.x

[23]   Arrieta, M.C., Madsen, K., Doyle, J. and Meddings, J. (2009) Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse. Gut, 58, 41-48. doi:10.1136/gut.2008.150888

[24]   Lee, Y.T., Sung, J.J., Poon, P., Lai, K.N. and Li, P.K. (1998) Association of HLA class-II genes and anti neutrophil cytoplasmic antibodies in Chinese patients with inflammatory bowel disease. Scandanavian Journal of Gastroenterology, 33, 623-627. doi:10.1080/00365529850171909

[25]   Mandriota, S.J., Jussila, L., Jeltsch, M., Compagni, A., Baetens, D., Prevo, R., Banerji, S., Huarte, J., Montesano, R., Jackson, D.G., Orci, L., Alitalo, K., Christofori, G. and Pepper, M.S. (2001) Vascular endothelial growth factor-C-mediated lymphan-giogenesis promotestumour metastasis. EMBO, 20, 672-682. doi:10.1093/emboj/20.4.672

[26]   McGuckin, M.A., Eri, R., Simms, L.A., Florin, T.H. and Radford-Smith, G. (2009) Intestinal barrier dysfunction in inflammatory bowel diseases. Inflammatory Bowel Disease, 15, 100-113. doi:10.1002/ibd.20539

[27]   Cummings, J.H., Macfarlane, G.T. and Macfarlane, S. (2003) Intestinal bacteria and ulcerative colitis. Current Issues in Intestinal Microbiology, 4, 9-20.

[28]   Dieleman, L.A., Goerres, M.S., Arends, A., Sprengers, D., Torrice, C., Hoentjen, F., Grenther, W.B. and Sartor, R.B. (2003) Lactob cillus GG prevents recurrence of colitis in HLA-B27 transgenic rats after antibiotic treatment. Gut, 52, 370-376. doi:10.1136/gut.52.3.370

[29]   Guarner, F. and Malagelada, J.R. (2003) Role of bacteria in experimental colitis. Best Practice & Research Clinical Gatroenterology, 17, 793-804. doi:10.1016/S1521-6918(03)00068-4

[30]   Kanauchi, O., Mitsuyama, K., Araki, Y. and Andoh, A. (2003) Modification of intestinal flora in the treatment of inflammatory bowel disease. Current Pharmaceutical Design, 9, 333-346. doi:10.2174/1381612033391883

[31]   Rath, H.C., Schultz, M., Freitag, R., Dieleman, L.A., Li, F., Linde, H.J., Scholmerich, J. and Sartor, R.B. (2001) Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice. Infection and Immunity, 69, 2277-2285. doi:10.1128/IAI.69.4.2277-2285.2001

[32]   Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Ed berg, S. and Medzhitov, R. (2004) Recognition of com mensal micro-flora by toll-like receptors is required for intestinal home-ostasis. Cell, 118, 229-241. doi:10.1016/j.cell.2004.07.002

[33]   Lahat, G., Halperin, D., Barazovsky, E., Shalit, I., Rabau, M., Klausner, J. and Fabian, I. (2007) Immunomodula tory effects of ciprofloxacin in TNBS-induced colitis in mice. Inflammatory Bowel Disease, 13, 557-565.

[34]   Videla, S., Vilaseca, J., Guarner, F., Salas, A., Treserra, F., Crespo, E., Antolin, M. and Malagelada, J.R. (1994) Role of Intestinal microflora in chronic inflammation and ulceration of the rat colon. Gut, 35, 1090-1097. doi:10.1136/gut.35.8.1090

[35]   Yigitler, C., Gulec, B., Aydogan, H., Ozcan, A., Kilinc, M., Yigit, T., Kozak, O. and Pekcan, M. (2004) Effect of mesalazine, metronidazole and gentamicin on bacterial translocation in experimental colitis. Journal of Gastroenterology and Hepatology, 19, 1179-1186. doi:10.1111/j.1440-1746.2004.03457.x

[36]   Hans, W., Scholmerich, J., Gross, V. and Falk, W. (2000) The role of the resident intestinal flora in acute and chronic dextran sulfate sodium-induced colitis in mice. European Journal of Gastroenterology & Hepatology, 12, 267-273. doi:10.1097/00042737-200012030-00002

[37]   Kang, S.S., Bloom, S.M., Norian, L.A., Geske, M.J., Flavell, R.A., Stappenbeck, T.S. and Allen, P.M. (2008) An antibiotic responsive mouse model of fulminant ulcerative coltis. Public Library of Science Medicine, 5, e41.

[38]   Hoentjen, F., Harmsen, H.J., Braat, H., Torrice, C.D., Mann, B.A., Sartor, R.B. and Dieleman, L.A. (2003) Antibiotics with a seletive aerobic or anaerobic spectrum have different therapeutic activities in various regions of the colon in interleukin 10 gene deficient mice. Gut, 52, 1721-1727. doi:10.1136/gut.52.12.1721

[39]   Werner, T. and Haller, D. (2007) Intestinal epithelial cell signaling and chronic inflammation: From the proteome to specific molecular mechanisms. Mutation Research, 622, 42-57. doi:10.1016/j.mrfmmm.2007.05.010

[40]   Binion, D.G., Otterson, M.F. and Rafiee, P. (2008) Curcumin inhi its VEGF-mediated angiogenesis in human intestinal mcrovascular endothelial cells through COX-2 and MAPK inhibition. Gut, 57, 1509-1517. doi:10.1136/gut.2008.152496

[41]   Goebel, S., Huang, M., Davis, W.C., Jennings, M., Siahaan, T.J., Alexander, J.S. and Kevil, C.G. (2006) VEGF A stimulation of leukocyte adhesion to colonic microvascular endothelium: Implications for inflammatory bowel disease. American Journal of Physiology-Gastro intestinal and Liver Physology, 290, G648-G654.

[42]   Deng, X., Tolstanova, G., Khomenko, T., Chen, L., Tarnawski, A., Szabo, S. and Sandor, Z. (2009) Mesalamine restores angiogenic balance in experimental ulcerative colitis by reducing expression of endostatin and angiostatin: Novel molecular mechanism for therapeutic action of mesalmine. Journal of Pharmacology and Experimental Therapeutics, 331, 1071-1078. doi:10.1124/jpet.109.158022

[43]   Ganta, V.C., Cromer, W., Mills, G.L., Traylor, J., Jennings, M., Daley, S., Clark, B., Mathis, J.M., Bernas, M., Boktor, M., Jordan, P., Witte, M. and Alexander, J.S. (2010) Angiopoietin-2 in experimental colitis. Inflammatory Bowel Disease, 16, 1029-1039. doi:10.1002/ibd.21150

[44]   Danese, S., Sans, M., Spencer, D.M., Beck, I., Donate, F., Plunkett, M.L., de la, M.C., Redline, R., Shaw, D.E., Levine, A.D., Mazar, A.P. and Fiocchi, C. (2007) Angiogenesis blockade as a new therapeutic approach to experimental colitis. Gut, 56, 855-862. doi:10.1136/gut.2006.114314