Mycobacterium avium subsp. paratuberculosis lipophilic antigen causes Crohn’s disease-type necrotizing colitis in Mice.pdf
RESEARCH Open Access Mycobacterium avium subsp. paratuberculosis lipophilic antigen causes Crohn’s disease-type necrotizing colitis in Mice Eiichi Momotani1*, Hiroshi Ozaki2, Masatoshi Hori2, Shizuo Yamamoto3, Takashi Kuribayashi3, Shigetoshi Eda4 and Masahiro Ikegami5 Abstract Background: A 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model was developed to investigate the pathogenesis and to evaluate a method of treating human Crohn’s disease. This experimental model rapidly induces colitis similar to human Crohn’s disease lesion in a reproducible manner. However, natural exposure of the human digestive tract to TNBS is unrealistic. A novel animal model based on realistic data is eagerly anticipated in future research on pathogenesis of CD. Method: We evaluated the potency of Map antigen molecules in an effort to develop a novel colitis model using a more realistic source than TNBS. We prepared the Map antigen by ethanol extraction and developed a mouse model in a manner similar to that of the well-known TNBS-induced colitis in mice. In the experiment, seven days after subcutaneous (SC) injection of the antigen into normal C57BL/6 mice, the same antigen in 50% ethanol was injected into the colon by the transanal route with a fine cannula. Results: On the fifth day after the transanal injection, histopathological examination revealed full-thickness necrotizing colitis with erosion and ulcers; severe infiltration with neutrophils, lymphocytes, macrophages, and perforation. However, no change was detected with each single Map-antigen injection. Conclusion: The present results provide a novel animal model for research on CD and may be the key to clarifying the relationship between CD and Map. This is the first evidence that mycobacterium antigen induces necrotizing colitis. Keywords: Mycobacterium, Paratuberculosis, Crohn’s disease, IBD, Mice, Necrotizing colitis, TNBS Background The number of studies attempting to detect clues to the mystery of Crohn’s disease (CD), a chronic intractable in- testinal disease, has recently increased (Lakatos 2009; Momotani et al. 2012; Simmons 2010). Althoug various symptomatic treatments and approaches to diet restric- tion half of CD patients require surgery within 10 years after diagnosis. The risk of postoperative recurrence is 44 to 55% after 10 years (Peyrin-Biroulet et al. 2010). The globally rising rate of pediatric CD is also a major issue (Benchimol et al. 2011; Jakobsen et al. 2008; Phavichitr et al. 2003). No convincing explanation of the pathogen- esis of CD currently exists; however, various environmen- tal factors (e.g., pathogenic or non-pathogenic microbes, lifestyle, hygiene factors, diet, and stress) have been sug- gested (Economou and Pappas 2008; Glasser and Darfeuille-Michaud 2008; Lakatos 2009; Momotani et al. 2012; Neuman and Nanau 2012). Responsible host genes such as the famous NOD2 and a genetic predisposition to CD have been suspected as well (Economou and Pap- pas 2008; Glasser and Darfeuille-Michaud 2008; Umeno et al. 2011; Vora et al. 2012). The focus in recent years has been on Mycobacterium avium subsp. paratuberculo- sis (Map) (Behr and Kapur 2008; Eltholth et al. 2009; Momotani et al. 2012), due to the reported pathological similarities of CD and paratuberculosis (Ptb), accumulat- ing reports of frequent detection of Map IS900 DNA, and much less isolation of Map from CD lesions (Abu- bakar et al. 2008; Chiodini 1989; Feller et al. 2007; Momotani et al. 2012). Detection of live Map and Map IS900 DNA in children with early-onset CD has been reported (Kirkwood et al. 2009). * Correspondence: eiichimomotani@gmail.com 1Research Area of Pathology and Pathophysiology, National Institute of Animal Health, 3-1-5 Kan-nondai, Tsukuba 305-0856, Japan Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Momotani et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Momotani et al. SpringerPlus 2012, 1:47 http://www.springerplus.com/content/1/1/47 Paratuberculosis is a chronic and progressive granuloma- tous enteritis that affects livestock and wild animals world- wide (Chiodini et al. 1984; Momotani et al. 2012; Nielsen and Toft 2009; Raizman et al. 2009; Stabel et al. 2009). However, differences between CD and Ptb have been pointed out (Momotani et al. 2012; Van Kruiningen 1999). An additional mystery is the “invisible Map” that sup- posedly grows in CD lesions (Momotani et al. 2012; Pierce 2009; Van Kruiningen 1999). This phenomenon has been explained by isolation of a cell-wall-deficient, spheroplastic form of Map from human CD lesions (Wall et al. 1993). However, even immunohistochemical staining for cytoplas- mic and cell-wall components of Map could not detect Map in CD lesions (Kobayashi et al. 1989; Momotani et al. 2012; Pierce 2009; Sartor 2005; Van Kruiningen 1999). In contrast to the hypothesis that Map infection causes CD, there are no reports of CD-like Ptb lesions in natural or ex- perimental infection with Map (Chiodini 1989; Chiodini et al. 1984). Furthermore, intestinal lesions in Ptb of cyno- molgus were very similar to those of bovine but differed histopathologically from human CD (McClure et al. 1987). An experimental colitis model using mice or rats with haptenizing agent 2,4,6-trinitrobenzene sulfonic acid (TNBS) yields pathological findings similar to those of human CD (Arita et al. 2005; Neurath et al. 2000; te Velde et al. 2006). However, natural exposure of the human digestive tract to TNBS is unrealistic. In contrast, exposure of the human digestive tract to Map antigen is realistic (Behr and Kapur 2008; Eltholth et al. 2009; Over et al. 2011), since frequently detected Map IS900 DNA in CD patients (Abubakar et al. 2008; Chiodini 1989; Feller et al. 2007; Momotani et al. 2012) is considered to be evidence of Map antigen exposure, rather than Map infection. In the present study, we prepared a lipophilic antigen of Map, and TNBS in the previous TNBS colitis model (Arita et al. 2005) was replaced with the Map antigen. Histopathological evaluation revealed severe necrotizing colitis that is very similar to that in the well- known TNBS colitis model. The present study proposes a new CD model and a novel hypothesis on the patho- genesis of human CD. Results Clinical findings and gross pathology During the experiment period, only a few mice exhibited in- activation and a rough coat. During the autopsy, thickening of the colon wall with congestion was observed in the 6 cases exhibited total score than 15 (Figure 1A). Histopathology Stacked bar graphs present histopathological findings regarding degree (Figure 1B), distribution of lesions (Figure 1B), and types of infiltrating cells (Figure 1C). All sections were stained with hematoxylin and eosin (H&E), and the magnification of photos is indicated as a bar. Group 1 Histopathological findings caused by TNBS are pre- sented in Figure 2. Three severe (cases T3-5, Figure 2A) and two mild (cases T1 and 2, Figure 2A) colitis cases were observed. Case T-4 exhibited the most severe full- thickness necrotizing colitis (Figure 2A-D). The most se- verely damaged tissue was shaped like an erupting vol- cano (Figure 2A, arrow). The colitis included various degrees of erosion, ulceration, and infiltration with neu- trophils, lymphocytes, and macrophages in laminapro- pria mucosa (Figure 2B-F). Atypical epithelial cells including irregularly shaped, vacuolated, and regenerat- ing cells were observed (Figure 2B-F). Epithelium was sometimes infiltrated with neutrophils (Figure 2C, arrows). Granulation (g), an early stage of fibrosis, was observed with ulcer formation (Figures 2B and C). Intes- tinal epithelial cells and crypt (c) structure on the necro- tizing area disappeared or were modified by inflammation and granulation (Figure 2B-F). Neutrophils were the predominant infiltrating cells (Figure 2C); how- ever, other cell types also contributed. Edema (ed) of the muscle layer (m) was observed (Figure 2A and B). Medium infiltration and edema (ed) in the lamina pro- pria mucosa and sub-mucosa (sm) were observed (Figure 2D). Erosion and ulceration were observed in cases T-2 (Figure 2E) and T-3 (Figure 2F). Group 2 Histopathological findings of group 2 are presented in Figure 3. The most severe findings (Figures 3A-F) were observed in case M100-2. Figures 3G and H are from case M100-3. Case M100-2 exhibited severe full- thickness destructive enteritis (Figures 3A-F). The most severe changes were deep ulcer and necrotizing enteritis (ne) (Figures 3A and B). Accumulation of inflammatory cells (aic), debris, and edema (e) were seen as a pseudo- membrane (Figures 3A and B). The normal structure of the mucosa completely disappeared (Figure 3, arrow); however, the severity differed from area to area for the same case (Figures 3A and B). In some areas of the colon, the structure of the epithelium was maintained, but infiltration and edema were characteristically observed in the muscle layer (ml) (Figures 3C and D). Cellular infiltration is observed between circular muscu- lar fibers (Figure 3E). Edema and cellular infiltration of the longitudinal muscle (lm) and the serosal membrane (Figures 3D and F) were observed. Fibrin deposition (f) was observed on the serosal membrane (sm) (Figure 3F). Full-thickness necrotizing enteritis was observed in case M100-3 (Figure 3G). The pseudomem- brane (pm) was also observed (Figure 3F). Infiltration in Momotani et al. SpringerPlus 2012, 1:47 Page 2 of 10 http://www.springerplus.com/content/1/1/47 The probability that people having some genetic predis- position will ingest live Map in food is minimal, but the chance of exposure by heat-killed Map antigen may be very frequent. Although the heating process of dairy products eliminates live Map (Stabel 2000; Stabel and Lambertz 2004; Stabel et al. 1997), it may not elimin- ate hazards to human health. Of course, we should not neglect the possibility of human Map infection (Hermon-Taylor 2009; Singh et al. 2010). Conclusions The present study proposed a novel mouse model for CD-like colitis and the ability of Map antigen to induce necrotizing colitis, which may be the key to understand- ing the relationship between CD and Map. This model may help clarify the pathogenesis of CD, as well as other diseases with a suspected etiological relationship to Map (e.g., irritable bowel syndrome (Scanu et al. 2007), mul- tiple sclerosis (Cossu et al. 2011), and type-1 diabetes mellitus (Paccagnini et al. 2009)). Also, this is the first evidence that mycobacterium antigen induces necrotiz- ing colitis. In addition, the authors recommend that people who may have a genetic predisposition (Econo- mou and Pappas 2008; Economou et al. 2004; Glasser and Darfeuille-Michaud 2008; Henderson et al. 2011; Lakatos 2009; Tsianos et al. 2012; Tuci et al. 2011; Umeno et al. 2011) to CD (i.e., if a relative has CD) should avoid dairy products possibly contaminated with the Map antigen (Eltholth et al. 2009; Foods 2010; Hermon-Taylor et al. 2000; Millar et al. 1996; Patel and Shah 2011) because no other measures for preventing CD are known. Methods Antigen preparation Mycobacterium avium subspecies paratuberculosis (ATCC 19698) was grown in Middle brook 7H9 liquid medium (Difco Laboratories, MD, USA) enriched with BBL Middle brook OADC (Becton Dickinson, Tokyo, Japan) and 2mg/L of mycobactin J (Allied Laboratory, MO, USA) for two weeks. Next, 90 ml of the culture was centrifuged at 2,200xg for 20min, the supernatant was removed, and the culture was re-suspended in phos- phate buffer saline (PBS). After washing twice with PBS, wet bacilli (1g wet weight) were collected. Surface lipo- philic antigen was isolated by a previous method (Speer et al. 2006). The bacilli were suspended in 12ml of 80% ethanol by vortex at room temperature for 1min. The suspension was centrifuged, and the resulting super- natant was collected. The dried material weighed 20mg; thus, the concentration was calculated as 1.7mg/ml in 80% ethanol. The material (Map-L antigen) was dis- solved with 50% ethanol and used as MAP-L100 antigen (0.68μg/μl). Antigens serially diluted 10 times were used as MAP-L10 (0.068μg/μl) and MAP-L1 antigen (0.0068μg/μl). Preparation of the culture and isolation of the antigen were carried out in laboratory certified as a biosafety level 2 (BSL2-TS-49) in NIAH. Experimental animals C57BL/6Cr Slc female mice at 10 weeks old were pur- chased from Japan SLC, Inc., and kept in a specific- pathogen-free (SPF) environment under the conditions described above. The mice were fed ad libitum during the experiments. Experiment procedure This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Animal Health. The protocol was approved by the Committee of the Ethics of Animal Experiments of the NIAH (Per- mit Numbers: 08–118, 09–130 and A10-025), and all efforts were made to minimize suffering. All treatments were performed under general anesthesia with Avertin (2,2,-Tribromoethanol) (Arita et al. 2005). The mice were divided into six groups and injected twice with different antigens (Figure 1A). The primary injection was performed subcutaneously. The secondary antigen injections to the colon were performed through the trans-anal route with a fine urinary catheter (Atom Multipurpose Tube, 1.35mm in diameter, Atom Medical Corporation, Tokyo). Feeding was stopped 24 h before the secondary injection. This experimental procedure was basically accorded to previously reported TNBS colitis model by Arita et al. (Arita et al. 2005), because of the the favorable reproducibility. Group 1 mice, TNBS positive control group were trea- ted subcutaneously with 2.5% TNBS in 50% ethanol and then injected with 10% TNBS in ethanol by the transanal route. All mice in groups 2, 3, and 4 were subcutane- ously injected with 25.5μg/150μl of MAP-L antigen in 50% ethanol (Map-L100 antigen). Seven days after treat- ment, the same antigen with three different concentra- tions (2.5 (MAP-L100), 0.25 (MAP-L10), and 0.025μg (MAP-L1)/150μl in 50% ethanol) were injected into the colon. Group 5 mice were pretreated subcutaneously with 50% ethanol, and then 25μg of MAP-L100 antigen per 150μl in 50% ethanol was injected into the colon. Group 6 mice were treated with 50% ethanol by SC in- jection and then by injection into the colon as a non- antigen control. Five days after the injection, the mice were put down by carbon dioxide gas. Colon tissues were sampled and fixed in 20% buffered formalin solu- tion for histopathological examination. The fixative was gently injected into the lumen of the intestine by syringe with a 21 gauge needle. Momotani et al. SpringerPlus 2012, 1:47 Page 8 of 10 http://www.springerplus.com/content/1/1/47 All experiment protocols used in this study were approved by the Ethics Review Committee for Animal Experimentation of NIAH (approval Nos. 08–118 and 09–130). Histopathology Tissues fixed for three days were trimmed to be round slices and embedded in paraffin blocks. Sections were cut 4μm thick and stained with hematoxylin and eosin (H&E) and then observed under a microscope. The find- ings were recorded, and the degree of changes were expressed as 0 to 3 and made stacked bar graphs. (Figures A1-3). Abbreviations CD: Crohn’s disease; UC: Ulcerative colitis; Map: Mycobacterium avium subsp. paratuberculosis; Ptb: Paratuberculosis; TNBS: 2,4,6-trinitrobenzene sulfonic acid; OADC: Oleic Acid-Albumin Fraction V-Dextrose-Catalase enrichment; SPF: Specific-pathogen-free. Competing interests The authors declare that they have no competing interests. Author’s contributions EM, HO, MH and MI conceived and designed the experiments. SE purified the antigen. EM, SE, SY and TK performed the experiments. EM, HO, MH, SE and MI analyzed the data. EM, HO, MH, and SE contributed reagents, materials, or analysis tools. EM, SE, SY and MI wrote the paper. All authors read and approved the final manuscript. Acknowledgements This work was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education No. 23240061(to EM) and No. 20228005 (to H O), and the Bio-oriented Technology Research Advancement Institute (BRAIN) (to EM). We thank Mr. M. Kobayashi and Ms. M. Shimada for preparing excellent histopathological sections. Author details 1Research Area of Pathology and Pathophysiology, National Institute of Animal Health, 3-1-5 Kan-nondai, Tsukuba 305-0856, Japan. 2Department of Veterinary Pharmacology Graduate School of Agriculture and Life Sciences, the University of Tokyo, Tokyo 113-8657, Japan. 3Laboratories of Immunology, School of Life and Environmental Science, Azabu University, Fuchinobe 1-17-71, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan. 4Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, the University of Tennessee, Knoxville, Tennessee 37996-1071, USA. 5Department of Pathology, the Jikei University School of Medicine, Minato-ku, Tokyo, Japan. Received: 12 September 2012 Accepted: 29 October 2012 Published: 8 November 2012 References Abubakar I, Myhill D, Aliyu SH, Hunter PR (2008) Detection of Mycobacterium avium subspecies paratuberculosis from patients with Crohn's disease using nucleic acid-based techniques: a systematic review and meta-analysis. 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Title: Mycobacterium avium subsp. ​ paratuberculosis lipophilic antigen causes Crohn’s disease-type necrotizing colitis in Mice ​
Authors: Eiichi Momotani, Hiroshi Ozaki, Masatoshi Hori, Shizuo Yamamoto, Takashi Kuribayashi, Shigetoshi Eda, Masahiro Ikegami ​
Publisher: SpringerPlus
Date: November 8, 2012 ​
Abstract: Background: A 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model was developed to investigate the pathogenesis and to evaluate a method of treating human Crohn’s disease. ​ This experimental model rapidly induces colitis similar to human Crohn’s disease lesion in a reproducible manner. ​ However, natural exposure of the human digestive tract to TNBS is unrealistic. ​ A novel animal model based on realistic data is eagerly anticipated in future research on pathogenesis of CD. ​
Method: We evaluated the potency of Map antigen molecules in an effort to develop a novel colitis model using a more realistic source than TNBS. ​ We prepared the Map antigen by ethanol extraction and developed a mouse model in a manner similar to that of the well-known TNBS-induced colitis in mice. ​ In the experiment, seven days after subcutaneous (SC) injection of the antigen into normal C57BL/6 mice, the same antigen in 50% ethanol was injected into the colon by the transanal route with a fine cannula. ​
Results: On the fifth day after the transanal injection, histopathological examination revealed full-thickness necrotizing colitis with erosion and ulcers; severe infiltration with neutrophils, lymphocytes, macrophages, and perforation. ​ However, no change was detected with each single Map-antigen injection. ​
Conclusion: The present results provide a novel animal model for research on CD and may be the key to clarifying the relationship between CD and Map. ​ This is the first evidence that mycobacterium antigen induces necrotizing colitis. ​