Mouse Model of Reversible Intestinal Inflammation
可逆性肠道炎症小鼠模型
发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2173 浏览次数: 11264
评审: Ivan ZanoniAlesssandro ArduiniMareta Ruseva
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参见作者原研究论文
The authors used this protocol in:
May 2016
Abstract
Current therapies to treat inflammatory bowel disease by dampening excessive inflammatory immune responses have had limited success (Reinisch et al., 2011; Rutgeerts et al., 2005; Sandborn et al., 2012). To develop new therapeutic interventions, there is a need for better understanding of the mechanisms that are operative during mucosal healing (Pineton de Chambrun et al., 2010). To this end, a reversible model of colitis was developed in which colitis induced by adoptive transfer of naïve CD4+ CD45RBhi T cells in lymphopenic mice can be reversed through depletion of colitogenic CD4+ T cells (Brasseit et al., 2016).
Keywords: Colitis (结肠炎)
Remission (缓解)
Relapsing disease (复发性疾病)
Mucosal healing (黏膜愈合)
Background
Our understanding of the pathogenesis of inflammatory bowel disease (IBD), which is a chronic inflammatory disorder of the intestine, has been greatly improved with the development of animal models aiming to recapitulate human disease (Khanna et al., 2014). Despite the identification of a wide array of immunological targets, current therapies have had limited success in treating IBD and limited knowledge is available about the mechanisms that are induced in the establishment of long-term remission and the associated mucosal healing (D’Haens et al., 2014). A major limitation, so far, has been the lack of animal models in which remission can be reproducibly induced in animals with established disease. In models of infection induced intestinal inflammation, pro-inflammatory and anti-inflammatory mechanisms can be operative simultaneously, implying that dissecting the role of different immune pathways during resolution of inflammation may be a challenge (Endt et al., 2010; Sonnenberg et al., 2011). In dextran sodium sulfate (DSS) induced colitis, DSS can be easily administered in the drinking water to either induce acute or chronic intestinal inflammation and this is followed by treatment with normal drinking water to study resolution of colitis. However, the kinetics and severity of disease is highly dependent on numerous factors including differences in the dose of DSS used and critically on the amount of DSS consumed which is virtually impossible to normalize between different animals of the same cage (Chassaing et al., 2014; Perše and Cerar, 2012). Using the T cell transfer mediated colitis model, it was elegantly shown that intestinal inflammation can be reversed by the adoptive transfer of CD45RBlo regulatory T cells (Treg) in colitic animals, resulting in remission induction within 10-14 weeks post Treg transfer (Mottet et al., 2003). The kinetics of remission induction however varies depending on the expansion of transferred Treg and it can be difficult to synchronize the onset of remission between animals of same experimental group. To overcome the unpredictable timing and extent of remission induction, we developed a new mouse model of reversible intestinal inflammation in which intestinal inflammation (induced by the adoptive transfer of naïve CD45RBhi T cells in lymphopenic animals) can be reversed by depletion of colitogenic CD4+ T cells in mice with established disease, resulting in reproducible induction of remission from colitis (Brasseit et al., 2016).
Materials and Reagents
- 50 ml tube (SARSTEDT, catalog number: 62.547.254 )
- 5 ml Falcon polystyrene (PS) round bottom tube (Corning, Falcon®, catalog number: 352058 )
- 100 µm nylon cell strainer (Corning, catalog number: 431752 )
- Glass slide Superfrost (Biosystems, catalog number: 85-0551-00 )
- 6-10 week old congenic CD90.1 or CD45.1 mice as donors (in-house bred mice on C57BL/6 background, initially purchased from THE JACKSON LABORATORY, USA)
Notes: - Congenic mice CD90.1 are used as donors to easily distinguish between donor (CD90.1) and recipient cells (CD90.2) but C57BL/6 mice can also be used as donors.
- It is possible to use donor mice older than 10 weeks old but the frequency of naïve CD4+ CD45RBhi T cells is expected to decrease with age.
- 10-16 week old Helicobacter positive C57BL/6 Rag2-/- or Rag1-/- mice (initially purchased from THE JACKSON LABORATORY, USA) housed under specific pathogen-free (SPF) conditions as recipients with a minimum of 20 g body weight
Notes: - Helicobacter negative SPF Rag2-/- (or Rag1-/-) mice may also be used as recipients. However, the kinetics of colitis induction are delayed and the variation in disease activity may greatly vary at a given time post CD4 T cell-mediated colitis induction in those Helicobacter negative mice.
- Positivity for the presence of Helicobacter can be assessed by PCR using genomic DNA extracted from fecal pellets as previously described by Brasseit et al., 2016.
- We have not observed any differences in the kinetics of colitis induction between male and female recipients.
- 0.5 M EDTA (Sigma-Aldrich, catalog number: 27285 ), in sodium form
- EasySepTM Mouse Streptavidin RapidSpheresTM Isolation Kit (STEMCELL Technologies, catalog number: 19860 )
- InVivoMAb anti-mouse CD4 (Clone GK1.5) (BioxCell, catalog number: BE0003-1 )
- Anti-mouse antibodies
Antibodies Company Catalog number Clone Dilution B220 Biotin BioLegend 103203 RA3-6B2 1:800 CDαBiotin BioLegend 100703 53-6.7 1:200 CD45RB FITC Affymetrix 11-0455-85 C363-16A 1:800 CD25 PE BioLegend 102008 PC61 1:800 CD4 APC-Cy7 BioLegend 100526 RM4-5 1:800 - Horse serum (HS) (Sigma-Aldrich, catalog number: H1270 )
- Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
- 4% buffered formalin (EMD Millipore, catalog number: 100496 )
- Ethanol (EMD Millipore, catalog number: 100983 )
- Xylene (VWR, catalog number: 28975 )
- Paraffin (Engelbrecht, catalog number: 17932 )
- Mayer’s hemalum (Hematoxylin) solution (EMD Millipore, catalog number: 109249 )
- Xylene based mounting medium Eukitt® (Sigma-Aldrich, catalog number: 03989 )
- Sodium chloride (NaCl) (EMD Millipore, catalog number: 106406 )
- di-Sodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) (EMD Millipore, catalog number: 106580 )
- di-Potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) (EMD Millipore, catalog number: 105099 )
- Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 31107 )
- Potassium hydrogen carbonate (KHCO3) (EMD Millipore, catalog number: 104854 )
- Eosin-Phloxine solution (VWR, catalog numbers: 341973R and 10047229 , respectively)
- Glacial acetic acid (EMD Millipore, catalog number: 100063 )
- Hydrochloric acid (EMD Millipore, catalog number: 100317 )
- Phosphate-buffered saline (PBS) (see Recipes)
- ACK red blood lysis buffer (see Recipes)
- Eosin-Phloxine solution (see Recipes)
- 0.5% HCl-ethanol (see Recipes)
Equipment
- Centrifuge
- Neubauer chamber (Hemocytometer)
- EasySepTM magnet (STEMCELL Technologies, catalog number: 18000 )
- BD FACS ARIA III (BD, model: BD FACS ARIA III )
- Tissue-Tek VIP 6 tissue processor (SAKURA, model: Tissue-Tek VIP 6 Vaccum Infiltration Processor )
- Tissue-Tek®uni-cassette® (SAKURA, catalog number: 4172 )
- Heating block
- Dissection kit (forceps and scissors)
- Cold plate (-10 °C)
- Microtome (Leica Biosystems, Wetzlar, Germany)
- Water bath
- Pipette
- CO2 chamber
Note: CO2 is used as an approved and recommended euthanasia method in accordance with the Swiss Federal and Cantonal animal experimental regulations.
Software
- Graphpad Prism 6 (La Jolla, CA; https://www.graphpad.com/scientific-software/prism/)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kwong Chung, C. K., Brasseit, J., Althaus-Steiner, E., Rihs, S. and Mueller, C. (2017). Mouse Model of Reversible Intestinal Inflammation. Bio-protocol 7(6): e2173. DOI: 10.21769/BioProtoc.2173.
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分类
免疫学 > 粘膜免疫学 > 消化道
细胞生物学 > 组织分析 > 组织分离
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