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Mouse Model of Reversible Intestinal Inflammation
可逆性肠道炎症小鼠模型   

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Mucosal Immunology
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

  1. 50 ml tube (SARSTEDT, catalog number: 62.547.254 )
  2. 5 ml Falcon polystyrene (PS) round bottom tube (Corning, Falcon®, catalog number: 352058 )
  3. 100 µm nylon cell strainer (Corning, catalog number: 431752 )
  4. Glass slide Superfrost (Biosystems, catalog number: 85-0551-00 )
  5. 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:
    1. 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.
    2. 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.
  6. 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:
    1. 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.
    2. 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.
    3. We have not observed any differences in the kinetics of colitis induction between male and female recipients.
  7. 0.5 M EDTA (Sigma-Aldrich, catalog number: 27285 ), in sodium form
  8. EasySepTM Mouse Streptavidin RapidSpheresTM Isolation Kit (STEMCELL Technologies, catalog number: 19860 )
  9. InVivoMAb anti-mouse CD4 (Clone GK1.5) (BioxCell, catalog number: BE0003-1 )
  10. 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

  11. Horse serum (HS) (Sigma-Aldrich, catalog number: H1270 )
  12. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  13. 4% buffered formalin (EMD Millipore, catalog number: 100496 )
  14. Ethanol (EMD Millipore, catalog number: 100983 )
  15. Xylene (VWR, catalog number: 28975 )
  16. Paraffin (Engelbrecht, catalog number: 17932 )
  17. Mayer’s hemalum (Hematoxylin) solution (EMD Millipore, catalog number: 109249 )
  18. Xylene based mounting medium Eukitt® (Sigma-Aldrich, catalog number: 03989 )
  19. Sodium chloride (NaCl) (EMD Millipore, catalog number: 106406 )
  20. di-Sodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) (EMD Millipore, catalog number: 106580 )
  21. di-Potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) (EMD Millipore, catalog number: 105099 )
  22. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 31107 )
  23. Potassium hydrogen carbonate (KHCO3) (EMD Millipore, catalog number: 104854 )
  24. Eosin-Phloxine solution (VWR, catalog numbers: 341973R and 10047229 , respectively)
  25. Glacial acetic acid (EMD Millipore, catalog number: 100063 )
  26. Hydrochloric acid (EMD Millipore, catalog number: 100317 )
  27. Phosphate-buffered saline (PBS) (see Recipes)
  28. ACK red blood lysis buffer (see Recipes)
  29. Eosin-Phloxine solution (see Recipes)
  30. 0.5% HCl-ethanol (see Recipes)

Equipment

  1. Centrifuge
  2. Neubauer chamber (Hemocytometer)
  3. EasySepTM magnet (STEMCELL Technologies, catalog number: 18000 )
  4. BD FACS ARIA III (BD, model: BD FACS ARIA III )
  5. Tissue-Tek VIP 6 tissue processor (SAKURA, model: Tissue-Tek VIP 6 Vaccum Infiltration Processor )
  6. Tissue-Tek®uni-cassette® (SAKURA, catalog number: 4172 )
  7. Heating block
  8. Dissection kit (forceps and scissors)
  9. Cold plate (-10 °C)
  10. Microtome (Leica Biosystems, Wetzlar, Germany)
  11. Water bath
  12. Pipette  
  13. 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

  1. Graphpad Prism 6 (La Jolla, CA; https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Selection of naïve CD4+ T cells
    1. On day 0, isolate spleens from donor CD90.1 mice. Use 1 spleen per 2 recipient Rag1-/- mice.
    2. Disaggregate spleens on a 100 µm nylon filter and rinse the filter with PBS/2% horse serum (HS) to obtain a single cell suspension.
    3. Spin down cell suspension at 370 x g for 5 min.
    4. Lyse the red blood cells by resuspending the cell pellet in 3 ml of ACK lysis buffer for 3 min.
    5. Wash cells with PBS/2%HS and resuspend the cell pellet in 1 ml of PBS/2% HS containing 5 mM EDTA.
      Note: The initial volume for cell resuspension is not critically important since the cells have to be counted and adjusted to 1 x 108 cells/ml, as indicated in the following step.
    6. Count the cell numbers using a hemocytometer and adjust the cell concentration to 1 x 108 cells/ml.
    7. Add 50 µl/ml of the provided rat serum (from EasySepTM Mouse Streptavidin RapidSpheresTM Isolation Kit) and incubate with biotinylated antibodies (anti-mouse B220 and CD8α). Mix the cells carefully by pipetting.
    8. Leave the tube containing the cell suspension on ice for 15 min.
      Note: Cells have to be mixed carefully by pipetting following addition of anti-mouse antibodies. The tube containing the cell resuspension can be left on ice without further mixing/inverting/shaking.
    9. Vortex the streptavidin RapidSpheresTM for 30 sec and add 85 µl/ml of RapidSpheresTM to the cell suspension.
      Note: No initial wash step of the RapidSpheresTM is required but vortexing the RapidSpheresTM is critical since they tend to settle at the bottom of the tube.
    10. Mix carefully by pipetting up and down and incubate at room temperature for 2.5 min.
    11. Bring the cell suspension to a total volume of 2.5 ml using PBS/2% HS containing 5 mM EDTA and transfer to a 5 ml PS tube.
    12. Place the 5 ml PS tube containing the cell suspension into the EasySepTM magnet for 2.5 min at room temperature.
    13. In one continuous motion, invert the magnet and carefully pour off the cell fraction (streptavidin RapidSpheresTM unbound fraction) into a new 5 ml tube, leaving the magnet and tube in an inverted position for 2-3 sec to ensure optimal transfer of the cell suspension.


      Figure 1. T cell enrichment method using splenocytes of donor mice 

    14. Wash cells in PBS/2% HS at 370 x g for 5 min.
    15. Stain cells in 1 ml of anti-mouse CD45RB FITC, CD25 PE and CD4 APC-Cy7. Mix the cells carefully by pipetting. Leave the tube containing the cell suspension on ice for 20 min without further mixing/inverting/shaking.
    16. Wash cells (enriched for CD4+ T cells) with PBS/2% HS prior to cell sorting for CD4+CD45RBhi T cells on a BD FACSARIA III cell sorter.


      Figure 2. Gating strategy for the selection of naïve CD45RBhi T cells from spleens of donor C57BL/6 congenic mice. Lymphocytes are first selected based on their forward and side scatter properties, followed by the exclusion of doublet cells. Regulatory and activated T cells are excluded by selecting CD25 negative lymphocytes from the singlets gate and finally, naïve CD4+ T cells are selected based on their high expression of CD4 and CD45RB.

    17. Collect sorted cells in 1 ml of HS (100% HS).
    18. Wash sorted CD4+CD45RBhi T cells in PBS for 5 min at 370 x g and resuspend in PBS at a concentration of 1 x 106 cells/ml.
      Note: Keep the cells on ice at all times. If sorted naïve CD4+ T cells cannot be injected right away, keep the cells in HS on ice. Only wash and resuspend the cells with PBS prior to injection.
    19. To induce colitis, inject C57BL/6 Rag2-/- mice with 2 x 105 CD4+CD45RBhi T cells in a volume of 200 µl intraperitoneally (i.p).
    20. Once the animals are colitic (see Procedure B), inject the animals three times with 250-500 µg of anti-CD4 depleting antibody with an interval of 72 h between each injection to induce remission.
      Note: Remission induction was initially performed using 500 µg of αCD4 depleting antibody, as described by Brasseit et al., 2016. In our experiments, we can still achieve the same level of CD4+ T cell depletion and similar kinetics of remission induction using 3 treatments of 250 µg αCD4 depleting antibody.
    21. Monitor the animals daily for weight change and normal activity, with relapse of colonic inflammation expected around 22-25 days (in Helicobacter positive recipients) after the first treatment with anti-CD4 depleting antibody.
      Note: To assess the development of colitis, monitor the mice daily since the kinetics of colitis induction is strongly dependent on the composition of the microbiota implying that disease progression may vary from animals of one SPF mouse house facility to the other. In our experience, clinical and histopathological signs of colitis are seen around day 9-10 post T cell transfer in Helicobacter positive mice and around day 30 post T cell transfer in Helicobacter negative mice. In Helicobacter positive recipient mice, clear weight loss (approximately 10%) and appearance of colitogenic CD4+ T cells in the blood occurs very rapidly from one day to the other. Moreover, we generally observe that the weight loss correlates strongly with the extent of intestinal inflammation in Helicobacter positive but not in Helicobacter negative recipients.

  2. Clinical and histopathological scoring
    1. Mice with active intestinal inflammation (clinical score ≥ 8) and with detectable CD4+ T cells in the blood are considered colitic. For the measurement of CD4+ T cells in the blood, collect 2-3 drops of blood by the tail vein in 1 ml of PBS/2% HS/5 mM EDTA. Spin down the cells by centrifugation at 370 x g for 5 min. Resuspend the cells in 100 µl of PBS/2% HS containing anti-mouse CD4 antibody (Refer to Materials and Reagents section for antibody dilution) by pipetting. Following incubation on ice for 15 min, wash the cells in PBS/2% HS and analyse on a flow cytometer.
    2. For clinical disease scoring, monitor the following parameters daily:
      1. Weight loss
      2. Stool consistency
      3. Appearance of blood in the stool
      4. Behavior


        Figure 3. Scoring system used to assess colitic animals following transfer of CD4+ CD45RBhi T cells in lymphopenic recipients. Mice with a clinical score of ≥ 8 were considered colitic (multiple combinations leading to a clinical score of 8 are possible).  

    3. For histological disease scoring, collect colon (middle) sections for hematoxylin and eosin (H&E) staining.


      Figure 4. Collection of colon tissue section for histology

    4. In a blinded manner, measure the following parameters (this should be done by an experienced pathologist). Refer to the publication by Brasseit et al., 2016 in Mucosal Immunology for more details:
      1. Cellular infiltrates in the lamina propria (Score from 0 to 3)
      2. Loss of goblet cells (Score from 0 to 3)
      3. Presence of crypt abscess (Score from 0 to 3)
      4. Epithelial erosion (Score from 0 to 1)
      5. Hyperemia (Score from 0 to 2)
      6. Thickness of the colonic mucosa (Score from 0 to 3)

  3. Paraffin embedding and H&E staining
    Note: The paraffin embedding and H&E staining are routinely carried out in our histology core lab facility using a fully automated procedure on a Tissue-Tex VIP 6 but the basic steps are described below.
    1. Collect 1 cm colonic tissue (middle section) in a uni-cassette® and fix in 4% buffered formalin overnight at room temperature.
    2. Process the tissue as follows (using an automated tissue processor):
      1. 4% buffered formalin for 1.25 h at 35 °C.
      2. Distilled water for 15 min at room temperature.
      3. 70% ethanol for 1.5 h at 35 °C.
      4. 80% ethanol for 1 h at 35 °C. 
      5. 95% ethanol for 1.5 h at 35 °C.
      6. 100% ethanol for 1 h at 35 °C (three changes of 1 h each).
      7. Xylene for 1 h at 35 °C (two changes of 1 h each).
      8. Paraffin for 45 min at 60 °C (four changes of 45 min each).
      Note: Changes of ethanol, xylene and paraffin are required to reduce carryover of reagents from previous steps.
    3. To embed in a paraffin block. Choose an appropriate mold that corresponds to the size of the cassette.
    4. Open the cassette and discard the lid.
    5. Add some liquid paraffin into the mold, which is placed on a heating block at 60 °C to prevent the paraffin from solidifying.
    6. Transfer the tissue into the mold using warm forceps such that the forceps can be used to transfer the tissue from the cassette to the mold without solidifying the paraffin.
    7. Place the mold on a cold plate (-10 °C) to start solidifying the paraffin and immediately press the tissue down with forceps.
    8. Place the tissue cassette on top of the mold and press firmly. Ensure that there is sufficient paraffin to cover the plastic cassette.
    9. Wait for the paraffin to solidify such that the paraffin block can easily come out of the mold.
    10. Cut 5 µm sections using a microtome. Briefly, insert the blade in the holder and adjust the angle of the blade to 5°. Place the paraffin block in the object clamp and adjust to the desired orientation. Cut a few sections (10 µm) to expose the tissue surface. Once the tissue is exposed and the tissue cuts are smooth, adjust the section thickness to 5 µm. Carefully pick up the 5 µm tissue sections with forceps and float them in a warm water bath at 37 °C. Collect the sections on a clean glass slide and dry on a heating plate at 40 °C for 20 min.
    11. To stain with hematoxylin and eosin, first deparaffinize the slides with xylene for 5 min.
    12. Rinse twice with xylene and rehydrate the slides by rinsing sequentially in:
      1. Absolute ethanol (two changes);
      2. 95% ethanol
      3. 70% ethanol 
      4. Distilled water
      Note: The exact volumes are not specified here since this will depend on the size of the glass-staining jar. We recommended that the slides have to be totally submerged in the specified reagent.
    13. Stain with Mayer’s hemalum solution for 7 min and rinse with tap water.
    14. Bleach with 0.5% HCl-ethanol for a few seconds (depending on the desired staining depth of the nucleus) and wash slide immediately with tap water followed by a bluing step in tap water for 10 min.
    15. Rinse with 70% ethanol.
    16. Counter stain using Eosin-Phloxine solution for 2-3 min. Rinse twice with 70% ethanol.
    17. Dehydrate the slides by rinsing sequentially in:
      1. 70% ethanol
      2. 95% ethanol
      3. Absolute ethanol (two changes in total)
      4. Xylene (two changes in total)
      Note: The exact volumes are not specified here since this will depend on the size of the glass-staining jar. We recommended that the slides have to be totally submerged in the specified reagent.
    18. Mount with Eukitt®

Data analysis

In the design of the experimental set-up, it is recommended to include 4-5 mice/group and perform 2-3 independent experiments. For data analysis, compare recipient mice that received naïve CD45RBhi T cells (experimental group) to those that received CD45RBlo T cells (control group, no colitis). To assess the statistical significance, use an unpaired t-test on GraphPad Prism 6 (La Jolla, CA).

Notes

  1. To ensure the best cell yield and viability, always work on ice unless indicated otherwise.
  2. To get rid of the supernatant following centrifugation, aspirate the supernatant and discard rather than pouring it off since cells may not adhere strongly to the bottom of the 5 ml PS tube.
  3. For cell resuspension, this should be performed gently using a pipette and not by vortexing.
  4. Prior to cell injection, mix the cells by inverting the tube up and down to ensure that all mice are injected with the same number of cells.
  5. For intraperitoneal injection, ensure that the needle is inserted properly in the peritoneal cavity prior to injecting the cells.
  6. If Rag-/- recipients are imported from another mouse house facility, it is highly advisable to house these mice for at least two weeks prior to naïve CD4+ T cell transfer to allow stabilization of the microbiota.

Recipes

  1. Phosphate-buffered saline (PBS)
    137 mM NaCl
    1.69 mM Na2HPO4·2H2O
    8.63 mM K2HPO4·3H2O
  2. ACK red blood lysis buffer
    150 mM NH4Cl
    10 mM KHCO3
    0.1 mM EDTA
  3. Eosin-Phloxine solution
    100 ml of 1% eosin stock solution
    10 ml of 1% phloxine stock solution
    780 ml of 95% ethanol
    4 ml glacial acetic acid
  4. 0.5% HCl-ethanol
    20 ml of 25% hydrochloric acid
    980 ml of 70% ethanol

Acknowledgments

This work was supported by the Swiss National Science Foundation Sinergia grant to Andrew Macpherson, Christoph Mueller, Wolf-Dieter Hardt and Daniela Finke as well as by the SNSF grant No. 31-138392 to C.M. The described protocol was first outlined in Brasseit et al., 2016.

References

  1. Brasseit, J., Althaus-Steiner, E., Faderl, M., Dickgreber, N., Saurer, L., Genitsch, V., Dolowschiak, T., Li, H., Finke, D., Hardt, W. D., McCoy, K. D., Macpherson, A. J., Corazza, N., Noti, M. and Mueller, C. (2016). CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis. Mucosal Immunol 9(3): 689-701.
  2. Chassaing, B., Aitken, J. D., Malleshappa, M., and Vijay-Kumar, M. (2014). Dextran sulfate sodium (DSS)-induced colitis in mice. Curr Protoc Immunol 104: Unit 15 25
  3. D’Haens, G. R ., Sartor, R. B ., Silverberg, M. S., Petersson, J. and Rutgeerts, P. (2014). Future directions in inflammatory bowel disease management. J Crohns Colitis 8(8):726-734
  4. Endt, K., Stecher, B., Chaffron, S., Slack, E., Tchitchek, N., Benecke, a., Van Maele, L., Sirard, J. C., Mueller, A. J., Heinkenwalder, M., Macpherson, A. J., Strugnell, R., Von Mering, C. and Hardt, W. D. (2010). The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea. PLoS Pathog 6(8): e1001097.
  5. Khanna, P. V., Shih, D. Q., Haritunians, T., McGovern, D. P. and Targan, S. (2014). Use of animal models in elucidating disease pathogenesis in IBD. Semin Immunopathol 36(5): 541-551.
  6. Mottet, C., Uhlig, H. H. and Powrie, F. (2003). Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol 170(8): 3939-3943.
  7. Perše, M. and Cerar, A (2012). Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol 2012: 718617.
  8. Pineton de Chambrun, G., Peyrin-Biroulet, L., Lemann, M. and Colombel, J. F. (2010). Clinical implications of mucosal healing for the management of IBD. Nat Rev Gastroenterol Hepatol 7(1): 15-29.
  9. Reinisch, W., Sandborn, W. J., Hommes, D. W., D’Haens, G., Hanauer, S., Schreiber, S., Panaccione, R., Fedorak, R. N., Tighe, M. B., Huang, B., Kampman, W., Lazar, A. and Thakkar, R. (2011). Adalimumab for induction of clinical remission in moderately to severely active ulcerative colitis: results of a randomised controlled trial. Gut 60(6): 780-787.
  10. Rutgeerts, P., Sandborn, W. J., Feagan, B. G., Reinisch, W., Olson, A., Johanns, J., Travers, S., Rachmilewitz, D., Hanauer, S. B., Lichtenstein, G. R., de Villiers, W. J., Present, D., Sands, B. E. and Colombel, J. F. (2005). Infliximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med 353(23): 2462-2476.
  11. Sandborn, W. J., van Assche, G., Reinisch, W., Colombel, J. F., D'Haens, G., Wolf, D. C., Kron, M., Tighe, M. B., Lazar, A. and Thakkar, R. B. (2012). Adalimumab induces and maintains clinical remission in patients with moderate-to-severe ulcerative colitis. Gastroenterology 142(2): 257-265 e251-253.
  12. Sonnenberg, G.F., Monticelli, L.A., Elloso, M.M., Fouser, L.A., and Artis, D. (2011). CD4+ lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 34(1): 122-134.

简介

目前通过抑制过度炎症免疫应答治疗炎症性肠病的治疗方法取得了有限的成功(Reinisch等人,2011; Rutgeerts等人,2005; Sandborn等人[ et al。,2012)。为了开发新的治疗干预措施,需要更好地了解粘膜愈合期间手术的机制(Pineton de Chambrun等,2010)。为此,开发了一种可逆模型的结肠炎,其中通过淋巴细胞小鼠中过早转移原始CD4 + / CD40RB T细胞诱导的结肠炎可以通过消除结肠发生CD4 + T细胞(Brasseit等,2016)。

背景随着发展旨在重现人类疾病的动物模型,我们对肠道炎症性肠疾病(IBD)的发病机制的理解已经大大改善(Khanna等人)。 ,2014)。尽管鉴定了广泛的免疫学目标,目前的治疗方法在治疗IBD方面取得的成功有限,而且有关知识可用于建立长期缓解和相关粘膜愈合时引起的机制(D'Haens >等,,2014)。到目前为止,一个主要的限制是缺乏动物模型,其中可以在具有既定疾病的动物中可再现地诱导缓解。在感染引起肠道炎症的模型中,促炎和抗炎机制可以同时运作,这意味着在解决炎症期间解剖不同免疫途径的作用可能是一个挑战(Endt等人。 ,2010; Sonnenberg等人,2011)。在葡聚糖硫酸钠(DSS)诱导的结肠炎中,DSS可以容易地在饮用水中施用以诱导急性或慢性肠道炎症,然后用正常饮用水治疗以研究结肠炎的解决。然而,疾病的动力学和严重性高度依赖于许多因素,包括所使用的DSS的剂量差异以及消费的DSS量的差异,这几乎不可能在同一笼子的不同动物之间正常化(Chassaing等人,2014;Perše和Cerar,2012)。使用T细胞转移介导的结肠炎模型,优选地表明,通过在结肠动物中过继转移CD45RB 调节性T细胞(Treg)可以逆转肠道炎症,导致10- Treg转移后14周(Mottet等人,2003)。缓解诱导的动力学根据转移的Treg的扩增而变化,并且可能难以同步相同实验组动物之间的缓解发作。为了克服不可预测的缓解诱导时间和程度,我们开发了一种新型的可逆肠炎症小鼠模型,其中肠道炎症(通过淋巴细胞动物中过早转移初始CD45RB T细胞诱导)可以是通过已确定的疾病的小鼠中的结肠发生CD4 + T细胞的消耗而逆转,导致可重复诱导的结肠炎缓解(Brasseit等,2016)。

关键字:结肠炎, 缓解, 复发性疾病, 黏膜愈合

材料和试剂

  1. 50ml管(SARSTEDT,目录号:62.547.254)
  2. 5 ml Falcon聚苯乙烯(PS)圆底管(Corning,Falcon ®,目录号:352058)
  3. 100μm尼龙细胞过滤器(Corning,目录号:431752)
  4. 玻璃滑块Superfrost(生物系统,目录号:85-0551-00)
  5. 6-10周龄的同源CD90.1或CD45.1小鼠作为供体(C57BL/6背景上的内部繁殖的小鼠,最初购自美国的JACKSON LABORATORY)
    注意:
    1. 使用同源小鼠CD90.1作为供体,以容易地区分供体(CD90.1)和受体细胞(CD90.2),但是也可以使用C57BL/6小鼠作为供体。
    2. 可以使用超过10周龄的供体老鼠,但是天真的CD4 CD45RB 预期T细胞随年龄增长而降低。
  6. 10-16周龄幽门螺杆菌阳性C57BL/6 Rag2 //> 或 Rag1 -/- 小鼠(最初购自美国的JACKSON LABORATORY,美国),以无特定病原体(SPF)条件作为受体,其体重至少为20克, 注意:
    1. > 小鼠也可以用作收件人。然而,结肠炎诱导的动力学被延迟,并且在那些幽门螺杆菌阴性小鼠中的CD4T细胞介导的结肠炎诱导之后的给定时间,疾病活动的变化可能会很大变化。
    2. 幽门螺杆菌存在的阳性可以通过PCR使用从粪便丸提取的基因组DNA进行评估,如Brasseit等人,2016所述。
    3. 我们没有观察到男性和女性接受者之间结肠炎诱导的动力学有任何差异。
  7. 0.5M EDTA(Sigma-Aldrich,目录号:27285),钠形式
  8. EasySep TM 小鼠链霉亲和素RapidSpheres TM隔离试剂盒(STEMCELL Technologies,目录号:19860)
  9. MAb抗小鼠CD4(克隆GK1.5)(BioxCell,目录号:BE0003-1)
  10. 抗小鼠抗体
    抗体
    公司
    目录号
    克隆
    稀释
    B220生物素
    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

  11. 马血清(HS)(Sigma-Aldrich,目录号:H1270)
  12. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  13. 4%缓冲福尔马林(EMD Millipore,目录号:100496)
  14. 乙醇(EMD Millipore,目录号:100983)
  15. 二甲苯(VWR,目录号:28975)
  16. 石蜡(Engelbrecht,目录号:17932)
  17. Mayer's hemalum(苏木精)溶液(EMD Millipore,目录号:109249)
  18. 二甲苯基安装介质Eukitt ®(Sigma-Aldrich,目录号:03989)
  19. 氯化钠(NaCl)(EMD Millipore,目录号:106406)
  20. 二钠磷酸氢二钠(Na 2 HPO 4·2H 2 O)(EMD Millipore,目录号:106580)
  21. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)(EMD Millipore,目录号:105099)
  22. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:31107)
  23. 碳酸氢钾(KHCO 3)(EMD Millipore,目录号:104854)
  24. 曙红 - 福洛辛溶液(VWR,目录号:341973R和10047229)分别为
  25. 冰醋酸(EMD Millipore,目录号:100063)
  26. 盐酸(EMD Millipore,目录号:100317)
  27. 磷酸盐缓冲盐水(PBS)(见食谱)
  28. ACK红血溶解缓冲液(参见食谱)
  29. 曙红 - 福洛辛溶液(见配方)
  30. 0.5%HCl - 乙醇(参见食谱)

设备

  1. 离心机
  2. Neubauer室(血细胞计数器)
  3. EasySep TM 磁铁(STEMCELL Technologies,目录号:18000)
  4. BD FACS ARIA III(BD,型号:BD FACS ARIA III)
  5. Tissue-Tek VIP 6组织处理器(SAKURA,型号:Tissue-Tek VIP 6 Vaccum Infiltration Processor)
  6. Tissue-Tek ® uni-cassette ®(SAKURA,目录号:4172)
  7. 加热块
  8. 解剖套件(镊子和剪刀)
  9. 冷板(-10°C)
  10. 切片机(Leica Biosystems,Wetzlar,Germany)
  11. 水浴
  12. 移液器
  13. CO 2
    注意:根据瑞士联邦和州动物实验规则,CO 2 被用作经批准和推荐的安乐死方法。

软件

  1. Graphpad Prism 6(La Jolla,CA; https://www。 graphpad.com/scientific-software/prism/

程序

  1. 选择天真的CD4 + T细胞
    1. 在第0天,将脾从供体CD90.1小鼠中分离出来。每2个接收者使用1个脾脏 小鼠。
    2. 在100μm尼龙过滤器上分解脾脏,并用PBS/2%马血清(HS)冲洗过滤器,以获得单细胞悬浮液。
    3. 以370×g旋转细胞悬浮液5分钟。
    4. 通过将细胞沉淀物重悬于3ml ACK裂解缓冲液中3分钟来溶解红细胞。
    5. 用PBS/2%HS洗涤细胞,并将细胞沉淀重新悬浮于含有5mM EDTA的1ml PBS/2%HS中。
      注意:细胞再悬浮的初始体积不是非常重要的,因为细胞必须被计数并调整到1×10 8细胞/ml,如下面的步骤所示。
    6. 使用血细胞计数器计数细胞数,并将细胞浓度调整至1×10 8细胞/ml。
    7. 加入50μl/ml所提供的大鼠血清(来自EasySep TM 小鼠链霉抗生物素蛋白RapidSpheres TM分离试剂盒),并与生物素化抗体(抗小鼠B220和CD8α)一起温育。通过移液小心地混合细胞。
    8. 将含有细胞悬浮液的管放在冰上15分钟。
      注意:添加抗小鼠抗体后,必须通过移液小心混合细胞。含有细胞再悬浮的管可以保留在冰上,无需进一步的混合/反转/摇动。
    9. 涡旋链霉亲和素RapidSpheres TM 30分钟,并向细胞悬液中加入85μl/ml的RapidSpheres TM。
      注意:不需要RapidSpheres TM的初始洗涤步骤,因为它们倾向于沉淀在管的底部,因此涡旋RapidSpheres TM 是至关重要的。 em>
    10. 通过上下移液仔细混合,并在室温下孵育2.5分钟。
    11. 使用含有5mM EDTA的PBS/2%HS将细胞悬浮液总体积为2.5ml,并转移至5ml PS管中。
    12. 将含有细胞悬浮液的5ml PS管放置在EasySep TM 磁体中2.5分钟。
    13. 在一个连续的运动中,将磁体反转并小心地将细胞部分(链霉抗生物素蛋白RapidSpheres TM未结合部分)倒入新的5ml管中,将磁体和管置于倒置位置2-3秒以确保细胞悬浮液的最佳转移。


      图1.使用供体小鼠脾细胞的T细胞富集方法

    14. 将PBS/2%HS中的细胞在370×g下洗涤5分钟。
    15. 在1ml抗小鼠CD45RB FITC,CD25 PE和CD4 APC-Cy7中染色细胞。通过移液小心地混合细胞。将含有细胞悬浮液的管放置在冰上20分钟,无需进一步混合/反转/摇动
    16. 在PBS/2%HS之前洗涤细胞(富含CD4 + T细胞),然后在CD4细胞上分离CD4 +/CD40R细胞BD FACSARIA III细胞分选机。


      图2.来自供体C57BL/6同源小鼠脾脏的初始CD45RB T细胞的选择策略首先根据其前向和侧向散射特性选择淋巴细胞,然后排除双胞胎细胞。通过从单门选择CD25阴性淋巴细胞排除调节性和活化的T细胞,最后,基于CD4和CD45RB的高表达选择初始CD4 + T细胞。

    17. 收集1 ml HS(100%HS)中的分选细胞。
    18. 在37℃下,将PBS中的分选的CD4 + CD45RB hi T细胞洗涤5分钟,并以1×10 4的浓度重悬于PBS中> 6 cells/ml。
      注意:始终将细胞保持在冰上。如果排序天真的CD4 + T细胞不能立即注射,将细胞保持在HS上。注射前只用PBS清洗和重悬细胞。
    19. 为了诱导结肠炎,将具有2×10 5个CD4 +细胞的C57BL/6Rag2 + CD45RB hi 腹膜内(ip)体积为200μl的T细胞。
    20. 一旦动物结肠(见方法B),每次注射间隔72小时,用250-500μg抗CD4耗竭抗体注射动物三次以诱导缓解。
      注意:如Brasseit等人,2016所述,首先使用500μg的αCD4消耗性抗体进行缓解诱导。在我们的实验中,我们仍然可以达到相同水平的CD4 + T细胞消耗和类似的缓解诱导动力学,使用3种250μgαCD4消耗抗体的处理。
    21. 在用抗CD4耗竭抗体进行第一次治疗后,每天监测动物的体重变化和正常活动,结肠炎症的复发预期在22-25天左右(在幽门螺杆菌阳性受体中)。
      注意:为了评估结肠炎的发展,每天监测小鼠,因为结肠炎诱导的动力学强烈依赖于微生物群的组成,意味着疾病进展可能因一个SPF小鼠家庭设施的动物而异。根据我们的经验,在幽门螺杆菌阳性小鼠T细胞转移的第9-10天左右和幽门螺杆菌阴性小鼠T细胞转移后大约第30天发现结肠炎的临床和组织病理学征象。在幽门螺杆菌阳性受体小鼠中,血液中明显的体重减轻(约10%)和凝血性CD4 + T细胞的出现非常迅速地从一天到另一天发生。此外,我们通常观察到体重减轻与幽门螺杆菌阳性但不是幽门螺杆菌阴性受体的肠道炎症程度强烈相关。

  2. 临床和组织病理评分
    1. 血液中具有活动性肠道炎症(临床评分≥8)和可检测的CD4 T细胞的小鼠被认为是结肠炎。为了测量血液中的CD4 + T细胞,通过尾静脉在1ml PBS/2%HS/5mM EDTA中收集2-3滴血液。通过在370×g离心5分钟将细胞旋转下来。通过移液将细胞重悬于含有抗小鼠CD4抗体的100μlPBS/2%HS(参见材料和试剂部分用于抗体稀释)。在冰上孵育15分钟后,以PBS/2%HS洗涤细胞,并在流式细胞仪上分析。
    2. 对于临床疾病评分,每天监测以下参数:
      1. 减肥
      2. 粪便一致性
      3. 血液中的粪便外观
      4. 行为


        图3.用于在淋巴细胞受体中转移CD4 + CD45RB T细胞后评估结肠动物的评分系统。临床评分为≥8被认为是结肠炎(可能导致临床分数为8的多重组合)。  

    3. 对于组织学疾病评分,收集结肠(中)切片用于苏木精和伊红(H& E)染色。


      图4.用于组织学的结肠组织切片的收集

    4. 以盲法测量以下参数(这应由经验丰富的病理学家完成)。更多信息,请参阅Brasseit等人的发表。。
      1. 细胞浸润在固有层中(得分从0到3)
      2. 杯状细胞丢失(分数从0到3)
      3. 存在隐窝脓肿(分数从0到3)
      4. 上皮侵蚀(从0分到1分)
      5. 嗜睡(0至2分)
      6. 结肠粘膜厚度(分数从0到3)

  3. 石蜡包埋和H& E染色
    注意:石蜡包埋和H& E染色通常在我们的组织学核心实验室设施中使用Tissue-Tex VIP 6上的全自动化程序进行,但基本步骤如下所述。
    1. 在单片盒中收集1cm结肠组织(中间部分),并在室温下固定在4%缓冲的福尔马林中。
    2. 如下处理组织(使用自动组织处理器):
      1. 4%缓冲福尔马林,在35℃下1.25小时。
      2. 蒸馏水在室温下15分钟。
      3. 70%乙醇在35℃下1.5小时。
      4. 80%乙醇在35°C下1小时。 
      5. 95%乙醇在35°C下1.5小时。
      6. 100%乙醇在35°C下1小时(每次1小时3次)
      7. 二甲苯在35℃下1小时(每次1小时两次)
      8. 石蜡在60℃下45分钟(四次变化45分钟)。
      注意:需要乙醇,二甲苯和石蜡的改变以减少以前步骤中的试剂携带量。
    3. 嵌入石蜡块。选择对应于磁带盒尺寸的合适的模具。
    4. 打开磁带并丢弃盖子。
    5. 将一些液体石蜡加入到模具中,将其放置在60℃的加热块上,以防止石蜡固化。
    6. 使用温镊将组织转移到模具中,使得镊子可以用于将组织从盒转移到模具而不固化石蜡。
    7. 将模具放在冷板(-10°C)上,开始固化石蜡,并立即用镊子将组织向下压。
    8. 将薄纸盒放在模具顶部并牢固按住。确保有足够的石蜡覆盖塑料盒。
    9. 等待石蜡固化,使石蜡块容易从模具中脱出。
    10. 使用切片机切割5μm切片。简单地说,将刀片插入支架并将刀片的角度调整到5°。将石蜡块放在物体夹中,并调整到所需的方向。切割几个部分(10μm)以暴露组织表面。一旦组织被暴露并且组织切割是光滑的,则将部分厚度调节到5μm。用镊子小心地取出5μm的组织切片并将其漂浮在37℃的温水浴中。在干净的玻璃载玻片上收集切片,并在加热板上在40℃下干燥20分钟。
    11. 用苏木精和曙红染色,首先用二甲苯将载玻片脱蜡5分钟。
    12. 用二甲苯冲洗两次,并通过以下顺序冲洗将水合玻片再次水化:
      1. 绝对乙醇(两个变化);
      2. 95%乙醇
      3. 70%乙醇 
      4. 蒸馏水
      注意:这里没有规定确切的体积,因为这将取决于玻璃染色罐的尺寸。我们建议幻灯片必须完全浸没在指定的试剂中。
    13. 用Mayer's hemalum溶液染色7分钟,用自来水冲洗。
    14. 用0.5%HCl - 乙醇漂白几秒钟(取决于所需的细胞核的染色深度),并立即用自来水洗涤载玻片,然后在自来水中进行10分钟的蓝色步骤。
    15. 用70%乙醇冲洗。
    16. 使用曙红 - 菲洛昔康溶液进行抗菌染色2-3分钟。用70%乙醇冲洗两次
    17. 通过以下顺序冲洗去除水滑梯:
      1. 70%乙醇
      2. 95%乙醇
      3. 绝对乙醇(总共两个变化)
      4. 二甲苯(总共两个变化)
      注意:这里没有规定确切的体积,因为这将取决于玻璃染色罐的尺寸。我们建议幻灯片必须完全浸没在指定的试剂中。
    18. 搭载Eukitt ®

数据分析

在实验设计的设计中,建议包括4-5只小鼠/组,进行2-3次独立实验。为了进行数据分析,将接受初始CD45RB T细胞(实验组)的受体小鼠与接受CD45RB细胞的对照组(对照组,无结肠炎)进行比较。为了评估统计学意义,请使用Graphpad Prism 6(La Jolla,CA)上的不配对的测试。

笔记

  1. 为了确保最佳的细胞产量和生存力,除非另有说明,否则始终在冰上工作
  2. 为了在离心后除去上清液,吸出上清液并丢弃,而不是将其倒出,因为细胞不能牢固地粘附到5ml的PS管的底部。
  3. 对于细胞再悬浮,这应该使用移液器轻轻地进行,而不是通过涡旋。
  4. 注射细胞之前,通过反转管上下混合细胞,以确保所有小鼠注射相同数量的细胞。
  5. 为了腹腔注射,在注射细胞之前,确保针头正确插入腹腔。
  6. 如果 -/- 收件人是从另一个鼠标房屋设施导入的,建议将这些老鼠放置至少两周之前,天真的CD4 + T细胞转移以允许微生物群的稳定化。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl
    1.69mM Na 2 HPO 4·2H 2 O/
    8.63mM K 2 HPO 4·3H 2 O
  2. ACK红血溶解缓冲液
    150mM NH 4 Cl
    10mM KHCO 3
    0.1 mM EDTA
  3. 曙红 - 氟洛芬溶液
    100ml 1%伊红储备液
    10ml 1%的福洛辛储备液
    780毫升95%乙醇 4毫升冰醋酸
  4. 0.5%HCl - 乙醇
    20毫升25%盐酸
    980毫升70%乙醇

致谢

这项工作得到了瑞士国家科学基金会Sinergia补助金给Andrew Macpherson,Christoph Mueller,Wolf-Dieter Hardt和Daniela Finke以及C.M.的SNSF拨款31-138392的支持。描述的方案首先在Brasseit等人,2016年概述。

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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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