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Aug 2018

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Induction and Analysis of Anti-CD40-induced Colitis in Mice
小鼠中抗-CD40抗体诱导的结肠炎的诱导和分析   

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Abstract

Colon inflammation or colitis affects more than 1 million people worldwide. Several pre-clinical models, including chemical-induced (i.e., DSS, TNBS) or pathogen-induced (i.e., Citrobacter rodentium) have been used to study mechanisms involved in the development and regulation of colitis. Anti-CD40 induced colitis model has gained acceptance to study the roles of innate immune cells during acute intestinal inflammation. Here we describe a rapid, robust and reproducible protocol to induce and analyze anti-CD40 mediated colitis in mice.

Keywords: Anti-CD40 (抗CD40抗体), Colitis (结肠炎), IBD (IBD), Innate immunity (先天免疫), Inflammation (炎症), IL-23 (IL-23), IL-22 (IL-22)

Background

Inflammatory Bowel Disease (IBD), including Crohn’s disease and Ulcerative colitis, affects about 1.5 million people in the United States (Ng et al., 2017). To better understand the mechanisms involved in the development and progression of IBD, a number of pre-clinical models (i.e., DSS, TNBS, anti-CD40, etc.) have been developed to address various aspects of immune response during tissue injury over the last two decades. CD40 is highly expressed by the colon lamina propria antigen presenting cells. We have demonstrated that activation of CD40 signaling using an agonist anti-CD40 antibody can trigger colitis in T and B cells deficient mice (here referred as Rag-/- mice) driven by excessive production of IL-23, IL-1β and IL-12 by myeloid cells (Uhlig et al., 2006). Anti-CD40 model is a unique model of colitis driven by IL-23-producing gut resident CX3CR1+ macrophages and IL-22-producing group 3 innate lymphoid cells (ILC3) (Bauche et al., 2018). This model of colitis is restricted to the proximal colon and is a potent model to study the role of innate immunity in colon inflammation. Here, we describe a robust and reproducible method to induce and analyze anti-CD40-induced colitis in mice. Anti-CD40-treated Rag2-/- mice lose up to 20% of their initial weight within three days post injection, and then return to their initial weight by Day 7 post induction (Figure 1A). Elevated levels of pro-inflammatory cytokines can be detected in the proximal colon as soon as Day 1 post induction (Cayatte et al., 2012) (Figure 1B) but maximal disease–characterized by massive infiltration of innate immune cells, loss of goblet cells and development of mitotic figures–is observed in the proximal colon at day 7 post disease induction (Figure 1C). Immune cell infiltration in the proximal colon and cytokine production, such as IL-22, by innate lymphoid cells can be measured by flow cytometry (Figure 1D).


Figure 1. Induction and analysis of the anti-CD40 colitis mouse model. A. Percentage of initial weight over a 7 days period. Initial weight is measured right before injection of isotype or anti-CD40 antibodies. B. Gene expression profile of the proximal colon at Day 7 post treatment. Data shows relative fold change over the isotype control. C. Representative photomicrographs of H&E stained colon section 7 days after injection of isotype (left) or anti-CD40 antibodies (right). IC: Immune cell infiltration; GC: Loss of Goblet cells; AB: Apoptotic body; MF: Mitotic figures. Scale bars = 250 μm. D. Representative dot plot of IL-22 production by proximal colon lamina propria ILC3 (gated on lineage-, CD90high, CD45int, RORγt+ cells) at Day 2 post treatment.

Materials and Reagents

  1. Materials
    1. Pipette tips (Thermo Scientific)
    2. Eppendorf tubes, 1.5 ml (Eppendorf, catalog number: 05-402-5)
    3. 1 ml sterile sub-Q syringe 26 G (BD, catalog number: 3095971)
    4. Sterile cell strainer 70 μm Nylon mesh (Fisher Scientific, catalog number: 22363548)
    5. 50 ml centrifuge tube (Fisher Scientific, catalog number: 05-539-13)
    6. 15 ml conical centrifuge tube (Fisher Scientific, catalog number: 339650)
    7. 15 ml sterile plastic tube (Fisher Scientific, catalog number: 14-956-1D)
    8. 5 ml Serological pipettes (Falcon, catalog number: 357543)
    9. 25 ml Serological pipettes (Fisher Brand, catalog number: 13-678-11)
    10. Petri dishes (Falcon, catalog number: 351029)
    11. Serum or plasma separation tubes (SARSTEDT, catalog number: 41.1378.005)
    12. Parafilm (Pechiney, catalog number: PM-996)
    13. V bottom 96-well plate (Costar, catalog number: 3894)
    14. 70 μm cell strainer

  2. Animals
    Eight to twelve weeks old Rag2-/- mice (Taconic, catalog number: RAGN12) were used to study anti-CD40 mediated colitis.

  3. Reagents
    1. Liquid nitrogen
    2. Anti-mouse CD45 APC-eFluor780 antibody (eBioscience, catalog number: 47-0451-82)
    3. Anti-mouse CD90.2 BV786 antibody (BD Bioscience, catalog number: 564365)
    4. Anti-mouse RORγt PerCP-eFluor710 antibody (eBioscience, catalog number: 46-6981-82)
    5. Anti-mouse NK1.1 Alexa Fluor 700 antibody (BD Bioscience, catalog number: 560515)
    6. Anti-mouse CD11c PE-Cy7 antibody (BD Bioscience, catalog number: 561022)
    7. Anti-mouse CD11b FITC antibody (BD Bioscience, catalog number: 557396)
    8. Anti-mouse IL-22 APC antibody (eBioscience, catalog number: 17-7222-82)
    9. Endotoxin-free Anti-CD40 antibody (Clone FKG45, BioXCell, catalog number: BE0016-2)
    10. Anti-Rat IgG2a isotype control (Clone 2A3, BioXCell, catalog number: BP0089)
    11. HyClone Phosphate Buffered Saline (DPBS), 1x (GE Healthcare, catalog number: SH30028.02)
    12. Hank’s buffered salt solution (HBSS)
    13. 10% Neutral Buffered Formalin (Thermo Fisher Scientific, catalog number: 245-685)
    14. Penicillin/streptomycin solution 100x (Corning, catalog number: 30-002C1)
    15. Sodium pyruvate 100 mM (Gibco, catalog number: 11360070)
    16. 2-Mercaptoethanol 55 mM (Gibco, catalog number: 21985023)
    17. Ultra Pure 0.5 μM EDTA pH 8.0 (Gibco, catalog number: 15575-038)
    18. HEPES buffer solution 1 M (Gibco, catalog number: 15630-080)
    19. Hyclone Standard Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: SH30088.03)
    20. Collagenase type VIII (Sigma-Aldrich, catalog number: C2139)
    21. DNase I (Sigma-Aldrich, catalog number: D5025)
    22. Dispase (Corning, catalog number: 354235)
    23. Percoll (GE Healthcare, catalog number: 17-0891-01)
    24. IMDM, GlutaMAX supplement (Thermo Fisher Scientific, catalog number: 31980097)
    25. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139)
    26. Ionomycin (Sigma-Aldrich, catalog number: I0634)
    27. Brefeldin A (BD Bioscience, catalog number: 555029)
    28. Stain Buffer (BD, Pharmingen, catalog number: 554656)
    29. Transcription Factor Buffer Set (BD, Pharmingen, catalog number: 562725)
    30. Fixable viability stain 510 (BD, Horizon, catalog number: 564406)
    31. Mouse BD Fc Block (BD, Pharmingen, catalog number 553141)
    32. Ethanol 70% (Fisher Scientific, catalog number: HC1500)
    33. Ethanol 95% (Fisher Scientific, catalog number: HC1300)
    34. Ethanol 100% (Fisher Scientific, catalog number: HC600)
    35. Xylene (Fisher Scientific, catalog number: HC700)
    36. Paraffin (Fisher Scientific, catalog number: 23-0210-400)
    37. Hematoxylin (MasterTech Scientific, catalog number: HXHHEGAL)
    38. Eosin (Thermo Scientific, catalog number: 71311)
    39. Collagenase D (Sigma, catalog number: C5138-1G)
    40. Epithelial cell dissociation solution (see Recipes)
    41. Enzyme digestion mix (see Recipes)
    42. Complete IMDM media (see Recipes)

Equipment

  1. Scalpel (Southmedic, catalog number: SMI1/73-0121)
  2. Pipettes (Thermo Scientific)
  3. Forceps and Scissors (Fine Science Tools)
  4. Sterile cell culture hood
  5. Centrifuge (Thermo Fisher Scientific)
  6. Shaking water bath (Precision)
  7. Brightfield Microscope
  8. LSRII Flow cytometer (BD Biosciences)
  9. Incubator
  10. Vortex (Scientific Industries)

Software

  1. FlowJo_V10 (FlowJo, LLC, https://www.flowjo.com)
  2. GraphPad Prism 7 (GraphPad, https://www.graphpad.com)
  3. Microsoft Excel (Microsoft)

Procedure

  1. Induction of colitis in Rag2-/- mice
    1. Dilute the anti-CD40 antibody or isotype control to 1 mg/ml in sterile 1x PBS.
    2. Inject 100 μg of anti-CD40 or isotype control antibody intraperitoneal (i.p.) or 50 μg intravenous injection (i.v.)–based on researcher’s technical expertise–with a 1 ml sterile sub-Q syringe 26 G. Each batch of anti-CD40 antibody should be verified to be endotoxin-free (by checking with the manufacturer) to avoid systemic immune activation.
      Notes:
      1. Every animal facility will have a specific microbial environment, so it is crucial to determine the dose of anti-CD40 empirically by performing an initial dose-titration (25-100 μg) (Figure 2). 
      2. A dose that is too high will result in death of the animals while an insufficient dose will lead to weak disease induction (characterized by inconsistent or poor colon inflammation) and variability in colitis development. 
      3. Mice are weighed every day for seven consecutive days and % weight loss is calculated in comparison to the initial weight on Day 0. Mice that lose more than 20% of initial body weight or demonstrate shaking or severe systemic responses are euthanized within 24 h.
      4. Weight loss kinetics of colitis may vary between i.p. and i.v. injection type. In our hands, mice recover weight more quickly after i.p. delivery. However, the route of injection does not alter the timing or severity of colitis development.
    3. At Days 2-7 post induction, euthanize mice by carbon dioxide asphyxiation according to the approved ethical protocol.
    4. Collect serum via cardiac puncture for the detection of systemic cytokines levels.


      Figure 2. Titration of the anti-CD40 antibody. Rag2-/- mice received a single dose of anti-CD40 antibody i.v. as indicated. Percentage of initial weight over a 7 days period. Initial weight is measured right before injection of anti-CD40 antibody.

  2. Isolation and preparation of colon tissue/cells for endpoint analysis
    1. Histopathological analysis by H&E staining
      1. Harvest colons and remove fecal content by gently dislodging from colon with forceps. Roll colon into a tissue embedding cassette with proximal end in the center and fix in 10% neutral buffered formalin overnight (the cassette needs to be submerged).
      2. Transfer the cassettes to 70% ethanol, embed in paraffin, section at 4-5 μm and then stain with hematoxylin for 10 min and eosin as previously described (Wang et al., 2017).
      3. Wash with running water until the water is clear.
      4. Transfer the slides to the Eosin solution and incubate for 3 min.
      5. Successively transfer the slides into 70% ethanol for 20 s, 90% ethanol for 20 s, 100% ethanol for 1 min and xylene for 3 min.
      6. Take out slides from xylene and dry them out in a fume hood.
      7. Mount and cover the slides.
    2. Gene expression profiling of the proximal colon
      1. At the desired time point after induction of colitis, mice are euthanized by carbon dioxide asphyxiation according to approved ethical protocol.
        Note: For analysis of genes encoding cytokines produced by the innate immune cells, we recommend euthanizing mice on Days 1 to 3 post induction. For analysis of genes expressed by epithelial cells in response to inflammation, we recommend euthanizing the mice on Day 7 post induction when the disease is maximal.
      2. Harvest a piece of proximal colon (about 0.5 cm long), remove the fecal content by gently dislodging it from colon with forceps, transfer into a sterile polypropylene tube and snap freeze in liquid nitrogen (Figure 3B). Since inflammatory infiltrate can be variable, it is important to cut a vertical section of the colon starting from the base of the cecum and descending into the proximal colon to ensure consistent molecular signature.
        Note: To avoid RNA degradation, samples must be snap frozen in liquid nitrogen within 2 min post euthanasia. Frozen proximal colons can be stored at -80 °C for at least 6 months.
      3. Extract RNA and run RT-PCR protocols as described in Bauche et al., 2018.
    3. Isolation of proximal colon lamina propria cells, ex-vivo stimulation and flow cytometry
      Isolation of proximal colon lamina propria cells
      1. At 2-7 days post induction, mice are killed by carbon dioxide asphyxiation according to approved ethical protocol.
      2. Harvest and collect the proximal colon in a 15 ml Falcon tube filled with 5 ml of 1x HBSS. 
      3. Remove the fat (Figure 3A).
      4. Transfer the colon into a Petri dish.
      5. Cut colon open lengthwise and wash by submerging and gently shaking in 50 ml of 1x HBSS until the tissue is clear of fecal matter. 
      6. Cut into 0.5 cm segments. 
      7. Place into a 50 ml Falcon tube with 20 ml of epithelial cell dissociation solution. 
      8. Wrap in parafilm, place sideways in a water bath at 37 °C for 20 min with medium shaking.
      9. Vortex well for 15 s.
      10. Filter through a 70 μm strainer, collect and transfer the undigested piece of colon tissues in a new 50 ml Falcon tube. 
      11. Rinse fragments with 20 ml 1x HBSS by gently vortexing for 10 s.
      12. Transfer colon fragments into a petri dish and chop into tiny pieces with a scalpel.
      13. Using a 25 ml Serological pipettes, transfer into a new 50 ml tube with 10 ml of enzyme digestion mix then incubate for 20 min in a water bath at 37 °C with shaking as above.
      14. Vortex for 15 s.
      15. Collect sup by filtering through a 70 μm cell strainer and put on ice. Pellet cells for 5 min at 375 x g, 4 °C.
      16. Prepare 100% Percoll solution (9 parts Percoll + 1 part 10x PBS).
      17. Prepare 40% and 80% Percoll solutions by diluting 100% Percoll with 1x PBS.
      18. In a 15 ml tube resuspend the pellet in 5 ml of 40% Percoll, using a 5 ml Serological pipettes, and underlay with 5 ml of 80% Percoll for each colon.
      19. Spin 670 x g for 20 min at 20 °C with no acceleration and no brake.
      20. Collect interface (white ring) between and the two layers of percoll into a 15 ml tube, wash with 10 ml of complete IMDM media and spin for 5 min at 375 x g, 4 °C. 
      21. Resuspend in 1 ml of complete IMDM and cells are ready to go.
      Note: The interface is characterized by a white ring and contained mainly colon lamina propria immune cells. Cell number and viability should be measured at this step.

      Ex-vivo stimulation
      1. For cytokines analysis, stimulate 2 x 106 viable cells/well in a 96-well plate for 4 h at 37 °C, 5% CO2 with 50 ng/ml PMA, 500 ng/ml Ionomycin and Brefeldin A (1/1,000) in 200 μl of complete IMDM.
      2. Spin for 5 min at 375 x g, 4 °C and proceed to flow cytometry staining.

      Flow cytometry staining
      1. Incubate cells in 100 μl of 1x PBS with 0.3 μl of live/dead fixable dead cell stains and 5 μl of mouse BD Fc block per well for 25 min at 4 °C in the dark.
      2. Wash with 100 μl of 1x PBS per well and spin for 5 min at 375 x g.
      For surface staining:
      1. Incubate cell suspension with 0.2 μg of anti-mouse conjugated antibodies (i.e., CD45, CD3, CD4) in 100 μl of BD stain buffer per well for 25 min at 4 °C in the dark.
      2. Wash with 100 μl of BD stain buffer and spin for 5 min at 375 x g.
      For intracellular staining:
      1. Resuspend cell suspension in 100 μl of 1x Fix/perm buffer (BD) and incubate for 45 min at 4 °C in the dark.
      2. Wash with 1x Perm/Wash buffer and spin for 5 min at 375 x g.
      3. Incubate cell suspension with 0.2 μg of anti-mouse conjugated antibodies (i.e., RORγt, IL-22) in 100 μl of 1x Perm/Wash buffer per well for 45 min at 4 °C in the dark.
      4. Wash with 1x Perm/Wash buffer and spin for 5 min at 375 x g
      5. Resuspend cells in 300 μl of 1x Perm/Wash buffer. Analyze and acquire data using a flow cytometer (we used LSRII flow cytometer from BD and analyzed data on FlowJo). Samples can be stored overnight at 4 °C in the dark.


        Figure 3. Preparation of colon tissue. A. Attached fat tissue (here shown by arrows) must be removed from the colon as fat can decrease the viability of isolated colon lamina propria. B. Taqman Sampling: The colon is separated from the cecum and carefully dissected from host by removing all the fat and connective tissue. The colon is cut from the proximal (cecal) end, down the midline and until the distal end. Flatten out the tissue, cut a 2-3 cm strip (pink square) and immediately snap freeze using polypropylene tubes in liquid nitrogen.

Data analysis

  1. Tissues are scored for severity of disease by a pathologist according to three criteria (each criterion is scored from 0 to 3, 0 = negative, 1 = mild, 2 = moderate and 3 = severe):
    Inflammation: when present is characterized by infiltration of large numbers (60%-70%) of mononuclear cells (macrophages and lymphocytes) and 30%-40% of neutrophils and band cells. The scoring of inflammation includes severity of infiltration, loss of glands, erosion, and dilatation of glandular lumina, presence of crypt abscess and degeneration of epithelial cells.
    Apoptosis: The prevalence of apoptotic bodies is scored on a scale of 0-3: 0 = negative; 1 = low; 2 = moderate; 3 = high.
    Regeneration: Regenerative changes assessed include scoring the prevalence of mitotic figures in the upper 1/3 of the mucosa, nuclear density (nuclear crowding) within individual glandular structures, regularity of the surface epithelium. Regeneration is scored on a scale of 0-3: 0 = negative; 1 = low; 2 = moderate; 3 = high.
  2. For gene expression profiling, average the normalized values from isotypes
    Calculate the fold change over the isotype control using the following formula:

    fold change = (Normalized value of the sample)/(Average normalized value of the isotype control group)

  3. For flow cytometry analysis, acquire at least 5 x 105 colon lamina propria cells
    1. The events are gated on FSC-A and SSC-A, then gated on FSC-A and FSC-H to remove doublets.
    2. Exclude cellular debris and dead cells (using LIVE/DEAD fixable stain).
    3. To study the phenotype of infiltrating immune cells, gate on CD45+ cells.
    4. For analysis of IL-22-producing ILC3, gate on live cells, lineage (CD11c, CD11b, NK1.1), CD90.2high, CD45int, RORγt+, IL-22+ cells. For further details, gating strategy can be found in previous publication (Bauche et al., 2018).
  4. Statistical analysis was performed using Prism 7. Unpaired t-test is used as statistical test. A P value < 0.05 is considered statistically significant.

Recipes

  1. Epithelial cell dissociation solution
    1x HBSS
    Penicillin/streptomycin (1,000 U/ml)
    5 mM EDTA
    10 mM HEPES
  2. Enzyme digestion mix
    1x HBSS
    Penicillin/streptomycin (1,000 U/ml)
    10% FBS
    2 mg/ml collagenase type VIII
    30 U/ml of DNase I
    50 U/ml of Dispase
    Alternately, 1 mg/ml of collagenase D (Sigma) can be used to preserve cell surface molecules when analyzing by FACS
  3. Complete IMDM media, 500 ml
    450 ml of IMDM, GlutaMAX supplement
    1,000 U/ml of Penicillin/streptomycin
    1 mM of Sodium pyruvate
    0.55 mM of 2-Mercaptoethanol
    50 ml of Heat inactivated FBS

Acknowledgments

We would like to thank Gil Asio and Danye Cheng for processing colon samples. We appreciate Lakshmanan Annamalai and Jennifer H. Yearley for their expertise in analyzing and scoring the histological samples. Wendy Blumenschein and Jeff Grein for running gene expression profile.

Competing interests

The authors declare no competing financial interests.

Ethics

Mice were maintained under specific pathogen-free conditions and kept in microisolators with filtered water at Merck Research Laboratories (MRL) animal facility, Palo Alto. All animal procedures were approved by the Institutional Animal Care and Use Committee of MRL in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC).

References

  1. Bauche, D., Joyce-Shaikh, B., Jain, R., Grein, J., Ku, K. S., Blumenschein, W. M., Ganal-Vonarburg, S. C., Wilson, D. C., McClanahan, T. K., Malefyt, R. W., Macpherson, A. J., Annamalai, L., Yearley, J. H. and Cua, D. J. (2018). LAG3+ regulatory t cells restrain interleukin-23-producing cx3cr1+ gut-resident macrophages during group 3 innate lymphoid cell-driven colitis. Immunity 49(2): 342-352: e345.
  2. Cayatte, C., Joyce-Shaikh, B., Vega, F., Boniface, K., Grein, J., Murphy, E., Blumenschein, W. M., Chen, S., Malinao, M. C., Basham, B., Pierce, R. H., Bowman, E. P., McKenzie, B. S., Elson, C. O., Faubion, W. A., Malefyt Rde, W., Kastelein, R. A., Cua, D., McClanahan, T. K. and Beaumont, M. (2012). Biomarkers of therapeutic response in the il-23 pathway in inflammatory bowel disease. Clin Transl Gastroenterol 3: e10.
  3. Ng, S. C., Shi, H. Y., Hamidi, N., Underwood, F. E., Tang, W., Benchimol, E. I., Panaccione, R., Ghosh, S., Wu, J. C. Y., Chan, F. K. L., Sung, J. J. Y. and Kaplan, G. G. (2017). Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet 390(10114): 2769-2778. 
  4. Uhlig, H. H., McKenzie, B. S., Hue, S., Thompson, C., Joyce-Shaikh, B., Stepankova, R., Robinson, N., Buonocore, S., Tlaskalova-Hogenova, H., Cua, D. J. and Powrie, F. (2006). Differential activity of IL-12 and IL-23 in mucosal and systemic innate immune pathology. Immunity 25(2): 309-318.
  5. Wang, C., Yue, F. and Kuang, S. (2017). Muscle histology characterization using H&E staining and muscle fiber type classification using immunofluorescence staining. Bio-protocol 7(10): e2279.

简介

结肠炎症或结肠炎影响全世界超过100万人。 一些临床前模型,包括化学诱导的( ie ,DSS,TNBS)或病原体诱导的( ie , Citrobacter rodentium ) 用于研究涉及结肠炎的发展和调节的机制。 抗CD40诱导的结肠炎模型已被接受用于研究先天免疫细胞在急性肠道炎症期间的作用。 在这里,我们描述了一种快速,稳健和可重复的方案,用于诱导和分析小鼠中抗CD40介导的结肠炎。
【背景】炎症性肠病(IBD),包括克罗恩病和溃疡性结肠炎,在美国影响约150万人(Ng 等人,,2017)。为了更好地了解IBD发展和进展所涉及的机制,许多临床前模型( ie ,DSS,TNBS,anti-CD40,等)都有在过去的二十年中,我们开发了用于解决组织损伤期间免疫应答的各个方面。 CD40由结肠固有层抗原呈递细胞高度表达。我们已经证明,使用激动剂抗CD40抗体激活CD40信号传导可引发T细胞和B细胞缺陷小鼠(此处称为 Rag - / - 小鼠)的结肠炎。由骨髓细胞过量产生IL-23,IL-1β和IL-12(Uhlig 等,,2006)。抗CD40模型是一种独特的结肠炎模型,由产生IL-23的肠道CX3CR1 + 巨噬细胞和产生IL-22的3组天然淋巴细胞(ILC3)驱动(Bauche et al 。,2018)。这种结肠炎模型仅限于近端结肠,是研究先天免疫在结肠炎症中作用的有效模型。在这里,我们描述了一种强大且可重复的方法来诱导和分析小鼠中抗CD40诱导的结肠炎。抗-CD40处理的 Rag 2 - / - 小鼠在注射后3天内失去其初始体重的20%,然后在第7天恢复其初始体重诱导后(图1A)。诱导后第1天,可以在近端结肠中检测到升高的促炎细胞因子水平(Cayatte et al。,2012)(图1B),但是最大的疾病 - 以先天的大量浸润为特征在疾病诱导后第7天,在近端结肠中观察到免疫细胞,杯状细胞的丢失和有丝分裂象的发展(图1C)。可以通过流式细胞术测量近端结肠中的免疫细胞浸润和先天性淋巴样细胞的细胞因子产生,例如IL-22(图1D)。


图1.抗CD40结肠炎小鼠模型的诱导和分析。 A. 7天内初始体重的百分比。在注射同种型或抗CD40抗体之前立即测量初始重量。 B.治疗后第7天近端结肠的基因表达谱。数据显示同种型对照的相对倍数变化。 C.注射同种型(左)或抗CD40抗体(右)后7天H&E染色结肠切片的代表性显微照片。 IC:免疫细胞浸润; GC:杯状细胞丢失; AB:凋亡小体; MF:有丝分裂数字。比例尺=250μm。 D.近端结肠固有层ILC3产生IL-22的代表性点图(谱系 - ,CD90 高,CD45 int ,RORγt处理后第2天 + 细胞)。

关键字:抗CD40抗体, 结肠炎, IBD, 先天免疫, 炎症, IL-23, IL-22

材料和试剂

  1. 材料
    1. 移液器吸头(Thermo Scientific)
    2. Eppendorf试管,1.5 ml(Eppendorf,目录号:05-402-5)
    3. 1 ml无菌sub-Q注射器26 G(BD,目录号:3095971)
    4. 无菌细胞过滤器70μm尼龙网(Fisher Scientific,目录号:22363548)
    5. 50毫升离心管(Fisher Scientific,目录号:05-539-13)
    6. 15毫升锥形离心管(Fisher Scientific,目录号:339650)
    7. 15毫升无菌塑料管(Fisher Scientific,目录号:14-956-1D)
    8. 5 ml Serological pipettes(Falcon,目录号:357543)
    9. 25 ml血清移液器(Fisher Brand,目录号:13-678-11)
    10. 培养皿(Falcon,目录号:351029)
    11. 血清或血浆分离管(SARSTEDT,目录号:41.1378.005)
    12. Parafilm(Pechiney,目录号:PM-996)
    13. V底96孔板(Costar,目录号:3894)
    14. 70μm细胞过滤器

  2. 动物
    8至12周龄 Rag 2 - / - 小鼠(Taconic,目录号:RAGN12)用于研究抗CD40介导的结肠炎。

  3. 试剂
    1. 液氮
    2. 抗小鼠CD45 APC-eFluor780抗体(eBioscience,目录编号:47-0451-82)
    3. 抗小鼠CD90.2 BV786抗体(BD Bioscience,目录编号:564365)
    4. 抗小鼠RORγtPerCP-eFluor710抗体(eBioscience,目录编号:46-6981-82)
    5. 抗小鼠NK1.1 Alexa Fluor 700抗体(BD Bioscience,目录号:560515)
    6. 抗小鼠CD11c PE-Cy7抗体(BD Bioscience,目录编号:561022)
    7. 抗小鼠CD11b FITC抗体(BD Bioscience,目录编号:557396)
    8. 抗小鼠IL-22 APC抗体(eBioscience,目录编号:17-7222-82)
    9. Endotoxin-free抗CD40抗体(克隆FKG45,BioXCell,目录号:BE0016-2)
    10. 抗大鼠IgG2a同种型对照(克隆2A3,BioXCell,目录号:BP0089)
    11. HyClone磷酸盐缓冲盐水(DPBS),1x(GE Healthcare,目录号:SH30028.02)
    12. 汉克的缓冲盐溶液(HBSS)
    13. 10%中性缓冲福尔马林(赛默飞世尔科技,目录号:245-685)
    14. 青霉素/链霉素溶液100x(Corning,目录号:30-002C1)
    15. 丙酮酸钠100 mM(Gibco,目录号:11360070)
    16. 2-Mercaptoethanol 55 mM(Gibco,目录号:21985023)
    17. 超纯0.5μMEDTApH 8.0(Gibco,目录号:15575-038)
    18. HEPES缓冲溶液1 M(Gibco,目录号:15630-080)
    19. Hyclone标准胎牛血清(FBS)(赛默飞世尔科技,目录号:SH30088.03)
    20. VIII型胶原酶(Sigma-Aldrich,目录号:C2139)
    21. DNase I(Sigma-Aldrich,目录号:D5025)
    22. Dispase(Corning,目录号:354235)
    23. Percoll(GE Healthcare,目录号:17-0891-01)
    24. IMDM,GlutaMAX补充剂(Thermo Fisher Scientific,目录号:31980097)
    25. Phorbol 12-myristate 13-acetate(PMA)(Sigma-Aldrich,目录号:P8139)
    26. 离子霉素(Sigma-Aldrich,目录号:I0634)
    27. Brefeldin A(BD Bioscience,目录号:555029)
    28. 染色缓冲液(BD,Pharmingen,目录号:554656)
    29. 转录因子缓冲液组(BD,Pharmingen,目录号:562725)
    30. 固定活力污渍510(BD,Horizon,目录号:564406)
    31. 小鼠BD Fc Block(BD,Pharmingen,目录号553141)
    32. 乙醇70%(Fisher Scientific,目录号:HC1500)
    33. 乙醇95%(Fisher Scientific,目录号:HC1300)
    34. 乙醇100%(Fisher Scientific,目录号:HC600)
    35. 二甲苯(Fisher Scientific,目录号:HC700)
    36. 石蜡(Fisher Scientific,目录号:23-0210-400)
    37. 苏木精(MasterTech Scientific,目录号:HXHHEGAL)
    38. Eosin(Thermo Scientific,目录号:71311)
    39. 胶原酶D(Sigma,目录号:C5138-1G)
    40. 上皮细胞解离解决方案(见食谱)
    41. 酶消化混合物(见食谱)
    42. 完整的IMDM媒体(参见食谱)

设备

  1. Scalpel(Southmedic,目录号:SMI1 / 73-0121)
  2. 移液器(Thermo Scientific)
  3. 镊子和剪刀(精细科学工具)
  4. 无菌细胞培养罩
  5. 离心机(赛默飞世尔科技)
  6. 摇水浴(精密)
  7. 明场显微镜
  8. LSRII流式细胞仪(BD Biosciences)
  9. 恒温箱
  10. Vortex(科学工业)

软件

  1. FlowJo_V10(FlowJo,LLC, https://www.flowjo.com )
  2. GraphPad Prism 7(GraphPad, https://www.graphpad.com )
  3. Microsoft Excel(Microsoft)

程序

  1. Rag 2 - / - 小鼠结肠炎的诱导
    1. 在无菌1x PBS中将抗CD40抗体或同种型对照稀释至1mg / ml。
    2. 根据研究人员的技术专长,用1 ml无菌sub-Q注射器26 G注射100μg抗CD40或同种型对照抗体腹膜内(ip)或50μg静脉注射(iv) - 每批抗CD40抗体应该被证实不含内毒素(通过与制造商核实)以避免全身免疫激活。
      注意:
      1. 每个动物设施都有特定的微生物环境,因此通过初始剂量滴定(25-100μg)凭经验确定抗CD40的剂量至关重要(图2)。
      2. 过高的剂量会导致动物死亡,而剂量不足会导致疾病诱导不良(结肠炎症不一致或不良)和结肠炎发展的变异。&nbsp;
      3. 每天对小鼠进行连续七天的称重,并计算与第0天的初始体重相比的体重减轻%。在24小时内对失去超过初始体重的20%或表现出摇晃或严重全身反应的小鼠实施安乐死。小时。
      4. 结肠炎的体重减轻动力学可能在i.p.之间变化。和i.v.注射类型。在我们手中,小鼠在i.p.后更快地恢复体重。交货。然而,注射途径不会改变结肠炎发展的时间或严重程度。
    3. 在诱导后第2-7天,根据批准的伦理方案通过二氧化碳窒息使小鼠安乐死。
    4. 通过心脏穿刺收集血清,用于检测全身细胞因子水平。


      图2.抗-CD40抗体的滴定。 Rag 2 - / - 小鼠静脉内接受单剂量的抗-CD40抗体。如..所示。 7天内初始重量的百分比。在注射抗CD40抗体之前立即测量初始重量。

  2. 用于终点分析的结肠组织/细胞的分离和制备
    1. H&amp; E染色的组织病理学分析
      1. 通过用镊子轻轻地从结肠中移出来收集结肠并去除粪便内容物。将结肠卷入组织包埋盒中,其近端位于中心,并在10%中性缓冲福尔马林中固定过夜(盒子需要浸没)。
      2. 将盒子转移到70%乙醇中,以4-5μm的浓度包埋在石蜡中,然后用苏木精染色10分钟,并如前所述用曙红染色(Wang et al。,2017)。
      3. 用自来水冲洗直至水清澈。
      4. 将载玻片转移至Eosin溶液并孵育3分钟。
      5. 依次将载玻片转移到70%乙醇中20秒,90%乙醇20秒,100%乙醇1分钟,二甲苯3分钟。
      6. 从二甲苯中取出载玻片,在通风橱中将其擦干。
      7. 安装并覆盖幻灯片。
    2. 近端结肠的基因表达谱分析
      1. 在诱导结肠炎后的所需时间点,根据批准的伦理方案,通过二氧化碳窒息使小鼠安乐死。
        注意:为了分析编码由先天免疫细胞产生的细胞因子的基因,我们建议在诱导后第1天至第3天对小鼠实施安乐死。为了分析由炎症引起的上皮细胞表达的基因,我们建议在诱导后第7天对疾病最大化时对小鼠进行安乐死。
      2. 收获一块近端结肠(约0.5厘米长),用镊子轻轻地从结肠中取出粪便,转移到无菌聚丙烯管中,在液氮中快速冷冻(图3B)。由于炎症浸润可以变化,因此从盲肠基部开始切割结肠的垂直部分并下降到近端结肠以确保一致的分子特征是很重要的。
        注意:为避免RNA降解,样品必须在安乐死后2分钟内在液氮中快速冷冻。冷冻近端结肠可以在-80°C下保存至少6个月。
      3. 如Bauche et al。,2018中所述提取RNA并运行RT-PCR方案。
    3. 近端结肠固有层细胞的分离,离体刺激和流式细胞术
      近端结肠固有层细胞的分离
      1. 诱导后2-7天,根据批准的伦理方案,通过二氧化碳窒息杀死小鼠。
      2. 在装有5毫升1x HBSS的15毫升Falcon试管中收集并收集近端结肠。&nbsp;
      3. 去除脂肪(图3A)。
      4. 将结肠转移到培养皿中。
      5. 将结肠纵向切开并浸没并在50ml 1x HBSS中轻轻摇动直至组织中没有粪便物质进行洗涤。&nbsp;
      6. 切成0.5厘米的部分。&nbsp;
      7. 置于含有20ml上皮细胞解离溶液的50ml Falcon管中。&nbsp;
      8. 用封口膜包裹,在37℃的水浴中侧向放置20分钟,中等摇动。
      9. 涡旋15秒。
      10. 通过70微米过滤器过滤,收集并转移未消化的结肠组织,放入新的50毫升falcon管中。&nbsp;
      11. 用20ml 1x HBSS冲洗片段,轻轻涡旋10秒。
      12. 将结肠碎片转移到培养皿中,用手术刀切成小块。
      13. 使用25ml血清移液管,转移到具有10ml酶消化混合物的新的50ml管中,然后在37℃的水浴中如上所述摇动孵育20分钟。
      14. 涡旋15秒。
      15. 通过70μm细胞过滤器过滤收集sup并置于冰上。在375 x g ,4℃下沉淀细胞5分钟。
      16. 制备100%Percoll溶液(9份Percoll + 1份10x PBS)。
      17. 通过用1x PBS稀释100%Percoll制备40%和80%Percoll溶液。
      18. 在15ml管中,使用5ml血清移液管将沉淀重悬于5ml 40%Percoll中,并在每个结肠中用5ml 80%Percoll垫层。
      19. 在20℃下旋转670 x g 20分钟,没有加速且没有制动。
      20. 将两层percoll之间的界面(白色环)收集到15ml管中,用10ml完全IMDM培养基洗涤并在375℃下旋转5分钟,4℃。&nbsp;
      21. 重悬于1毫升完全IMDM中,细胞准备就绪。
      注意:界面的特征是白色环,主要包含结肠固有层免疫细胞。应在此步骤测量细胞数和活力。

      离体刺激
      1. 对于细胞因子分析,在96孔板中在37℃,5%CO 2 50 ng / ml刺激2 x 10 6 活细胞/孔在200μl完全IMDM中,PMA,500ng / ml离子霉素和布雷菲德菌素A(1 / 1,000)。
      2. 在375 x g ,4℃下旋转5分钟并进行流式细胞术染色。

      流式细胞仪染色
      1. 将细胞在100μl含有0.3μl活/死可固定死细胞染色剂的1x PBS中孵育,每孔加入5μl小鼠BD Fc嵌段,在黑暗中于4°C孵育25分钟。
      2. 每孔用100μl1xPBS洗涤并在375 x g 下旋转5分钟。
      表面染色:
      1. 将细胞悬浮液与0.2μg抗小鼠缀合的抗体(即,CD45,CD3,CD4)在每孔100μlBD染色缓冲液中孵育25分钟,在黑暗中进行25分钟。
      2. 用100μlBD染色缓冲液洗涤并在375 x g 下旋转5分钟。
      用于细胞内染色:
      1. 将细胞悬浮液重悬于100μl1xFix / perm缓冲液(BD)中,并在4℃下在黑暗中孵育45分钟。
      2. 用1x Perm /洗涤缓冲液洗涤并在375 x g 下旋转5分钟。
      3. 将细胞悬浮液与0.2μg抗小鼠缀合的抗体(即,RORγt,IL-22)在每孔100μl的1x Perm / Wash缓冲液中孵育45分钟,在黑暗中于4℃孵育45分钟。
      4. 用1x Perm /洗涤缓冲液洗涤并在375 x g 下旋转5分钟。&nbsp;
      5. 将细胞重悬于300μl的1x Perm / Wash缓冲液中。使用流式细胞仪分析和获取数据(我们使用来自BD的LSRII流式细胞仪并分析FlowJo上的数据)。样品可以在4°C的黑暗中储存过夜。


        图3.结肠组织的制备。 :一种。必须从结肠中除去附着的脂肪组织(此处用箭头表示),因为脂肪会降低分离的结肠固有层的活力。 B. Taqman取样:将结肠与盲肠分开,通过去除所有脂肪和结缔组织,从宿主中仔细解剖。从近端(盲肠)端切下结肠,沿中线向下切直到远端。将纸巾弄平,切成2-3厘米的条状(粉红色方形),并立即用液氮中的聚丙烯管快速冷冻。

数据分析

  1. 病理学家根据三个标准对组织进行疾病严重程度评分(每个标准评分为0至3,0分=阴性,1 =轻度,2 =中度,3 =严重):
    炎症:当存在的特征在于大量(60%-70%)的单核细胞(巨噬细胞和淋巴细胞)和30%-40%的中性粒细胞和带状细胞的浸润。炎症的评分包括浸润的严重程度,腺体的损失,腺体腔的侵蚀和扩张,隐窝脓肿的存在和上皮细胞的退化。
    细胞凋亡:凋亡小体的患病率评分为0-3:0 =阴性; 1 =低; 2 =中等; 3 =高。
    再生:评估的再生变化包括评估粘膜上1/3中有丝分裂象的流行程度,个体腺体结构内的核密度(核拥挤),表面上皮的规律性。再生评分为0-3:0 =阴性; 1 =低; 2 =中等; 3 =高。
  2. 对于基因表达谱,平均来自同种型的标准化值
    使用以下公式计算同种型对照的倍数变化:

    倍数变化=(样品的标准化值)/(同种型对照组的平均标准化值)

  3. 对于流式细胞术分析,获得至少5×10 5个结肠固有层细胞
    1. 事件在FSC-A和SSC-A上进行门控,然后在FSC-A和FSC-H上进行门控以去除双峰。
    2. 排除细胞碎片和死细胞(使用LIVE / DEAD可固定染色剂)。
    3. 为了研究浸润性免疫细胞的表型,在CD45 + 细胞上进行门控。
    4. 用于分析产生IL-22的ILC3,活细胞上的门,谱系(CD11c,CD11b,NK1.1) - ,CD90.2 high ,CD45 int ,RORγt + ,IL-22 + 细胞。有关更多详细信息,可以在之前的出版物中找到门控策略(Bauche et al。,2018)。
  4. 使用Prism 7进行统计分析。未配对的 t - 测试用作统计测试。 P 值&lt; 0.05被认为具有统计学意义。

食谱

  1. 1.上皮细胞解离解决方案
    1x HBSS
    青霉素/链霉素(1,000 U / ml)
    5 mM EDTA
    10mM HEPES
  2. 酶消化混合物
    1x HBSS
    青霉素/链霉素(1,000 U / ml)
    10%FBS
    2 mg / ml VIII型胶原酶
    30 U / ml的DNase I
    50 U / ml的Dispase
    或者,当通过FACS分析时,1mg / ml的胶原酶D(Sigma)可用于保存细胞表面分子
  3. 完整的IMDM媒体,500毫升
    450毫升IMDM,GlutaMAX补充剂
    1,000 U / ml的青霉素/链霉素
    1 mM丙酮酸钠
    0.55mM的2-巯基乙醇
    50毫升热灭活FBS

致谢

我们要感谢Gil Asio和Danye Cheng处理结肠样本。我们感谢Lakshmanan Annamalai和Jennifer H. Yearley在分析和评分组织学样本方面的专业知识。 Wendy Blumenschein和Jeff Grein用于运行基因表达谱。

利益争夺

作者声明没有竞争性的经济利益。

伦理

将小鼠维持在无特定病原体的条件下,并在Merck Research Laboratories(MRL)动物设施Palo Alto中用过滤水保持在微型隔离器中。所有动物程序均由MRL的机构动物护理和使用委员会根据实验动物护理评估和认证协会(AALAC)的指导批准。

参考

  1. Bauche,D.,Joyce-Shaikh,B.,Jain,R.,Grein,J.,Ku,KS,Blumenschein,WM,Ganal-Vonarburg,SC,Wilson,DC,McClanahan,TK,Malefyt,RW,Macpherson, AJ,Annamalai,L.,Yearley,JH和Cua,DJ(2018)。 LAG3 + 调节性细胞抑制产生白细胞介素-23的cx3cr1 Immunity 49(2):342-352:e345。
  2. Cayatte,C.,Joyce-Shaikh,B.,Vega,F.,Boniface,K.,Grein,J.,Murphy,E.,Blumenschein,WM,Chen,S.,Malinao,MC,Basham,B., Pierce,RH,Bowman,EP,McKenzie,BS,Elson,CO,Faubion,WA,Malefyt Rde,W.,Kastelein,RA,Cua,D.,McClanahan,TK and Beaumont,M。(2012)。 炎症性肠病中il-23途径治疗反应的生物标志物。 Clin Transl Gastroenterol 3:e10。
  3. Ng,SC,Shi,HY,Hamidi,N.,Underwood,FE,Tang,W.,Benchimol,EI,Panaccione,R.,Ghosh,S.,Wu,JCY,Chan,FKL,Sung,JJY和Kaplan, GG(2017)。 在21 st 世纪的炎症性肠病的全球发病率和患病率:对基于人群的研究进行系统评价。 Lancet 390(10114):2769-2778。&nbsp;
  4. Uhlig,HH,McKenzie,BS,Hue,S.,Thompson,C.,Joyce-Shaikh,B.,Stepankova,R.,Robinson,N.,Buonocore,S.,Tlaskalova-Hogenova,H.,Cua,DJ和Powrie,F。(2006)。 IL-12和IL-23在粘膜和系统性先天免疫病理中的差异活性。 Immunity 25(2):309-318。
  5. Wang,C.,Yue,F。和Kuang,S。(2017)。 使用免疫荧光染色,使用H&amp; E染色和肌纤维类型分类进行肌肉组织学表征。 Bio -protoco l 7(10):e2279。

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Joyce-Shaikh, B., Cua, D. J. and Bauché, D. (2019). Induction and Analysis of Anti-CD40-induced Colitis in Mice. Bio-protocol 9(3): e3153. DOI: 10.21769/BioProtoc.3153.
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