Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, The University of California Los Angeles, Los Angeles
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Koon, H. W., Ho, S., Cheng, M., Ichikawa, R., Pothoulakis, C. Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation. J. Vis. Exp. (68), e4208, doi:10.3791/4208 (2012).
To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.
1. Before-you-start Technical Considerations
|Group||Bone marrow donor to recipient||Treatment|
|A||BM exchange||CD45.1 WT to CD45.2 KO||DSS colitis|
|B||Water normal control|
|C||BM exchange||CD45.2 KO to CD45.1 WT||DSS colitis|
|D||Water normal control|
|E||Sham||CD45.1 WT to CD45.1 WT||DSS colitis|
|F||Water normal control|
|G||Sham||CD45.2 KO to CD45.2 KO||DSS colitis|
|H||Water normal control|
* A brief illustration of experimental protocol is shown in Figure 1.
2. Recipient Mice Irradiation
3. Donor Bone Marrow Extraction
4. Counting Bone Marrow Donor Cells
5. Infusion to Recipient Mice
6. Induction of Colitis and Evaluation of Colitis
7. Quality Inspection of Bone Marrow Transplantation by Flow Cytometry
#1 No antibody
#2 30 μl FITC isotype control + 30 μl PE isotype control + 15 μl CD16/32 blocking
#3 30 μl FITC CD45.1 Ab + 15 μl CD16/32 blocking
#4 30 μl PE CD45.2 Ab + 15 μl CD16/32 blocking
#5 30 μl FITC CD45.1 Ab + 30 μl PE CD45.2 Ab + 15 μl CD16/32 blocking
|Add nothing to blood or bone marrow sample #1|
|Add 5 μl of antibody mixture #2 to each blood or bone marrow sample #2|
|Add 3 μl of antibody mixture #3 to each blood or bone marrow sample #3|
|Add 3 μl of antibody mixture #4 to each blood or bone marrow sample #4|
|Add 5 μl of antibody mixture #5 to each blood or bone marrow sample #5|
If the gene-of-interest plays a significant role in immune cells during development of colitis, the mice receiving bone marrow of different genotype through bone marrow transplantation (WT to KO or KO to WT) should have an altered response to DSS colitis. One of the most important parameters for determining the severity of colitis is the H&E staining of the colonic tissues. Colonic tissue structure changes and signs of inflammation can be quantitatively evaluated by H&E histology scoring system. Criteria for H&E histology scoring for DSS colitis model can found here 2. Alternatively, chemical induced colitis can also be induced by trinitrobenzene sulfonic acid (TNBS). Methods of TNBS colitis induction and H&E histology scoring criteria can be found in a previous publication 3. Histology score difference between groups can be analyzed by student's t-tests.
The mice may have significantly ameliorated or worsened colitis when compared to mice with sham bone marrow transplantation (WT to WT or KO to KO). If the gene-of-interest plays a significant role in colitis via bone marrow derived cells, the mice with exchanged bone marrow should respond significantly different from the mice with sham bone marrow transplantation.
For DSS colitis model, histology change may be evaluated by histology scoring (see Figure 4). Significant change of histology score may indicate the development of colitis mediated by the gene-of-interest in bone marrow derived cells. For example, cathelicidin is an anti-microbial and anti-inflammatory peptide gene (gene-of-interest in our case) 4. Without bone marrow transplantation, cathelicidin KO mice generally developed worse colitis than wild-type mice did in response to DSS. Transfusion of wild-type (WT) bone marrow to cathelicidin knockout (KO) mice leads to ameliorated colitis while transfusion of bone marrow from cathelicidin knockout (KO) mice to wild-type (WT) mice leads to worsened colitis when exposed to DSS (Figure 2).
To verify expression of gene-of-interest, mRNA expression of gene-of-interest (e.g. cathelicidin) from donor wild-type bone marrow should be detectable in the colonic (or other) tissues of the cathelicidin deficient knockout recipient mice after bone marrow transplantation. It is also interesting to know whether the expression of gene-of-interest in wild-type recipient mice is reduced after donation of knockout mice bone marrow.
Successful engraftment of donor bone marrow is represented by dominant ratio of donor CD isotype over recipient CD isotype in both peripheral blood cells and bone marrow of recipient mice. Bone marrow shows best labeling of CD45.1 and CD45.2 in flow cytometry as blood has a lot of unstained cells (Figure 2 and 3).
Figure 1. The experimental protocol of bone marrow transplantation.
Figure 2. Flow cytometry data of bone marrow of recipient mice after bone marrow transplantation. The Y-axis shows FITC-labeled CD45.1 signal and X-axis shows PE-labeled CD45.2 signal. Successful bone transplantation is defined by the change of CD45.1 or CD45.2 genotype in both bone marrow and blood of the recipient mice. Click here to view larger figure.
Figure 3. Flow cytometry data of blood of recipient mice after bone marrow transplantation. The Y-axis shows FITC-labeled CD45.1 signal and X-axis shows PE-labeled CD45.2 signal. Successful bone transplantation is defined by the change of CD45.1 or CD45.2 genotype in both bone marrow and blood of the recipient mice. Click here to view larger figure.
Figure 4. Evaluation of colitis in mice after bone marrow transplantation. (A) Sample H&E images of colons with normal histology and DSS colitis. (B) Histology scores. Successful induction of DSS colitis can be confirmed by significant increase of histology score by day 5 of DSS treatment. After the exchange of bone marrow to different genotype, the histology score should change significantly. This suggests the altered course of colitis by the gene-of-interest expressing in the bone marrow derived cells. Data are represented by mean ± standard error of means.
This bone marrow transplantation approach is suitable for immunology research of colitis, infection, cancer, obesity and other diseases. This bone marrow transplantation experiment is needed when the gene-of-interest is expressed in both bone marrow derived and non-bone marrow derived cells and the gene-of-interest is suspected to mediate disease by cells from either population. For example, antimicrobial peptide cathelicidin is shown to modulate acute colitis. But it is expressed in both epithelial cells and immune cells (such as macrophages). Then we used bone transplantation to define which population of cells modulates acute colitis in mice. The colitis severity was significantly altered after the change of bone marrow cathelicidin genotypes via bone marrow transplantation. Then we can conclude that cathelicidin expressed in bone marrow derived cells plays a significant role in modulating acute colitis in response to DSS.
Bone marrow derived cells typically include red blood cells, white blood cells and platelets. Bone marrow transplantation changes the genotype of these cells but not others. Laterally speaking, this experiment can only differentiate the role of gene-of-interest in bone marrow derived cells versus non-bone marrow derived cells. But this experiment cannot further define which population of bone marrow derived cells mediates the colitis as all red blood cells, white blood cells and platelets are derived from bone marrow. The fate of bone marrow derived cells migrated to colons during colitis is an active and controversial area of research. After bone marrow transplantation and induction of murine colitis, it is likely that some of bone marrow derived cells exist as lymphoid cells and myofibroblasts in the colons 5. Another report suggested that bone marrow derived cells serve as endothelial progenitor cells for neovascularization in recovery process 6. There is also evidence showing bone marrow derived cells are associated with epithelial cell differentiation in patients with colitis 7. We cannot exclude the possibility that some of bone marrow derived cells may differentiate to cells other than typical immune cells and play modulating roles in colitis development. Nevertheless, bone marrow derived monocyte/macrophage population is important for the protection against chemical induced colonic mucosal damage 8,9. This report is consistent with our finding that cathelicidin expressed from bone marrow derived cells modulates DSS colitis while cathelicidin is secreted from monocytes/macrophages 4.
On the other hand, there are many ways to track the success of bone marrow transplantation. The flow cytometry analysis of CD45.1 and CD45.2 genotypes can provide a quantitative method to determine the proportion of donor bone marrow stem cell derived cells in the recipient mice. Bone marrow derived cells can differentiate into multiple kinds of cells 10. When flow cytometry analysis is not feasible, it is possible to use male (XY chromosome) donor mice and female (XX chromosome) recipient mice 11. The donor mice will carry unique Y chromosomes in the body of XX only female recipient mice. Y chromosome can be identified by fluorescent in situ hybridization 11. In addition, immunohistochemistry of CD45.1, CD45.2 and/or the gene-of-interest in the tissues may be necessary to visualize the donor bone marrow derived cells.
But CD45 flow cytometry analysis and Y chromosome in situ hybridization procedures are usually done after the mice were sacrificed. To monitor the location of donor cells in the recipient mice being used to study chronic diseases like cancer without sacrificing the mice, it is possible to use green fluorescent protein transgenic mice as donor mice 12. Therefore, the donor bone marrow derived cells can be tracked in the body of the recipient mice under non-invasive high resolution optical imaging under transient anesthesia and this can be done repeatedly.
Not all mice have successful bone marrow transplantation 13. We observed ~10-20% of mice die in the first 2 weeks after irradiation due to anemia or infection. Therefore, more mice than needed should be prepared at the start of experiment. For example, you should prepare 10 mice per group at the start of experiment if you expect 8 mice per group available at the end of the colitis experiment. Also, make sure you remove the dead mice as soon as possible. The speed of immune reconstitution is correlated to the number to hematopoietic stem cells in bone marrow infused into the recipient mice 14. Therefore, it is crucial to have sufficient number of live bone marrow cells (1 x 107 cells per mouse) infused into recipient mice for successful bone marrow transplantation.
Recipient mice after irradiation have compromised immunity. Donor mice dissection and bone marrow preparation procedures should be done in the same standard as cell culture experiments. Use of sterile instruments and containers, aseptic handling techniques should be applied. Throughout all experiments, PBS contains 1% Penicillin-streptomycin and 10 U/ml heparin should be used during the handling of bone marrows. All reagents are cell culture grade. Additional technical discussion can be found in reference 13.
No conflicts of interest declared.
This work was funded by Pilot and Feasibility Study grant from UCLA-CURE Center, the Crohn's and Colitis Foundation of America Career Development Award (#2691) and National Institute of Health NIDDK K01 (DK084256) funding to Hon Wai Koon.
Bone marrow irradiation operation was assisted by Bernard Levin and Scott Kitchen of UCLA Center for AIDS Research Mouse/Human Chimera Core facility. Flow cytometry operation was assisted by UCLA Vector Core facility.
|Cell staining buffer||Biolegend||#420201|
|10X RBC lysis buffer||Biolegend||#420301|
|FITC mouse isotype control||Biolegend||#400207|
|PE mouse isotype control||Biolegend||#400211|
|PE anti-mouse CD45.2||Biolegend||#109807|
|FITC anti-mouse CD45.1||Biolegend||#110705|
|Anti-mouse CD16/32 blocking||Biolegend||#101301|
|40 μm cell strainer||Fisherbrand||#22363547|
|PBS 1X||MP biomedicals||#1860454|
|Sulfatrim (SMZ)||Qualitest||NC9242720 (fisher)|
|Lysol IC||Andwin Scientific||NC9745686 (fisher)|
|Mouse restrainer||Braintree Scientific||#TV-150|
|Irradiator||J.L. Shepherd and Associates||Mark I 68A|
|Flow cytometer||BD||BD FACSCanto II|
|Flow cytometer test-tube||Falcon||#352052|
|Hemoccult ICT||Beckman Coulter||G0328QW|