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CAR promotes hematopoietic regeneration
Competitive reconstitution analysis
CD45.2 donor bone marrow cells (3×105 unless otherwise indicated) were mixed with an equal number of freshly isolated CD45.1 competitor BM cells, and the mixture was injected intra- venously via the retro-orbital route into 6- to 9-week-old CD45.1 mice that had been previously irradiated with a total dose of 10 Gy, essentially as we described.18-20 BM cells (1x106) collected from primary recipients were used for the secondary transplantation. In order to measure reconstitution of transplant- ed mice, peripheral blood was collected at the indicated times after transplantation and the presence of CD45.1+ and CD45.2+ cells in lymphoid and myeloid compartments was measured.
5-fluorouracil treatment
5-FU was administered intraperitoneally to female wild-type (WT) or CAR conditional knockout (cKO) mice at a dose of 150, 250, or 300 mg/kg. In order to test Notch signaling, DAPT (Selleckchem, cat. #S2215) was injected at 10 mg/kg. Differences in survival of the two groups were analyzed using a log-rank test.
Retrovirus infection
LNX1 and LNX2 were cloned from cDNA of mouse intestine. The dominant negative forms (DN-LNX1 and DN-LNX2) were sub cloned 21 and retroviral plasmids MSCV-DN-LNX1-IRES- GFP and MSCV-DN-LNX2-IRES-GFP were constructed using standard methods. The retroviral plasmids with PCL-ECO (2:1) were transfected using PolyJet (SignaGen) into 293 T cells. The resulting retroviral supernatant was collected 48-72 hours later and used for infection. At 6 days after 5-FU injection, the Lin- cells from the BM of CAR cKO mice were isolated, and the Lin- cells were resuspended in viral supernatants (1×105 cells/mL) with 4 mg/mL polybrene and centrifuged at 2,000 rpm for 2 hours before culturing for 24 hours in StemSpan (StemCell Technologies) in the presence of 10 ng/mL stem cell factor, 20 ng/mL thrombopoietin, and 10 ng/mL interleukin 3. Cells were then resuspended in viral supernatant for another round of infec- tion. After 24 hours, green fluorescent protein positive (GFP+) cells were isolated for repopulation assay.
Statistics
Statistical significance of differences was assessed using 2- tailed Student’s t-test. Animal survival analysis was assessed with long-rank test. A P-value of 0.05 or less was considered sig- nificant. Values are reported as mean ± standard error of the mean.
Results
CAR expression is elevated in stressed hematopoietic stem cells
We compared CAR mRNA levels in HSC, hematopoi- etic progenitors, and differentiated hematopoietic cells before and after stress. As reported previously,22 CAR is expressed in differentiated cells and not stem cells or hematopoietic progenitors with the exception of granu- locyte-monocyte progenitor (GMP) cells (Figure 1A). Shortly after treatment of mice with 5-FU or after BM transplantation, CAR expression was greatly enhanced in phenotypical LT-HSC (identified as Lin-Sca1+cKit+CD135- CD34- or LSKFC) compared to untreated mice (Figure 1B). CAR expression returned to the original low level at the later time point (Figure 1B). However, ST-HSC, mul- tipotent hematopoietic progenitors (MPP) and
hematopoietic progenitors did not increase CAR expres- sion after 5-FU treatment (Online Supplementary Figure 1). In order to prove CAR is expressed on plasma membrane of HSC, we stained BM cells from 5-FU treated mice with anti-mCAR. Real-time qantitative polymerase chain reaction (RT-qPCR) and CAR cKO BM cells both prove the specific binding of the anti-mCAR (Online Supplementary Figure 2; Figure 1B). The surface CAR expression was detected in LSK population after 5-FU treatment (Figure 1C). Competitive repopulation assay with sorted CAR negative and positive expressed LSKFC cells from 5-FU treated mice indicates that both subpop- ulations had similar repopulation abilities (Figure 1D and E; Online Supplementary Figure 3). These results imply that CAR plays a role in HSC during regeneration induced by stress.
CAR supports hematopoietic regeneration after injury
CAR deletion in mice is embryonically lethal.23 In order to study the function of CAR, CARloxp/loxp mice were cross- bred with UBC-CreERT2 mice. In the resulting mice, global knockout (KO) of CAR can be induced with tamox- ifen treatment; these mice are thereafter referred to as CAR conditional KO (cKO) mice. The hematopoietic sys- tem is damaged by treatment with the chemotherapy drug 5-FU.24 CAR cKO mice were more sensitive to 5-FU treatment and died faster. After administration of 300 mg/kg 5-FU, 60% of CAR cKO mice died within 2 weeks, whereas all WT mice (which were UBC- CreERT2/CARwt/wt mice with tamoxifen treatment) sur- vived (Figure 2A). This indicates that CAR is important in the response to injury of the hematopoietic system. In order to compare kinetics of hematopoietic system recov- ery in WT and cKO mice, we treated mice with 250 mg/kg 5-FU, a dose that does not cause death of the WT mice. The complete blood count (CBC) data indicated CAR cKO mice had a slower recovery, although there was not a significant difference in hematopoietic cell counts between WT and CAR cKO mice before 5-FU treatment. On day 8 after 5-FU treatment, total white blood cell (WBC) and individual lineage cell numbers (including neutrophils, monocytes, eosinophils, basophils, and lymphocytes) had decreased relative to pretreatment levels. Numbers rebounded more rapidly in WT mice than in the CAR cKO mice. On day 14 post 5- FU treatment there were an average of 12.8x103 ±1.5 WBC per μL in WT mice and only 6.2x103±0.4 WBC per μl in cKO mice (Figure 2B). On day 17 post treatment, the CAR cKO mice and WT mice had equivalent WBC counts of about 12x103 per μL. A significant difference in neu- trophil numbers between WT and CAR cKO mice was detected on day 12 (Figure 2B). Red blood cell (RBC) num- bers decreased more slowly than WBC counts and the lowest cell counts were detected on day 14 in both WT and CAR cKO mice. Although there were no significant differences detected before day 14, the WT mice had sig- nificant more RBC than cKO mice in peripheral blood between day 14 and 17 (Figure 2B). Specifically, Mac1+ cells and B220+ cells had significantly faster recovery in WT mice than in CAR cKO mice after 5-FU treatment (Online Supplementary Figure 4). These results indicate that CAR cKO mice produce hematopoietic blood cells more slowly after 5-FU treatment, which explains why mice lacking CAR die after a dose of 5-FU that is sub-lethal dose to WT mice.
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