1. Volume 23, Issue 2, Pages 339-351 (February 2015)
Targeted 25-hydroxyvitamin D3 1α-hydroxylase Adoptive Gene Therapy Ameliorates DSS-induced Colitis Without Causing Hypercalcemia in Mice 
Bo Li, David J Baylink, Michael H Walter, Kin-Hing William Lau, Xianmei Meng, Jun Wang, Andriy Cherkas, Xiaolei Tang, Xuezhong Qin 
Molecular Therapy 
Volume 23, Issue 2, Pages (February 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Engraftment of CD11b+/Gr1+ monocytes at the inflamed colon of mice with dextran sulfate sodium (DSS)-induced colitis. (a and b) DSS colitis in 8-week-old C57BL/6 mice was induced as described in Materials and Methods. Gr1+ monocytes were isolated from C57BL/6 mice, labeled with far-red dye and injected, via tail vein, into colitis mice (5 × 106 cells per mouse) at day 7 of the DSS treatment. At day 8 or 10 (1 or 3 days post-cell infusion), mice were perfused with phosphate-buffered saline and the tissue samples were collected for imaging. (a) Ex vivo imaging of far-red dye labeled monocytes in frozen tissue sections at day 1 post-cell infusion. (b) Ex vivo imaging of far-red dye labeled monocytes in frozen tissue sections at day 3 post-cell infusion. Arrows indicate labeled monocytes in organs of injected mice. Images show ×100 original magnifications.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Mac1 promoter was able to yield high levels of transgene expression in activated macrophages. (a) The lentiviral vector contains a central polypurine tract (cPPT) for efficient nuclear entry, and self-inactivating deletion in the U3 region of the LTRs. The cis-acting partial Gag sequence (Gag) and Rev-response element (RRE) sequences are also indicated. WPRE (woodchuck hepatitis post-transcriptional regulatory element) element is included to enhance marker gene expression. (b) Sca1+ HSCs were isolated from bone marrow cells of C57BL/6 mice, transduced with the lentiviral vector Mac1-GFP-PGK-mCherry, and injected via tail vein into recipient mice. Five weeks post-bone marrow transplantation, peritoneal macrophages and splenocytes were isolated and further treated with 50 ng/ml phorbol 12-myristate 13-acetate for 16 hours. Expression of the marker gene, GFP, was visualized under fluorescent microscopy (×200 magnification). (c) Bone marrow CD11b+Gr1+ monocytes were isolated from the bone marrow transplants and then treated with macrophage colony-stimulating factor for 7 days. Expression of the GFP marker gene was visualized under fluorescent microscopy at day 3 and day 7 (×200 magnification).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Adoptive transfer of CD11b+/Gr1+ monocytes overexpressing CYP27B1 ameliorated dextran sulfate sodium (DSS)-induced colitis without causing hypercalcemia. (a) Six-week-old C57BL/6 mice were transplanted with Sca1+ HSCs transduced with the Mac1-CYP (Mac1-hCYP27B1-PGK-mCherry) or the Mac1-GFP (Mac1-GFP-PGK-mCherry) lentiviral vector. At the age of 8 weeks, each recipient mouse was treated with DSS to induce colitis. At day 7 of the DSS treatment, mice were injected via tail vein with 4 × 106 Gr1+ monocytes that were isolated from the bone marrow transplants. (b) Body weights were recorded during disease induction and recovery period. (c) Survival rate was recorded daily. (d) At day 13 (6 days post-monocytes infusion), mice were sacrificed and the colon length was measured. (e) Serum concentrations of calcium were measured 6 days post-monocytes infusion. Data from one of the two independent experiments are shown as means ± SEM (n = 5–7). (f) Representative H&E staining of distal colon cross-sections. All images are shown at 20× magnification. (g) Histological score was measured in cross-sections of the distal colon. Data are presented as means ± SEM (n = 10–12) pooled from two independent experiments. (h) Mucosal regeneration index was determined at cross-sections of the distal colon (Material and Methods). Data of the representative of the two independent experiments are presented as means ± SEM (n = 4–5). *P < 0.05, **P < 0.01, or ***P < versus DSS-treated mice. Mo, monocytes.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Adoptive transfer of CD11b+/Gr1+ M2 macrophages overexpressing CYP27B1 improved IBD pathology. (a and b) M2 macrophages were transduced with the indicated lentiviral vectors and were incubated with 2.5 μmol/l 25(OH)D3 for 20 hours (Materials and Methods). (a) Human CYP27B1 mRNA level was measured by real-time qRT-PCR. Data are shown as means ± SEM (n = 4). ***P < (b) 1,25(OH)2D3 concentration in the conditioned media pooled from triplicate samples was measured by radioimmunoassay. (c) Colitis induction in 8-week-old C57BL/6 mice and M2 macrophages adoptive transfer were performed described in Material and Methods. On day 13 (6 days post-macrophages infusion), mice were sacrificed, and the colon samples were collected. Representative H&E staining images of the frozen distal colon sections were shown. All images are shown at 20× magnification. (d) Histological score of colon sections was determined as described in Materials and Methods. Data are presented as means ± SEM (n = 5). (e) Mucosal regeneration index was determined as described in Material and Methods. Data are presented as means ± SEM (n = 4–5). **P < 0.01, or ***P < versus dextran sulfate sodium-treated mice. M2Ø, M2 macrophages.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Adoptive transfer of CD11b+/Gr1+ monocytes overexpressing CYP27B1 reduced the expression of inflammatory cytokines in the colon of mice with dextran sulfate sodium (DSS)-induced colitis. The experimental design was described in Figure 3. (a) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the mRNA levels of proinflammatory cytokines produced mainly by macrophages/dendritic cells. (b) Real-time qRT-PCR analysis of the mRNA level of IFN-γ, a pathogenic Th1-specific cytokine in the colon. (c) Real-time qRT-PCR analysis of the mRNA level of IL-17a, a pathogenic Th17-specific cytokine in the colon. Data of a representative experiment of two independent experiments are presented as means ± SEM (n = 4–6). *P < 0.05, **P < 0.01, or ***P < versus DSS-treated mice. Mo, monocytes.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Adoptive transfer of CD11b+/Gr1+ monocytes overexpressing CYP27B1 significantly increased expression of the protective Th2 cytokines. The experimental design was described in Figure 3. Colon and lymph node samples were collected 6 days post-monocytes infusion. (a) mRNA levels of Th2 cytokines in colon were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). (b) mRNA levels of Th2 cytokines in lymph nodes were measured by real-time qRT-PCR. Data are presented as means ± SEM (n = 4–6). *P < 0.05, **P < 0.01 versus dextran sulfate sodium-treated mice. Mo, monocytes.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Evidence that adoptive transfer of CD11b+/Gr1+ monocytes overexpressing CYP27B1 ameliorated dextran sulfate sodium (DSS)-induced colitis in part through restoring epithelial barrier function. (a and b) The experimental design was described in Figure 3. Colon tissues were collected 6 days post-monocytes infusion. (a,b) The mRNA level of two tight junction genes, claudin-1 and ZO-1, in colon was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data are presented as means ± SEM (n = 4–6). (c) Monolayers of Caco-2 cells were pretreated with or without 10 nmol/l 1,25(OH)2D3 for 48 hours. 5% DSS was then added to the media for 10 hours before the analysis of gene expression. The mRNA level of ZO-1 and claudin-1 in cultured epithelial cells was determined by real-time qRT-PCR. Data are presented as means ± SEM (n = 4). (d) The effects of 1,25(OH)2D3 on paracellular permeability of epithelial cell monolayers. Epithelial monolayers were obtained by culturing Caco-2 cells on filters in transwells and were treated as described in panel c. The permeability to fluorescein isothiocyanate-conjugated dextran is presented as % of the value in cells treated with DSS only. Data are presented as means ± SEM (n = 3). *P < 0.05, **P < 0.01, or ***P < versus DSS-treated group.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
A proposed model of the mechanism by which adoptive CYP27B1 gene therapy acts to ameliorate dextran sulfate sodium-induced colitis. Infused exogenous Gr1+ monocytes or M2 macrophages overexpressing CYP27B1 guided by the Mac1 promoter are recruited to the inflamed colon, where they are activated to express exogenous CYP27B1 to synthesize 1,25(OH)2D3 locally. 1,25(OH)2D3 produced by the activated exogenous macrophages then acts on T cells to cause a switch from the pathogenic Th1-Th17 response to the protective Th2 response. 1,25(OH)2D3 also acts on infiltrating macrophages leading to reduced expression of proinflammatory cytokines. As a result of the reduced inflammation, mucosal regeneration is expedited. In addition, 1,25(OH)2D3 also promotes mucosal regeneration via increasing epithelial tight junction protein synthesis and restoring epithelial barrier function.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions