HO-1 Activation In Bone-Mesenchymal Stem Cells For Treatment and Prevention Of Acute Graft-Versus-Host Disease In Mice
Authors: Ma Dan , Wang Jishi , Li Yan , Fang Qin , Sun Jia , Hu Xiuying 
Published: October 2019

Abstract

Objective
Bone marrow mesenchymal stem cells (BMSCs) were confirmed that have great potential in decreasing acute graft-versus-host disease(aGVHD) after allogeneic hematopoietic stem cells transplantation(allo-HSCT) in various clinical trails. However, the immuno-modulatory effects of BMSCs are not always successfully achieved in vivo. In this study, we aimed to proved that activation of heme oxygenase-1(HO-1) in BMSCs could significantly enhance the capacity on decreasing aGVHD in vivo and explored the relative mechanism.

Methods
We cloned mice’s HO-1 cDNA from mice bone marrow and constructed recombinant lenti-virus vectors (Lentivirus-V5-D-TOPO-HO-1-EGFP/Lentivirus-V5-D-TOPO-EGFP), which titer was 1×1011pfu/mL. These mouse BMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction. Then recombinant lenti-virus vectors were transferred into mouse BMSCs, and the expression of EGFP and HO-1 were detected by fluorescence microscope, RT-PCR and Western blot respectively. We established mice’s aGVHD model after allo-HSCT. Four groups were separated in vivo test (Group A: aGVHD control; Group B: aGVHD model injected into mouse BMSCs; Group C: aGVHD model injected into mouse BMSCs transfected with EGFP; Group D: aGVHD model injected into mBMSCs transfected with HO-1 gene). The survival time, body weight and clinical score of aGVHD in mice model were monitored. Liver, intestine and lung in each group were obtained for histological examination. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ and TNF-„p were also determined using a Cytometric Bead Array.

Results
In vitro, over-expression of HO-1 promoted the proliferation of BMSCs, the cell proliferation rate of BMSC transfected with lenti-virus-HO-1 was 1.25 folder higher over natural BMSCs(p<0.05).
In vivo, the immuno-suppressive capacity of BMSCs expressing HO-1 in a major histo-compatibility complex (MHC)-mismatched mouse model of bone marrow (BM) transplantation from C57BL/6 donors to BALB/c recipients. Treatment with HO-1 over-expressing BMSCs obviously decreased the animal mortality rate,60% mice in group BMSCs suffered from aGVHD and died, while 70% mice in group BMSCs-HO-1 were still alive, and expressed less clinical and pathological aGVHD scores. In addition, compared with other groups, the level of negative regulative cytokines, IL-2, IL-6, IFN-γ and TNF-„p in recipients injected with BMSCs-HO-1, were significantly decreased (FCBMSC-HO-1/BMSC=cytokines concentration detected in group infused BMSCs-HO-1/group infused BMSCs, if FCBMSC-HO-1/BMSC<1, means the cytokines concentration detected in group infused BMSCs-HO-1 decreased. Our study indicated that: FCBMSC-HO-1/BMSC of IL-2: 0.4±0.1; IL-6: 0.7±0.1; IFN-γ: 0.7±0.05; TNF-„p: 0.8±0.02. p<0.05), while those positively regulative cytokines, IL-4 and IL-10 were increased(FCBMSC-HO-1/BMSC>1, means the cytokines concentration detected in group infused BMSCs-HO-1 increased. Our study indicated that: FCBMSC-HO-1/BMSC of IL-4: 1.3±0.15; IL-10: 1.5±0.1; p<0.05). In field of Th1/Th2 cells proliferation, the value of CD8+ and CD4+ T cells and the ratio of Th1/Th2 T cell subsets decreased(CTBMSC-HO-1/BMSC= cells counts of group infused BMSCs-HO-1/group infused BMSCs, CTBMSC-HO-1/BMSC<1, means cells counts of group infused BMSCs-HO-1 decreased. Our data showed that, CTBMSC-HO-1/BMSC of CD8+ T cells: 0.25±0.09; CD4+ T cells:0.47±0.06; Th1/Th2 T cell subsets: 0.32±0.05; p<0.05) , at the same time the proportion of CD4+CD25+ T cells increased both in spleen lymphocytes in vivo after allo-HSCT with BMSCs expressing HO-1 compared with conventional allo-HSCT(CTBMSC-HO-1/BMSC of CD4+ CD25+ Foxp3+ cells:13.4±2.1; p<0.01. CTBMSC-HO-1/BMSC>1,means cells counts of group infused BMSCs-HO-1 increased).

Conclusion
our report strongly reveals that activation of HO-1 could enhance the ability of BMSCs to effectively alleviate aGVHD. The mechanism involved that the enhanced homing ability of BMSCs, immuno-suppressive ability to decrease negatively regulative cytokines, while to increase positive part. At last, BMSCs transfected lenti-virus-HO-1 expressed the potential ability to induce Treg cells proliferation.

Disclosures:
No relevant conflicts of interest to declare.