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Immunosuppressive mechanisms of human bone marrow derived mesenchymal stromal cells in BALB/c host graft versus host disease murine models.

Robles JD, Liu YP, Cao J, Xiang Z, Cai Y, Manio M, Tang EH, Chan GC - Exp Hematol Oncol (2015)

Bottom Line: Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs.Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.These mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, The University of Hong Kong Li Ka Shing Faculty of Medicine, Queen Mary Hospital, 102 Pokfulam Rd., HKSAR, PRC.

ABSTRACT

Background: Mesenchymal stromal cells (MSCs) are proven to have immunosuppressive functions via various mechanisms. These mechanisms were demonstrated by administering bone marrow derived human MSCs (hMSCs) to graft versus host disease (GVHD) murine models.

Methods: BALB/c host mice were irradiated prior to receiving C57BL/6 donor T cell depleted bone marrow (TCDBM) cells (negative control) and donor CD4+ T lymphocyte with (treatment group) or without hMSCs (positive control). The presence of hMSCs in target tissues and lymphoid organs was documented by using in vivo imaging and measuring the expression of EphB2 and ephrin-B2 by RTqPCR. Survival rate and GVHD score were also monitored. Tissue sections were obtained for histopathologic analysis. Flow cytometry was used to document donor T cell alloreactivity and expression of CCR5, CXCR3 and CCR7. ELISA was utilized to determine levels of proinflammatory cytokines, RANTES (CCL5) and phosphorylated STAT 5A/B. RTqPCR was performed to quantify expression of CCL3 and CXCL9. Western blotting was performed to qualitatively measure iNOS expression.

Results: Survival rate and GVHD score improved with hMSC treatment. Pathologic changes of GVHD were abrogated. Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs. Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.

Conclusions: These mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.

No MeSH data available.


Related in: MedlinePlus

Chemokine expression in host spleen and target organs. (A) RANTES expression. (B) Expression of CCL3 and CXCL9. (C-D) Expression of CCR5, CXCR3 and CCR7. Lethally irradiated BALB/c host mice were given intravenous injections of 2 × 10^6 TCDBM cells from C57BL/6 donors with or without 0.25 × 10 ^6 CD4+ T cells from donors and 1 × 10 ^6 hMSCs. (A) Host spleen and colon were harvested 6d after transplantation and RANTES expression measured using ELISA (*p = 0.0356). Data were combined from 2 independent experiments with 3-4 mice in each experiment. (B) Expression of CCL3 and CXCL9 6d after transplantation in BALB/c host spleen, colon and lung were measured by RT-qPCR. Data were combined from 2 independent experiments with 4-6 mice. (C,D) Expression of CCR5, CXCR3 and CCR7 6d after transplantation in BALB/c host spleen were measured by using trypan blue cell exclusion assay and flow cytometry and expressed in absolute numbers of CD3+ CCR5+, CD3+ CXCR3+ (C) and CD3+ CCR7+ (D) splenocytes. Data were combined from 2 independent experiments with 2-4 mice in each experiment (*p<0.005).
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Fig6: Chemokine expression in host spleen and target organs. (A) RANTES expression. (B) Expression of CCL3 and CXCL9. (C-D) Expression of CCR5, CXCR3 and CCR7. Lethally irradiated BALB/c host mice were given intravenous injections of 2 × 10^6 TCDBM cells from C57BL/6 donors with or without 0.25 × 10 ^6 CD4+ T cells from donors and 1 × 10 ^6 hMSCs. (A) Host spleen and colon were harvested 6d after transplantation and RANTES expression measured using ELISA (*p = 0.0356). Data were combined from 2 independent experiments with 3-4 mice in each experiment. (B) Expression of CCL3 and CXCL9 6d after transplantation in BALB/c host spleen, colon and lung were measured by RT-qPCR. Data were combined from 2 independent experiments with 4-6 mice. (C,D) Expression of CCR5, CXCR3 and CCR7 6d after transplantation in BALB/c host spleen were measured by using trypan blue cell exclusion assay and flow cytometry and expressed in absolute numbers of CD3+ CCR5+, CD3+ CXCR3+ (C) and CD3+ CCR7+ (D) splenocytes. Data were combined from 2 independent experiments with 2-4 mice in each experiment (*p<0.005).

Mentions: Chemokine receptors expressed by donor T cells and chemokine ligands expressed by GVHD target tissues play a critical role in donor T cell alloreactivity in GVHD [19]. In terms of the ligands, a significant decrease in expression of RANTES (CCL5) in host splenocytes (p = 0.0356) but not in the colon (p = 0.3214) was noted (Figure 6A). Analysis via RT-qPCR of chemokine ligands CCL3 and CXCL9 was also performed. Suppression of these ligands in the host spleen, colon and lungs was noted with hMSC treatment (Figure 6B). Moreover, the number of cells expressing the corresponding receptors CCR5 (receptor for CCL3 and RANTES) and CXCR3 (receptor for CXCL9) are also significantly decreased in the spleen with hMSC treatment (Figure 6C).Figure 6


Immunosuppressive mechanisms of human bone marrow derived mesenchymal stromal cells in BALB/c host graft versus host disease murine models.

Robles JD, Liu YP, Cao J, Xiang Z, Cai Y, Manio M, Tang EH, Chan GC - Exp Hematol Oncol (2015)

Chemokine expression in host spleen and target organs. (A) RANTES expression. (B) Expression of CCL3 and CXCL9. (C-D) Expression of CCR5, CXCR3 and CCR7. Lethally irradiated BALB/c host mice were given intravenous injections of 2 × 10^6 TCDBM cells from C57BL/6 donors with or without 0.25 × 10 ^6 CD4+ T cells from donors and 1 × 10 ^6 hMSCs. (A) Host spleen and colon were harvested 6d after transplantation and RANTES expression measured using ELISA (*p = 0.0356). Data were combined from 2 independent experiments with 3-4 mice in each experiment. (B) Expression of CCL3 and CXCL9 6d after transplantation in BALB/c host spleen, colon and lung were measured by RT-qPCR. Data were combined from 2 independent experiments with 4-6 mice. (C,D) Expression of CCR5, CXCR3 and CCR7 6d after transplantation in BALB/c host spleen were measured by using trypan blue cell exclusion assay and flow cytometry and expressed in absolute numbers of CD3+ CCR5+, CD3+ CXCR3+ (C) and CD3+ CCR7+ (D) splenocytes. Data were combined from 2 independent experiments with 2-4 mice in each experiment (*p<0.005).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Chemokine expression in host spleen and target organs. (A) RANTES expression. (B) Expression of CCL3 and CXCL9. (C-D) Expression of CCR5, CXCR3 and CCR7. Lethally irradiated BALB/c host mice were given intravenous injections of 2 × 10^6 TCDBM cells from C57BL/6 donors with or without 0.25 × 10 ^6 CD4+ T cells from donors and 1 × 10 ^6 hMSCs. (A) Host spleen and colon were harvested 6d after transplantation and RANTES expression measured using ELISA (*p = 0.0356). Data were combined from 2 independent experiments with 3-4 mice in each experiment. (B) Expression of CCL3 and CXCL9 6d after transplantation in BALB/c host spleen, colon and lung were measured by RT-qPCR. Data were combined from 2 independent experiments with 4-6 mice. (C,D) Expression of CCR5, CXCR3 and CCR7 6d after transplantation in BALB/c host spleen were measured by using trypan blue cell exclusion assay and flow cytometry and expressed in absolute numbers of CD3+ CCR5+, CD3+ CXCR3+ (C) and CD3+ CCR7+ (D) splenocytes. Data were combined from 2 independent experiments with 2-4 mice in each experiment (*p<0.005).
Mentions: Chemokine receptors expressed by donor T cells and chemokine ligands expressed by GVHD target tissues play a critical role in donor T cell alloreactivity in GVHD [19]. In terms of the ligands, a significant decrease in expression of RANTES (CCL5) in host splenocytes (p = 0.0356) but not in the colon (p = 0.3214) was noted (Figure 6A). Analysis via RT-qPCR of chemokine ligands CCL3 and CXCL9 was also performed. Suppression of these ligands in the host spleen, colon and lungs was noted with hMSC treatment (Figure 6B). Moreover, the number of cells expressing the corresponding receptors CCR5 (receptor for CCL3 and RANTES) and CXCR3 (receptor for CXCL9) are also significantly decreased in the spleen with hMSC treatment (Figure 6C).Figure 6

Bottom Line: Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs.Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.These mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, The University of Hong Kong Li Ka Shing Faculty of Medicine, Queen Mary Hospital, 102 Pokfulam Rd., HKSAR, PRC.

ABSTRACT

Background: Mesenchymal stromal cells (MSCs) are proven to have immunosuppressive functions via various mechanisms. These mechanisms were demonstrated by administering bone marrow derived human MSCs (hMSCs) to graft versus host disease (GVHD) murine models.

Methods: BALB/c host mice were irradiated prior to receiving C57BL/6 donor T cell depleted bone marrow (TCDBM) cells (negative control) and donor CD4+ T lymphocyte with (treatment group) or without hMSCs (positive control). The presence of hMSCs in target tissues and lymphoid organs was documented by using in vivo imaging and measuring the expression of EphB2 and ephrin-B2 by RTqPCR. Survival rate and GVHD score were also monitored. Tissue sections were obtained for histopathologic analysis. Flow cytometry was used to document donor T cell alloreactivity and expression of CCR5, CXCR3 and CCR7. ELISA was utilized to determine levels of proinflammatory cytokines, RANTES (CCL5) and phosphorylated STAT 5A/B. RTqPCR was performed to quantify expression of CCL3 and CXCL9. Western blotting was performed to qualitatively measure iNOS expression.

Results: Survival rate and GVHD score improved with hMSC treatment. Pathologic changes of GVHD were abrogated. Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs. Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.

Conclusions: These mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.

No MeSH data available.


Related in: MedlinePlus