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Alleviation of skin inflammation after Lin(-) cell transplantation correlates with their differentiation into myeloid-derived suppressor cells.

Jeong Ryu S, Ju JM, Kim W, Bum Kim M, Hee Oh K, Sup Lee D, Lee H, Eun Oh J, Soo Park K, Young Choi E - Sci Rep (2015)

Bottom Line: Longitudinal in vivo imaging and flow cytometry analyses revealed that Lin(-) cells home directly to inflamed skin within 6 h, where they undergo extensive expansion with the peak on day 14 post-transplantation, and preferential differentiation into CD11b(+)Ly6G(int)Ly6C(+) cells by day 7.Cells with phenotypic profiles of neutrophils, macrophages, and DCs appeared in inflamed skin on day 14.Progenies of transplanted Lin(-) cells showed similar kinetics of expansion and myeloid differentiation in BM.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
To understand the cellular mechanism underlying the therapeutic effects exerted by hematopoietic stem cell transplantation in the repair of tissue damage, we investigated the in vivo dynamics of bone marrow (BM) lineage-negative (Lin(-)) cells transplanted into mice with hyper sensitivity dermatitis. Longitudinal in vivo imaging and flow cytometry analyses revealed that Lin(-) cells home directly to inflamed skin within 6 h, where they undergo extensive expansion with the peak on day 14 post-transplantation, and preferential differentiation into CD11b(+)Ly6G(int)Ly6C(+) cells by day 7. Cells with phenotypic profiles of neutrophils, macrophages, and DCs appeared in inflamed skin on day 14. Progenies of transplanted Lin(-) cells showed similar kinetics of expansion and myeloid differentiation in BM. However, differentiation into CD11b(+)Ly6G(int)Ly6C(+) cells in the inflamed skin on day 7 was more skewed toward CD115(+) cells (≥60%) with immune suppressive function and higher expression levels of iNOS, arginase, and IL-10, compared with those in the BM. Transplantation of Lin(-) cells reduced the levels of Cd3 transcript and CD4(+)/CD8(+) cells in inflamed skin. These results demonstrate differentiation of transplanted Lin(-) cells into myeloid-derived suppressor cells in inflamed skin to be the basis of the alleviation of skin inflammation after Lin(-) cell transplantation.

No MeSH data available.


Related in: MedlinePlus

Differentiation of CD45.1+Lin− cells into CD115+ MDSCs in inflamed skin.(a) Flow cytometric analysis of CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− or Lin+ cells on day 7 post-transplantation. CD45.1+ cells were analyzed by flow cytometry and representative data are shown. Percentage values of CD115+ cells within the CD11b+Ly6GintLy6C+ and CD11b+Ly6GhiLy6C+ cell populations are plotted. (b,c) Immune suppression assays of CD45.1+ cells isolated from the inflamed skin or BM (b), or those from skin in the presence (+) or absence (−) of L-NMMA (1 mM). CFSE dilution by CFSE-labeled CD3+ T cells was analyzed via flow cytometry after stimulation in the presence sorted CD45.1+ cells. (d) qRT-PCR analysis of CD45.1+ cells isolated from the skin and BM. Relative levels of transcripts expressed by CD45.1+ cells in the BM and skin of mice transplanted with Lin− cells and Lin+ cells were compared. Delta-delta Ct values were normalized to those obtained from amplification of β-actin and were expressed as fold changes compared with gene profiles of Lin− cells in inflamed skin. (e) Flow cytometric analysis for expression of CD11b and NOS2 by CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− cells on day 7. Representative flow cytometric data are shown, and the percentage values of NOS2+ or arginase-1+ cells within the CD45.1+ cell populations are plotted. (f) qRT-PCR analysis using RNA extracted from the inflamed skin of Lin− or Lin+ cell recipients, along with negative control mice that received PBS (no Tpl; no transplantation). Relative transcript levels are shown. Normalized values are expressed as fold changes compared with gene profiles of the no transplantation sample. (g) Flow cytometric analysis of skin-infiltrating cells after staining with anti-CD4-PE, anti-CD8-PE, anti-B220-APC, anti-CD45.1-eFlour 450 antibodies on day 7 post-transplantation. Representative flow cytometric data are shown after CD45.1− cell gating. Data shown (a–g) are representative of two independent experiments (n = 3–5 mice/group/experiment). Data (a,d–g) are presented as means ±SEM. P values were determined using two-tailed unpaired Student’s t-tests; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
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f7: Differentiation of CD45.1+Lin− cells into CD115+ MDSCs in inflamed skin.(a) Flow cytometric analysis of CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− or Lin+ cells on day 7 post-transplantation. CD45.1+ cells were analyzed by flow cytometry and representative data are shown. Percentage values of CD115+ cells within the CD11b+Ly6GintLy6C+ and CD11b+Ly6GhiLy6C+ cell populations are plotted. (b,c) Immune suppression assays of CD45.1+ cells isolated from the inflamed skin or BM (b), or those from skin in the presence (+) or absence (−) of L-NMMA (1 mM). CFSE dilution by CFSE-labeled CD3+ T cells was analyzed via flow cytometry after stimulation in the presence sorted CD45.1+ cells. (d) qRT-PCR analysis of CD45.1+ cells isolated from the skin and BM. Relative levels of transcripts expressed by CD45.1+ cells in the BM and skin of mice transplanted with Lin− cells and Lin+ cells were compared. Delta-delta Ct values were normalized to those obtained from amplification of β-actin and were expressed as fold changes compared with gene profiles of Lin− cells in inflamed skin. (e) Flow cytometric analysis for expression of CD11b and NOS2 by CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− cells on day 7. Representative flow cytometric data are shown, and the percentage values of NOS2+ or arginase-1+ cells within the CD45.1+ cell populations are plotted. (f) qRT-PCR analysis using RNA extracted from the inflamed skin of Lin− or Lin+ cell recipients, along with negative control mice that received PBS (no Tpl; no transplantation). Relative transcript levels are shown. Normalized values are expressed as fold changes compared with gene profiles of the no transplantation sample. (g) Flow cytometric analysis of skin-infiltrating cells after staining with anti-CD4-PE, anti-CD8-PE, anti-B220-APC, anti-CD45.1-eFlour 450 antibodies on day 7 post-transplantation. Representative flow cytometric data are shown after CD45.1− cell gating. Data shown (a–g) are representative of two independent experiments (n = 3–5 mice/group/experiment). Data (a,d–g) are presented as means ±SEM. P values were determined using two-tailed unpaired Student’s t-tests; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: The CD11b+Ly6GintLy6C+ phenotype with proliferative potential is consistent with their classification as MDSCs, which have been well characterized to have an immunosuppressive function in tumor environments22. Mouse MDSCs are known to express CD11530. Thus, we examined the expression of CD115 by the Ly6GintLy6C+ cells of transplanted CD45.1+Lin− cell origin in the inflamed skin and BM on day 7, after which disease scores decrease (Fig. 1c), implying a high probability that Lin− progenies are exerting suppressive function about the time point. The proportion of CD115+ cells in the predominant Ly6GintLy6C+ cell population was significantly elevated (62.4% on average) in the inflamed skin, with little decrease in the proportion of CD115+ cells among the minor Ly6GhiLy6C+ cell population (24.45%; Fig. 7a). Unexpectedly, however, proportions of CD115+ cells were significantly lower (27% on average) among the Ly6GintLy6C+ cells in the BM. These results show that, despite the similarity in the overall phenotypes of the Ly6GintLy6C+ cells between the inflamed skin and BM, differentiation into CD115+ cells was preferential in the inflamed skin. As an additional control, Lin+ cells in inflamed skin comprised low proportions of CD115+ cells (19.3% on average).


Alleviation of skin inflammation after Lin(-) cell transplantation correlates with their differentiation into myeloid-derived suppressor cells.

Jeong Ryu S, Ju JM, Kim W, Bum Kim M, Hee Oh K, Sup Lee D, Lee H, Eun Oh J, Soo Park K, Young Choi E - Sci Rep (2015)

Differentiation of CD45.1+Lin− cells into CD115+ MDSCs in inflamed skin.(a) Flow cytometric analysis of CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− or Lin+ cells on day 7 post-transplantation. CD45.1+ cells were analyzed by flow cytometry and representative data are shown. Percentage values of CD115+ cells within the CD11b+Ly6GintLy6C+ and CD11b+Ly6GhiLy6C+ cell populations are plotted. (b,c) Immune suppression assays of CD45.1+ cells isolated from the inflamed skin or BM (b), or those from skin in the presence (+) or absence (−) of L-NMMA (1 mM). CFSE dilution by CFSE-labeled CD3+ T cells was analyzed via flow cytometry after stimulation in the presence sorted CD45.1+ cells. (d) qRT-PCR analysis of CD45.1+ cells isolated from the skin and BM. Relative levels of transcripts expressed by CD45.1+ cells in the BM and skin of mice transplanted with Lin− cells and Lin+ cells were compared. Delta-delta Ct values were normalized to those obtained from amplification of β-actin and were expressed as fold changes compared with gene profiles of Lin− cells in inflamed skin. (e) Flow cytometric analysis for expression of CD11b and NOS2 by CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− cells on day 7. Representative flow cytometric data are shown, and the percentage values of NOS2+ or arginase-1+ cells within the CD45.1+ cell populations are plotted. (f) qRT-PCR analysis using RNA extracted from the inflamed skin of Lin− or Lin+ cell recipients, along with negative control mice that received PBS (no Tpl; no transplantation). Relative transcript levels are shown. Normalized values are expressed as fold changes compared with gene profiles of the no transplantation sample. (g) Flow cytometric analysis of skin-infiltrating cells after staining with anti-CD4-PE, anti-CD8-PE, anti-B220-APC, anti-CD45.1-eFlour 450 antibodies on day 7 post-transplantation. Representative flow cytometric data are shown after CD45.1− cell gating. Data shown (a–g) are representative of two independent experiments (n = 3–5 mice/group/experiment). Data (a,d–g) are presented as means ±SEM. P values were determined using two-tailed unpaired Student’s t-tests; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
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f7: Differentiation of CD45.1+Lin− cells into CD115+ MDSCs in inflamed skin.(a) Flow cytometric analysis of CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− or Lin+ cells on day 7 post-transplantation. CD45.1+ cells were analyzed by flow cytometry and representative data are shown. Percentage values of CD115+ cells within the CD11b+Ly6GintLy6C+ and CD11b+Ly6GhiLy6C+ cell populations are plotted. (b,c) Immune suppression assays of CD45.1+ cells isolated from the inflamed skin or BM (b), or those from skin in the presence (+) or absence (−) of L-NMMA (1 mM). CFSE dilution by CFSE-labeled CD3+ T cells was analyzed via flow cytometry after stimulation in the presence sorted CD45.1+ cells. (d) qRT-PCR analysis of CD45.1+ cells isolated from the skin and BM. Relative levels of transcripts expressed by CD45.1+ cells in the BM and skin of mice transplanted with Lin− cells and Lin+ cells were compared. Delta-delta Ct values were normalized to those obtained from amplification of β-actin and were expressed as fold changes compared with gene profiles of Lin− cells in inflamed skin. (e) Flow cytometric analysis for expression of CD11b and NOS2 by CD45.1+ cells in the inflamed skin and BM of recipients of CD45.1+Lin− cells on day 7. Representative flow cytometric data are shown, and the percentage values of NOS2+ or arginase-1+ cells within the CD45.1+ cell populations are plotted. (f) qRT-PCR analysis using RNA extracted from the inflamed skin of Lin− or Lin+ cell recipients, along with negative control mice that received PBS (no Tpl; no transplantation). Relative transcript levels are shown. Normalized values are expressed as fold changes compared with gene profiles of the no transplantation sample. (g) Flow cytometric analysis of skin-infiltrating cells after staining with anti-CD4-PE, anti-CD8-PE, anti-B220-APC, anti-CD45.1-eFlour 450 antibodies on day 7 post-transplantation. Representative flow cytometric data are shown after CD45.1− cell gating. Data shown (a–g) are representative of two independent experiments (n = 3–5 mice/group/experiment). Data (a,d–g) are presented as means ±SEM. P values were determined using two-tailed unpaired Student’s t-tests; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: The CD11b+Ly6GintLy6C+ phenotype with proliferative potential is consistent with their classification as MDSCs, which have been well characterized to have an immunosuppressive function in tumor environments22. Mouse MDSCs are known to express CD11530. Thus, we examined the expression of CD115 by the Ly6GintLy6C+ cells of transplanted CD45.1+Lin− cell origin in the inflamed skin and BM on day 7, after which disease scores decrease (Fig. 1c), implying a high probability that Lin− progenies are exerting suppressive function about the time point. The proportion of CD115+ cells in the predominant Ly6GintLy6C+ cell population was significantly elevated (62.4% on average) in the inflamed skin, with little decrease in the proportion of CD115+ cells among the minor Ly6GhiLy6C+ cell population (24.45%; Fig. 7a). Unexpectedly, however, proportions of CD115+ cells were significantly lower (27% on average) among the Ly6GintLy6C+ cells in the BM. These results show that, despite the similarity in the overall phenotypes of the Ly6GintLy6C+ cells between the inflamed skin and BM, differentiation into CD115+ cells was preferential in the inflamed skin. As an additional control, Lin+ cells in inflamed skin comprised low proportions of CD115+ cells (19.3% on average).

Bottom Line: Longitudinal in vivo imaging and flow cytometry analyses revealed that Lin(-) cells home directly to inflamed skin within 6 h, where they undergo extensive expansion with the peak on day 14 post-transplantation, and preferential differentiation into CD11b(+)Ly6G(int)Ly6C(+) cells by day 7.Cells with phenotypic profiles of neutrophils, macrophages, and DCs appeared in inflamed skin on day 14.Progenies of transplanted Lin(-) cells showed similar kinetics of expansion and myeloid differentiation in BM.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
To understand the cellular mechanism underlying the therapeutic effects exerted by hematopoietic stem cell transplantation in the repair of tissue damage, we investigated the in vivo dynamics of bone marrow (BM) lineage-negative (Lin(-)) cells transplanted into mice with hyper sensitivity dermatitis. Longitudinal in vivo imaging and flow cytometry analyses revealed that Lin(-) cells home directly to inflamed skin within 6 h, where they undergo extensive expansion with the peak on day 14 post-transplantation, and preferential differentiation into CD11b(+)Ly6G(int)Ly6C(+) cells by day 7. Cells with phenotypic profiles of neutrophils, macrophages, and DCs appeared in inflamed skin on day 14. Progenies of transplanted Lin(-) cells showed similar kinetics of expansion and myeloid differentiation in BM. However, differentiation into CD11b(+)Ly6G(int)Ly6C(+) cells in the inflamed skin on day 7 was more skewed toward CD115(+) cells (≥60%) with immune suppressive function and higher expression levels of iNOS, arginase, and IL-10, compared with those in the BM. Transplantation of Lin(-) cells reduced the levels of Cd3 transcript and CD4(+)/CD8(+) cells in inflamed skin. These results demonstrate differentiation of transplanted Lin(-) cells into myeloid-derived suppressor cells in inflamed skin to be the basis of the alleviation of skin inflammation after Lin(-) cell transplantation.

No MeSH data available.


Related in: MedlinePlus