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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 myeloid and granulocytic lineages in the inflamed skin and BM of the dermatitis mice.(a) Flow cytometric analysis of Lin− or Lin+ cells purified for transplantation. (b) Flow cytometric analysis of CD45.1+ cells in the inflamed skin of dermatitis mice transplanted with CD45.1+Lin− or Lin+ cells (1 × 106) at various time points after transplantation. CD45.1+ cells isolated from the skin were analyzed for expression of CD11b, Ly6G, Ly6C, F4/80, and CD11c on the designated days post-transplantation. Representative data gated for CD45.1+ cells are shown. Percentage values of each population within the CD45.1+CD11b+ cells and absolute cell numbers are shown. (c) DNCB-treated, CD45.1+Lin− cell-transplanted, and FTY-720-treated mice were injected with 1 mg BrdU daily for 3 days. FTY-720 was added to drinking water continuously until day 4 starting from day 1 post-transplantation. Leukocytes were isolated from the skin and stained with anti CD45.1-eFluor 450, anti-CD11b APC, anti-Ly6G PE and anti-BrdU-FITC antibodies. Representative data gated for CD45.1+BrdU+ cells are shown (d) Flow cytometric analysis of CD45.1+ cells in the BM. Data were analyzed as described above; representative data gated for CD45.1+ cells are shown. Data (a-d) are representative of three independent experiments (n = 5 mice/group/experiment). Data (B,d) 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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f6: Differentiation of CD45.1+Lin− cells into myeloid and granulocytic lineages in the inflamed skin and BM of the dermatitis mice.(a) Flow cytometric analysis of Lin− or Lin+ cells purified for transplantation. (b) Flow cytometric analysis of CD45.1+ cells in the inflamed skin of dermatitis mice transplanted with CD45.1+Lin− or Lin+ cells (1 × 106) at various time points after transplantation. CD45.1+ cells isolated from the skin were analyzed for expression of CD11b, Ly6G, Ly6C, F4/80, and CD11c on the designated days post-transplantation. Representative data gated for CD45.1+ cells are shown. Percentage values of each population within the CD45.1+CD11b+ cells and absolute cell numbers are shown. (c) DNCB-treated, CD45.1+Lin− cell-transplanted, and FTY-720-treated mice were injected with 1 mg BrdU daily for 3 days. FTY-720 was added to drinking water continuously until day 4 starting from day 1 post-transplantation. Leukocytes were isolated from the skin and stained with anti CD45.1-eFluor 450, anti-CD11b APC, anti-Ly6G PE and anti-BrdU-FITC antibodies. Representative data gated for CD45.1+BrdU+ cells are shown (d) Flow cytometric analysis of CD45.1+ cells in the BM. Data were analyzed as described above; representative data gated for CD45.1+ cells are shown. Data (a-d) are representative of three independent experiments (n = 5 mice/group/experiment). Data (B,d) 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: To better characterize the myeloid lineage of Lin− progenies, we used flow cytometry to examine CD45.1+ cells in the inflamed skin on days 5 and 7 post-transplantation of CD45.1+Lin− cells when the ear inflammation was severe, and on day 14 when the skin inflammation subsided significantly (Fig. 1), supposing that healing effects of Lin− cell transplantation would be exerted before inflammation begins to subside. Lin− cells isolated from CD45.1+ donor mice were negative for CD11b and Gr-1 (Ly6G and Ly6C; Fig. 6a), as well as B220, CD3, Ter119 and CD11c expression (data not shown). However, when analyzed on day 5 post-transplantation, all of the CD45.1+ cells of donor origin were CD11b+ (Fig. 6b), consistent with a myeloid cell lineage. A large majority (≥80%) of CD11b+ cells exhibited a Ly6GintLy6C+ phenotype on days 5 and 7 post-transplantation. However, on day 14, the proportions of Ly6GintLy6C+ cells were reduced dramatically (33.6% on average), although their absolute numbers were not changed significantly, compared to those on day 7. Instead, Ly6GhiLy6ClowF4/80− neutrophils were detected at larger proportions (43%), with their numbers being significantly increased, along with Ly6GlowLy6Clow (9.6%) and Ly6G−Ly6Clow (7.4%) cells, which were composed of F4/80+ macrophages and CD11c+ dendritic cells2229.


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 myeloid and granulocytic lineages in the inflamed skin and BM of the dermatitis mice.(a) Flow cytometric analysis of Lin− or Lin+ cells purified for transplantation. (b) Flow cytometric analysis of CD45.1+ cells in the inflamed skin of dermatitis mice transplanted with CD45.1+Lin− or Lin+ cells (1 × 106) at various time points after transplantation. CD45.1+ cells isolated from the skin were analyzed for expression of CD11b, Ly6G, Ly6C, F4/80, and CD11c on the designated days post-transplantation. Representative data gated for CD45.1+ cells are shown. Percentage values of each population within the CD45.1+CD11b+ cells and absolute cell numbers are shown. (c) DNCB-treated, CD45.1+Lin− cell-transplanted, and FTY-720-treated mice were injected with 1 mg BrdU daily for 3 days. FTY-720 was added to drinking water continuously until day 4 starting from day 1 post-transplantation. Leukocytes were isolated from the skin and stained with anti CD45.1-eFluor 450, anti-CD11b APC, anti-Ly6G PE and anti-BrdU-FITC antibodies. Representative data gated for CD45.1+BrdU+ cells are shown (d) Flow cytometric analysis of CD45.1+ cells in the BM. Data were analyzed as described above; representative data gated for CD45.1+ cells are shown. Data (a-d) are representative of three independent experiments (n = 5 mice/group/experiment). Data (B,d) 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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f6: Differentiation of CD45.1+Lin− cells into myeloid and granulocytic lineages in the inflamed skin and BM of the dermatitis mice.(a) Flow cytometric analysis of Lin− or Lin+ cells purified for transplantation. (b) Flow cytometric analysis of CD45.1+ cells in the inflamed skin of dermatitis mice transplanted with CD45.1+Lin− or Lin+ cells (1 × 106) at various time points after transplantation. CD45.1+ cells isolated from the skin were analyzed for expression of CD11b, Ly6G, Ly6C, F4/80, and CD11c on the designated days post-transplantation. Representative data gated for CD45.1+ cells are shown. Percentage values of each population within the CD45.1+CD11b+ cells and absolute cell numbers are shown. (c) DNCB-treated, CD45.1+Lin− cell-transplanted, and FTY-720-treated mice were injected with 1 mg BrdU daily for 3 days. FTY-720 was added to drinking water continuously until day 4 starting from day 1 post-transplantation. Leukocytes were isolated from the skin and stained with anti CD45.1-eFluor 450, anti-CD11b APC, anti-Ly6G PE and anti-BrdU-FITC antibodies. Representative data gated for CD45.1+BrdU+ cells are shown (d) Flow cytometric analysis of CD45.1+ cells in the BM. Data were analyzed as described above; representative data gated for CD45.1+ cells are shown. Data (a-d) are representative of three independent experiments (n = 5 mice/group/experiment). Data (B,d) 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: To better characterize the myeloid lineage of Lin− progenies, we used flow cytometry to examine CD45.1+ cells in the inflamed skin on days 5 and 7 post-transplantation of CD45.1+Lin− cells when the ear inflammation was severe, and on day 14 when the skin inflammation subsided significantly (Fig. 1), supposing that healing effects of Lin− cell transplantation would be exerted before inflammation begins to subside. Lin− cells isolated from CD45.1+ donor mice were negative for CD11b and Gr-1 (Ly6G and Ly6C; Fig. 6a), as well as B220, CD3, Ter119 and CD11c expression (data not shown). However, when analyzed on day 5 post-transplantation, all of the CD45.1+ cells of donor origin were CD11b+ (Fig. 6b), consistent with a myeloid cell lineage. A large majority (≥80%) of CD11b+ cells exhibited a Ly6GintLy6C+ phenotype on days 5 and 7 post-transplantation. However, on day 14, the proportions of Ly6GintLy6C+ cells were reduced dramatically (33.6% on average), although their absolute numbers were not changed significantly, compared to those on day 7. Instead, Ly6GhiLy6ClowF4/80− neutrophils were detected at larger proportions (43%), with their numbers being significantly increased, along with Ly6GlowLy6Clow (9.6%) and Ly6G−Ly6Clow (7.4%) cells, which were composed of F4/80+ macrophages and CD11c+ dendritic cells2229.

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