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Burn injury dictates the phenotype of CMP‐derived DCs. Burn injury not only hampers DC production but also dictates the phenotype to pDCs. (A) Histograms representative of FACS results for ic‐GATA1 and <t>‐MafB</t> and membrane‐associated MHC‐II, CD11c, and F480 in CMP → DCs from sham (blue) and burn (red) mice. CD11c, ic‐GATA1, and MHC‐II expressions was reduced significantly in burn vs. sham CMP → DC cultures. F480 (negative) and MafB (no change) were measured to exclude any possible MØ contamination in DC cultures. (B, left) Live/Dead gating shows that ∼80% of cultured cells were viable in both sham and burn. (B, middle) B220+CD11c+ double‐positive cells were gated to characterize pDCs. A 2.5‐fold increase in pDCs was found in burn (16%) compared with sham (44%). (B, right) MHC‐II is decreased significantly in burn and is independent of MafB expression in CMP → DCs. (C and D) Confocal images of CMP → DCs, triple staining with fluorescent antibodies for anti‐MHC‐II (red) GATA1 (green), and DAPI for nucleus (blue) are shown. Low expression of MHC‐II and GATA1 in burn (D) vs. sham (C) is seen in the intensities of green and red colors individually and in merged images, respectively. Zeiss LSM 510 laser‐scanning microscope (Carl Zeiss MicroImaging GmbH) was used to view with C‐Apochromat 40×_1.20 water immersion, and images were acquired using Zeiss LSM 510, version 4.2, SP1 software.
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Burn injury dictates the phenotype of CMP‐derived DCs. Burn injury not only hampers DC production but also dictates the phenotype to pDCs. (A) Histograms representative of FACS results for ic‐GATA1 and ‐MafB and membrane‐associated MHC‐II, CD11c, and F480 in CMP → DCs from sham (blue) and burn (red) mice. CD11c, ic‐GATA1, and MHC‐II expressions was reduced significantly in burn vs. sham CMP → DC cultures. F480 (negative) and MafB (no change) were measured to exclude any possible MØ contamination in DC cultures. (B, left) Live/Dead gating shows that ∼80% of cultured cells were viable in both sham and burn. (B, middle) B220+CD11c+ double‐positive cells were gated to characterize pDCs. A 2.5‐fold increase in pDCs was found in burn (16%) compared with sham (44%). (B, right) MHC‐II is decreased significantly in burn and is independent of MafB expression in CMP → DCs. (C and D) Confocal images of CMP → DCs, triple staining with fluorescent antibodies for anti‐MHC‐II (red) GATA1 (green), and DAPI for nucleus (blue) are shown. Low expression of MHC‐II and GATA1 in burn (D) vs. sham (C) is seen in the intensities of green and red colors individually and in merged images, respectively. Zeiss LSM 510 laser‐scanning microscope (Carl Zeiss MicroImaging GmbH) was used to view with C‐Apochromat 40×_1.20 water immersion, and images were acquired using Zeiss LSM 510, version 4.2, SP1 software.

Journal: Journal of Leukocyte Biology

Article Title: Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness

doi: 10.1189/jlb.1A0815-377R

Figure Lengend Snippet: Burn injury dictates the phenotype of CMP‐derived DCs. Burn injury not only hampers DC production but also dictates the phenotype to pDCs. (A) Histograms representative of FACS results for ic‐GATA1 and ‐MafB and membrane‐associated MHC‐II, CD11c, and F480 in CMP → DCs from sham (blue) and burn (red) mice. CD11c, ic‐GATA1, and MHC‐II expressions was reduced significantly in burn vs. sham CMP → DC cultures. F480 (negative) and MafB (no change) were measured to exclude any possible MØ contamination in DC cultures. (B, left) Live/Dead gating shows that ∼80% of cultured cells were viable in both sham and burn. (B, middle) B220+CD11c+ double‐positive cells were gated to characterize pDCs. A 2.5‐fold increase in pDCs was found in burn (16%) compared with sham (44%). (B, right) MHC‐II is decreased significantly in burn and is independent of MafB expression in CMP → DCs. (C and D) Confocal images of CMP → DCs, triple staining with fluorescent antibodies for anti‐MHC‐II (red) GATA1 (green), and DAPI for nucleus (blue) are shown. Low expression of MHC‐II and GATA1 in burn (D) vs. sham (C) is seen in the intensities of green and red colors individually and in merged images, respectively. Zeiss LSM 510 laser‐scanning microscope (Carl Zeiss MicroImaging GmbH) was used to view with C‐Apochromat 40×_1.20 water immersion, and images were acquired using Zeiss LSM 510, version 4.2, SP1 software.

Article Snippet: Gene silencing (siRNA) CMPs (5 × 10 4 ) were rinsed with Opti‐MEM (Thermo Fisher Scientific Life Sciences) to remove any residual FBS and then transfected with 40 pmole/10 6 cells of MafB siRNA (sc‐35840; Santa Cruz Biotechnology, Santa Cruz, CA, USA) by the addition of X‐tremeGENE siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany) following kit instructions.

Techniques: Derivative Assay, Membrane, Cell Culture, Expressing, Staining, Laser-Scanning Microscopy, Software

MafB‐dependent erythromyeloid bifurcation of CMPs following burn injury. Freshly sorted CMPs from sham and burn mice were analyzed for membrane‐bound M‐CSFR and ic‐MafB expressions. (A). CMPs from burn mice (upper right) had more MafB+M‐CSFR+ cells compared with sham mice (upper left). MafB silencing also reduced M‐CSFR expression in sham CMPs from 38.8 to 12.5% (lower left) and burn CMPs from 55.6 to 28% (lower right), respectively. Results emphasize the direct role of MafB on M‐CSFR expression in CMPs. (B) Representative confocal images of MafB (green) in sham and burn CMPs (upper panels) and burn CMPs + MafB siRNA at 60 h after transfection (lower right panel). Histogram in lower left panel represents the MFI of MafB obtained by flow cytometry. B, Burn; B+si, MafB siRNA; S, sham. (C) CMPs from sham and burn mice were placed in an intermediate cocktail for 5 d. Myeloid and erythroid commitment bias were checked using anti‐CD11B (CR3A) and anti‐CD71 (TfR), as shown in the histograms, with CD11B and CD71 on the x‐axes, respectively. CD11B expression was the highest in burn CMPs (blue line) compared with sham (red line) and is reversed to sham levels after MafB silencing in burn CMPs (green line). Conversely, MafB silencing increased TfR (green line), which was otherwise down‐regulated following burn (blue line) compared with sham (red). (D) The cultures were extended in an Epo‐containing erythroid cocktail for 4 more days. Representative FACS plots from each condition, sham and burn, with and without siRNA MafB assessing erythroid commitment with CD71 on the y‐axis and Ter119 on the x‐axis. (E) Mean results are depicted in box plots enumerating the percentage of CD71+ cells (upper) and MFI of Tfrs (lower) in CMPs from sham and burn mice with scramble siRNA and MafB siRNA. *P < 0.0001 vs. sham; ^P < 0.01 vs. PBD #7. ctRNA, Control RNA.

Journal: Journal of Leukocyte Biology

Article Title: Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness

doi: 10.1189/jlb.1A0815-377R

Figure Lengend Snippet: MafB‐dependent erythromyeloid bifurcation of CMPs following burn injury. Freshly sorted CMPs from sham and burn mice were analyzed for membrane‐bound M‐CSFR and ic‐MafB expressions. (A). CMPs from burn mice (upper right) had more MafB+M‐CSFR+ cells compared with sham mice (upper left). MafB silencing also reduced M‐CSFR expression in sham CMPs from 38.8 to 12.5% (lower left) and burn CMPs from 55.6 to 28% (lower right), respectively. Results emphasize the direct role of MafB on M‐CSFR expression in CMPs. (B) Representative confocal images of MafB (green) in sham and burn CMPs (upper panels) and burn CMPs + MafB siRNA at 60 h after transfection (lower right panel). Histogram in lower left panel represents the MFI of MafB obtained by flow cytometry. B, Burn; B+si, MafB siRNA; S, sham. (C) CMPs from sham and burn mice were placed in an intermediate cocktail for 5 d. Myeloid and erythroid commitment bias were checked using anti‐CD11B (CR3A) and anti‐CD71 (TfR), as shown in the histograms, with CD11B and CD71 on the x‐axes, respectively. CD11B expression was the highest in burn CMPs (blue line) compared with sham (red line) and is reversed to sham levels after MafB silencing in burn CMPs (green line). Conversely, MafB silencing increased TfR (green line), which was otherwise down‐regulated following burn (blue line) compared with sham (red). (D) The cultures were extended in an Epo‐containing erythroid cocktail for 4 more days. Representative FACS plots from each condition, sham and burn, with and without siRNA MafB assessing erythroid commitment with CD71 on the y‐axis and Ter119 on the x‐axis. (E) Mean results are depicted in box plots enumerating the percentage of CD71+ cells (upper) and MFI of Tfrs (lower) in CMPs from sham and burn mice with scramble siRNA and MafB siRNA. *P < 0.0001 vs. sham; ^P < 0.01 vs. PBD #7. ctRNA, Control RNA.

Article Snippet: Gene silencing (siRNA) CMPs (5 × 10 4 ) were rinsed with Opti‐MEM (Thermo Fisher Scientific Life Sciences) to remove any residual FBS and then transfected with 40 pmole/10 6 cells of MafB siRNA (sc‐35840; Santa Cruz Biotechnology, Santa Cruz, CA, USA) by the addition of X‐tremeGENE siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany) following kit instructions.

Techniques: Membrane, Expressing, Transfection, Flow Cytometry, Control

Schematic summary. Tripotential lineage commitment of CMPs into RBC, MØ, and DC is respecified by the altered transcription factor MafB /GATA1 axis following burn injury.

Journal: Journal of Leukocyte Biology

Article Title: Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness

doi: 10.1189/jlb.1A0815-377R

Figure Lengend Snippet: Schematic summary. Tripotential lineage commitment of CMPs into RBC, MØ, and DC is respecified by the altered transcription factor MafB /GATA1 axis following burn injury.

Article Snippet: Gene silencing (siRNA) CMPs (5 × 10 4 ) were rinsed with Opti‐MEM (Thermo Fisher Scientific Life Sciences) to remove any residual FBS and then transfected with 40 pmole/10 6 cells of MafB siRNA (sc‐35840; Santa Cruz Biotechnology, Santa Cruz, CA, USA) by the addition of X‐tremeGENE siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany) following kit instructions.

Techniques: