The Immune Response to Skin Trauma Is Dependent on the Etiology of Injury in a Mouse Model of Burn and Excision Samantha M. Valvis, Jason Waithman, Fiona.

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The Immune Response to Skin Trauma Is Dependent on the Etiology of Injury in a Mouse Model of Burn and Excision Samantha M. Valvis, Jason Waithman, Fiona M. Wood, Mark W. Fear, Vanessa S. Fear Journal of Investigative Dermatology Volume 135, Issue 8, Pages 2119-2128 (August 2015) DOI: 10.1038/jid.2015.123 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Temporal cytokine, chemokine, and hematology changes in burn and excision injury. Mice received sham (control), burn, or excision injury, and sera or whole blood were collected at the indicated time points for immune assay or hematology, respectively. Inflammatory cytokines IL-6, TNFα, and IL-10 (a), Chemokines monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1α (MIP1α), macrophage inflammatory protein 1β (MIP1β), KC/CXCL1, GCSF, and Eotaxin (b), and T-cell-modulating cytokines IL-3, IL-12p70, IL-4, IL-5, and IL-13 (c). Bar graphs indicate control (white bars), burn injury (hatched bars), and excision-injury (gray bars). Results are shown as mean+SEM. n=5–8 mice per group for at least two independent experiments. *P<0.05 compared with control; #P<0.05 compared with burn injury group; **P<0.01 compared with control; ##P<0.01 compared with burn injury. Hematology profile for monocytes (d) and neutrophils (d). Bars indicate control (white bars), burn injury (hatched bars), and excision injury (gray bars). Results are shown as median and range (0–100th percentile). n=6–8 mice per group and at least two independent experiments. *P<0.05 compared with control; #P<0.05 compared with burn injury group; **P<0.01 compared with control; ##P<0.01 compared with burn injury. GCSF, granulocyte colony–stimulating factor; TNF-α, tumor necrosis factor-α. Journal of Investigative Dermatology 2015 135, 2119-2128DOI: (10.1038/jid.2015.123) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dendritic cell responses in local draining lymph nodes after burn injury and excision injury. Mice received sham (control), burn, or excision injury, and ILNs were harvested to single suspensions at the indicated time points. Dendritic cells were gated CD11c+ MHC II+ and further gated for subpopulations using CD8α, then CD11b and CD103 (a). Graphs show ILN total cell number (b), total DCs, CD8a+ DC, and CD8a− DC number (c), and the number of CD8a− DC that was CD103+, CD11bhi, or CD11blo (d). Lymph node percentage frequency of total DCs, the percentage frequency of CD8a+ DC and CD8a− DC in total DCs (e), and the percentage frequency of CD103+ DC, CD11bhi DC, and CD11blo DC in the CD8a− DC compartment (f) are as indicated. Expression of MHC II as determined by MFI is presented for total DCs and DC gated CD8a+ and CD8a− (g). Data indicate MHC II MFI on CD8a− DC population further gated for CD103+ DC, CD11bhi DC, or CD11blo DC sub-populations (h). Bars indicate control (white bars), burn injury (hatched bars), and excision injury (gray bars). Results are shown as median and range (0–100th percentile). n=5–8 mice per group, for at least two independent experiments. *P<0.05 compared with control; #P<0.05 compared with burn injury group; **P<0.01 compared with control; ##P<0.01 compared with burn injury. ILN, inguinal lymph node; MFI, Mean Fluorescence Intensity; MHC, major histocompatibility complex. Journal of Investigative Dermatology 2015 135, 2119-2128DOI: (10.1038/jid.2015.123) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CD4 and CD8 cellular response to burn injury and excision injury. Mice received sham (control) burn or excision injury, and ILNs were harvested to single suspensions at the indicated time points. Dot plots indicate flow cytometric gating strategy for CD4+ cells that were further gated on Ki67hi, CD25+, or CD25+FoxP3+ cells (a). ILN CD4+, CD4+CD25+, and CD4 Treg (CD25+FoxP3+) cell number and the percentage frequency of Ki67hi, CD25+, and Treg (CD25+FoxP3+) in CD4+ cell population are indicated (b). CD8+ cells gating strategy is indicated in dot plots showing further gating on Ki67hi cells (c). ILN CD8+ and CD8+CD25+ cell number and the percentage frequency of Ki67hi and CD25+ cells in CD8 cell population are shown (d). Bars indicate control (white bars), burn injury (hatched bars), and excision injury (gray bars). Results are shown as median and range (0–100th percentile)., n=5–8 mice per group, for at least two independent experiments. *P<0.05 compared with control; #P<0.05 compared with burn injury group; **P<0.01 compared with control; ##P<0.01 compared with burn injury. ILN, inguinal lymph node; Treg, T regulatory. Journal of Investigative Dermatology 2015 135, 2119-2128DOI: (10.1038/jid.2015.123) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Innate and adaptive immune responses in the remodeling phase of burn injury and excision injury. Mice received sham (control), burn, or excision injury. Sera or whole blood were collected at day 84, and IL-10, WCC, lymphocytes, neutrophils and eosinophils were measured (a). ILNs were harvested to single suspensions at the indicated time points and total DCs, CD8a+ DC, CD8a− DC, and CD8a− DC subpopulations CD103+, CD11bhi, and CD11blo numbers determined (b) as per previous gating strategy. Expression of MHC II as determined by MFI is presented for total DCs, and DC gated CD8a+ and CD8a−, and for CD8a− DC population further gated for CD103+ DC, CD11bhi DC, or CD11blo DC sub-populations (c). ILN CD4+, CD4+CD25+, and CD4 Treg (CD25+FoxP3+) cell number and the percentage frequency of Ki67hi, CD25+, and Treg (CD25+FoxP3+) in CD4+ cell population are indicated (d). ILN CD8+ and CD8+CD25+ cell number and the percentage frequency of Ki67hi and CD25+ cells in CD8 cell population are shown (e). Bars indicate control (white bars), burn injury (hatched bars), and excision injury (gray bars). Results are shown as median and range (0–100th percentile). n=5–8 mice per group, for at least two independent experiments. *P<0.05 compared with control; #P<0.05 compared with burn injury group; **P<0.01 compared with control; ##P<0.01 compared with burn injury. ILN, inguinal lymph node; MFI, Mean Fluorescence Intensity; MHC, major histocompatibility complex; Treg, T regulatory; WCC, white cell count. Journal of Investigative Dermatology 2015 135, 2119-2128DOI: (10.1038/jid.2015.123) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Burn injury leads to suppressed acute splenic T-cell cytokine responses. Spleen cells (3 × 105cells per ml) were isolated and stimulated with 10 ng ml−1 concavalin A and cytokine levels determined in supernatant at 48 hours. Bars indicate control (white bars) and burn injury day 7 post injury (hatched bars). Results are shown as median and range (0–100th percentile, n=5 mice per group). At Day 14, there was no significant difference between burn injury and control spleen cultures stimulated with concavalin A (data not shown). Journal of Investigative Dermatology 2015 135, 2119-2128DOI: (10.1038/jid.2015.123) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions