Fig 1.
Exacerbated skin inflammation and ulceration in mice treated with combination of PDGF-BB + bleomycin.
(A) Outline of experimental design. Intradermal injection of saline, PDGF-BB alone, bleomycin alone, or PDGF-BB + bleomycin were performed daily for 21 consecutive days on female mice at 8 weeks of age. (B) Representative photographs of skin lesions on backs of treated mice. Frequency of ulceration was calculated based on visual examination of H&E stained tissue sections. (C) Representative skin section of treated mice stained by H&E, trichrome, anti-CD31, or anti-IBA-1 immunohistochemistry (scale bar, 500 μm). (D-G) Quantification of dermal thickness (D), percent area CD31 (E) or percent area IBA-1 (F) immunoreactivity, or total collagen (E) as determined by image analysis as described in the methods. Data is representative of at least two experiments with at least 5 mice/group/experiment. *P < 0.05, **P < 0.01, ***P < 0.001, versus saline or between bracketed comparisons shown (student’s t test).
Fig 2.
Inflammatory response is skewed towards a Type I IFN-dependent response with increased recruitment of monocytes.
(A) Relative mRNA expression levels of the ISGs: MX1, RSAD2, IRF7, or MDA5 (top) or proinflammatory mediators: IL1B, IL6, TNF, or CCL2 from lesional skin homogenates of skin biopsies performed by Q-PCR on day 3. (B-G) Single-cell suspensions of skin biopsies obtained from treated mice and analyzed by flow cytometry. (B) Representative dot plot of total CD45-postive leukocytes co-stained with CD11b and Ly6C. (C-G) Proportion of total live cells obtained from each biopsy that stained positive for markers of: (C) monocytes (CD45+CD11b+Ly6Chi), (D) tissue resident macrophages (CD45+CD11b+F4/80+Ly6C-), (E) plasmacytoid dendritic cells (CD45+CD11b-CD317+B220+), (F) endothelial cells (CD45-CD31+), or (G) stromal cells (CD45-PDGFRβ+). Data is representative of at least two experiments with at least 5 mice/group/experiment. *P < 0.05, **P < 0.01, ***P < 0.001, versus saline or between bracketed comparisons shown (student’s t test).
Fig 3.
Myeloid cells are a prominent source of IFNβ upon treatment with the combination of PDGF-BB + bleomycin.
Wildtype mice were treated with saline, PDGF-BB alone, bleomycin alone, or PDGF-BB + bleomycin were assessed for production of IFNβ by intracellular staining on day 3 post-treatment. (A) Representative flow cytometric plots of intracellular staining of IFNβ+ cells from single-cell suspensions obtained from skin biopsies are shown. (B) Proportion of total live cells positive for IFNβ. (C) Representative flow cytometric plots of IFNβ-producing leukocytes (gated IFNβ+). (D) Proportion of IFNβ+ cells that are leukocytes as defined by expression of CD45. (E) Representative flow cytometric plots of IFNβ-producing monocytes (gated IFNβ+CD45+). (D) Absolute number of IFNβ-producing monocytes as defined by co-expression of CD11b and Ly6C (gated IFNβ+CD45+). Data is representative of at least two experiments with at least 5 mice/group/experiment. *P < 0.05, **P < 0.01, ***P < 0.001, versus saline or for bracketed comparisons shown (student’s t test).
Fig 4.
IFNAR activation is necessary for blood vessel stabilization and recruitment of monocyte/macrophage cells in PDGF-BB + bleomycin-treated mice.
(A) Representative photographs of skin lesions on backs of treated wildtype or IFNAR-deficient mice. Frequency of ulceration was calculated based on visual examination of H&E stained tissue sections. (B) Representative skin section of treated mice stained by H&E, trichrome, anti-IBA-1, or anti-CD31 immunohistochemistry (scale bar for H&E, trichrome, and anti-IBA-1: 500 μm, scale bar for anti-CD31: 300 μm). (C-F) Quantification of dermal thickness (C), percent area CD31 (E) or percent area IBA-1 (F) immunoreactivity, or total collagen (D) staining as determined by image analysis as described in the methods. *P < 0.05, **P < 0.01, ***P < 0.001, versus saline or for bracketed comparisons shown (student’s t test).
Fig 5.
Model of chronic activation of PDGFRβ in a model of skin fibrosis.
Chronic activation of PDGFRβ in a model of skin fibrosis promotes vascular activation (ICAM1 and ANGPT2) and Type I IFN-dependent inflammatory response (ISGs). PDGFRβ activation significantly increases the numbers of IFNβ-producing inflammatory monocytes recruited to the tissue. PDGF-BB-dependent functions of tissue injury, blood vessel maintenance, and monocyte/macrophage cell recruitment in the skin were found to be dependent on IFNAR signaling (highlighted in green) in this model of dermal fibrosis.