Inducible deletion of the Blimp-1 gene in adult epidermis causes granulocyte-dominated chronic skin inflammation in mice

Abstract

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor important for the differentiation and function of several types of immune cells. Because skin serves as a physical barrier and acts as an immune sentinel, we investigated whether Blimp-1 is involved in epidermal immune function. We show that Blimp-1 expression is reduced in skin lesions of some human eczema samples and in stimulated primary keratinocytes. Epidermal-specific deletion of PR domain containing 1, with ZNF domain (Prdm1), the gene encoding Blimp-1, in adult mice caused spontaneously inflamed skin characterized by massive dermal infiltration of neutrophils/macrophages and development of chronic inflammation associated with higher levels of cytokines/chemokines, including granulocyte colony-stimulating factor (G-CSF), and enhanced myelopoiesis in bone marrow. Deletion of Prdm1 in the epidermis of adult mice also led to stronger inflammatory reactions in a tape-stripping test and in a disease model of contact dermatitis. The elevated G-CSF produced by keratinocytes after deletion of Prdm1 in vitro was mediated by the transcriptional activation of FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1). Systemic increases in G-CSF contributed to the inflammatory responses, because deletion of the G-CSF gene [colony stimulating factor 3, (Csf3)] prevented neutrophilia and partially ameliorated the inflamed skin in Prdm1-deficient mice. Our findings indicate a previously unreported function for Blimp-1 in restraining steady-state epidermal barrier immunity.

Fig. 1.

Expression of Blimp-1 in human epidermis and stimulated keratinocytes. (A–E) Immunohistochemical staining of human skin sections with isotype control antibody, mouse IgG (A) or with anti–Blimp-1 (B–E) or from individuals with normal skin (A and B), dyshidrotic eczema (C), autoeczematization dermatitis (D), or nummular eczema (E). Each dashed line denotes the epidermis–dermis boundary. Arrows indicate Blimp-1⁺ cells. (Scale bar: 100 μm.) (F) Relative expression of Blimp-1 from unclassified eczema skin samples (n = 19) compared with normal skin. Eczema skin sections that contained areas of both increased and decreased Blimp-1 were categorized as complex. (G) Immunoblots showing reduced Blimp-1 protein in human (Left) and mouse (Right) primary keratinocytes treated with LPS (5 μg/mL) or TNF-α (50 ng/mL), or untreated (−), for 24 h; results are representative of three experiments.

Fig. 2.

Deletion of epidermal Prdm1 in adult mice leads to spontaneous development of skin inflammation and sensitized skin. (A) Deletion of Prdm1 alleles at 2 mo after i.p. injection of 4OHT followed by immunoblotting with anti–Blimp-1 of cultured keratinocytes derived from tail skin of Ctrl or CKO mice. (B–K) At 6 mo after 4OHT injection, H&E staining of neck skin sections from CKO mice (C and E) showed inflamed skin compared with control (Ctrl) mice (B and D). Immunohistochemical (F and G) or immunofluorescence (H–K) staining of neck skin from Ctrl (F, H, and J) and CKO (G, I, and K) mice using antibodies specific for Gr-1 (F and G), CD4 (H and I), and F4/80 (J and K). (L–N) At 2 mo after 4OHT injection, Ctrl and CKO mice were used for contact hypersensitivity tests. H&E staining shows ear sections of Ctrl (L) and CKO (M) mice after rechallenging with DNFB for 24 h. The extent of ear swelling was determined by comparing changes in ear thickness before and after challenges (N) (Ctrl, n = 8; CKO, n = 9). (O–R) H&E staining of back skin sections from Ctrl and CKO mice at 2 d after receiving 10 tape-stripping treatments (treated); control skin areas are designated as “untouched.” Results are representative of three independent experiments. **P < 0.01. Images in D and E are enlarged fields of the boxed areas of B and C, respectively. The dotted line in H–K denotes the epidermis–dermis boundary. (Scale bars: 200 μm in B and C; 100 μm in L, M, and O–R; 50 μm in D, E, and H–K; 15 μm in F and G.)

Fig. 3.

Enhanced neutrophil-dominated inflammatory reactions in CKO mice. (A) The numbers of leukocytes measured with an automated hematology analyzer in Ctrl and CKO mice at 6 mo after 4OHT injection. The mean number of each cell type is indicated by a horizontal line (Ctrl, n = 15; CKO, n = 18). (B) Flow cytometry analysis of the frequencies of Gr-1⁺Mac-1⁺ cells in BM cells and splenocytes (SP) of Ctrl and CKO mice. The numbers shown represent the percentage of Gr-1⁺Mac-1⁺ neutrophils in each upper-right quadrant. (C) Heat map of microarray analysis using indicated keratinocytes cultured with 4OHT or EtOH. Data were normalized to Prdm1^f/f (f/f), K5-CreER(Cre)⁻ + EtOH group, and the 12 genes with the greatest changes are shown. (D) RNA from keratinocytes cultured as described in C was used for qRT-PCR analysis of cytokine/chemokine gene expression. (E) The Luminex assay was used to detect the increased levels of G-CSF and CXCL-1 in serum of CKO mice at 6 mo after 4OHT injection. Results are mean ± SEM (n = 3, 4, and 6 in B, D, and E, respectively). *P < 0.05; **P < 0.01; ***P < 0.005.

Fig. 4.

Blimp-1 indirectly regulates G-CSF expression by suppressing Fos and Fosl1. (A) Keratinocytes from neonatal mice were cultured and transduced with lentiviral vectors expressing Blimp-1–GFP or GFP for 2 d. The expression of the indicated genes was analyzed with qRT-PCR. Data were normalized to levels of the internal control, Actb, and are presented as the ratio of the indicated genes in Blimp-1–GFP to GFP-expressing keratinocytes. (B) qRT-PCR of Fos and Fosl1 mRNA levels using RNA prepared from keratinocytes cultured as described in Fig 3C. (C) A dual-luciferase reporter assay conducted in primary keratinocytes to compare the activities of the Csf3 promoter containing either the WT or the mutated (Mut) AP-1–binding element. (D and E) Dual-luciferase reporter assays of Csf3 promoter activities. Firefly luciferase reporter driven by the WT Csf3 promoter was used in a cotransfection experiment with Blimp-1 (D) or the indicated expression vectors (E) into primary keratinocytes. Here “−” indicates transfected with empty vector. (F) qRT-PCR of Csf3 mRNA from keratinocytes treated with EtOH or 4OHT at 5 d after transduction with lentiviral vectors producing control shRNA (shCtrl), shRNA against Fos (shFos), shRNA against Fosl1 (shFosl1), or shRNA against both Fos and Fosl1 (shF+F). Here “1” and “2” represent two different shRNAs used. Data are mean ± SEM (n ≥3 in all panels). *P < 0.05.

Fig. 5.

G-CSF contributes to inflammatory diseases in CKO mice. (A) The number of neutrophils in peripheral blood of the indicated mice at 5–6 mo after 4OHT injection was measured with an automated hematology analyzer. Data are mean ± SEM (n = 4). ***P < 0.005. (B) Populations of Gr-1⁺Mac-1⁺ neutrophils in BM and splenocytes (SP) of the indicated mice shown in a representative dot plot from two independent experiments. (C) H&E staining of neck skin from the indicated mice at 6 mo after 4OHT injection. The enlarged images in the boxed areas of the upper panels are shown in the lower panels. Arrowheads indicate lymphocytes. (Scale bars: 100 μm in upper panels; 15 μm in lower panels.)