LPS induces CD40 gene expression through the activation of NF-κB and STAT-1α in macrophages and microglia by Hongwei Qin, Cynthia A. Wilson, Sun Jung Lee, Xueyan Zhao, and Etty N. Benveniste Blood Volume 106(9):3114-3122 November 1, 2005 ©2005 by American Society of Hematology

LPS induces CD40 expression in a dose- and time-dependent manner. LPS induces CD40 expression in a dose- and time-dependent manner. (A) RAW264.7 cells were treated with medium or varying concentrations of LPS (0.1-1000 ng/mL) or IFN-γ (10 ng/mL) for 8 hours, then total RNA was isolated and analyzed by ribonuclease protection assay (RPA) for CD40, TNF-α, and GAPDH mRNA. The basal level of the untreated sample was set as 1.0, and fold induction on LPS or IFN-γ treatment was compared with that. (B) RAW264.7 cells were treated in the absence or presence of LPS or IFN-γ 36 hours, then stained with either anti-CD40 or isotype-matched control antibody. Cells were subjected to fluorescence-activated cell sorting (FACS) analysis. Samples were analyzed by measuring MFI. Fold change in CD40 MFI value was calculated and shown as the mean ± SD of 3 experiments. (C) RAW264.7 and EOC13 cells were treated with 10 ng/mL LPS for up to 48 hours, then RNA was isolated and subjected to RPA analysis for CD40 and GAPDH mRNA. Fold induction on LPS treatment was calculated as in panel A. (D) RAW264.7 cells were treated in the absence or presence of LPS (10 ng/mL) for up to 48 hours. CD40 protein expression was analyzed by FACS analysis. Fold change of LPS-induced CD40 MFI value was calculated and shown as the mean ± SD of 3 experiments. (E) RAW264.7 cells were treated in the absence or presence of LPS (10 ng/mL) for up to 48 hours. Protein lysates were prepared and subjected to immunoblotting with anti-CD40 antibody, then stripped, and reprobed with antiactin antibody as a loading control. UN indicates untreated. Representative of 3 experiments. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology

LPS induces CD40 expression in primary macrophages and microglia. LPS induces CD40 expression in primary macrophages and microglia. Human primary macrophages (A) or murine primary microglia (B) were treated with LPS (10 ng/mL) for 36 hours, and then cells were subjected to FACS analysis for CD40 protein expression. ISO indicates isotype antibody. In addition, cells were treated with LPS for 4 and 8 hours, then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA expression. The cell numbers are mapped on the y-axis. Representative of 2 experiments. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology

NF-κB and GAS elements are important for LPS-induced activation of the CD40 promoter. NF-κB and GAS elements are important for LPS-induced activation of the CD40 promoter. (A) Deletion constructs of the human CD40 promoter. (B) RAW264.7 cells were transiently transfected with 0.2 μg of the indicated constructs, then treated with medium or LPS (10 ng/mL) for 12 hours and analyzed for luciferase activity. Values were normalized to total protein, and fold induction was calculated by dividing the LPS treatment values by UN levels. Data are presented as mean ± SD of 3 experiments. (C) Site-directed mutant constructs of NF-κB and GAS elements in the human CD40 promoter. (D) RAW264.7 cells were transiently transfected with 0.2 μg of the indicated constructs, allowed to recover for 4 hours, then were treated with medium or LPS (10 ng/mL) for 12 hours and analyzed for luciferase activity. Fold induction was calculated and presented as mean ± SD of 3 experiments. (E) RAW264.7 cells were transiently cotransfected with the WT CD40 promoter construct (0.2 μg) and expression vectors containing WT or double negative (DN) of IKK-α or IKK-β cDNA (0.1 μg), then they were treated with LPS (10 ng/mL) for 12 hours and analyzed for luciferase activity. Differences in the amount of DNA were adjusted with the empty vector pcDNA3. Data are presented as mean ± SD of 3 experiments. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology

NF-κB and STAT-1α signaling pathways are activated by LPS. (A) RAW264 NF-κB and STAT-1α signaling pathways are activated by LPS. (A) RAW264.7 cells were incubated in the absence or presence of LPS (10 ng/mL) for up to 4 hours. NF-κB and STAT-1α signaling pathways are activated by LPS. (A) RAW264.7 cells were incubated in the absence or presence of LPS (10 ng/mL) for up to 4 hours. Protein lysates were prepared and subjected to immunoblotting with anti–phospho-IKK-α/βSer180/Ser181, anti–phospho-IκBαSer32, and anti–phospho-NF-κB p65Ser536, stripped and reprobed with anti–NF-κB p65 and antiactin as loading controls. (B) RAW264.7 cells were incubated in medium or LPS (10 ng/mL) for up to 4 hours, lysed, and assayed for expression of IRF-3 protein. Actin protein expression was used as a loading control. (C) RAW264.7 or EOC13 cells were treated with LPS (10 ng/mL) for up to 12 hours, then RNA was isolated and subjected to RPA analysis for IFN-β and GAPDH mRNA expression. (D) RAW264.7 cells were treated with LPS (10 ng/mL) for up to 12 hours, then supernatants were collected and subjected to ELISA analysis for IFN-β protein expression. Data are presented as the mean ± SD of 3 experiments. (E) RAW264.7 cells were incubated in medium or LPS (10 ng/mL) for up to 4 hours, then cell lysates were prepared and subjected to immunoblotting with anti–phospho-STAT-1αTyr701 and anti–phospho-STAT-1αSer727, stripped, and reprobed with anti–STAT-1α and antiactin as loading controls. (F) RAW264.7 cells were treated with medium or LPS (10 ng/mL) in the absence or presence of 10 μg/mL isotype antibody or IFN-β–neutralizing antibody for 4 hours. Cell lysates were prepared and subjected to immunoblotting with anti–phospho-STAT-1αTyr701. Total STAT-1α and actin protein expression were used as loading controls. Representative of 3 experiments. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology

Endogenous IFN-β is important for optimal LPS-induced CD40 expression. Endogenous IFN-β is important for optimal LPS-induced CD40 expression. (A) RAW264.7 or EOC13 cells were treated with medium or LPS (10 ng/mL) in the presence of 10 μg/mL isotype antibody or neutralizing antibodies against IFN-γ, IFN-α, or IFN-β for 4 hours (RAW264.7 cells) or 8 hours (EOC13 cells). RNA was harvested and analyzed by RPA for CD40 and GAPDH mRNA. Representative of 3 experiments. (B) RAW264.7 cells were treated with medium or LPS (10 ng/mL) in the absence or presence of 10 μg/mL isotype antibody or IFN-β–neutralizing antibody for 36 hours. CD40 protein expression was detected by flow cytometry. Samples were analyzed by measuring MFI. Fold change in CD40 MFI value was calculated and shown as the mean ± SD of 3 experiments. (C) Primary microglia from WT and STAT-1α–deficient mice were treated with LPS (10 ng/mL) for 4 hours, and then mRNA was analyzed by RPA for CD40 and GAPDH expression. (D) Primary microglia from WT and STAT-1α–deficient mice were treated with LPS for 36 hours, then cells were subjected to FACS analysis for CD40 protein expression. Fold change in CD40 MFI value was calculated and shown as the mean ± SD of 3 experiments. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology

LPS stimulates nuclear translocation and recruitment of transcriptional activators to the CD40 promoter and histone modifications. LPS stimulates nuclear translocation and recruitment of transcriptional activators to the CD40 promoter and histone modifications. (A) RAW264.7 cells were incubated in the absence or presence of LPS (10 ng/mL) for up to 4 hours. Cytoplasmic and nuclear fractions were prepared and assayed with anti–phospho-NF-κB p65Ser536 and anti–phospho-STAT-1αTyr701. The cytoplasmic and nuclear fractions were also probed with anti–caspase-3, anti–c-Jun, anti–NF-κB p65, or anti–STAT-1α antibodies. (B) RAW264.7 cells were treated with LPS (10 ng/mL) for up to 4 hours, then the cells were crosslinked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with anti–NF-κB p65, anti–NF-κB p50, anti–STAT-1α, and anti–Pol II antibody, or normal rabbit IgG. The basal level of the untreated sample was set as 1.0, and fold induction on LPS treatment was compared with that. Representative of 3 to 4 experiments. (C) RAW264.7 cells were treated with LPS (10 ng/mL) for up to 4 hours, then the cells were crosslinked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against histone acetylation, methylation, and phosphorylation. Fold induction on LPS treatment was calculated as in panel B. Representative of 3 experiments. (D) EOC13 cells were treated with LPS (10 ng/mL) for up to 4 hours, then the cells were crosslinked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against NF-κB p65, anti–NF-κB p50, STAT-1α, Ac-H3, and Ac-H4. Fold induction on LPS treatment was calculated as in panel B. Representative of 3 experiments. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology

Proposed model of LPS-induced CD40 gene expression. Proposed model of LPS-induced CD40 gene expression. LPS activates NF-κB, which leads to subsequent nuclear translocation and binding of p65 and p50 to NF-κB elements in the CD40 promoter. Concurrently, LPS induces IRF-3 expression, which activates IFN-β expression. This leads to the subsequent activation of STAT-1α, which then dimerizes, translocates into the nucleus, and binds to GAS elements in the CD40 promoter with delayed kinetics compared with NF-κB p65 and p50. Concurrent with NF-κB and STAT-1α recruitment, LPS leads to modifications in H3 and H4 and to recruitment of RNA Pol II. The sequential recruitment of transcription factors and Pol II to the CD40 promoter, in conjunction with permissive histone modifications, results in transcriptional activation of the CD40 gene. See “Discussion” for details. MD2 indicates the cofactor for TLR4; TIRAP, Toll-IL-1R domain-containing adapter protein; TYK2, tyrosine kinase-2. Hongwei Qin et al. Blood 2005;106:3114-3122 ©2005 by American Society of Hematology