LPS induces CD40 gene expression through the activation of NF-kappaB and STAT-1alpha in macrophages and microglia

Abstract

CD40 is expressed on various immune cells, including macrophages and microglia. Aberrant expression of CD40 is associated with autoimmune inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. Interaction of Toll-like receptor-4 (TLR4) with the Gram-negative bacteria endotoxin lipopolysaccharide (LPS) results in the induction of an array of immune response genes. In this study, we describe that LPS is a strong inducer of CD40 expression in macrophages and microglia, which occurs at the transcriptional level and involves the activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 1alpha (STAT-1alpha). LPS-induced CD40 expression involves the endogenous production of the cytokine interferon-beta (IFN-beta), which contributes to CD40 expression by the activation of STAT-1alpha. Blocking IFN-beta-induced activation of STAT-1alpha by IFN-beta-neutralizing antibody reduces LPS-induced CD40 gene expression. Furthermore, LPS induces acetylation and phosphorylation of histones H3 and H4 and the recruitment of NF-kappaB, STAT-1alpha, and RNA polymerase II on the CD40 promoter in vivo in a time-dependent manner, all events important for CD40 gene transcription. These results indicate that both LPS-induced NF-kappaB activation and endogenous production of IFN-beta that subsequently induces STAT-1alpha activation play critical roles in the transcriptional activation of the CD40 gene by LPS.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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/β^{Ser180/Ser181}, anti–phospho-IκBα^Ser32, and anti–phospho-NF-κB p65^Ser536, 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-1α^Tyr701 and anti–phospho-STAT-1α^Ser727, 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-1α^Tyr701. Total STAT-1α and actin protein expression were used as loading controls. Representative of 3 experiments.

Figure 5.

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.

Figure 6.

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 p65^Ser536 and anti–phospho-STAT-1α^Tyr701. 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.

Figure 7.

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.