Transcription of the gene encoding TNF-α is increased by IL-1β in rat and human islets and β-cell lines

Abstract

Synthesis and secretion of immunomodulatory proteins, such as cytokines and chemokines, controls the inflammatory response within pancreatic islets. When this inflammation does not resolve, destruction of pancreatic islet β-cells leads to diabetes mellitus. Production of the soluble mediators of inflammation, such as TNF-α and IL-1β, from resident and invading immune cells, as well as directly from islet β-cells, is also associated with suboptimal islet transplantation outcomes. In this study, we found that IL-1β induces rapid increases in TNF-α mRNA in rat and human islets and the 832/13 clonal β-cell line. The surge in transcription of the TNF-α gene required the inhibitor of kappa B kinase beta (IκKβ), the p65 subunit of the NF-κB and a signal-specific recruitment of RNA polymerase II to the gene promoter. Of note was the increased intracellular production of TNF-α protein in a manner consistent with mRNA accumulation in response to IL-1β, but no detectable secretion of TNF-α into the media. Additionally, TNF-α specifically induces expression of CD11b, but not CD11c, on neutrophils, which could contribute to the inflammatory milieu and diabetes progression. We conclude that activation of the NF-κB pathway in pancreatic β-cells leads to rapid intracellular production of the pro-inflammatory TNF-α protein through a combination of specific histone covalent modifications and NF-κB signaling pathways.

Keywords: Cytokine; Diabetes mellitus; Inflammation; Islet; NF-κB; Transcription.

Figure 1. Cytokine-mediated induction of TNFα mRNA in rat and human islets and a β-cell line

A. 832/13 cells were untreated (NT) or treated with 1 ng/mL IL-1β for the indicated times. B. Isolated rat islets were untreated (NT) or treated with 10 ng/mL IL-1β for the 1, 2 or 3h. C. Isolated rat islets were treated for 6 h with 10 ng/mL IL-1β, 100 U/mL IFN-γ or both cytokines in combination. **, P < 0.01, *, P < 0.05. D. 832/13 cells were treated with either 1 ng/mL IL-1β or IL-1β + 100 U/mL IFN-γ for 0.5, 1, 2 or 3 h. *, P < 0.05, #, P < 0.1. E. Following a 3 h stimulation with 1 ng/mL IL-1β (pre-exposure response induce by IL-1β is set at 100%), 832/13 cells were exposed to Actinomycin D (to block transcription) in the presence or absence of 100 U/mL IFN-γ. Total RNA was isolated at 0, 0.5, 1 and 2 h. F. Human islets were untreated (NT) or stimulated with IL-1β for 3 h.*, P < 0.05. A-F. TNFα transcript abundance was normalized to the housekeeping gene Ribosomal S9 (RS9). Data are shown as means ± SEM from three individual experiments.

Figure 2. Cytokines induce production of TNFα in 832/13 cells and TNF-α enhances surface expression of Cd11b, but not Cd11c, in bone-marrow derived neutrophils

A, B. 832/13 cells were treated with IL-1β (1 ng/mL), IFN-γ (100 U/mL) or the combination for 3, 6 or 12 h. TNFα secretion into the media (A) and cellular TNFα content (B) were quantified by ELISA and normalized to total protein. ***, P < 0.001 vs. NT, **, P < 0.01 (grey bars vs. NT), n.s. = not significant. ELISA assays were performed on three separate occasions. Data are expressed as means ± SEM. C,D. Isolated murine bone marrow neutrophils were exposed to 1ng/ml of TNFα for 30min (TNF) or media alone (Con). Cells were stained with antibodies to CD11c APC or CD11b PE and analyzed by flowcytometry. Results shown are representative of three biological replicates.

Figure 3. IL-1β-dependent stimulation of TNFα requires IκKβ

A. 832/13 cells were pre-treated for 1 h with either DMSO (vehicle control) or the indicated concentrations of the IκKβ inhibitor TPCA. Cells were subsequently treated for 3 h with 1 ng/mL IL-1β. Relative TNFα mRNA abundance was normalized to RS9. **, P < 0.01 vs. DMSO, *, P < 0.01 vs. DMSO. B. 832/13 cells were transfected with siRNA duplexes targeting either a scrambled control sequence (siScramble) or IκKβ (siIκKβ). After 48 h exposure to siRNA, cells were harvested and mRNA levels of IκKβ were quantified. **, P <0 .01. C. 832/13 cells were transfected with siScramble (siScr), siIκKβ or siIκKβ or siIκKα. After 48 h culture with the indicated siRNA duplexes, cells were harvested and an immunoblot performed to determine the cellular abundance of IκKβ; Actin was used as the loading control. The immunoblot shown is representative of two independent experiments. D. 832/13 cells were transfected with siScramble and siIκKβ duplexes. After 48 h, cells were stimulated with 1 ng/mL IL-1β for 3 h. TNFα mRNA level was quantified and normalized to RS9. **, P < 0.01. For mRNA experiments, three individual replicates were generated and expressed as means ± SEM.

Figure 4. NF-κB subunit p65, but not p50, is required for IL-1β-dependent activation of the TNFα gene

A. 832/13 cells were transduced with adenoviruses overexpressing βGalactosidase (βGAL) or IκBα Superrepressor (SR). Following a 24 h transduction with the indicated adenoviruses, cells were stimulated for 15 or 30 min with 1 ng/mL IL-1β. An immunoblot was performed using whole cell lysates and antibodies against IκBα using actin as the loading control. B. 832/13 cells were transduced with adenoviruses overexpressing βGAL or IκBα SR. Following a 24 h exposure to adenoviruses, cells were stimulated for 3 h with 1 ng/mL IL-1β. **, P < 0.01. C, D. . 832/13 cells were transfected with siRNA targeted to p65 and incubated for 48 h. C. 48 h post-transfection, whole cell lysates were blotted to determine abundance of p65. Actin served as the loading control. D. After 48 h exposure to siRNA cells were stimulated for 3 h with 1 ng/mL IL-1β. ***, P < 0.001, n.s. = not significant. B-D. mRNA levels of TNFα were measured and normalized to RS9. Immunoblots were performed on two separate occasions and a representative image is shown. Data are shown as means ± SEM from three independent experiments.

Figure 5. IL-1β recruits p65 to the TNFα promoter and promotes changes in histone methylation and RNA Pol II phosphorylation

A. Schematic representation of the TNFα promoter and coding regions. Arrows indicate regions amplified by PCR using recovered DNA as a template. B-G. 832/13 cells were treated with 1 ng/mL IL-1β for the indicated times. ChIP assays were performed using antisera to, methylated histone H3 (lysine 4; B and lysine 9; C), p65 (D) ,total Pol II (E) and Pol II CTD-phospho Serine 5 (F) and Serine 2 (G) on both the TNFα promoter and coding regions. ***, P < 0.001 vs. NT, **, P < 0.01 vs. NT, *, P < 0.05 vs. NT. ChIP signal is shown relative to IgG control as the means ± SEM from 3-4 individual experiments.