A distal enhancer in Il12b is the target of transcriptional repression by the STAT3 pathway and requires the basic leucine zipper (B-ZIP) protein NFIL3

Abstract

Deregulated IL-12 and IL-23 production from activated myeloid lineage cells is a key driver of numerous T cell-dependent autoimmune and inflammatory diseases. IL-12 and IL-23 share a common p40 subunit encoded by Il12b, which is negatively regulated at the transcriptional level by the STAT3 (signal transducer and activator of transcription 3)-activating anti-inflammatory cytokine IL-10. We found that IL-10 targets an enhancer 10 kb upstream of the Il12b transcriptional start site. Within the enhancer, a single 10-bp site is required for the inhibitory effects of IL-10 and is bound by NFIL3 (nuclear factor, interleukin 3-regulated), a B-ZIP transcription factor. Myeloid cells lacking NFIL3 produce excessive IL-12p40 and increased IL-12p70. Thus, the STAT3-dependent expression of NFIL3 is a key component of a negative feedback pathway in myeloid cells that suppresses proinflammatory responses.

FIGURE 1.

IL-10 restricts Il12b transcription by a mechanism that targets an enhancer. A, fusion constructs between the enhancer (depicted as hyper-sensitive site, HSS) and the Il12b promoter. Each construct was flanked by duplicated β-globin insulators (INS). Four basic constructs (EnPr1, 2, 3, and Pr1) are shown. B–E, luciferase reporter activity in stably transfected RAW264.7 macrophages are shown following stimulation with LPS or LPS plus IL-10 for the times indicated. Note that the activity of the basal Pr1 reporter is ∼5-fold less that when the enhancer is attached. Data are presented as mean relative luciferase units (RLU) plus the S.D. and are representative of 10 experiments using stable transfections and five experiments using transient transfections. F, sequence conservation between species around the region of minimal IL-10 responsiveness within the Il12b enhancer (yellow).

FIGURE 2.

NFIL3 mRNA and protein levels are regulated by IL-10 signaling. A and B, BMDMs (A) and PDMs (B) from C57BL/6 mice (n = 4) were stimulated with LPS or LPS plus IL-10 over time. RNA was collected and analyzed by qRT-PCR for the indicated targets, and the data are presented as the mean ± S.E. C, BMDMs from Stat3^+/+;Tie2-cre (WT, n = 2) or Stat3^flox/flox;Tie2-cre (n = 2) were stimulated with LPS over time, and NFIL3 mRNA amounts were detected by qRT-PCR. Data are presented as the mean NFIL3 expression relative to GAPDH expression plus the S.D. D, Gut lamina propria and splenic CD11b⁺ cells were collected. RNA was analyzed by qRT-PCR for the indicated targets, and the data are presented as the mean plus the S.D.

FIGURE 3.

A NFIL3 binding site is identified in the enhancer region of Il12b. A, sequence of the consensus NFIL3 binding site. B and C, binding of NFIL3 to the enhancer. B, the sequence of gel-shift oligonucleotides used is shown, with each sequential 5- or 6-bp mutation (Mutants A–G). C, IVTT NFIL3 was generated and bound to ³²P-labeled oligonucleotide probes and then complexes were resolved by electrophoresis. Binding data are representative of three experiments.

FIGURE 4.

Overexpression of NFIL3 inhibits Il12b transcription. A, specificity of anti-NFIL3 antibodies. NFIL3 retroviral producer cell lines were analyzed by immunoblot with two anti-NFIL3 antibodies, V-19 and C-18. Empty vector YFP⁺ lines were used as a negative control, and NFIL3 IVTT was used as a positive control. The asterisk denotes a nonspecific band used as a loading control. B and C, BMDMs from Il10^−/− (B) and C57BL/6 (C) mice were stimulated with LPS with or without IL-10. Protein lysates were immunoblotted for NFIL3 or Grb2 as a loading control. D, RAW264.7 macrophages were transiently cotransfected with 0.2 μg, 1 μg, or 5 μg of NFIL3/pcDNA3.1 and 5 μg of the EnPr2 luciferase reporter. Cells were stimulated in duplicate with LPS, and luciferase activity was measured at the times indicated. Data are presented as the mean relative luciferase units (RLU) plus the S.D. and are representative of two experiments. E, RAW264.7 macrophages were transiently transfected with 2 μg of NFIL3/pcDNA3.1, stimulated with LPS or LPS plus IL-10 in triplicate for the indicated times, and then IL-12p40 mRNA was detected by qRT-PCR. Data are presented as the mean IL-12p40 expression relative to GAPDH expression plus the S.D. Data are representative of two experiments.

FIGURE 5.

NFIL3 is required for inhibition of IL-12p40. A, BMDMs and PDMs from Nfil3^−/− mice or littermate WT controls (n = 3) were stimulated with LPS, and RNA was analyzed by qRT-PCR for expression of IL-12p40 and IL-6. Data are presented as the individual mRNA levels, including the means, normalized to GAPDH. B, supernatants from cells stimulated for 8 h with LPS (L) and with LPS plus IL-10 (L + 10) were analyzed for secreted IL-12p40 by ELISA. Data are presented as the individual protein levels (solid lines depict the respective means). C and D) CD11c⁺ DCs from Nfil3^−/− and littermate WT controls (n = 3) were stimulated with LPS (C) or curdlan (Cur.) (D) with and without IL-10 for 24 h. Secreted IL-12p40, IL-12p70, and IL-23 amounts were determined by ELISA. E, DCs from above were stimulated for 4 h with LPS or curdlan alone or plus IL-10, and qRT-PCR was used to measure IL-12p40 and IL-23p19 transcripts. Data from C–E are presented as the mean protein levels or mRNA levels normalized to GAPDH plus the S.E. All data are representative of at least two experiments unless otherwise noted. *, **, and *** represent significance, where p < 0.05, < 0.01, and < 0.001, respectively, by Student's t test.

FIGURE 6.

NFIL3 regulates systemic IL-12p40 levels in vivo. A, Nfil3^−/− and littermate WT controls were administered a sublethal LPS challenge (20 mg/kg), and serum IL-12p40 and IL-6 protein levels were detected by ELISA at 4 and 18 h post-challenge. Data are presented as the individual serum protein levels, including the means. B, Nfil3^−/− and littermate WT controls were infected with T. gondii. Serum IL-12p40, IFN-γ, and IL-6 protein levels were detected by ELISA 3 and 7 days post-infection. Data are presented as the individual serum protein levels, including the means. Data are representative of three independent experiments. *, p < 0.05 by Student's t test.