IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages

Abstract

IL-10 plays a central nonredundant role in limiting inflammation in vivo. However, the mechanisms involved remain to be resolved. Using primary human macrophages, we found that IL-10 inhibits selected inflammatory genes, primarily at a level of transcription. At the TNF gene, this occurs not through an inhibition of RNA polymerase II (Pol II) recruitment and transcription initiation but through a mechanism targeting the stimulation of transcription elongation by cyclin-dependent kinase (CDK) 9. We demonstrated an unanticipated requirement for a region downstream of the TNF 3' untranslated region (UTR) that contains the nuclear factor κB (NF-κB) binding motif (κB4) both for induction of transcription by lipopolysaccharide (LPS) and its inhibition by IL-10. IL-10 not only inhibits the recruitment of RelA to regions containing κB sites at the TNF gene but also to those found at other LPS-induced genes. We show that although IL-10 elicits a general block in RelA recruitment to its genomic targets, the gene-specific nature of IL-10's actions are defined through the differential recruitment of CDK9 and the control of transcription elongation. At TNF, but not NFKBIA, the consequence of RelA recruitment inhibition is a loss of CDK9 recruitment, preventing the stimulation of transcription elongation.

Figure 1.

IL-10 inhibits proinflammatory gene transcription. (A) Cells were treated with LPS with or without IL-10 for 30 or 120 min. RNA was isolated and used in quantitative RT-PCR with probes specific for the indicated mRNA. (B) Position of PCR primers for detecting downstream Pol II at the TNF, IL6, NFKBIA, and NFKBIZ genes. (C) Cells were stimulated for the indicated times with LPS, followed by ChIP using Pol II antibodies (filled bars) or a rabbit isotype control (open bars) at downstream regions of the TNF and IL6 genes. (D) Cells were treated with LPS with or without IL-10 for 30 or 120 min and Pol II was measured by ChIP at the downstream regions of TNF, IL6, NFKBIZ, and NFKBIA. Data in A and C are expressed as mean ± SD of triplicate measurements for a single donor, representative of three independent experiments using different donors. Data in D are pooled data from three donors and are expressed as mean ± SEM. Statistical significance was assessed using the paired Student’s t test. *, P < 0.05; NS (not significant).

Figure 2.

IL-10 inhibits transcription elongation at TNF. (A and B) Cells were treated with LPS with or without IL-10 for 30 min followed by ChIP using Pol II, phospho-Ser2–specific, or rabbit isotype control antibodies at the TSS or downstream regions of the TNF (A) or NFKBIA (B) genes. (C) Cells were left unstimulated or preincubated for 30 min with 500 nM flavopiridol, followed by LPS stimulation for 30 min, and then subjected to ChIP. (D) ChIP assay of CDK9 recruitment to the start of the TNF or NFKBIA genes upon stimulation with LPS with or without IL-10. Data are expressed as mean ± SD of triplicate measurements for a single donor, representative of three independent experiments using different donors.

Figure 3.

IL-10 inhibits the recruitment of RelA to proinflammatory genes. (A–C) Adenovirus reporters based on the human TNF gene were infected into macrophages and stimulated with LPS with or without IL-10 for 4 h, after which luciferase assays were performed. Cells were either transfected with scrambled control oligonucleotides (siControl) or RelA-specific siRNA (siRelA; A), and cytoplasmic extracts were subjected to Western blotting using anti-RelA or tubulin antibodies to evaluate the extent of RelA knockdown (B) or cells were stimulated for the indicated times with LPS and total RNA was isolated and used as template in a quantitative RT-PCR gene expression assay for TNF mRNA (C). (D) Position of primers used in ChIP assays for the detection of RelA recruitment to the human TNF gene. NF-κB sites are indicated below the diagram, with the PCR targets represented by black lines with the length of the amplicons indicated above. (E) Cells were stimulated with LPS with or without IL-10 for 60 min, after which ChIP assays were performed using RelA-specific antibodies. Data are expressed as mean ± SD of triplicate measurements for a single donor representative of eight (A) or three (C and E) independent experiments using different donors.

Figure 4.

IL-10 inhibition of elongation at TNF is RelA dependent. (A and B) Cells were transfected with a siControl or siRelA oligonucleotides. Cells were stimulated with LPS with or without IL-10 for 1 h, after which ChIP was performed using RelA-specific antibodies and primers designed to the κB4 site of TNF (A) or Pol II–specific antibodies and primers specific for the TNF TSS or downstream regions (B). (C) Cells were treated with or without LPS for 1 h and ChIP was performed using Pol II–specific (left), phosphor-Ser2 (middle), or CDK9 (right) antibodies and primers specific for the TNF TSS+1. Data are expressed as mean ± SD of triplicate measurements for a single donor representative of three independent experiments using different donors.