The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription

Abstract

The antiinflammatory cytokine IL-10 inhibits the production of multiple, diverse inflammatory mediators from activated macrophages and dendritic cells, a process requiring STAT3 activation. However, the mechanisms involved in the broad inhibitory effects of IL-10 are controversial. I eliminated the contribution of the major confounding variable to understanding the antiinflammatory response, the 3' UTR region of inflammatory mediator genes, through knock-in mutation and analysis of the effects of IL-10 on transcription rate of inflammatory genes. IL-10 activates STAT3 to act indirectly by selectively inhibiting gene transcription independent of general effects on NF-kappaB or posttranscriptional mRNA processing through a process that reduces the overall transcriptional rate of specific genes.

Fig. 1.

IL-10 inhibits the rate of transcription at LPS-induced inflammatory genes. (a) Total RNA was isolated from stimulated macrophages and subjected to denaturing agarose gel electrophoresis to assess RNA quality followed by Northern blotting using probes specific for IL-10 targets whose expression levels have been previously shown to decrease after costimulation with LPS and IL-10. Shown is the same Northern blot probed with cDNA probes for TNF-α or IL-1α. Note that IL-10 strongly inhibits total mRNA levels. (b) PT RT-PCR assays were performed for the target genes shown by using normalized DNase-treated RNA samples from IL-10^-/- BMDMs stimulated with 10 ng/ml IL-10 (black triangles), 100 ng/ml LPS (red triangles), or LPS and IL-10 (green triangles) for 0, 30, 60, 120, and 240 min. RNA was reverse-transcribed in the presence (Upper) or absence (Lower) of reverse transcriptase (RT) and subjected to SYBR green real-time PT RT-PCR. Results are representative of three independent experiments.

Fig. 2.

Analysis of the effects of IL-10 on IκB and IKK activity. (a) PT RT-PCR analysis of the effects of IL-10 on LPS-mediated induction of IκBα transcription. IL-10^-/- BMDMs stimulated with 10 ng/ml IL-10 (black circles), 100 ng/ml LPS (green triangles) or LPS and IL-10 (red triangles) for 0, 30, 60, 120, and 240 min. RNA was reverse-transcribed and subjected to SYBR green real-time PT RT-PCR. (b) Immunoblotting analysis of IκBα and IκBβ levels in primary macrophages after stimulation with LPS and IL-10. Note that IκB proteins levels drop after proteosome-mediated destruction of each protein followed by resynthesis. (c) (Left and Center) IKK activity assays showing that IL-10 does not affect the ability of immunoprecipitated IKK to phosphorylate a GST-IκBα 1-55 purified recombinant fusion protein. (Right) The same assay using a mutant form of the substrate.

Fig. 3.

Construction and analysis of mice in which the Tnfa locus has been modified to replace the endogenous 3′ UTR with the Gapdh 3′ UTR. (a) Schematic diagram of the modified locus after targeting and subsequent cre-lox-mediated elimination of the pgk-neo cassette. (b) PCR analysis of mice carrying the wild-type or Tnfa 3′ UTR::Gapdh 3′ UTR alleles. (c) Northern blotting analysis using a TNF-α cDNA probe of macrophages from wild-type or Tnfa 3′ UTR::Gapdh 3′ UTR mice treated with LPS or LPS and IL-10 under conditions described in the legend to Fig. 1. Note that the modification of the Tnfa locus causes two transcripts to be made. Equivalent total RNA levels are shown by ethidium bromide staining. (d and e) Representative conventional real-time RT-PCR analysis from RNA samples isolated from stimulated BMDMs from wild type for Tnfa 3′ UTR::Gapdh 3′ UTR mice. BMDMs were stimulated with IL-10 (black circles), LPS (red triangles), or LPS and IL-10 (green triangles) as described in the legend to Fig. 1. Note the scale difference between the two genotypes reflected the higher levels of the stabilized TNF-α mRNA in BMDMs from Tnfa 3′ UTR::Gapdh 3′ UTR mice.

Fig. 4.

The IL-10-mediated antiinflammatory effect requires new protein synthesis. (a and b) PT RT-PCR analysis of IL-10 targets in the absence (a) or presence (b) of 10 μg/ml cycloheximide (CHX). Total RNA was isolated from IL-10^-/- macrophages stimulated as described in Fig. 1 (LPS, green triangles; LPS and IL-10, red triangles) and subjected to PT RT-PCR. Note that induction of IL-6 by LPS requires new protein synthesis and that the requirement for new protein synthesis by IL-10 to block this gene cannot be determined. Data are representative of two independent experiments performed under identical conditions. (c) Northern blotting analysis of the effects of cycloheximide on IL-10-mediated inhibition of TNF-α mRNA in Tnfa 3′ UTR::Gapdh 3′ UTR BMDMs. Data are representative of three independent experiments. (d and e) Quantitative analysis by RT-PCR of the effects of IL-10 on Tnfa 3′ UTR::Gapdh 3′ UTR mRNA levels. Shown is conventional RT-PCR analysis for TNF-α using a FAM-labeled probe. BMDMs were stimulated with LPS (green triangles) or LPS and IL-10 (red triangles) as described in the legend to Fig. 1. Data were normalized by using β-actin as a standard. Data are representative of three independent experiments.