Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10.

IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-alpha, a potent proinflammatory mediator that is the target for multiple anti-TNF-alpha clinical strategies in Crohn's disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-alpha and IL-1alpha in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3' untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.

Recruitment of Stat1 to chromatin is required for interferon-induced serine phosphorylation of Stat1 transactivation domain.

The transcription factor Stat1 plays an essential role in responses to interferons (IFNs). Activation of Stat1 is achieved by phosphorylation on Y701 that is followed by nuclear accumulation. For full transcriptional activity and biological function Stat1 must also be phosphorylated on S727. The molecular mechanisms underlying the IFN-induced S727 phosphorylation are incompletely understood. Here, we show that both Stat1 Y701 phosphorylation and nuclear translocation are required for IFN-induced S727 phosphorylation. We further show that Stat1 mutants lacking the ability to stably associate with chromatin are poorly serine-phosphorylated in response to IFN-gamma. The S727 phosphorylation of these mutants is restored on IFN-beta treatment that induces the formation of the ISGF3 complex (Stat1/Stat2/Irf9) where Irf9 represents the main DNA binding subunit. These findings indicate that Stat1 needs to be assembled into chromatin-associated transcriptional complexes to become S727-phosphorylated and fully biologically active in response to IFNs. This control mechanism, which may be used by other Stat proteins as well, restricts the final activation step to the chromatin-tethered transcription factor.

Molecular mechanisms of the anti-inflammatory functions of interferons.

Interferons are pleiotropic cytokines with important proinflammatory functions required in defence against infections with bacteria, viruses and multicellular parasites. In recent years, fundamental functions of interferons in other processes such as cancer immunosurveillance, immune homeostasis and immunosuppression have been established. In addition, anti-inflammatory roles of interferons are well-documented in several inflammatory disease models in the mouse, most importantly in experimental autoimmune encephalomyelitis that resembles multiple sclerosis in humans. While the beneficial effects of interferons in such disease models are known, the molecular mechanisms remain poorly understood. Only recently a few molecular principles for the anti-inflammatory properties of interferons at the cellular level have been revealed. They include the ability of interferons to reduce the expression of the receptors for the inflammation-related cytokines IL-1 and IL-4, or to increase the expression of the potent anti-inflammatory genes tristetraprolin and Twist. However, the individual contribution of these anti-inflammatory responses to the overall beneficial effects of interferons in inflammatory diseases is still an open question. Also, the reason for the apparently limited number of tissues that are susceptible to the anti-inflammatory functions of interferons remains enigmatic. This review summarizes the present knowledge of the anti-inflammatory effects of interferons, and describes the currently known molecular mechanisms that may help explain the benefits of interferon signalling in several inflammatory diseases.

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9.

Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.

Interferons limit inflammatory responses by induction of tristetraprolin.

Interferons (IFNs) are cytokines with pronounced proinflammatory properties. Here we provide evidence that IFNs also play a key role in decline of inflammation by inducing expression of tristetraprolin (Ttp). TTP is an RNA-binding protein that destabilizes several AU-rich element-containing mRNAs including TNFalpha. By promoting mRNA decay, TTP significantly contributes to cytokine homeostasis. Now we report that IFNs strongly stimulate expression of TTP if a costimulatory stress signal is provided. IFN-induced expression of Ttp depends on the IFN-activated transcription factor STAT1, and the costimulatory stress signal requires p38 MAPK. Within the Ttp promoter we have identified a functional gamma interferon-activated sequence that recruits STAT1. Consistently, STAT1 is required for full expression of Ttp in response to LPS that stimulates both p38 MAPK and, indirectly, interferon signaling. We demonstrate that in macrophages IFN-induced TTP protein limits LPS-stimulated expression of several proinflammatory genes, such as TNFalpha, IL-6, Ccl2, and Ccl3. Thus, our findings establish a link between interferon responses and TTP-mediated mRNA decay during inflammation, and propose a novel immunomodulatory role of IFNs.

IFN regulatory factor 3-dependent induction of type I IFNs by intracellular bacteria is mediated by a TLR- and Nod2-independent mechanism.

Like viruses, intracellular bacteria stimulate their host cells to produce type I IFNs (IFN-alpha and IFN-beta). In our study, we investigated the signals and molecules relevant for the synthesis of and response to IFN by mouse macrophages infected with Listeria monocytogenes. We report that IFN-beta is the critical immediate-early IFN made during infection, because the synthesis of all other type I IFN, expression of a subset of infection-induced genes, and the biological response to type I IFN was lost upon IFN-beta deficiency. The induction of IFN-beta mRNA and the IFN-beta-dependent sensitization of macrophages to bacteria-induced death, in turn, was absolutely dependent upon the presence of the transcription factor IFN regulatory factor 3 (IRF3). IFN-beta synthesis and signal transduction occurred in macrophages deficient for TLR or their adaptors MyD88, TRIF, or TRAM. Expression of Nod2, a candidate receptor for intracellular bacteria, increased during infection, but the protein was not required for Listeria-induced signal transduction to the Ifn-beta gene. Based on our data, we propose that IRF3 is a convergence point for signals derived from structurally unrelated intracellular pathogens, and that L. monocytogenes stimulates a novel TLR- and Nod2-independent pathway to target IRF3 and the type I IFN genes.