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-␥. The S727 phosphorylation of these mutants is restored on IFN-␤ 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 chromatinassociated 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.kinase ͉ transcription T he Stat (signal transducers and activators of transcription) proteins are major cytokine-activated transcription factors that play a vital role in the biology of the hematopoietic and immune systems (1). Triggering of the cytokine receptor causes Stat tyrosine phosphorylation by the receptor-associated Jak tyrosine kinases causing the Stat homo-or heterodimers to accumulate in the nucleus and bind DNA. In addition, several Stat proteins are serine-phosphorylated in the C-terminal transactivation domain. Generation of knockin mice bearing alanine instead of serine at position 727, the site of Stat1 and Stat3 serine phosphorylation, and in vivo reconstitution experiments using Stat4 mutated at the phosphorylation site S721 proved the importance of these modifications for the transcriptional activity and biological function (2-4). Stat1 is activated in response to type I and type II interferons (IFNs) by phosphorylation at both Y701 and S727. On type I IFN (IFN-␣ and IFN-␤) stimulation Stat1 is assembled in the ISGF3 complex (Stat1, Stat2, and Irf9 heterotrimer) and, to a lesser extent, in Stat1 homodimers. The type II IFN-␥ activates primarily Stat1 homodimers. Several studies revealed that Stat1 complexes are, to a considerable part, preassembled before IFN stimulation (5-7). Phosphorylation of Y701 triggers Stat1 to accumulate in the nucleus in an importin-␣5-dependent manner (8, 9). Residues within the Stat1 N terminus as well as in the DNA binding domain were shown to be critical for the IFN-induced nuclear translocation (10-13). Stat1 can also shuttle between the cytoplasm and nucleus independently of IFN stimulation and Y701 phosphorylation (11). Both types of IFNs require S727-phosphorylated Stat1 for biological responses (14-16). The identity of the IFN-induced S727 kinase is not fully resolved. Th...

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.

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 ofMAPKs and NF-B, 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.Group A streptococcus (GAS 4 ; Streptococcus pyogenes) is an important human Gram-positive pathogen responsible for a wide spectrum of infections, ranging from mild diseases (e.g. tonsillitis) to serious illness (e.g. necrotizing fasciitis, sepsis, or severe poststreptococcal sequelae) (1). The persistence of GAS in the human population and the severity of some GAS diseases are the result of activities of a number of virulence factors that enable the pathogen to escape immune surveillance or, on contrary, induce an overreaction of the immune system (2, 3). Although GAS is generally regarded as an extracellular pathogen, recent findings suggest that GAS can survive (although not multiply) within various host cells, such as neutrophils, macrophages, epithelial cells, and fibroblasts (4 -7). The surviving bacteria may serve as a reservoir for recurrent GAS diseases.Immune responses to bacteria are initiated by recognition of bacterial components called pathogen-associated molecular patterns through host cell-encoded pattern recognition receptors (PRRs) (8, 9). Typically, pathogen-associated molecular patterns are components of the bacterial cell wall (e.g. lipopolysaccharide and lipoteichoic acid), but they may also be derived from the inside of bacteria (e.g. DNA). The primary function of PRRs is to trigger signaling cascades that activate antimicrobial defense programs. The best studied class of PRRs is the Toll-like receptor (TLR) family, which consists of 13 transmembrane glycoprote...