Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/ signal transducer and activator of transcription (JAK/ STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-␣. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-␤ (IFN-␤), which is able to exert auto-and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NFB signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NFB pathway.

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IntroductionInnate immunity represents the first line of defense against invading pathogens. Tissue-resident immune cells, including macrophages and dendritic cells (DCs), are the first immune cells to encounter infectious agents. Recognition of infecting pathogens within these cells is performed by now well-known pattern recognition receptors responding to conserved microbial structures. 1 Among these receptors, toll-like receptors (TLRs) have been identified as playing a pivotal role and indeed, both macrophages and dendritic cells express TLRs. [2][3][4] In macrophages, TLR stimulation activates the antimicrobial weaponry, including increased phagocytosis 5 ; in dendritic cells, migration into lymph nodes, antigen presentation, cytokine secretion, and subsequent activation of adaptive immunity 6 are enhanced. Interestingly, both of these primary pathogen-encountering cells can develop from monocytes, which thus serve as common progenitors. Differentiation is regulated by cytokines from the environment and immune cells among which macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophagecolony-stimulating factor (GM-CSF) are of ultimate importance.Although it is well accepted that TLR stimulation activates macrophages and dendritic cells, there is only limited information as to a role of TLRs during differentiation processes. Thus, it is largely unknown whether TLR stimulation affects the replenishment of tissue-resident macrophages and dendritic cells from monocytes. This question is of significance as it might suggest a role for infectious stimuli to influence and guide the cellular composition of inflammatory responses.It has been reported that migration of dendritic cells from local tissue to lymph nodes can be inhibited by intradermal administration of either whole Salmonella typhimurium or simply by LPS in a TLR-4-dependent manner. 7 Another study showed that administration of LPS during the early stage of differentiation of human monocytes to DCs diminished the generation of CD1a ϩ DCs. 8 This was confirmed by another group demonstrating that this blocking effect of LPS depends on activation of mitogen-activated protein kinase p38. 9 However, the exact mechanisms of these inhibitory effects still remain to be identified. Secreted cytokines such as IL-10 9 have been suggested but seem to play only a minor role. 7 Myeloid dendritic cell differentiation depends on the activity of GM-CSF, a cytokine that signals through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), especially JAK2 and STAT5. 10 Suppressor of cytokine signaling proteins (SOCS) have been identified as intracellular feedback inhibitors of JAK/STAT signaling. 11-13 Furthermore, SOCS-1 has been shown to negatively regulate JAK2 and GM-CSF signaling by inhibiting phosphorylation 14 as well as proteasomal targeting 15 of JAK2. Accordingly, SOCS-1 knockout mice, in spite of a lethal phenotype due to hypersensitivity 16,17 toward IFN-␥, also exhibit hyperresponsiveness to GM-CSF. 18,19 We and othe...