Tumor necrosis factor-α (TNFα) was cloned over 2 decades ago and its identification in part led to the discovery of a super family of tumor necrosis factors (TNFs) and their receptors. TNFα signals through two transmembrane receptors, TNFR1 and TNFR2, and regulates a number of critical cell functions including cell proliferation, survival, differentiation, and apoptosis. Macrophages are the major producers of TNFα and interestingly are also highly responsive to TNFα. Aberrant TNFα production and TNF receptor signaling have been associated with the pathogenesis of several diseases, including rheumatoid arthritis, Crohn’s disease, atherosclerosis, psoriasis, sepsis, diabetes, and obesity. TNFα has been shown to play a pivotal role in orchestrating the cytokine cascade in many inflammatory diseases and because of this role as a “master-regulator” of inflammatory cytokine production, it has been proposed as a therapeutic target for a number of diseases. Indeed anti-TNFα drugs are now licensed for treating certain inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. In this review we discuss the discovery of TNFα and its actions especially in regulating macrophage biology. Given its importance in several human diseases, we also briefly discuss the role of anti-TNFα therapeutics in the treatment of inflammatory diseases.

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-κB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-κB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.

SUMMARY Iron is an essential cofactor with unique redox properties. Iron regulatory proteins 1 and 2 (IRP1/2) have been established as important regulators of cellular iron homeostasis, but little is known about the role of other pathways in this process. Here we report that the mammalian target of rapamycin (mTOR) regulates iron homeostasis by modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. Mechanistic studies identify tristetraprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA. We also show that TTP is strongly induced by iron chelation, promotes downregulation of iron-requiring genes in both mammalian and yeast cells, and modulates survival in low-iron states. Taken together, our data uncover a link between metabolic, inflammatory, and iron regulatory pathways, and point towards the existence of a yeast-like TTP-mediated iron conservation program in mammals.

Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in Raw264.7 macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene, macrophage inflammatory protein-1β. Consistent with these results, over-expression of GRK2 or 5 enhances TNFα-induced NFκB activity. In addition, we show that GRK2 and 5 interact with IκBα via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and 5 in vitro. Furthermore, we also find that GRK5 but not GRK2 phosphorylates IκBα at the same amino acid residues (Ser32/36) as that of IKKβ. Interestingly, associated with these results, knockdown of IKKβ in Raw264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and 5 are important and novel mediators of a non-traditional IκBα-NFκB signaling pathway.

Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3′-untranslated regions (3′UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3′UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases.AU-rich elements | mRNA stability | inflammation | deadenylation T ristetraprolin (TTP) is the prototype of a small family of RNA binding proteins that can bind to adenylate-uridylate (AU)-rich elements (AREs) in the 3′-UTR (3′UTR) of its target mRNAs and promote their rapid turnover (1, 2). TTP-deficient mice developed a chronic systemic inflammatory syndrome (3) that was prevented by interfering with the action of TNF (3-5). Tnf mRNA was then identified as a direct target of TTP-mediated destabilization (4, 6); its increase in stability in the TTP KO mice leads to the commensurate overproduction of TNF protein (4, 5).TTP mRNA expression exhibits a pattern characteristic of immediate-early response genes in several cell types, with low-toundetectable levels of expression under basal conditions, and a rapid and transient induction upon stimulation (4,7,8). The transient nature of this induction is largely due to the instability of the TTP mRNA itself, part of which is thought to be due to AREs located within the 3′UTR of TTP mRNA (4,8,9). Indeed, TTP has been suggested to bind to its own AREs and autoregulate its expression through a negative feedback loop (9). Although expression of TTP protein in these systems is also rapidly inducible, the protein is more stable than the mRNA after induction, often persisting at high level...