Macrophages activated by the gram negative bacterial product lipopolysaccharide (LPS) switch their core metabolism from oxidative phosphorylation to glycolysis1. Inhibition of glycolysis with 2-deoxyglucose (2DG) suppressed LPS-induced Interleukin-1 beta (IL-1β) but not Tumour necrosis factor alpha (TNFα) in macrophages. A comprehensive metabolic map of LPS-activated macrophages revealed up-regulation of glycolytic and down-regulation of mitochondrial genes, which correlated directly with the expression profiles of altered metabolites. LPS strongly increased the TCA cycle intermediate succinate. Glutamine-dependent anerplerosis was the major source of succinate with the ‘Gamma-Aminobutyric Acid (GABA)-shunt’ pathway also playing a role. LPS-induced succinate stabilized Hypoxia-inducible factor 1α (HIF-1α), an effect inhibited by 2DG, with IL-1β as an important target. LPS also increases succinylation of several proteins. Succinate is therefore identified as a metabolite in innate immune signalling which leads to enhanced IL-1β production during inflammation.

Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca 2؉ flux, activate NF-B, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-B signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).

[Keywords: Gene chip; gene transcription; IL-6; Jak-Stat; NF B] Supplemental material is available at http://www.genesdev.org.

Interleukin 1 (IL-1) is a protein with several biological activities regulating host defense and immune responses. We report here the isolation of human IL-1 cDNA. It encodes a precursor polypeptide of 269 amino acids (30,747 Mr). mRNA isolated by hybridization to this cDNA was translated in a reticulocyte cell-free system, yielding immunoprecipitable IL-1. Furthermore, this hybrid-selected mRNA was injected into Xenopus laevis oocytes, which subsequently secreted biologically active IL-1. The cDNA nucleotide sequence suggests that IL-1 is initially translated as a precursor molecule that is subsequently processed into the 15,000-20,000 Mr protein usually associated with IL-1 activity.Several aspects of immunological function and host response to infection and injury are attributable to various proteins released from stimulated mononuclear phagocytes (1). These include the following activities: leukocytic pyrogen (LP), a mediator of fever; leukocytic endogenous mediator (LEM), an inducer of several components of the acute-phase response; lymphocyte activating factor (LAF), which augments both lymphocyte proliferation and lymphokine production; and mononuclear cell factor (MCF), which induces prostaglandin E2 and collagenase synthesis in synovial cells. LP and LAF activities copurify and share common physical characteristics (2-4). Similarly, there is evidence that LP and LEM copurify (5) and that LAF and MCF are closely related. The term interleukin 1 (IL-1) is now used to describe these varied biological activities, although it is unclear whether IL-1 represents a single substance or a family of related molecules (1). We report here the cloning and sequence of a cDNA synthesized by reverse transcription of poly(A)+ RNA isolated from adherent human monocytes stimulated with endotoxin. mRNA isolated by hybridization to the cDNA clone directed the synthesis and secretion of biologically active IL-1 when injected into Xenopus laevis oocytes. The nucleotide sequence and in vitro translation suggest that human IL-1 is initially synthesized as a precursor with a molecular weight of 30,747.MATERIALS AND METHODS bound to oligo(dT)-cellulose. Translation Systems. Rabbit reticulocyte lysate was prepared, optimized, and treated with micrococcal nuclease as described (7). Each translation mixture contained 1 ,ug of poly(A)+ RNA in the presence of 100 ,uCi (1 uCi = 37 kBq) of [35S]methionine per ml. After incubation for 1 hr at 37°C, samples were immunoprecipitated (8) by incubation with 20 ,ld of rabbit anti-human IL-1 polyclonal serum (9) for 18 hr followed by the addition of 100 ,ul of IgGsorb (The Enzyme Center, Boston, MA). Antigens were then solubilized by the addition of 20 ,ul of electrophoresis buffer (10), boiled for 5 min, and electrophoresed in a 17.5% polyacrylamide gel (15 x 17 x 0.15 cm) (10) at 35 mA for 5 hr. Gels were treated with EN3HANCE (DuPont), dried, and exposed to photographic film for 24-72 hr.Poly(A)+ RNA samples to be assayed for biological activity were micro-injected into Xenopus oocyte...

The process through which multipotential hematopoietic cells commit to distinct lineages involves the induction of specific transcription factors. PU.1 (also known as Spi-1) and GATA-1 are transcription factors essential for the development of myeloid and erythroid lineages, respectively. Overexpression of PU.1 and GATA-1 can block differentiation in lineages in which they normally are downregulated, indicating that not only positive but negative regulation of these factors plays a role in normal hematopoietic lineage development. Here we demonstrate that a region of the PU.1 Ets domain (the winged helix-turn-helix wing) interacts with the conserved carboxyl-terminal zinc finger of GATA-1 and GATA-2 and that GATA proteins inhibit PU.1 transactivation of critical myeloid target genes. We demonstrate further that GATA inhibits binding of PU.1 to c-Jun, a critical coactivator of PU.1 transactivation of myeloid promoters. Finally, PU.1 protein can inhibit both GATA-1 and GATA-2 transactivation function. Our results suggest that interactions between PU.1 and GATA proteins play a critical role in the decision of stem cells to commit to erythroid vs. myeloid lineages.