A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells

Dendritic cells (DC) produce interleukin-12 (IL-12) in response to Toll-like receptor (TLR) activation. Two major TLR signaling pathways participate in the response to pathogens: the nuclear factor-kappaB (NF-kappaB)-dependent pathway leading to inflammatory cytokine secretion including IL-12 and the interferon (IFN)-dependent pathway inducing type I IFN and IFN-regulated genes. Here we show that the two pathways cooperate and are likely both necessary for inducing an optimal response to pathogens. R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly(I:C) (TLR3 ligand) or lipopolysaccharide (LPS; TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-beta mRNA accumulation by mouse bone marrow-derived DC (BM-DC). Strikingly, IL-12p70 but not IL-12p40 secretion was strongly reduced in BM-DC from STAT1(-/-) and IFNAR(-/-) mice. STAT1 tyrosine-phosphorylation, IL-12p35, and IFN-beta mRNA accumulation were strongly inhibited in IFNAR(-/-) BM-DC activated with the TLR ligand combinations. Similar observation were obtained in human TLR8-expressing monocyte-derived DC (moDC) using neutralizing anti-IFNAR2 antibodies, although results also pointed to a possible involvement of IFN-lambda1 (also known as IL-29). This suggests that TLR engagement on DC induces endogenous IFNs that further synergize with the NF-kappaB pathway for optimal IL-12p70 secretion. Moreover, analysis of interferon regulatory factors (IRF) regulation in moDC suggests a role for IRF7/8 in mediating IRF3-independent type I IFN and possibly IL-12p35 synthesis in response to TLR7/8.     

Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma- production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells

Natural killer cell stimulatory factor or interleukin 12 (NKSF/IL-12) is a heterodimeric cytokine produced by monocytes/macrophages, B cells, and possibly other accessory cell types primarily in response to bacteria or bacterial products. NKSF/IL-12 mediates pleiomorphic biological activity on T and NK cells and, alone or in synergy with other inducers, is a powerful stimulator of interferon gamma (IFN- gamma) production. IL-10 is a potent inhibitor of monocyte-macrophage activation, that inhibits production of tumor necrosis factor alpha (TNF-alpha), IL-1 and also IFN-gamma from lymphocytes acting at the level of accessory cells. Because TNF-alpha and IL-1 are not efficient inducers of IFN-gamma, the mechanism by which IL-10 inhibits IFN-gamma production is not clear. In this paper, we show that IL-10 is a potent inhibitor of NKSF/IL-12 production from human peripheral blood mononuclear cells activated with Staphylococcus aureus or lipopolysaccharide (LPS). Both the production of the free NKSF/IL-12 p40 chain and the biologically active p70 heterodimer are blocked by IL- 10. NKSF/IL-12 p40 chain mRNA accumulation is strongly induced by S. aureus or LPS and downregulated by IL-10, whereas the p35 mRNA is constitutively expressed and only minimally regulated by S. aureus, LPS, or IL-10. Although IL-10 is able to block the production of NKSF/IL-12, a powerful inducer of IFN-gamma both in vitro and in vivo, the mechanism of inhibition of IFN-gamma by IL-10 cannot be explained only on the basis of inhibition of NKSF/IL-12 because IL-10 can partially inhibit IFN-gamma production induced by NKSF/IL-12, and also, the IFN-gamma production in response to various stimuli in the presence of neutralizing antibodies to NKSF/IL-12. Our findings that antibodies against NKSF/IL-12, TNF-alpha, or IL-1 beta can significantly inhibit IFN-gamma production in response to various stimuli and that NKSF/IL-12 and IL-1 beta can overcome the IL-10-mediated inhibition of IFN-gamma, suggest that IL-10 inhibition of IFN-gamma production is primarily due to its blocking production from accessory cells of the IFN-gamma- inducer NKSF/IL-12, as well as the costimulating molecule IL-1 beta.     

Interferon-gamma and bacterial lipopolysaccharide act synergistically on human neutrophils enhancing interleukin-8, interleukin-1beta, tumor necrosis factor-alpha, and interleukin-12 p70 secretion and phagocytosis via upregulation of toll-like receptor 4

In human neutrophils, interferon (IFN)-gamma enhanced the expression of toll-like receptor 4 (TLR4), a crucial component of the signaling receptor complex for bacterial lipopolysaccharide (LPS). Lipopolysaccharide alone did not affect TLR4 expression, but costimulation with IFN-gamma and LPS induced higher levels of TLR4 expression than stimulation with IFN-gamma alone. Using the protein synthesis inhibitor cycloheximide and measuring the expression of CD35 in neutrophils stimulated with IFN-gamma and LPS alone or in combination, we could demonstrate that IFN-gamma enhances TLR4 by de novo protein synthesis, whereas the addition of LPS acts synergistically by enhancing vesicular mobilization to the cell surface. Costimulation with IFN-gamma and LPS induced neutrophil activation and enhanced secretion of the cytokines, interleukin (IL)-8, IL-1beta, tumor necrosis factor-alpha, and IL-12 p70, and phagocytosis of latex beads, processes that were blocked by a monoclonal antibody specific for TLR4. These data suggest that IFN-gamma primes neutrophils to respond to LPS.     

Inhibitors of ATP-binding cassette transporters suppress interleukin-12 p40 production and major histocompatibility complex II up-regulation in macrophages

ATP-binding cassette (ABC) transporters are a large family of proteins whose role is to translocate various substances across biological membranes. They include the Tangier disease protein ABC1, sulfonylurea receptors (SUR), multidrug resistance protein (MDR), and cystic fibrosis transmembrane regulator (CFTR). In the current study, we investigated the involvement of ABC transporters in the regulation of lipopolysaccharide (LPS) and/or interferon (IFN)-gamma-induced interleukin (IL)-12 p40 and tumor necrosis factor (TNF)-alpha production, nitric oxide formation, as well as major histocompatibility complex II up-regulation in macrophages. The general ABC transporter inhibitor glibenclamide suppressed both IL-12 p40 and nitric oxide production. However, glibenclamide failed to affect the production of TNF-alpha. The selective ABC1 inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and sulfobromophthalein mimicked the suppressive effect of glibenclamide on IL-12 p40 production. On the other hand, both the MDR inhibitor verapamil and CFTR blocker 2,2'-iminodibenzoic acid failed to suppress the production of IL-12 p40. Furthermore, selective inhibitors and activators of SURs were without effect. In agreement with the pharmacological data, macrophages expressed mRNA for ABC1, but not SURs or CFTR. Intracellular levels of IL-12 p40 were decreased by glibenclamide, suggesting that glibenclamide does not affect IL-12 p40 secretion. The effect of glibenclamide did not involve an interference with the activation of the p38 and p42/44 mitogen-activated protein kinases or c-Jun kinase. Glibenclamide also suppressed IFN-gamma-induced up-regulation of major histocompatibility complex II. Taken together, our results indicate that ABC proteins regulate LPS and/or IFN-gamma-induced macrophage activation.     

The chemotherapeutic agent DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis

Vascular disrupting agents (VDAs) represent a novel approach to the treatment of cancer, resulting in the collapse of tumor vasculature and tumor death. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a VDA currently in advanced phase II clinical trials, yet its precise mechanism of action is unknown despite extensive preclinical and clinical investigations. Our data demonstrate that DMXAA is a novel and specific activator of the TANK-binding kinase 1 (TBK1)-interferon (IFN) regulatory factor 3 (IRF-3) signaling pathway. DMXAA treatment of primary mouse macrophages resulted in robust IRF-3 activation and approximately 750-fold increase in IFN-beta mRNA, and in contrast to the potent Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), signaling was independent of mitogen-activated protein kinase (MAPK) activation and elicited minimal nuclear factor kappaB-dependent gene expression. DMXAA-induced signaling was critically dependent on the IRF-3 kinase, TBK1, and IRF-3 but was myeloid differentiation factor 88-, Toll-interleukin 1 receptor domain-containing adaptor inducing IFN-beta-, IFN promoter-stimulator 1-, and inhibitor of kappaB kinase-independent, thus excluding all known TLRs and cytosolic helicase receptors. DMXAA pretreatment of mouse macrophages induced a state of tolerance to LPS and vice versa. In contrast to LPS stimulation, DMXAA-induced IRF-3 dimerization and IFN-beta expression were inhibited by salicylic acid. These findings detail a novel pathway for TBK1-mediated IRF-3 activation and provide new insights into the mechanism of this new class of chemotherapeutic drugs.     

Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha

Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guerin (BCG) model we assessed whether early IL- 12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti- tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.     

Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice

Leishmania major promastigotes were found to avoid activation of mouse bone marrow-derived macrophages (BMM0) in vitro for production of cytokines that are typically induced during infection with other intracellular pathogens. Coexposure of BMM0 to the parasite and other microbial stimuli resulted in complete inhibition of interleukin (IL) 12 (p40) mRNA induction and IL-12 release. In contrast, mRNA and protein levels for IL-1(alpha), IL-1(beta), tumor necrosis factor (TNF) alpha, and inducible NO synthase (iNOS) were only partially reduced, and signals for IL-10 and monocyte chemoattractant protein (MCP-1/JE) were enhanced. The parasite could provide a detectable trigger for TNF- alpha and iNOS in BMM0 primed with interferon (IFN) gamma, but still failed to induce IL-12. Thus IL-12 induction is selectively impaired after infection, whereas activation pathways for other monokine responses remain relatively intact. Selective and complete inhibition of IL-12(p40) induction was observed using BMM0 from either genetically susceptible or resistant mouse strains, as well as IL-10 knockout mice, and was obtained using promastigotes from cutaneous, visceral, and lipophosphoglycan-deficient strains of Leishmania. The impaired production of the major physiological inducer of IFN-gamma is suggested to underlie the relatively prolonged interval of parasite intracellular survival and replication that is typically associate with leishmanial infections, including those producing self-limiting disease.