ABSTRACT
Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1ß processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1ß maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1ß secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1ß, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1ß cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1ß by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1ß, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.
Subject(s)
Caspase 8/immunology , Dendritic Cells/immunology , Histone Deacetylase Inhibitors/pharmacology , Interleukin-1beta/metabolism , Macrophages/immunology , Animals , Bone Marrow Cells , Carrier Proteins , Caspase 1/genetics , Caspase 1/immunology , Caspase Inhibitors/pharmacology , Caspases/genetics , Caspases, Initiator , Cells, Cultured , Colitis/chemically induced , Dextran Sulfate , Histone Deacetylases/immunology , Inflammasomes/immunology , Lipopolysaccharides , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 ProteinABSTRACT
Both hypoxic and inflammatory conditions activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here, we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of cosubstrate oxygen versus deprivation of co-factor iron).
Subject(s)
Ferritins/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Inflammation/metabolism , Iron/metabolism , Prolyl Hydroxylases/metabolism , Animals , Chromatography, High Pressure Liquid , Lipopolysaccharides/toxicity , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxygen/metabolism , Protein Processing, Post-Translational , Signal Transduction/physiology , Spectrophotometry, Atomic , Tandem Mass SpectrometryABSTRACT
Immune cells regulate a hypertonic microenvironment in the skin; however, the biological advantage of increased skin Na(+) concentrations is unknown. We found that Na(+) accumulated at the site of bacterial skin infections in humans and in mice. We used the protozoan parasite Leishmania major as a model of skin-prone macrophage infection to test the hypothesis that skin-Na(+) storage facilitates antimicrobial host defense. Activation of macrophages in the presence of high NaCl concentrations modified epigenetic markers and enhanced p38 mitogen-activated protein kinase (p38/MAPK)-dependent nuclear factor of activated T cells 5 (NFAT5) activation. This high-salt response resulted in elevated type-2 nitric oxide synthase (Nos2)-dependent NO production and improved Leishmania major control. Finally, we found that increasing Na(+) content in the skin by a high-salt diet boosted activation of macrophages in a Nfat5-dependent manner and promoted cutaneous antimicrobial defense. We suggest that the hypertonic microenvironment could serve as a barrier to infection.
Subject(s)
Anti-Infective Agents/pharmacology , Leishmaniasis, Cutaneous/drug therapy , Leishmaniasis, Cutaneous/metabolism , Macrophages/metabolism , Skin/metabolism , Sodium/metabolism , Animals , Enzyme Activation/physiology , Humans , Leishmania major/drug effects , Macrophages/drug effects , Mice , NFATC Transcription Factors/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Skin/drug effects , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Dendritic cells (DC) and macrophages (MΦ) play a pivotal role in antimicrobial defense, in the regulation of immune responses, and in maintaining tissue homeostasis. The analysis of DC and MΦ function relies on primary cells albeit these cells are known to be difficult to transfect. This makes the use of small interfering RNA (siRNA) for targeted manipulation of gene expression by RNA interference difficult. In the following chapter, we provide a detailed protocol for the successful transfer of siRNA via electroporation into a defined population of mouse bone marrow-derived MΦ or DC that does not cause toxicity to the myeloid cells or nonspecific alterations of their biological functions. Factors that influence the transfection and knockdown rate will be highlighted.