We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.
Abstract Low concentrations of heparin (<1 unit/ml) inhibit by 80% the activity of leukocyte acid phosphatase. Leukocyte β-glucuronidase is less sensitive to heparin, and alkaline phosphatase and lysozyme are unaffected. The possible inhibitory effects of heparin should be considered in all measurements of leukocyte enzymatic activities.
Myeloid-derived suppressor cells (MDSCs) increase late sepsis immunosuppression and mortality in mice. We reported that microRNA (miR) 21 and miR-181b expression in Gr1+CD11b+ myeloid progenitors increase septic MDSCs in mice by arresting macrophage and dendritic cell differentiation. Here, we report how sepsis regulates miR-21 and miR-181b transcription. In vivo and in vitro binding studies have shown that C/EBPα transcription factor, which promotes normal myeloid cell differentiation, binds both miRNA promoters in Gr1+CD11b+ cells from sham mice. In contrast, in sepsis Gr1+CD11b+ MDSCs miR-21 and miR-181b promoters bind both transcription factors Stat3 and C/EBPβ, which co-imunoprecipitate as a single complex. Mechanistically, transcription factor Rb phosphorylation supports Stat3 and C/EBPβ accumulation at both miRNA promoters, and C/EBPβ or Stat3 depletion by siRNA in sepsis Gr1+CD11b+ MDSCs inhibits miR-21 and miR-181b expression. To further support this molecular path for MDSC accumulation, we found that Stat3 and C/EBP binding at miR-21 or miR-181b promoter was induced by IL-6, using a luciferase reporter gene transfection into naive Gr1+CD11b+ cells. Identifying how sepsis MDSCs are generated may inform new treatments to reverse sepsis immunosuppression.
Activating macrophage NLRP3 inflammasome can promote excessive inflammation, leading to severe cell and tissue damage and organ dysfunction. Here, we showed that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuated macrophage NLRP3 inflammasome activation. Broad rewiring of intracellular metabolism and enhanced autophagic flux occurred in inflammasome-activated macrophages, but neither was necessary for the PDHK-regulated reduction of NLRP3 inflammasome activity. PDHK inhibition protected against inflammation-induced mitochondrial fragmentation and cristae remodeling and improved mitochondrial function by repurposing mitochondria from ROS production to ATP generation. Inhibition of PDHK increased the expression of the mitochondrial fusion protein optic atrophy-1 (OPA1). Suppression of OPA1 partially reversed the effect of PDHK inhibition on NLRP3 inflammasome activation. In conclusion, our study suggests that inhibition of PDHK dampens macrophage NLRP3 inflammasome activation during acute inflammation by ameliorating mitochondrial damage in a mechanism separate from its canonical role as a metabolic regulator.
The addition of either d - or l -amino acids fails to increase hexose monophosphate shunt activity of resting or phagocytizing neutrophils. This is presumptive evidence against a major role for amino acid oxidase in the bactericidal activity of the cell.
Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.
Incubation of the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine with human neutrophils produced a rapid elevation of the intracellular level of cyclic adenosine monophosphate (cAMP). This cAMP spike occurred within 30 s after exposure and returned to normal by 5 min.
Sustained silencing of potentially autotoxic acute proinflammatory genes like tumor necrosis factor alpha (TNFalpha) occurs in circulating leukocytes following the early phase of severe systemic inflammation. Aspects of this gene reprogramming suggest the involvement of epigenetic processes. We used THP-1 human promonocytes, which mimic gene silencing when rendered endotoxin-tolerant in vitro, to test whether TNFalpha proximal promoter nucleosomes and transcription factors adapt to an activation-specific profile by developing characteristic chromatin-based silencing marks. We found increased TNFalpha mRNA levels in endotoxin-responsive cells that was preceded by dissociation of heterochromatin-binding protein 1alpha, demethylation of nucleosomal histone H3 lysine 9 (H3(Lys(9))), increased phosphorylation of the adjacent serine 10 (H3(Ser(10))), and recruitment of NF-kappaB RelA/p65 to the TNFalpha promoter. In contrast, endotoxin-tolerant cells repressed production of TNFalpha mRNA, retained binding of heterochromatin-binding protein 1alpha, sustained methylation of H3(Lys(9)), reduced phosphorylation of H3(Ser(10)), and showed diminished binding of NF-kappaB RelA/p65 to the TNFalpha promoter. Similar levels of NF-kappaB p50 occurred at the TNFalpha promoter in the basal state, during active transcription, and in the silenced phenotype. RelB, which acts as a repressor of TNFalpha transcription, remained bound to the promoter during silencing. These results support an immunodeficiency paradigm where epigenetic changes at the promoter of acute proinflammatory genes mediate their repression during the late phase of severe systemic inflammation.
