Quiz Ref ID Clonal hematopoiesis of indeterminate potential (CHIP) is defined as the presence of an expanded somatic blood cell clone in persons without other hematological abnormalities.^1-3 The presence of CHIP was shown to increase with age and is significantly associated with the risk for coronary heart disease.^2,4 Quiz Ref ID The most commonly mutated genes in CHIP are the DNA methyltransferase DNMT3A and the DNA demethylase TET2, which both epigenetically control gene expression. Functionally, these 2 predominant CHIP driver genes are important regulators of inflammation by augmenting monocyte adhesion and stimulated macrophage activity.^5,6 Accordingly, loss of TET2 function in hematopoietic cells accelerates atherosclerosis and promotes inflammasome activation in mice,^4,7 whereas loss of DNMT3A experimentally altered myeloid cell function and promoted upregulation of chemokines.^8,9

Macrophages are present throughout the human body, constitute important immune effector cells, and have variable roles in a great number of pathological, but also physiological, settings. It is apparent that macrophages need to adjust their activation profile toward a steadily changing environment that requires altering their phenotype, a process known as macrophage polarization. Formation of reactive oxygen species (ROS), derived from NADPH-oxidases, mitochondria, or NO-producing enzymes, are not necessarily toxic, but rather compose a network signaling system, known as redox regulation. Formation of redox signals in classically versus alternatively activated macrophages, their action and interaction at the level of key targets, and the resulting physiology still are insufficiently understood. We review the identity, source, and biological activities of ROS produced during macrophage activation, and discuss how they shape the key transcriptional responses evoked by hypoxia-inducible transcription factors, nuclear-erythroid 2-p45-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor-γ. We summarize the mechanisms how redox signals add to the process of macrophage polarization and reprogramming, how this is controlled by the interaction of macrophages with their environment, and addresses the outcome of the polarization process in health and disease. Future studies need to tackle the option whether we can use the knowledge of redox biology in macrophages to shape their mediator profile in pathophysiology, to accelerate healing in injured tissue, to fight the invading pathogens, or to eliminate settings of altered self in tumors. Antioxid. Redox Signal. 19, 595–637.

Hypoxia inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits, serves as a key regulator of metabolic adaptation to hypoxia. The amount of HIF-1α protein is regulated either by attenuating von Hippel-Lindau protein (pVHL)-dependent ubiquitination and subsequent 26 S proteasomal degradation or by enhancing cap-dependent mRNA translation, presumably involving a phosphatidyinositol 3-kinase (PI3K)/Akt-regulated pathway. In addition, it became apparent that Hsp90 protects HIF-1α from oxygen-independent degradation. Here we present evidence that PI3K/Akt is required for heat shock proteins to stabilize HIF-1α. In pVHL-deficient renal cell carcinoma cells, PI3K inhibition by LY294002 and wortmannin or transfection of either a dominant negative PI3K or a kinase-dead Akt mutant substantially lowered constitutively expressed HIF-1α without altering HIF-1α mRNA. Inhibitors of mitogen-activated protein kinase kinase (MAPKK) such as PD98059 or the p38 MAPK inhibitor SB203580 showed no interference. Considering that PI3K inhibitors down-regulated heat shock protein 90 (Hsp90) as well as Hsp70 expression and moreover attenuated heat- or hypoxia-induced Hsp70 as well as hypoxia-induced Hsp90 up-regulation we conclude that PI3K inhibition promoted degradation of HIF-1α indirectly by reducing steady state concentrations of Hsp90 and/or Hsp70. HIF-1α co-immunoprecipitated with Hsp90/Hsp70 and direct binding of Hsp70 to the oxygen-dependent degradation domain (ODD) of HIF-1α was proven by a pull-down assay and a peptide array. PI3K-mediated degradation of HIF-1α was confirmed in HEK 293 cells under hypoxia, suggesting that heat shock proteins constitute an integral component for HIF-1α accumulation. We conclude that PI3K/Akt contributes to HIF-1α stabilization by provoking expression of heat shock proteins.

Peroxisome proliferator-activated receptor gamma (PPARgamma) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARgamma expression. Considering that regulation of PPARgamma expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARgamma1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3'-untranslated regions (UTRs), we analyzed the impact of the PPARgamma-3'-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARgamma-3'-UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3'-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARgamma1 mRNA decay, whereas miR-27b overexpression decreased PPARgamma1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARgamma1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARgamma1 mRNA. Understanding molecular mechanisms decreasing PPARgamma might help to better appreciate inflammatory diseases.

Hypoxia-inducible factor-1 (HIF-1) is a regulator of metabolic adaptation to hypoxia. It is now appreciated that HIF-1α accumulation is achieved under normoxic conditions by various factors, such as TNF-α. Here, it was our intention to gain insight into the signaling mechanisms used by TNF-α to stimulate HIF-1α. In tubular LLC-PK₁or human embryonic kidney cells, TNF-α induced accumulation of HIF-1α protein but not HIF-1α mRNA. Blocking nuclear factor (NF)-κB with sulfasalazine or expression of an IκB superrepressor attenuated HIF-1α accumulation, whereas transfection of active p50/p65-NF-κB subunits mimicked a TNF-α response. Experiments with actinomycin D and cycloheximide also pointed to a transcriptional and translational process in facilitating the TNF-α response. Interestingly, and in contrast to established hypoxic signaling concepts, TNF-α elicited HIF-1α accumulation in a ubiquitinated form that still bound the von Hippel-Lindau (pVHL) protein. These data indicate that HIF-1α accumulation by TNF-α demands the NF-κB pathway, preserves ubiquitination of HIF-1α, and allows the HIF-1α-pVHL interaction.