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Institute
In cells the interorganelle communication comprises vesicular and non-vesicular mechanisms. Non-vesicular material transfer predominantly takes place at regions of close organelle apposition termed membrane contact sites and is facilitated by a growing number of specialized proteins. Contacts of the endoplasmic reticulum (ER) and mitochondria are now recognized to be essential for diverse biological processes such as calcium homeostasis, phospholipid biosynthesis, apoptosis, and autophagy. In addition to these universal roles, ER-mitochondria communication serves also cell type-specific functions. In this review, we summarize the current knowledge on ER-mitochondria contacts in cells of the innate immune system, especially in macrophages. We discuss ER- mitochondria communication in the context of macrophage fatty acid metabolism linked to inflammatory and ER stress responses, its roles in apoptotic cell engulfment, activation of the inflammasome, and antiviral defense.
Inflammatory activation of astroglia adds to the pathology of various neurological diseases. Astrocytes respond to microglia-derived cytokines such as interleukin-1α (IL-1α) with enhanced inflammatory signaling. This provokes pro-inflammatory gene expression of, among others, the eicosanoid-generating enzyme prostaglandin endoperoxide synthase 2 (Ptgs2). Whereas metabolic regulation of innate immune cell inflammatory responses is intensely studied, pathways related to how metabolism modulates inflammatory signaling in astrocytes are underexplored. Here, we examined how mitochondrial oxidative phosphorylation affects inflammatory responses towards IL-1α and tumor necrosis factor α in neonatal rat astrocytes. Blocking respiratory complex I and III or adenosine triphosphate (ATP) synthase did not affect activation of inflammatory signaling by IL-1α, but did elicit differential effects on inflammatory gene mRNA expression. Remarkably, mRNA and protein expression of Ptgs2 by IL-1α was consistently up-regulated when oxidative phosphorylation was inhibited. The increase of Ptgs2 resulted from mRNA stabilization. Mitochondrial inhibitors also increased IL-1α-triggered secretion of eicosanoids, such as prostaglandin E2, prostaglandin F2α, and 6-keto-prostaglandin F1α, as assessed by liquid chromatography/mass spectrometry. Mechanistically, attenuating oxidative phosphorylation elevated adenosine monophosphate (AMP) and activated AMP-activated protein kinase (AMPK). AMPK silencing prevented Ptgs2 up-regulation by mitochondrial inhibitors, while AMPK activators recapitulated Ptgs2 mRNA stability regulation. Our data indicate modulation of astrocyte inflammatory responses by oxidative metabolism, with relevance towards eicosanoid production.
Macrophages exposed to the Th2 cytokines interleukin (IL) IL-4 and IL-13 exhibit a distinct transcriptional response, commonly referred to as M2 polarization. Recently, IL-4-induced polarization of murine bone marrow-derived macrophages (BMDMs) has been linked to acetyl-CoA levels through the activity of the cytosolic acetyl-CoA-generating enzyme ATP-citrate lyase (ACLY). Here, we studied how ACLY regulated IL-4-stimulated gene expression in human monocyte-derived macrophages (MDMs). Although multiple ACLY inhibitors attenuated IL-4-induced target gene expression, this effect could not be recapitulated by silencing ACLY expression. Furthermore, ACLY inhibition failed to alter cellular acetyl-CoA levels and histone acetylation. We generated ACLY knockout human THP-1 macrophages using CRISPR/Cas9 technology. While these cells exhibited reduced histone acetylation levels, IL-4-induced gene expression remained intact. Strikingly, ACLY inhibitors still suppressed induction of target genes by IL-4 in ACLY knockout cells, suggesting off-target effects of these drugs. Our findings suggest that ACLY may not be the major regulator of nucleocytoplasmic acetyl-CoA and IL-4-induced polarization in human macrophages. Furthermore, caution should be warranted in interpreting the impact of pharmacological inhibition of ACLY on gene expression.
Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.
Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is an established pharmacological activator of AMP-activated protein kinase (AMPK). Both, AICAR and AMPK were reported to attenuate inflammation. However, AICAR is known for many AMPK-independent effects, although the mechanisms remain incompletely understood. Here we report a potent suppression of lipopolysaccharide (LPS)-induced inflammatory gene expression by AICAR in primary human macrophages, which occurred independently of its conversion to AMPK-activating 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate. Although AICAR did not interfere with activation of cytosolic signalling cascades and nuclear translocation of nuclear factor - κB (NFκB) by LPS, it prevented the recruitment of NFκB and RNA polymerase II to target gene promoters. AICAR also inhibited signal transducer and activator of transcription 3 (STAT3)-dependent induction of interleukin (IL) IL-6 and IL-10 targets, while leaving STAT6 and HIF1α-dependent gene expression in IL-4 and dimethyloxalylgylcine-treated macrophages intact. This points to a transcription factor-specific mode of action. Attenuated gene expression correlated with impaired NFκB and STAT3, but not HIF-binding in electrophoretic mobility shift assays in vitro. Conclusively, AICAR interferes with DNA binding of NFκB and STAT3 to modulate inflammatory responses.
Obesity-associated insulin resistance is driven by inflammatory processes in response to metabolic overload. Obesity-associated inflammation can be recapitulated in cell culture by exposing macrophages to saturated fatty acids (SFA), and endoplasmic reticulum (ER) stress responses essentially contribute to pro-inflammatory signalling. AMP-activated protein kinase (AMPK) is a central metabolic regulator with established anti-inflammatory actions. Whether pharmacological AMPK activation suppresses SFA-induced inflammation in a human system is unclear. In a setting of hypoxia-potentiated inflammation induced by SFA palmitate, we found that the AMP-mimetic AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) potently suppressed upregulation of ER stress marker mRNAs and pro-inflammatory cytokines. Furthermore, AICAR inhibited macrophage ER stress responses triggered by ER-stressors thapsigargin or tunicamycin. Surprisingly, AICAR acted independent of AMPK or AICAR conversion to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate (ZMP) while requiring intracellular uptake via the equilibrative nucleoside transporter (ENT) ENT1 or the concentrative nucleoside transporter (CNT) CNT3. AICAR did not affect the initiation of the ER stress response, but inhibited the expression of major ER stress transcriptional effectors. Furthermore, AICAR inhibited autophosphorylation of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), while activating its endoribonuclease activity in vitro. Our results suggest that AMPK-independent inhibition of ER stress responses contributes to anti-inflammatory and anti-diabetic effects of AICAR.
Loss of HIF-1α in macrophages attenuates AhR/ARNT-mediated tumorigenesis in a PAH-driven tumor model
(2016)
Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.