Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages.

During macrophage activation, expression of IL-1R-associated kinase (IRAK)-M is induced to suppress TLR-mediated responses and is a hallmark of endotoxin tolerance. Endotoxin tolerance requires tight regulation of genes occurring at the transcriptional and epigenetic levels. To identify novel regulators of IRAK-M, we used RAW 264.7 macrophages and performed a targeted RNA interference screen of genes encoding chromatin-modifying enzymes, signaling molecules, and transcription factors involved in macrophage activation. Among these, the transcription factor CCAAT/enhancer binding protein (C/EBP)ß, known to be involved in macrophage inactivation, was necessary for the induction of IRAK-M expression. Chromatin immunoprecipitation showed that C/EBPß was recruited to the IRAK-M promoter following LPS stimulation and was indispensable for IRAK-M transcriptional activation. Among histone 3-modifying enzymes, our screen showed that knockdown of the histone 3 lysine 27 (H3K27) methyltransferase and part of the polycomb recessive complex 2, enhancer of Zeste 2, resulted in IRAK-M overexpression. In contrast, knockdown of the H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat X chromosome suppressed the induction of IRAK-M in response to LPS stimulation. Accordingly, we demonstrated that H3K27 on the IRAK-M promoter is trimethylated in unstimulated cells and that this silencing epigenetic mark is removed upon LPS stimulation. Our data propose a mechanism for IRAK-M transcriptional regulation according to which, in the naive state, polycomb recessive complex 2 repressed the IRAK-M promoter, allowing low levels of expression; following LPS stimulation, the IRAK-M promoter is derepressed, and transcription is induced to allow its expression.

Neorogioltriol and Related Diterpenes from the Red Alga Laurencia Inhibit Inflammatory Bowel Disease in Mice by Suppressing M1 and Promoting M2-Like Macrophage Responses.

Macrophages are central mediators of inflammation, orchestrating the inflammatory response through the production of cytokines and nitric oxide. Macrophages obtain pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, which can be modulated by soluble factors, including natural products. Despite the crucial protective role of inflammation, chronic or deregulated inflammation can lead to pathological states, such as autoimmune diseases, metabolic disorders, cardiovascular diseases, and cancer. In this case, we studied the anti-inflammatory activity of neorogioltriol (1) in depth and identified two structurally related diterpenes, neorogioldiol (2), and O11,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3), with equally potent activity. We investigated the mechanism of action of metabolites 1⁻3 and found that all three suppressed macrophage activation and promoted an M2-like anti-inflammatory phenotype by inducing expression of Arginase1, MRC1, IRAK-M, the transcription factor C/EBPß, and the miRNA miR-146a. In addition, they suppressed iNOS induction and nitric oxide production. Importantly, treatment of mice with 2 or 3 suppressed DSS-induced colitis by reducing tissue damage and pro-inflammatory cytokine production. Thus, all these three diterpenes are promising lead molecules for the development of anti-inflammatory agents targeting macrophage polarization mechanisms.

The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

Coordinated Regulation of miR-155 and miR-146a Genes during Induction of Endotoxin Tolerance in Macrophages.

Endotoxin tolerance occurs to protect the organism from hyperactivation of innate immune responses, primarily mediated by macrophages. Regulation of endotoxin tolerance occurs at multiple levels of cell responses and requires significant changes in gene expression. In the process of macrophage activation, induced expression of microRNA (miR)-155 and miR-146a contributes to the regulation of the inflammatory response and endotoxin tolerance. In this article, we demonstrate that expression of both miRNAs is coordinately regulated during endotoxin tolerance by a complex mechanism that involves monoallelic interchromosomal association, alterations in histone methyl marks, and transcription factor binding. Upon activation of naive macrophages, Histone3 was trimethylated at lysine4 and NFκBp65 was bound on both miR-155 and miR-146a gene loci. However, at the stage of endotoxin tolerance, both miR gene loci were occupied by C/EBPß, NFκBp50, and the repressive Histone3 marks trimethylation of K9 of H3. DNA fluorescence in situ hybridization experiments revealed monoallelic interchromosomal colocalization of miR-155 and miR-146a gene loci at the stage of endotoxin tolerance, whereas RNA-DNA-fluorescence in situ hybridization experiments showed that the colocalized alleles were silenced, suggesting a common repression mechanism. Genetic ablation of Akt1, which is known to abrogate endotoxin tolerance, abolished induction of loci colocalization and C/EBPß binding, further supporting that this mechanism occurs specifically in endotoxin tolerance. Overall, this study demonstrates that two miRNAs are coordinately regulated via gene colocalization at the three-dimensional chromatin space, same transcriptional machinery, and similar Histone3 methylation profile, contributing to the development of endotoxin tolerance.

The downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 axis and by human cytomegalovirus (HCMV) associated factors allows the activation of the HCMV major IE promoter and the transition to productive infection.

Earlier studies had suggested that epigenetic mechanisms play an important role in the control of human cytomegalovirus (HCMV) infection. Here we show that productive HCMV infection is indeed under the control of histone H3K27 trimethylation. The histone H3K27 methyltransferase EZH2, and its regulators JARID2 and NDY1/KDM2B repress GFI1, a transcriptional repressor of the major immediate-early promoter (MIEP) of HCMV. Knocking down EZH2, NDY1/KDM2B or JARID2 relieves the repression and results in the upregulation of GFI1. During infection, the incoming HCMV rapidly downregulates the GFI1 mRNA and protein in both wild-type cells and in cells in which EZH2, NDY1/KDM2B or JARID2 were knocked down. However, since the pre-infection levels of GFI1 in the latter cells are significantly higher, the virus fails to downregulate it to levels permissive for MIEP activation and viral infection. Following the EZH2-NDY1/KDM2B-JARID2-independent downregulation of GFI1 in the early stages of infection, the virus also initiates an EZH2-NDY1/ΚDM2Β-JARID2-dependent program that represses GFI1 throughout the infection cycle. The EZH2 knockdown also delays histone H3K27 trimethylation in the immediate early region of HCMV, which is accompanied by a drop in H3K4 trimethylation that may contribute to the shEZH2-mediated repression of the major immediate early HCMV promoter. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program.

Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice.

Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2(-/-) mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2(-/-) mice compared with WT mice. Alveolar macrophages from acid-injured Akt2(-/-) mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2(-/-) mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2(-/-) mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2(-/-) macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization.

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2(-/-) mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1(-/-) mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2(-/-) macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2(-/-) macrophages, and its target C/EBPß appear to play a key role in this process. C/EBPß, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Neuronal Mitophagy: Friend or Foe?

Maintenance of neuronal homeostasis is a challenging task, due to unique cellular organization and bioenergetic demands of post-mitotic neurons. It is increasingly appreciated that impairment of mitochondrial homeostasis represents an early sign of neuronal dysfunction that is common in both age-related neurodegenerative as well as in neurodevelopmental disorders. Mitochondrial selective autophagy, known as mitophagy, regulates mitochondrial number ensuring cellular adaptation in response to several intracellular and environmental stimuli. Mounting evidence underlines that deregulation of mitophagy levels has an instructive role in the process of neurodegeneration. Although mitophagy induction mediates the elimination of damaged mitochondria and confers neuroprotection, uncontrolled runaway mitophagy could reduce mitochondrial content overstressing the remaining organelles and eventually triggering neuronal cell death. Unveiling the molecular mechanisms of neuronal mitophagy and its intricate role in neuronal survival and cell death, will assist in the development of novel mitophagy modulators to promote cellular and organismal homeostasis in health and disease.

Case Report: Intracranial Hypertension Secondary to Guillain-Barre Syndrome.

Guillain-Barre Syndrome (GBS), a common cause of acute flaccid paralysis, is characterized by a rapidly progressive, usually symmetric weakness of the extremities. Headache and intracranial hypertension (ICHT) are very rare complications of GBS. Herein we report our current case of an obese girl with typical signs of GBS associated with autonomic dysfunction, cranial nerve deficits and increased intracranial pressure (ICP). We also perform a systematic study presenting and discussing previous case reports of GBS associated with ICHT, papilledema or hydrocephalus, highlighting the differences of the current case compared to previous studies. Although intracranial hypertension is a rare complication of pediatric GBS, clinicians should promptly detect it. Obesity may be a predisposing factor, given the strong association between idiopathic intracranial hypertension (IIH) and weight gain. Neurological evaluation, fundus examination and low threshold for intracranial imaging should be an integral part of medical practice in case of obesity, headache or visual changes in GBS patients.