Pro-inflammatory Gene Promoters Research Articles

Sulforaphane impedes mitochondrial reprogramming and histone acetylation in polarizing M1 (LPS) macrophages

M1 (LPS) macrophages are characterized by a high expression of pro-inflammatory mediators, and distinct metabolic features that comprise increased glycolysis, a broken TCA cycle, or impaired OXPHOS with augmented mitochondrial ROS production. This study investigated whether the phytochemical sulforaphane (Sfn) influences mitochondrial reprogramming during M1 polarization, as well as to what extent this can contribute to Sfn-mediated inhibition of M1 marker expression in murine macrophages. The use of extracellular flux-, metabolite-, and immunoblot analyses as well as fluorescent dyes indicative for mitochondrial morphology, membrane potential or superoxide production, demonstrated that M1 (LPS/Sfn) macrophages maintain an unbroken TCA cycle, higher OXPHOS rate, boosted fusion dynamics, lower membrane potential, and less superoxide production in their mitochondria when compared to control M1 (LPS) cells. Sustained OXPHOS and TCA activity but not the concomitantly observed high dependency on fatty acids as fuel appeared necessary for M1 (LPS/Sfn) macrophages to reduce expression of nos2, il1β, il6 and tnfα. M1 (LPS/Sfn) macrophages also displayed lower nucleo/cytosolic acetyl-CoA levels in association with lower global and site-specific histone acetylation at selected pro-inflammatory gene promoters than M1 (LPS), evident in colorimetric coupled enzyme assays, immunoblot and ChIP-qPCR analyses, respectively. Supplementation with acetate or citrate was able to rescue both histone acetylation and mRNA expression of the investigated M1 marker genes in Sfn-treated cells. Overall, Sfn preserves mitochondrial functionality and restricts indispensable nuclear acetyl-CoA for histone acetylation and M1 marker expression in LPS-stimulated macrophages.

Butyrate limits inflammatory macrophage niche in NASH

Immune cell infiltrations with lobular inflammation in the background of steatosis and deregulated gut-liver axis are the cardinal features of non-alcoholic steatohepatitis (NASH). An array of gut microbiota-derived metabolites including short-chain fatty acids (SCFA) multifariously modulates NASH pathogenesis. However, the molecular basis for the favorable impact of sodium butyrate (NaBu), a gut microbiota-derived SCFA, on the immunometabolic homeostasis in NASH remains elusive. We show that NaBu imparts a robust anti-inflammatory effect in lipopolysaccharide (LPS) stimulated or classically activated M1 polarized macrophages and in the diet-induced murine NASH model. Moreover, it impedes monocyte-derived inflammatory macrophage recruitment in liver parenchyma and induces apoptosis of proinflammatory liver macrophages (LM) in NASH livers. Mechanistically, by histone deactylase (HDAC) inhibition NaBu enhanced acetylation of canonical NF-κB subunit p65 along with its differential recruitment to the proinflammatory gene promoters independent of its nuclear translocation. NaBu-treated macrophages thus exhibit transcriptomic signatures that corroborate with a M2-like prohealing phenotype. NaBu quelled LPS-mediated catabolism and phagocytosis of macrophages, exhibited a differential secretome which consequently resulted in skewing toward prohealing phenotype and induced death of proinflammatory macrophages to abrogate metaflammation in vitro and in vivo. Thus NaBu could be a potential therapeutic as well as preventive agent in mitigating NASH.

SARS-CoV-2 restructures host chromatin architecture.

Some viruses restructure host chromatin, influencing gene expression, with implications for disease outcome. Whether this occurs for SARS-CoV-2, the virus causing COVID-19, is largely unknown. Here we characterized the 3D genome and epigenome of human cells after SARS-CoV-2 infection, finding widespread host chromatin restructuring that features widespread compartment A weakening, A-B mixing, reduced intra-TAD contacts and decreased H3K27ac euchromatin modification levels. Such changes were not found following common-cold-virus HCoV-OC43 infection. Intriguingly, the cohesin complex was notably depleted from intra-TAD regions, indicating that SARS-CoV-2 disrupts cohesin loop extrusion. These altered 3D genome/epigenome structures correlated with transcriptional suppression of interferon response genes by the virus, while increased H3K4me3 was found in the promoters of pro-inflammatory genes highly induced during severe COVID-19. These findings show that SARS-CoV-2 acutely rewires host chromatin, facilitating future studies of the long-term epigenomic impacts of its infection.

Maternal obesity blunts antimicrobial responses in fetal monocytes.

Maternal pre-pregnancy (pregravid) obesity is associated with adverse outcomes for both mother and offspring. Amongst the complications for the offspring is increased susceptibility and severity of neonatal infections necessitating admission to the intensive care unit, notably bacterial sepsis and enterocolitis. Previous studies have reported aberrant responses to LPS and polyclonal stimulation by umbilical cord blood monocytes that were mediated by alterations in the epigenome. In this study, we show that pregravid obesity dysregulates umbilical cord blood monocyte responses to bacterial and viral pathogens. Specifically, interferon-stimulated gene expression and inflammatory responses to respiratory syncytial virus (RSV) and E. coli were significantly dampened, respectively . Although upstream signaling events were comparable, translocation of the key transcription factor NF-κB and chromatin accessibility at pro-inflammatory gene promoters following TLR stimulation was significantly attenuated. Using a rhesus macaque model of western style diet-induced obesity, we further demonstrate that this defect is detected in fetal peripheral monocytes and tissue-resident macrophages during gestation. Collectively, these data indicate that maternal obesity alters metabolic, signaling, and epigenetic profiles of fetal monocytes leading to a state of immune paralysis during late gestation and at birth.

The Inhibition of LPS-Induced Oxidative Stress and Inflammatory Responses Is Associated with the Protective Effect of (-)-Epigallocatechin-3-Gallate on Bovine Hepatocytes and Murine Liver.

This study aimed to evaluate whether (-)-epigallocatechin-3-gallate (EGCG) alleviates hepatic responses to lipopolysaccharide (LPS)-induced inflammation and oxidation. Isolated bovine hepatocytes and BALB/c mice were used for LPS challenge and EGCG pretreatment experiments in vitro and in vivo. LPS-challenged (6 μg/mL) hepatocytes exhibited increased levels of NF-κB (p65 and IκBα) and MAPK (p38, ERK, JNK) phosphorylation as well as increased binding activity of p65 to target pro-inflammatory gene promoters, and these effects were suppressed by pretreatment with 50 μM EGCG. Moreover, the reduction in Nrf2 signaling and antioxidant enzyme activities induced by LPS stimulation were reversed upon EGCG treatment. In vivo experiments demonstrated the protective role of EGCG in response to GalN/LPS-induced mortality and oxidative damage. Together, our results suggest that EGCG is hepatoprotective via inhibition of MAPK/NF-κB signaling and activation of the Nrf2 cascade. This information might help design strategies for counteracting hepatitis in ruminants and monogastric animals.

Lysine methyltransferase G9a is an important modulator of trained immunity.

ObjectivesHistone methyltransferase G9a, also known as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2), mediates H3K9 methylation which is associated with transcriptional repression. It possesses immunomodulatory effects and is overexpressed in multiple types of cancer. In this study, we investigated the role of G9a in the induction of trained immunity, a de facto innate immune memory, and its effects in non‐muscle‐invasive bladder cancer (NMIBC) patients treated with intravesical Bacillus Calmette‐Guérin (BCG).MethodsEHMT2 expression was assessed upon induction of trained immunity by RNA sequencing and Western blotting. G9a inhibitor BIX‐01294 was used to investigate the effect on trained immunity responses in vitro. Subsequent cytokine production was measured by ELISA, epigenetic modifications were measured by ChIP‐qPCR, Seahorse technology was used to measure metabolic changes, and a luminescence assay was used to measure ROS release. RNA sequencing was performed on BIX‐01294‐treated monocytes ex vivo.ResultsThe expression of EHMT2 mRNA and protein decreased in monocytes during induction of trained immunity. G9a inhibition by BIX‐01294 induced trained immunity and amplified trained immunity responses evoked by various microbial ligands in vitro. This was accompanied by decreased H3K9me2 at the promoters of pro‐inflammatory genes. G9a inhibition was also associated with amplified ex vivo trained immunity responses in circulating monocytes of NMIBC patients. Additionally, altered RNA expression of inflammatory genes in monocytes of NMIBC patients was observed upon ex vivo G9a inhibition. Furthermore, intravesical BCG therapy decreased H3K9me2 at the promoter of pro‐inflammatory genes.ConclusionInhibition of G9a is important in the induction of trained immunity, and G9a may represent a novel therapeutic target in NMIBC patients.

New live attenuated tuberculosis vaccine MTBVAC induces trained immunity and confers protection against experimental lethal pneumonia.

Among infectious diseases, tuberculosis is the leading cause of death worldwide, and represents a serious threat, especially in developing countries. The protective effects of Bacillus Calmette-Guerin (BCG), the current vaccine against tuberculosis, have been related not only to specific induction of T-cell immunity, but also with the long-term epigenetic and metabolic reprogramming of the cells from the innate immune system through a process termed trained immunity. Here we show that MTBVAC, a live attenuated strain of Mycobacterium tuberculosis, safe and immunogenic against tuberculosis antigens in adults and newborns, is also able to generate trained immunity through the induction of glycolysis and glutaminolysis and the accumulation of histone methylation marks at the promoters of proinflammatory genes, facilitating an enhanced response after secondary challenge with non-related bacterial stimuli. Importantly, these findings in human primary myeloid cells are complemented by a strong MTBVAC-induced heterologous protection against a lethal challenge with Streptococcus pneumoniae in an experimental murine model of pneumonia.

PRMT1 Is Recruited via DNA-PK to Chromatin Where It Sustains the Senescence-Associated Secretory Phenotype in Response to Cisplatin

Protein arginine methyltransferase 1 (PRMT1) is overexpressed in various human cancers and linked to poor response to chemotherapy. Various PRMT1 inhibitors are currently under development; yet, we do not fully understand the mechanisms underpinning PRMT1 involvement in tumorigenesis and chemoresistance. Using mass spectrometry-based proteomics, we identified PRMT1 as regulator of arginine methylation in ovarian cancer cellstreated with cisplatin. We showed that DNA-dependent protein kinase (DNA-PK) binds to and phosphorylates PRMT1 in response to cisplatin, inducing its chromatin recruitment and redirecting its enzymatic activity toward Arg3 of histone H4 (H4R3). On chromatin, the DNA-PK/PRMT1 axis induces senescence-associated secretory phenotype through H4R3me2a deposition at pro-inflammatory gene promoters. Finally, PRMT1 inhibition reduces the clonogenic growth of cancer cells exposed to low doses of cisplatin, sensitizing them to apoptosis. While unravelling the role of PRMT1 in response to genotoxic agents, our findings indicate the possibility of targeting PRMT1 to overcome chemoresistance in cancer.

Histone deacetylase 2 is essential for LPS‐induced inflammatory responses in macrophages

The role of specific histone deacetylase (HDAC) proteins in regulating the lipopolysaccharide (LPS)‐induced inflammatory response and its underlying mechanisms are unclear. Here, HDAC2, a class I HDAC family protein, is essential for the LPS‐triggered inflammatory response in macrophages. LPS stimulation increases HDAC2 expression in macrophages. Knockdown of HDAC2 decreases the expression of proinflammatory genes, such as IL‐12, TNF‐α and iNOS following stimulation with LPS. The adoptive transfer of HDAC2 knockdown macrophages attenuates the LPS‐triggered innate inflammatory response in vivo, and these mice are less sensitive to endotoxin shock and Escherichia coli‐induced sepsis. Mechanistically, the c‐Jun protein is the main target of HDAC2‐mediated LPS‐induced production of proinflammatory cytokines. Moreover, HDAC2 knockdown increases the expression of c‐Jun, which directly binds the promoters of proinflammatory genes and forms nuclear receptor corepressor complexes to inhibit the transcription of proinflammatory genes in macrophages. These effects are rescued by c‐Jun expression. According to the chromatin immunoprecipitation analysis, HDAC2 also selectively suppresses c‐Jun expression by directly binding to its promoter and modifying histone acetylation after LPS stimulation. Our findings define a new function and mechanism of the HDAC2/c‐Jun signaling network that regulates the LPS‐induced immune response in macrophages.

HADC5 deacetylates MKL1 to dampen TNF-α induced pro-inflammatory gene transcription in macrophages.

Macrophage-dependent inflammatory response on the one hand functions as a key line of defense in host immunity but on the other hand underlies the pathogenesis of a host of human pathologies when aberrantly activated. Our previous investigations have led to the identification of megakaryocytic leukemia 1 (MKL1) as a key co-factor of NF-κB/p65 participating in TNF-α induced pro-inflammatory transcription in macrophages. How post-translational modifications contribute to the modulation of MKL1 activity remains an underexplored subject matter. Here we report that the lysine deacetylase HDAC5 interacts with and deacetylates MKL1 in cells. TNF-α treatment down-regulates HDAC5 expression and expels HDAC5 from the promoters of pro-inflammatory genes in macrophages. In contrast, over-expression of HDAC5 attenuates TNF-α induced pro-inflammatory transcription. Mechanistically, HDAC5-mediated MKL1 deacetylation disrupts the interaction between MKL1 and p65. In addition, deacetylation of MKL1 by HDAC5 blocks its nuclear translocation in response to TNF-α treatment. In conclusion, our work has identified an important pathway that contributes to the regulation of pro-inflammatory response in macrophages.

Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.

A negative feedback loop of ICER and NF-κB regulates TLR signaling in innate immune responses.

The NF-κB pathway has important roles in innate immune responses and its regulation is critical to maintain immune homeostasis. Here, we report a newly discovered feedback mechanism for the regulation of this pathway by TLR ligands in macrophages. Lipopolysaccharide (LPS) induced the expression of ICER via p38-mediated activation of CREB in macrophages. ICER, in turn, inhibited the transcriptional activity of NF-κB by direct interaction with the p65 subunit of NF-κB. Deficiency in ICER elevated binding of NF-κB to promoters of pro-inflammatory genes and their subsequent gene expression. Mice deficient in ICER were hypersensitive to LPS-induced endotoxic shock and showed propagated inflammation. Whereas ICER expression in ICER KO bone marrow transplanted mice rescued the ultra-inflammation phenotype, expression of a p65 binding-deficient ICER mutant failed to do so. Our results thus establish p38-CREB-ICER as key components of a negative feedback mechanism necessary to regulate TLR-driven inflammation.

Oxidized Guanine Base Lesions Function in 8-Oxoguanine DNA Glycosylase-1-mediated Epigenetic Regulation of Nuclear Factor κB-driven Gene Expression

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression.

Epigenetic Regulation of Early- and Late-Response Genes in Acute Pancreatitis.

Chromatin remodeling seems to regulate the patterns of proinflammatory genes. Our aim was to provide new insights into the epigenetic mechanisms that control transcriptional activation of early- and late-response genes in initiation and development of severe acute pancreatitis as a model of acute inflammation. Chromatin changes were studied by chromatin immunoprecipitation analysis, nucleosome positioning, and determination of histone modifications in promoters of proinflammatory genes in vivo in the course of taurocholate-induced necrotizing pancreatitis in rats and in vitro in rat pancreatic AR42J acinar cells stimulated with taurocholate or TNF-α. Here we show that the upregulation of early and late inflammatory genes rely on histone acetylation associated with recruitment of histone acetyltransferase CBP. Chromatin remodeling of early genes during the inflammatory response in vivo is characterized by a rapid and transient increase in H3K14ac, H3K27ac, and H4K5ac as well as by recruitment of chromatin-remodeling complex containing BRG-1. Chromatin remodeling in late genes is characterized by a late and marked increase in histone methylation, particularly in H3K4. JNK and p38 MAPK drive the recruitment of transcription factors and the subsequent upregulation of early and late inflammatory genes, which is associated with nuclear translocation of the early gene Egr-1 In conclusion, specific and strictly ordered epigenetic markers such as histone acetylation and methylation, as well as recruitment of BRG-1-containing remodeling complex are associated with the upregulation of both early and late proinflammatory genes in acute pancreatitis. Our findings highlight the importance of epigenetic regulatory mechanisms in the control of the inflammatory cascade.

Over‐expression of prothymosin‐α antagonizes TGFβ signalling to promote the development of emphysema

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor-β (TGFβ) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFβ signalling and reduced histone deacetylase (HDAC) activity through epigenetic up-regulation of histone acetylation in the promoters of pro-inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFβ signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non-histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFβ-Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down-regulates TGFβ-Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R-Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3-Smad4 complex to the TIMP-3 promoter, resulting in reduced TIMP-3 expression. These effects were detected in ProT-over-expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP-3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)-induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFβ signalling that contributes to emphysema pathogenesis.

Chronic Intermittent Hypoxia Promotes Epigenetic Regulation of Inflammatory Gene Promoters in Microglia

Microglial inflammatory activities contribute to chronic neuroinflammation and cognitive impairments in virtually all CNS disorders. Chronic intermittent hypoxia (CIH), a hallmark of sleep disordered breathing, causes significant hippocampal and cortical neuron loss and subsequent cognitive decline and impaired vigilance. Previously, we demonstrated an upregulation of microglial pro-inflammatory activities early in CIH neuropathology in rodent models. CIH also increased the expression of the Jumonji C family histone demethylase, JMJD3, in microglia in vitro and in vivo. The expression correlated with reduced global histone H3K27 tri-methylation levels, suggesting the potential for increased gene transcription during CIH in immortalized microglia. Therefore, we tested the hypothesis that epigenetic regulation at pro-inflammatory gene promoters contributes to microglial inflammatory activity following early CIH exposure. We used ChIP qPCR to detect JMJD3 binding patterns, in addition to H3K27 tri-methylation patterns, at inflammatory gene promoters in immortalized N9 microglia exposed to 21% O2 (normoxia) or 10 min cycles of 21% O2 and 1% O2 (CIH). Preliminary results demonstrate that CIH increases JMJD3 binding at the inflammatory genes interleukin-6 and cyclooxygenase-2 compared to normoxia. We further show high H3K27 trimethylation at inflammatory genes in normoxic samples, consistent with minimal inflammatory gene transcription in the healthy CNS. These studies are the first to investigate the effects of histone modifying enzymes during CIH in any cell type, and to suggest a mechanistic underpinning for CIH-induced neuroinflammation. Supported by NIH R01HL111598.

Akirin2 is critical for inducing inflammatory genes by bridging IκB-ζ and the SWI/SNF complex.

Transcription of inflammatory genes in innate immune cells is coordinately regulated by transcription factors, including NF-κB, and chromatin modifiers. However, it remains unclear how microbial sensing initiates chromatin remodeling. Here, we show that Akirin2, an evolutionarily conserved nuclear protein, bridges NF-κB and the chromatin remodeling SWI/SNF complex by interacting with BRG1-Associated Factor 60 (BAF60) proteins as well as IκB-ζ, which forms a complex with the NF-κB p50 subunit. These interactions are essential for Toll-like receptor-, RIG-I-, and Listeria-mediated expression of proinflammatory genes including Il6 and Il12b in macrophages. Consistently, effective clearance of Listeria infection required Akirin2. Furthermore, Akirin2 and IκB-ζ recruitment to the Il6 promoter depend upon the presence of IκB-ζ and Akirin2, respectively, for regulation of chromatin remodeling. BAF60 proteins were also essential for the induction of Il6 in response to LPS stimulation. Collectively, the IκB-ζ-Akirin2-BAF60 complex physically links the NF-κB and SWI/SNF complexes in innate immune cell activation. By recruiting SWI/SNF chromatin remodellers to IκB-ζ, transcriptional coactivator for NF-κB, the conserved nuclear protein Akirin2 stimulates pro-inflammatory gene promoters in mouse macrophages during innate immune responses to viral or bacterial infection.

MRTF-A mediates LPS-induced pro-inflammatory transcription by interacting with the COMPASS complex.

Chronic inflammation underscores the pathogenesis of a range of human diseases. Lipopolysaccharide (LPS) elicits strong pro-inflammatory responses in macrophages through the transcription factor NF-κB. The epigenetic mechanism underlying LPS-induced pro-inflammatory transcription is not fully understood. Herein, we describe a role for myocardin-related transcription factor A (MRTF-A, also known as MKL1) in this process. MRTF-A overexpression enhanced NF-κB-dependent pro-inflammatory transcription, whereas MRTF-A silencing inhibited this process. MRTF-A deficiency also reduced the synthesis of pro-inflammatory mediators in a mouse model of colitis. LPS promoted the recruitment of MRTF-A to the promoters of pro-inflammatory genes in an NF-κB-dependent manner. Reciprocally, MRTF-A influenced the nuclear enrichment and target binding of NF-κB. Mechanistically, MRTF-A was necessary for the accumulation of active histone modifications on NF-κB target promoters by communicating with the histone H3K4 methyltransferase complex (COMPASS). Silencing of individual members of COMPASS, including ASH2, WDR5 and SET1 (also known as SETD1A), downregulated the production of pro-inflammatory mediators and impaired the NF-κB kinetics. In summary, our work has uncovered a previously unknown function for MRTF-A and provided insights into the rationalized development of anti-inflammatory therapeutic strategies.

Cigarette Smoke Induces Distinct Histone Modifications in Lung Cells: Implications for the Pathogenesis of COPD and Lung Cancer

Cigarette smoke (CS)-mediated oxidative stress induces several signaling cascades, including kinases, which results in chromatin modifications (histone acetylation/deacetylation and histone methylation/demethylation). We have previously reported that CS induces chromatin remodeling in pro-inflammatory gene promoters; however, the underlying site-specific histone marks formed in histones H3 and H4 during CS exposure in lungs in vivo and in lung cells in vitro, which can either drive gene expression or repression, are not known. We hypothesize that CS exposure in mouse and human bronchial epithelial cells (H292) can cause site-specific posttranslational histone modifications (PTMs) that may play an important role in the pathogenesis of CS-induced chronic lung diseases. We used a bottom-up mass spectrometry approach to identify some potentially novel histone marks, including acetylation, monomethylation, and dimethylation, in specific lysine and arginine residues of histones H3 and H4 in mouse lungs and H292 cells. We found that CS-induced distinct posttranslational histone modification patterns in histone H3 and histone H4 in lung cells, which may be considered as usable biomarkers for CS-induced chronic lung diseases. These identified histone marks (histone H3 and histone H4) may play an important role in the epigenetic state during the pathogenesis of smoking-induced chronic lung diseases, such as chronic obstructive pulmonary disease and lung cancer.

Progression of Chronic Liver Inflammation and Fibrosis Driven by Activation of c-JUN Signaling in Sirt6 Mutant Mice

The human body has a remarkable ability to regulate inflammation, a biophysical response triggered by virus infection and tissue damage. Sirt6 is critical for metabolism and lifespan; however, its role in inflammation is unknown. Here we show that Sirt6-null (Sirt6(-/-)) mice developed chronic liver inflammation starting at ∼2 months of age, and all animals were affected by 7-8 months of age. Deletion of Sirt6 in T cells or myeloid-derived cells was sufficient to induce liver inflammation and fibrosis, albeit to a lesser degree than that in the global Sirt6(-/-) mice, suggesting that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6(-/-) mice showed increased MCP-1, IL-6, and TNFα expression levels and were hypersensitive to LPS stimulation. Mechanistically, SIRT6 interacts with c-JUN and deacetylates histone H3 lysine 9 (H3K9) at the promoter of proinflammatory genes whose expression involves the c-JUN signaling pathway. Sirt6-deficient macrophages displayed hyperacetylation of H3K9 and increased occupancy of c-JUN in the promoter of these genes, leading to their elevated expression. These data suggest that Sirt6 plays an anti-inflammatory role in mice by inhibiting c-JUN-dependent expression of proinflammatory genes.