Macrophage Inflammatory Gene Expression Research Articles

Reprogramming of the LXRα Transcriptome Sustains Macrophage Secondary Inflammatory Responses.

Macrophages regulate essential aspects of innate immunity against pathogens. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for host defense. The liver X receptors (LXRα, LXRβ) are ligand-dependent nuclear receptors that direct gene expression important for cholesterol metabolism and inflammation, but little is known about the individual roles of LXRα and LXRβ in antimicrobial responses. Here, the results demonstrate that induction of LXRα transcription by prolonged exposure to lipopolysaccharide (LPS) supports inflammatory gene expression in macrophages. LXRα transcription is induced by NF-κB and type-I interferon downstream of TLR4 activation. Moreover, LPS triggers a reprogramming of the LXRα cistrome that promotes cytokine and chemokine gene expression through direct LXRα binding to DNA consensus sequences within cis-regulatory regions including enhancers. LXRα-deficient macrophages present fewer binding of p65 NF-κB and reduced histone H3K27 acetylation at enhancers of secondary inflammatory response genes. Mice lacking LXRα in the hematopoietic compartment show impaired responses to bacterial endotoxin in peritonitis models, exhibiting reduced neutrophil infiltration and decreased expansion and inflammatory activation of recruited F4/80lo-MHC-IIhi peritoneal macrophages. Together, these results uncover a previously unrecognized function for LXRα-dependent transcriptional cis-activation of secondary inflammatory gene expression in macrophages and the host response to microbial ligands.

The early macrophage response to pathogens requires dynamic regulation of the nuclear paraspeckle

To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli.

Divergent effects of itaconate isomers on Coxiella burnetii growth in macrophages and in axenic culture.

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture.

FRI011 Exploring The Contribution Of PARP1-mediated Site-specific ADP-Ribosylation Of C/EBPb In Macrophages To Diet-induced Obesity

Abstract Disclosure: A.L. Whitaker: None. R. Gupte: None. T.S. Nandu: None. Inflammation is an important component of the pathological effects of obesity, which are mediated in part by adipose-associated macrophages (TAMs). Signal-regulated post-translational modifications in the TAMs help modulate the transcriptional programs that mediate biological outcomes. ADP-ribosylation is a posttranslational modification that regulates various cellular processes, including macrophage activation. ADP-ribosylation is the covalent attachment of ADP-ribose units on proteins by poly(ADP-ribose) polymerase (PARP) enzymes, mainly PARP1 in the nucleus. We have shown that ADP-ribosylation of key transcription factors (e.g., C/EBPβ and STAT1α) by PARP1 regulates adipogenesis, as well as pro-inflammatory responses in macrophages. Here we aim to investigate the role of PARP1-mediated ADP-ribosylation of C/EBPβ in inflammation and obesity using monocyte-specific mouse models. We have found that knockout (KO) of PARP1 in the monocyte/macrophage lineage (i.e., using a LysM-Cre driver) results in dramatic weight gain in mice with a nearly 2-fold increase in fat body mass in animals fed a high fat diet. Further analysis revealed decreases in adipose-associated macrophages and adipose hypertrophy, as well as increased hepatic lipid accumulation. Our findings exemplify the critical role that PARP1 in macrophages plays in maintaining homeostasis in key metabolic tissues. Moreover, our results indicate that regulation by PARP1 in macrophages contributes to resistance of diet-induced obesity through tissue-collaborative mechanisms. Our ongoing experiments suggest that PARP1 regulates LPS-induced inflammatory gene expression in macrophages. PARP1 controls LPS-mediated gene expression, in part, by ADP-ribosylating two sites (Glu 135 and Glu 139) in the regulatory domain of C/EBPβ; mutation of these sites inhibits PARP1-mediated PARylation of C/EBPβ and LPS-mediated gene expression through C/EBPβ. Additionally, treatment with a PARP inhibitor decreases LPS-mediated gene expression associated with upstream C/EBPβ enhancers. Moreover, treatment with a PARP inhibitor also decreases histone H3 lysine 27 acetylation (H3K27ac) at these C/EBPβ enhancers. To investigate the physiological impact of loss of C/EBPβ ADP-ribosylation in macrophages, we have generated a knock-in mouse model for inducible monocyte-specific expression of a C/EBPβ ADP-ribosylation site mutant (CebpbE135A/E139A). This is the first physiological model for the study of site-specific ADP-ribosylation in vivo. Using this model, we are investigating the impact of site-specific ADP-ribosylation of C/EBPβ in mice fed a high fat diet. With these studies, we hope to reveal the specific roles of PARP1-mediated ADP-ribosylation in diet-induced obesity. Presentation: Friday, June 16, 2023

Abstract P3189: Vcam-1-mediated Mitochondrial Biogenesis In Macrophages Exacerbates Inflammation And Atherosclerosis

Atherosclerotic cardiovascular disease is a leading cause of adult mortality worldwide. Macrophages play a key role in atherogenesis by sustaining the local inflammatory response, facilitating accumulation of immune cells in atheromas that contribute to plaque development. Mitochondrial metabolism plays a key role in governing inflammatory gene expression in macrophages. Yet, the role of mitochondrial biogenesis and mitochondrial DNA synthesis in regulating macrophage inflammation is not known. Vascular cell adhesion protein 1 (VCAM-1) expressed by endothelial cells mediates monocyte adhesion and extravasation in developing atherosclerotic plaques. We observed increased VCAM-1 expression in human and murine plaque macrophages. However, the function of this cell adhesion molecule expressed by macrophages is not known. Here, we evaluated if and how myeloid-VCAM-1 drive mitochondrial biogenesis, inflammation, and atherosclerosis pathology. Increased VCAM-1 expression in plaque macrophages correlated with oxidative DNA damage and mitochondrial volume. To understand the role of myeloid-Vcam1 in atherogenesis, we generated Apoe -/- LyzM cre/+ VCAM-1 fl/fl mice. Consistently, Apoe -/- mice lacking Vcam1 in macrophages exhibited reduced atherosclerosis severity and inflammation. Vcam1-deficient macrophages exhibited decreased inflammation, oxidative phosphorylation, mitochondrial biogenesis, and mitochondrial DNA synthesis genes, including Cmpk2. Cmpk2 deletion in macrophages after oxidized LDL treatment reduced inflammatory mediators that aggravate atherosclerosis. RNA sequencing analysis of Vcam-1 -deficient plaque macrophages and analysis of macrophages lacking Cmpk2 identified Fcor and Lyz1 as the target genes of Vcam1 and Cmpk2 . Interestingly, atherosclerotic plaque macrophages deficient of Sting , which mediates inflammatory signaling in response to oxidized mitochondrial DNA, had increased levels of Fcor and Lyz1 . Our data suggest that VCAM-1 in macrophages signals via CMPK2 and POLG to promote mitochondrial biogenesis, oxidation, and fragmentation, mediating atherogenesis.

Assessment of circulating extracellular vesicles from calorie‐restricted mice and humans in ischaemic injury models

AbstractCalorie restriction (CR) and fasting affect lifespan, disease susceptibility and response to acute injury across multiple animal models, including ischaemic injuries such as myocardial infarction or kidney hypoxia. The cargo and function of circulating extracellular vesicles (EV) respond to changes in host physiology, including exercise, injury, and other interventions. Thus, we hypothesised that EVs induced following CR may reflect some of the beneficial properties of CR itself. In a pilot study, EVs were isolated from mice following 21 days of 30 % CR, and from eight human donors after 72 h water‐only fasting. EV size, concentration and morphology were profiled by NTA, western blot and cryoEM, and their function was assessed using multiple assays related to ischaemic diseases. We found that EVs from post‐fasting samples better protected cardiac cells from hypoxia/reperfusion (H/R) injury compared to pre‐fasting EVs. However, there was no difference when used to treat H/R‐injured kidney epithelial cells. Post‐fasting derived EVs slowed the rate of fibroblast migration and slightly reduced macrophage inflammatory gene expression compared to pre‐fasting derived EVs. Lastly, we compared miRNA cargos of pre‐ and post‐fasting human serum EVs and found significant changes in a small number of miRNAs. We conclude that fasting appears to influence EV cargo and function, with varied effects worthy of further exploration.

Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses

Atherosclerosis is a chronic inflammatory disease with high morbidity and mortality rates worldwide. Doublecortin‐like kinase 1 (DCLK1), a microtubule‐associated protein kinase, is involved in neurogenesis and human cancers. However, the role of DCLK1 in atherosclerosis remains undefined. In this study, we identified upregulated DCLK1 in macrophages in atherosclerotic lesions of ApoE−/− mice fed an HFD and determined that macrophage‐specific DCLK1 deletion attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA sequencing analysis indicated that DCLK1 mediates oxLDL‐induced inflammation via NF‐κB signaling pathway in primary macrophages. Coimmunoprecipitation followed by LC–MS/MS analysis identified IKKβ as a binding protein of DCLK1. We confirmed that DCLK1 directly interacts with IKKβ and phosphorylates IKKβ at S177/181, thereby facilitating subsequent NF‐κB activation and inflammatory gene expression in macrophages. Finally, a pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation both in vitro and in vivo. Our findings demonstrated that macrophage DCLK1 promotes inflammatory atherosclerosis by binding to IKKβ and activating IKKβ/NF‐κB. This study reports DCLK1 as a new IKKβ regulator in inflammation and a potential therapeutic target for inflammatory atherosclerosis.

Metformin, Empagliflozin, and Their Combination Modulate Ex-Vivo Macrophage Inflammatory Gene Expression.

Type-2 Diabetes Mellitus is a complex, chronic illness characterized by persistent high blood glucose levels. Patients can be prescribed anti-diabetes drugs as single agents or in combination depending on the severity of their condition. Metformin and empagliflozin are two commonly prescribed anti-diabetes drugs which reduce hyperglycemia, however their direct effects on macrophage inflammatory responses alone or in combination are unreported. Here, we show that metformin and empagliflozin elicit proinflammatory responses on mouse bone-marrow-derived macrophages with single agent challenge, which are modulated when added in combination. In silico docking experiments suggested that empagliflozin can interact with both TLR2 and DECTIN1 receptors, and we observed that both empagliflozin and metformin increase expression of Tlr2 and Clec7a. Thus, findings from this study suggest that metformin and empagliflozin as single agents or in combination can directly modulate inflammatory gene expression in macrophages and upregulate the expression of their receptors.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Abstract MP27: Endothelial Nogo-B Modulates Ischemia Induced Collateral Artery Remodeling

Nogo-B, a member of the reticulon 4 family of proteins, is the dominant isoform expressed in endothelial cells (EC). We have shown that Nogo-B is necessary for blood flow recovery in ischemia. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. However, whether endothelial Nogo-B regulates arteriogenesis and/or angiogenesis is unknown. We generated an inducible EC-specific mouse overexpressing Nogo-B (Ng ECtg ) and investigated the arteriogenesis and angiogenesis in limb ischemia. Ischemia increased endothelial and serum Nogo-B expression. Blood flow recovery was markedly diminished after femoral artery ligation (FAL) in Ng ECtg compared to WT mice, in association with lower collateral density accessed by micro-CT arteriography. There was no reduction in capillary density or decrease in smooth muscle/pericyte and macrophage recruitment in Ng ECtg mice, suggesting EC Nogo-B overexpression regulates arteriogenesis but not angiogenesis. While Ng ECtg mice have normal number of native collateral artery, the early remodeling of the collateral artery was impaired after ischemia. Furthermore, in line with impaired remodeling, Ng ECtg mice have decreased functional (exercise) hyperemia response in the non-ischemic limb in vivo, and resistant arteries from Ng ECtg have diminished flow induced vasodilatation ex vivo compared to WT mice. Moreover, endothelial reconstitution of Nogo-B in global Nogo knockout background (NogoKO ECrc ) restored blood flow recovery after ischemia in vivo, further suggesting the EC specific function of Nogo-B in arteriogenesis. Mechanistically, we have shown that EC isolated from Ng ECtg mice have decreased nitric oxide release. Nogo-B overexpression negatively regulates eNOS phosphorylation, expression and golgi localization in EC. Future parabiosis study is warranted to elucidate the contribution of paracrine or EC intrinsic Nogo-B in modulating arteriogenesis and angiogenesis. In conclusion, our data showed for the first time that endothelial Nogo-B plays important roles in regulating collateral artery remodeling and blood flow through, at least in part, regulating NO bioavailability.

Microbial Phagocytic Receptors and Their Potential Involvement in Cytokine Induction in Macrophages.

Phagocytosis is an essential process for the uptake of large (>0.5 µm) particulate matter including microbes and dying cells. Specialized cells in the body perform phagocytosis which is enabled by cell surface receptors that recognize and bind target cells. Professional phagocytes play a prominent role in innate immunity and include macrophages, neutrophils and dendritic cells. These cells display a repertoire of phagocytic receptors that engage the target cells directly, or indirectly via opsonins, to mediate binding and internalization of the target into a phagosome. Phagosome maturation then proceeds to cause destruction and recycling of the phagosome contents. Key subsequent events include antigen presentation and cytokine production to alert and recruit cells involved in the adaptive immune response. Bridging the innate and adaptive immunity, macrophages secrete a broad selection of inflammatory mediators to orchestrate the type and magnitude of an inflammatory response. This review will focus on cytokines produced by NF-κB signaling which is activated by extracellular ligands and serves a master regulator of the inflammatory response to microbes. Macrophages secrete pro-inflammatory cytokines including TNFα, IL1β, IL6, IL8 and IL12 which together increases vascular permeability and promotes recruitment of other immune cells. The major anti-inflammatory cytokines produced by macrophages include IL10 and TGFβ which act to suppress inflammatory gene expression in macrophages and other immune cells. Typically, macrophage cytokines are synthesized, trafficked intracellularly and released in response to activation of pattern recognition receptors (PRRs) or inflammasomes. Direct evidence linking the event of phagocytosis to cytokine production in macrophages is lacking. This review will focus on cytokine output after engagement of macrophage phagocytic receptors by particulate microbial targets. Microbial receptors include the PRRs: Toll-like receptors (TLRs), scavenger receptors (SRs), C-type lectin and the opsonic receptors. Our current understanding of how macrophage receptor stimulation impacts cytokine production is largely based on work utilizing soluble ligands that are destined for endocytosis. We will instead focus this review on research examining receptor ligation during uptake of particulate microbes and how this complex internalization process may influence inflammatory cytokine production in macrophages.

PolyADP-Ribosylation of NFATc3 and NF-κB Transcription Factors Modulate Macrophage Inflammatory Gene Expression in LPS-Induced Acute Lung Injury

Pulmonary macrophages play a critical role in the recognition of pathogens, initiation of host defense via inflammation, clearance of pathogens from the airways, and resolution of inflammation. Recently, we have shown a pivotal role for the nuclear factor of activated T-cell cytoplasmic member 3 (NFATc3) transcription factor in modulating pulmonary macrophage function in LPS-induced acute lung injury (ALI) pathogenesis. Although the NFATc proteins are activated primarily by calcineurin-dependent dephosphorylation, here we show that LPS induces posttranslational modification of NFATc3 by polyADP-ribose polymerase 1 (PARP-1)-mediated polyADP-ribosylation. ADP-ribosylated NFATc3 showed increased binding to iNOS and TNFα promoter DNA, thereby increasing downstream gene expression. Inhibitors of PARP-1 decreased LPS-induced NFATc3 ribosylation, target gene promoter binding, and gene expression. LPS increased NFAT luciferase reporter activity in lung macrophages and lung tissue that was inhibited by pretreatment with PARP-1 inhibitors. More importantly, pretreatment of mice with the PARP-1 inhibitor olaparib markedly decreased LPS-induced cytokines, protein extravasation in bronchoalveolar fluid, lung wet-to-dry ratios, and myeloperoxidase activity. Furthermore, PARP-1 inhibitors decreased NF-кB luciferase reporter activity and LPS-induced ALI in NF-кB reporter mice. Thus, our study demonstrates that inhibiting NFATc3 and NF-кB polyADP-ribosylation with PARP-1 inhibitors prevented LPS-induced ALI pathogenesis.

Bespoken Nanoceria: An Effective Treatment in Experimental Hepatocellular Carcinoma.

Background and AimsDespite the availability of new‐generation drugs, hepatocellular carcinoma (HCC) is still the third most frequent cause of cancer‐related deaths worldwide. Cerium oxide nanoparticles (CeO2NPs) have emerged as an antioxidant agent in experimental liver disease because of their antioxidant, anti‐inflammatory, and antisteatotic properties. In the present study, we aimed to elucidate the potential of CeO2NPs as therapeutic agents in HCC.Approach and ResultsHCC was induced in 110 Wistar rats by intraperitoneal administration of diethylnitrosamine for 16 weeks. Animals were treated with vehicle or CeO2NPs at weeks 16 and 17. At the eighteenth week, nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO2NPs on tumor progression and animal survival was investigated. Hepatic tissue MS‐based phosphoproteomics as well as analysis of principal lipid components were performed. The intracellular uptake of CeO2NPs by human ex vivo perfused livers and human hepatocytes was analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory gene expression. Nanoceria treatment increased liver apoptotic activity, while proliferation was attenuated. Phosphoproteomic analysis revealed that CeO2NPs affected the phosphorylation of proteins mainly related to cell adhesion and RNA splicing. CeO2NPs decreased phosphatidylcholine‐derived arachidonic acid and reverted the HCC‐induced increase of linoleic acid in several lipid components. Furthermore, CeO2NPs reduced serum alpha‐protein levels and improved the survival of HCC rats. Nanoceria uptake by ex vivo perfused human livers and in vitro human hepatocytes was also demonstrated.ConclusionsThese data indicate that CeO2NPs partially revert the cellular mechanisms involved in tumor progression and significantly increase survival in HCC rats, suggesting that they could be effective in patients with HCC.

Lysophosphatidylcholine acyltransferase 2 (LPCAT2) co-localises with TLR4 and regulates macrophage inflammatory gene expression in response to LPS

Despite extensive investigations, an effective treatment for sepsis remains elusive and a better understanding of the inflammatory response to infection is required to identify potential new targets for therapy. In this study we have used RNAi technology to show, for the first time, that the inducible lysophosphatidylcholine acyltransferase 2 (LPCAT2) plays a key role in macrophage inflammatory gene expression in response to stimulation with bacterial ligands. Using siRNA- or shRNA-mediated knockdown, we demonstrate that, in contrast to the constitutive LPCAT1, LPCAT2 is required for macrophage cytokine gene expression and release in response to TLR4 and TLR2 ligand stimulation but not for TLR-independent stimuli. In addition, cells transfected to overexpress LPCAT2 exhibited increased expression of inflammatory genes in response to LPS and other bacterial ligands. Furthermore, we have used immunoprecipitation and Western blotting to show that in response to LPS, LPCAT2, but not LPCAT1, rapidly associates with TLR4 and translocates to membrane lipid raft domains. Our data thus suggest a novel mechanism for the regulation of inflammatory gene expression in response to bacterial stimuli and highlight LPCAT2 as a potential therapeutic target for development of anti-inflammatory and anti-sepsis therapies.

Myeloid-specific Asxl2 deletion limits diet-induced obesity by regulating energy expenditure.

We previously established that global deletion of the enhancer of trithorax and polycomb (ETP) gene, Asxl2, prevents weight gain. Because proinflammatory macrophages recruited to adipose tissue are central to the metabolic complications of obesity, we explored the role of ASXL2 in myeloid lineage cells. Unexpectedly, mice without Asxl2 only in myeloid cells (Asxl2ΔLysM) were completely resistant to diet-induced weight gain and metabolically normal despite increased food intake, comparable activity, and equivalent fecal fat. Asxl2ΔLysM mice resisted HFD-induced adipose tissue macrophage infiltration and inflammatory cytokine gene expression. Energy expenditure and brown adipose tissue metabolism in Asxl2ΔLysM mice were protected from the suppressive effects of HFD, a phenomenon associated with relatively increased catecholamines likely due to their suppressed degradation by macrophages. White adipose tissue of HFD-fed Asxl2ΔLysM mice also exhibited none of the pathological remodeling extant in their control counterparts. Suppression of macrophage Asxl2 expression, via nanoparticle-based siRNA delivery, prevented HFD-induced obesity. Thus, ASXL2 controlled the response of macrophages to dietary factors to regulate metabolic homeostasis, suggesting modulation of the cells' inflammatory phenotype may impact obesity and its complications.

Anti-Inflammatory Effects of HDL (High-Density Lipoprotein) in Macrophages Predominate Over Proinflammatory Effects in Atherosclerotic Plaques.

HDL (high-density lipoprotein) infusion reduces atherosclerosis in animal models and is being evaluated as a treatment in humans. Studies have shown either anti- or proinflammatory effects of HDL in macrophages, and there is no consensus on the underlying mechanisms. Here, we interrogate the effects of HDL on inflammatory gene expression in macrophages. Approach and Results: We cultured bone marrow-derived macrophages, treated them with reconstituted HDL or HDL isolated from APOA1Tg;Ldlr-/- mice, and challenged them with lipopolysaccharide. Transcriptional profiling showed that HDL exerts a broad anti-inflammatory effect on lipopolysaccharide-induced genes and proinflammatory effect in a subset of genes enriched for chemokines. Cholesterol removal by POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) liposomes or β-methylcyclodextrin mimicked both pro- and anti-inflammatory effects of HDL, whereas cholesterol loading by POPC/cholesterol-liposomes or acetylated LDL (low-density lipoprotein) before HDL attenuated these effects, indicating that these responses are mediated by cholesterol efflux. While early anti-inflammatory effects reflect reduced TLR (Toll-like receptor) 4 levels, late anti-inflammatory effects are due to reduced IFN (interferon) receptor signaling. Proinflammatory effects occur late and represent a modified endoplasmic reticulum stress response, mediated by IRE1a (inositol-requiring enzyme 1a)/ASK1 (apoptosis signal-regulating kinase 1)/p38 MAPK (p38 mitogen-activated protein kinase) signaling, that occurs under conditions of extreme cholesterol depletion. To investigate the effects of HDL on inflammatory gene expression in myeloid cells in atherosclerotic lesions, we injected reconstituted HDL into Apoe-/- or Ldlr-/- mice fed a Western-type diet. Reconstituted HDL infusions produced anti-inflammatory effects in lesion macrophages without any evidence of proinflammatory effects. Reconstituted HDL infusions in hypercholesterolemic atherosclerotic mice produced anti-inflammatory effects in lesion macrophages suggesting a beneficial therapeutic effect of HDL in vivo.

Lactational High-Fat Diet Exposure Programs Metabolic Inflammation and Bone Marrow Adiposity in Male Offspring.

Overnutrition during critical windows of development plays a significant role in life-long metabolic disease risk. Early exposure to excessive nutrition may result in altered programming leading to increased susceptibility to obesity, inflammation, and metabolic complications. This study investigated the programming effects of high-fat diet (HFD) exposure during the lactation period on offspring adiposity and inflammation. Female C57Bl/6J dams were fed a normal diet or a 60% HFD during lactation. Offspring were weaned onto a normal diet until 12 weeks of age when half were re-challenged with HFD for 12 weeks. Metabolic testing was performed throughout adulthood. At 24 weeks, adipose depots were isolated and evaluated for macrophage profiling and inflammatory gene expression. Males exposed to HFD during lactation had insulin resistance and glucose intolerance as adults. After re-introduction to HFD, males had increased weight gain and worsened insulin resistance and hyperglycemia. There was increased infiltration of pro-inflammatory CD11c+ adipose tissue macrophages, and bone marrow was primed to produce granulocytes and macrophages. Bone density was lower due to enhanced marrow adiposity. This study demonstrates that maternal HFD exposure during the lactational window programs offspring adiposity, inflammation, and impaired glucose homeostasis.

Inhibitory selectivity among class I HDACs has a major impact on inflammatory gene expression in macrophages

Histone deacetylases (HDACs) play an important role in cancer, degenerative diseases and inflammation. The currently applied HDAC inhibitors in the clinic lack selectivity among HDAC isoforms, which limits their application for novel indications such as inflammatory diseases. Recent, literature indicates that HDAC 3 plays an important role among class I HDACs in gene expression in inflammation. In this perspective, the development and understanding of inhibitory selectivity among HDACs 1, 2 and 3 and their respective influence on gene expression need to be characterized to facilitate drug discovery. Towards this aim, we synthesized nine structural analogues of the class I HDAC inhibitor Entinostat and investigated their selectivity profile among HDACs 1, 2 and 3. We found that we can explain the observed structure activity relationships by small structural and conformational differences between HDAC 1 and HDAC 3 in the ‘lid’ interacting region. Cell-based studies indicated, however, that application of inhibitors with improved HDAC 3 selectivity did not provide an anti-inflammatory response in contrast to expectations from biochemical evidence in literature. Altogether, in this study, we identified structure activity relationships among class I HDACs and we connected isoform selectivity among class I HDACs with pro- and anti-inflammatory gene transcription in macrophages.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Dose-response assessment of chemically modified curcumin in experimental periodontitis.

CMC2.24, a novel tri-ketonic chemically modified compound based on natural di-ketonic curcumin, has been shown to reduce bone loss and inflammatory mediators in experimental periodontitis, however, a potential dose-response relationship was not determined. The purpose of this study was to assess the effects of different doses of CMC2.24 on inflammation and bone resorption in vivo and also to describe on the effects of CMC2.24 on macrophage response. CMC2.24 was administered daily to animals for 28 days by oral gavage, at the following doses: 0 (control), 1, 3, 10, and 30mg/kg of body weight. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) into the gingival tissues. Outcomes assessed were bone resorption, detection of tartrate-resistant acid phosphatase, and determination of gene expression. In vitro, macrophages (RAW264.7) were treated with different concentrations of CMC2.24: 1, 3, 10, and 30 μM and then subjected to different activation stimuli. Gene expression, phagocytic activity, production of reactive oxygen species (ROS) and cytokine production were evaluated. CMC2.24 inhibited bone resorption, osteoclastogenesis, and tumor necrosis factor (TNF)-α expression in vivo. These beneficial responses reached maximum levels at a dose of 1mg/kg, i.e. no dose-dependent effect. In vitro, CMC2.24 reduced the production of TNF-α and interleukin-10, inhibited phagocytic activity and stimulated production of ROS. A dose-dependent effect was observed only for ROS production. Low doses of CMC2.24 (1mg/kg/day) administered orally were sufficient to significantly inhibit alveolar bone resorption associated with the experimental periodontal disease; whereas in vitro macrophage inflammatory gene expression and phagocytosis were reduced, whereas production of ROS was stimulated.