Inflammatory Activation Of Macrophages Research Articles

The Essential Role of FoxO1 in the Regulation of Macrophage Function.

Macrophages are widely distributed in various tissues and organs. They not only participate in the regulation of innate and adaptive immune response, but also play an important role in tissue homeostasis. Dysregulation of macrophage function is closely related to the initiation, development and prognosis of multiple diseases, including infection and tumorigenesis. Forkhead box transcription factor O1 (FoxO1) is an important member among the forkhead box transcription factor family. Through directly binding to the promoter regions of downstream target genes, FoxO1 is implicated in cell proliferation, apoptosis, metabolic activities and other biological processes. In this review, we summarized the regulatory role of FoxO1 in macrophage phagocytosis, migration, differentiation and inflammatory activation. We also emphasized that macrophage reciprocally modulated FoxO1 activity via a post-translational modification (PTM) dominant manner.

Accumulation of Intracellular Ferrous Iron in Inflammatory-Activated Macrophages.

Macrophages are important innate immune cells which can be polarized into heterogeneous populations. The inflammatory-activated M1 cells are known to be involved in all kinds of inflammatory diseases, which were also found to be associated with dysregulation of iron metabolism. While iron overload is known to induce M1 polarization, the valence states of iron and its intracellular dynamics during macrophage inflammatory activation have not been identified. In this study, THP-1-derived macrophages were polarized into M1, M2a, M2b, M2c, and M2d cells, and intracellular ferrous iron (Fe(II)) was measured by our previously developed ultrasensitive Fe(II) fluorescent probe. Significant accumulation of Fe(II) was only observed in M1 cells, which was different from the alterations of total iron. Time-dependent change of intracellular Fe(II) during the inflammatory activation was also consistent with the expression shifts of transferrin receptor CD71, ferrireductase Steap3, and Fe(II) exporter Slc40a1. In addition, accumulation of Fe(II) was also found in the colon macrophages of mice with ulcerative colitis, which was positively correlated to inflammatory phenotypes, including the productions of NO, IL-1β, TNF-α, and IL-6. Collectively, these results demonstrated the specific accumulation of Fe(II) in inflammatory-activated macrophages, which not only enriched our understanding of iron homeostasis in macrophages, but also indicated that Fe(II) could be further developed as a potential biomarker for inflammatory-activated macrophages.

Efficacy of plasma exchange on top of standard immunosuppressive therapy in adult autoimmune inflammatory rheumatic diseases-associated macrophage activation syndrome, a single center real-world analysis

BackgroundThere is a lack of documented real-world evidence about the efficacy of current therapeutics for autoimmune inflammatory rheumatic diseases (AIIRD)-associated adult macrophage activation syndrome (MAS). ObjectiveTo analyze the efficacy of different treatments, especially plasma exchange (PE), in AIIRD-associated MAS. MethodsAmong 5775 patients with AIIRD in Tongji Hospital from 2014 to 2020, 62 AIIRD-associated MAS cases were collected. Unadjusted logistic regression, least absolute shrinkage and selection operator (LASSO), and inverse probability of treatment weight (IPTW) analyses were used to characterize the clinical features and potential factors related to the prognosis. Paired t-test was used to compared the changes of inflammatory indicators before and after PE treatment. ResultsThe baseline data was defined as the data collected at the onset of MAS, and all of the 62 patients were diagnosed as AIIRD before MAS onset. The prevalance rate of MAS in AIIRD was 1.1%, and the most common types of AIIRD were systemic lupus erythematosus (45.2%) and adult-onset Still's disease (33.9%). All 62 MAS patients received glucocorticoids, 87.1% patients used at least one immunosuppressive agent, and 54.8% received PE. LASSO regression indicates a positive effect of PE on the basis of variables. After PE treatment, serum levels of multiple inflammatory cytokines were rapidly reduced, accompanied by improvements in clinical symptoms and laboratory indecies including ferritin, lactate dehydrogenase, and C-reactive protein. LASSO regression indicates that PE treatment was associated with a marked reduction of mortality (from 53.6% to 11.8%), with a hazard ratio (HR) of 0.148 (p < 0.001) after adjustment for confounding factors using IPTW analysis. ConclusionWith the background therapy of glucocorticoids and immunosuppressive agents, PE is an effective approach to rapidly clear inflammatory cytokines and reduce mortality of AIIRD-associated MAS. Clinical implicationThis study provided real-world information on the efficacy of PE in AIIRD-associated MAS.

Thiamine pretreatment improves endotoxemia-related liver injury and cholestatic complications by regulating galactose metabolism and inhibiting macrophage activation

BackgroundAs a major metabolic site, the liver is an important target organ of endotoxemia. High serum lipopolysaccharide (LPS) levels can cause hepatocyte necrosis and produce cholestasis, which results in severe liver injury. Contrastingly, thiamine (THA) has shown anti-inflammatory effects against severe infections and may be indicated for systemic endotoxemia treatment. Therefore, the present study was conducted to investigate the effective treatment of endotoxemia-induced liver injury with THA and the possible molecular mechanisms.Methodology/Principal Findings: in vivo, We established two models of endotoxemia-induced liver injury at the in vivo level using LPS and bile duct ligation (BDL) + LPS, administering prophylactic THA intraperitoneally to mice. In vitro, the effects of THA on RAW264.7 and THP-1 administration of LPS-induced inflammatory macrophage activation were observed. Metabolomic analysis screening and subsequent validation experiments were also performed. THA has different degrees of preventive therapeutic effects on different causes of endotoxemia-induced liver injury, as evidenced by a decreased alanine aminotransferase (ALT) and decreased inflammatory factors. This study aimed to clarify the specific mechanism. We subsequently found that THA reduced the inflammatory macrophages produced by RAW264.7 and THP-1 in response to LPS. Additionally, THA reduced galactose liver accumulation and improved glucose metabolism. Moreover, Galectin-3 (Gal-3), as a point of interaction between macrophage activation and galactose metabolism mechanisms, was observed to inhibit Gal-3 expression by THA at both in vivo and in vitro levels.Conclusions.This study revealed that THA may be a viable prophylactic treatment option for the prevention of liver injury occurring in endotoxemia, which is associated with its effects on the modulation of Gal-3 to improve the inflammatory response and the inhibition of galactose metabolism. Additional evidence is provided for its clinical application.

Characterization of Anti-Cancer Activities of Violacein: Actions on Tumor Cells and the Tumor Microenvironment.

Natural products have been shown to serve as promising starting points for novel anti-cancer drugs. In this study, the anti-cancer activities of the purple compound violacein, initially isolated from Chromobacterium violaceum, were investigated. To highlight the crucial role of the tumor microenvironment on the effectiveness of cancer therapies, this study includes effects on macrophages as prototypic cells of the microenvironment in addition to the investigation of tumor-centric activities. Using 2D and 3D cell culture models, automated live-cell microscopy, and biochemical analyses, violacein was demonstrated to inhibit tumor cell proliferation and migration. The violacein-triggered tumor cell death was further associated with caspase 3-like activation and ATP release. Stimuli released from dead cells resulted in inflammatory activation of macrophages, as shown by NF-κB reporter cell assays, macrophage morphology, and gene expression analysis. Moreover, macrophages deficient in the inflammasome component Nlrp3 were found to be significantly less sensitive towards treatment with violacein and doxorubicin. Taken together, this study provides new insights into the biological activity of violacein against cancer. In addition, the in vitro data suggest immunogenic features of induced cell death, making violacein an interesting candidate for further studies investigating the compound as an inducer of immunogenic cell death.

Diallyl disulfide ameliorates ethanol-induced liver steatosis and inflammation by maintaining the fatty acid catabolism and regulating the gut-liver axis

Diallyl disulfide (DADS) has been suggested to possess hepatoprotection against alcoholic liver disease (ALD) by a couple of pilot studies, while the underlying mechanisms remain largely unknown. This study aimed to investigate the hepatoprotective effects of DADS against ethanol-induced liver steatosis and early inflammation by using the chronic-plus-binge mice model and cultured J774A.1 macrophages and AML12 hepatocytes. We found that DADS significantly attenuated ethanol-induced elevation of serum aminotransferase activities, accumulation of liver triglyceride, hepatocytes apoptosis, oxidative stress, infiltration of macrophages and neutrophils, and proinflammatory polarization of macrophages in mice livers. In addition, chronic-plus-binge drinking induced apparent intestinal mucosa damage and disturbance of gut microbiota, endotoxemia, and activation of hepatic NF-κB signaling and NLRP3 inflammasome, which was inhibited by DADS. In vitro studies using cocultured AML12/J774A.1 cells showed that DADS suppressed ethanol/LPS–induced cell injury and inflammatory activation of macrophages. Furthermore, DADS ameliorated ethanol-induced decline of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1 (CPT1), and phosphorylated AMP-activated protein kinase (AMPK) protein levels in mice livers and AML12 cells. These results demonstrate that DADS could prevent ethanol-induced liver steatosis and early inflammation by regulating the gut-liver axis and maintaining fatty acid catabolism.

Modeling macrophage response to periodontal infections in vitro

Periodontal disease (PD) is a chronic inflammatory disorder characterized by the destruction of connective tissue, tooth loss and systemic infections. Numerous virulence factors derived from chronic opportunistic pathogens such as Porphyromonas gingivalis are responsible for enhanced microbial colonization, stimulation of inflammatory responses, and suppression of host defense response. Individuals with PD present with a complex array of inflammatory mediators, and cellular infiltration by neutrophils, lymphocytes and monocytes/macrophages. Although macrophages comprise 5–30% of cells in the cellular infiltrate of human periodontal lesions, how macrophage skewing contributes to PD, and more specifically in the downstream development of a macrophage immune response to oral bacteria are unclear. We, including others have investigated the activation of monocytes/macrophages, and discovered the means of type I vs. type II inflammatory activation vs. deactivation of macrophages both in vitro and in vivo. We have shown previously that P. gingivalis infection induces M1 macrophage formation, whereas opsonized‐P. gingivalis challenge was able to switch to M2‐like phenotype demonstrating macrophage plasticity. However, the mechanism of modulation and kinetics of macrophage inflammatory reactions during the process is unclear. Prior research in the field has failed to appreciate that it is the same monocyte/macrophage cell population which is initially polarized towards an effector “inflammatory” program and subsequently re‐polarized to the deactivation program not just in PD but in any inflammation. The overarching goal of our research is to understand how macrophage skewing contributes to PD, and the downstream development of immune response to PD‐associated bacteria towards development of new macrophage‐based therapeutics. Since macrophage polarization dynamics cannot be studied in an animal model, a reliable in vitro system is needed to provide fundamental insights into macrophage biology. We have developed an in vitro model that recapitulates different phases of the inflammatory response (recruitment, initiation, development and resolution), based on purified human peripheral blood monocytes exposed to a battery of microenvironmental cues mimicking macrophage polarization/depolarization in vivo and we have created a kinetic profile of polarization by RNA analysis. Further we have applied our model to macrophage/gingival fibroblast coculture system to study kinetics of polarization. And by comparing gene clusters that are differentially expressed in M1 and M2, we have successfully recapitulated the macrophage polarization kinetics of acute inflammatory and resolution phases during periodontal inflammation and validated the key inflammatory and transcriptional factors using RT‐PCR at different phases. In conclusion, we developed an in vitro model which can be further employed to test novel macrophage‐based therapeutics to treat periodontal disease.

Inflammatory Activation and Nitric Oxide Oxidation in Exertional Dyspnea in Southwest Asia Deployed Military Veterans

Post deployment personnel of the Southwest Asia theater of war present with significant exertional dyspnea. While deployed, these service men and women were exposed to a variety of inhaled toxicants including geological dust, burn pit smoke, and heavy metals. Upon examination, a large portion of these veterans display near‐normal pulmonary function and gas exchange in the lung, making diagnosis and treatment difficult. The aim of this study is to define the physiological and biochemical profiles of individuals with unexplained exertional dyspnea. We hypothesized that exposure to airborne toxicants has led to a chronic activated inflammatory state in the systemic vasculature, resulting in increased oxidative stress and reducing the bioavailability of nitric oxide (NO), contributing to the symptoms of dyspnea.Eligible veterans were tested for lung function by spirometry including diffusing capacity for carbon monoxide (DLco). Resting blood samples were taken and immediately separated into plasma and cellular components. Granuloyctes were removed from whole blood by magnetic separation, and subsequent leukocytes were collected and gated on expression of the myeloid marker CD45. Within CD45+ cells, flow cytometry was used to analyze the expression of inflammatory macrophage activation markers CD87, CD11b, CD163, HLA‐DR, CD195, CD192, CD14, CD16. Circulating levels of nitrite and nitrate (oxidation products of NO) were measured in plasma via reductive chemiluminescence, and the ratio of nitrate/nitrite (NOx ratio) was used as a marker for reduced bioavailability of NO. Increased expression of inflammatory marker CD87 in CD11b+ cells was positively correlated with expression of CD163 in HLA‐DR+ and CD14 in CD16+ (rho = .54, p &lt;.01; rho= .36, p= .047; n =30), but not CD195 in CD192+ (rho =‐.3, p =.1). Similar observations were observed in granulocytic cells regarding CD66b expression. This correlation between activation markers within the circulating myeloid‐derived cells is consistent with the presence of monocyte activation in the systemic vasculature. NOx levels were within normally observable bounds, however, the NOx ratio was negatively correlated with FEV1/FVC (a marker of lung obstruction) but not with %predicted DLco(rho =‐.56, p =.016; rho =.01, p = .96, n = 18). The reduction in FEV1/FVC with increased NOx ratio suggests potential loss of endothelial function, which may be contributing to exertional dyspnea and is currently under investigation.

Bone marrow granulocytes downregulate IL-1β and TNF production and the microbicidal activity of inflammatory macrophages.

Macrophages play critical roles in inflammation and defense against pathogens, as well as in the return to tissue homeostasis. Macrophage subpopulations displaying antagonistic phenotypes are generally classified as proinflammatory M1, implicated in antipathogen and antitumoral activities, or as anti-inflammatory M2, associated with tissue repair. Granulocytic and monocytic myeloid-derived suppressor cells recruited from the bone marrow to tissues and phagocytosis of apoptotic neutrophils can attenuate macrophage microbicidal activity. Here, we showed that bone marrow neutrophils, but not thioglycollate-recruited neutrophils, directly suppress the responses of macrophages that were previously committed to an inflammatory phenotype. Cocultures of inflammatory macrophages with bone marrow CD11b+Ly6Ghi granulocytes led to reduced release of IL-1β, TNF-α, and IL-6 by macrophages after lipopolysaccharide stimulation. The suppressive activity was unrelated to granulocyte apoptosis or to secreted factors and required cell-to-cell contact. The suppressive effect was paralleled by reduction in the nuclear levels of the NF-κB p65 subunit, but not of the p50 subunit. Furthermore, bone marrow granulocytes decreased the phagocytic activity of macrophages and their capacity to kill intracellular Escherichia coli. Taken together, these results show that bone marrow granulocytes can function as suppressors of the proinflammatory activity and microbial-killing responses of macrophages.

LncRNA NEAT1 Potentiates SREBP2 Activity to Promote Inflammatory Macrophage Activation and Limit Hantaan Virus Propagation.

As the global prototypical zoonotic hantavirus, Hantaan virus (HTNV) is prevalent in Asia and is the leading causative agent of severe hemorrhagic fever with renal syndrome (HFRS), which has profound morbidity and mortality. Macrophages are crucial components of the host innate immune system and serve as the first line of defense against HTNV infection. Previous studies indicated that the viral replication efficiency in macrophages determines hantavirus pathogenicity, but it remains unknown which factor manipulates the macrophage activation pattern and the virus-host interaction process. Here, we performed the transcriptomic analysis of HTNV-infected mouse bone marrow-derived macrophages and identified the long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1), especially the isoform NEAT1-2, as one of the lncRNAs that is differentially expressed at the early phase. Based on coculture experiments, we revealed that silencing NEAT1-2 hinders inflammatory macrophage activation and facilitates HTNV propagation, while enhancing NEAT1-2 transcription effectively restrains viral replication. Furthermore, sterol response element binding factor-2 (SREBP2), which controls the cholesterol metabolism process, was found to stimulate macrophages by promoting the production of multiple inflammatory cytokines upon HTNV infection. NEAT1-2 could potentiate SREBP2 activity by upregulating Srebf1 expression and interacting with SREBP2, thus stimulating inflammatory macrophages and limiting HTNV propagation. More importantly, we demonstrated that the NEAT1-2 expression level in patient monocytes was negatively correlated with viral load and HFRS disease progression. Our results identified a function and mechanism of action for the lncRNA NEAT1 in heightening SREBP2-mediated macrophage activation to restrain hantaviral propagation and revealed the association of NEAT1 with HFRS severity.

Ketogenesis acts as an endogenous protective programme to restrain inflammatory macrophage activation during acute pancreatitis.

SummaryBackgroundInnate immunity and metabolites link to the pathogenesis and severity of acute pancreatitis (AP). However, liver metabolism and its role in immune response and AP progression remain elusive. We investigated the function of liver metabolism in the pathogenesis of AP.MethodsCirculating ketone body β-hydroxybutyrate (βOHB) levels were determined in AP clinical cohorts and caerulein-induced AP (CER-AP) mouse models receiving seven (Cer*7) or twelve (Cer*12) injection regimens at hourly intervals. Liver transcriptomics and metabolomics were compared between CER-AP (Cer*7) and CER-AP (Cer*12). Inhibition of fatty acid β-oxidation (FAO)-ketogenesis, or supplementation of βOHB was performed in mouse models of AP. The effect and mechanism of βOHB were examined in vitro.FindingsElevated circulating βOHB was observed in patients with non-severe AP (SAP) but not SAP. These findings were replicated in CER-AP (Cer*7) and CER-AP (Cer*12), which manifested as limited and hyperactive immune responses, respectively. FAO-ketogenesis was activated in CER-AP (Cer*7), while impaired long-chain FAO and mitochondrial function were observed in the liver of CER-AP (Cer*12). Blockage of FAO-ketogenesis (Cpt1a antagonism or Hmgcs2 knockdown) worsened, while supplementation of βOHB or its precursor 1,3-butanediol alleviated the severity of CER-AP. Mechanistically, βOHB had a discernible effect on pancreatic acinar cell damage, instead, it greatly attenuated the activation of pancreatic and systemic proinflammatory macrophages via class I histone deacetylases.InterpretationOur findings reveal that hepatic ketogenesis is activated as an endogenous protective programme to restrain AP progression, indicating its potential therapeutic value.FundingThis work was supported by the National Natural Science Foundation of China, Shanghai Youth Talent Support Programme, and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant.

BET Protein Inhibition Regulates Macrophage Chromatin Accessibility and Microbiota-Dependent Colitis.

IntroductionIn colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice.MethodsWe performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10-/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization.ResultsTreatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo resulted in a mild reduction in colitis severity as compared with vehicle-treated mice.ConclusionWe identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis.

Microvesicle-Derived miRNAs Regulate Proinflammatory Macrophage Activation in the Lung Following Ozone Exposure.

Ozone is a ubiquitous air pollutant that causes lung damage and altered functioning. Evidence suggests that proinflammatory macrophages contribute to ozone toxicity. Herein, we analyzed the role of extracellular vesicles (EVs) and microRNA (miRNA) cargo in ozone-induced macrophage activation. Exposure of mice to ozone (0.8 ppm, 3 h) resulted in increases in bronchoalveolar lavage fluid EVs, which were comprised predominantly of microvesicles (MVs). NanoFACS analysis revealed that MVs generated following both air and ozone exposure was largely from CD45+ myeloid cells; these MVs were readily taken up by macrophages. Functionally, MVs from ozone, but not air treated mice, upregulated mRNA expression of inflammatory proteins in macrophages including inducible nitric oxide synthase (iNOS), CXCL-1, CXCL-2, and interleukin (IL)-1β. The miRNA profile of MVs in bronchoalveolar lavage fluid (BALF) was altered after ozone exposure; thus, increases in miR-21, miR-145, miR320a, miR-155, let-7b, miR744, miR181, miR-17, miR-92a, and miR-199a-3p were observed, whereas miR-24-3p and miR-20 were reduced. Ingenuity pathway analysis revealed that these miRNAs regulate pathways that promote inflammatory macrophage activation, and predicted that let-7a-5p/let-7b, miR-24-3p, miR-21-5p, miR-17, and miR-181a-5p are key upstream regulators of inflammatory proteins. After ozone exposure, miR-199a-3p, but not precursor miR-199a-3p, was increased in lung macrophages, indicating that it is derived from MV-mediated delivery. Furthermore, lung macrophage mRNA expression of IL-1β was upregulated after administration of MVs containing miR-199a-3p mimic but downregulated by miR-199a-3p inhibitor. Collectively, these data suggest that MVs generated following ozone exposure contribute to proinflammatory macrophage activation via MV-derived miRNAs including miR-199a-3p. These findings identify a novel pathway regulating macrophage inflammatory responses to inhaled ozone.

IRAK-M deletion aggravates acute inflammatory response and mitochondrial respiratory dysfunction following myocardial infarction: A bioinformatics analysis

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Interleukin-1 receptor associated kinase (IRAK)-M is a regulator of Toll-like receptor mediated inflammatory responses and plays an important role in the pathophysiologic processes of acute MI. We aimed to explore the effect of IRAK-M on regulating biological function and molecular interactions post-MI through bioinformatics analysis. Datasets from the Gene Expression Omnibus database were used to identify characteristics of IRAK-M expression in MI patients. The expression of IRAK-M was upregulated in MI patients and altered in a time-dependent manner during MI progression. Enrichment analysis showed that biological processes related to inflammatory response and leukocyte activation were markedly activated in MI patients with upregulated IRAK-M. Furthermore, we constructed MI model using wildtype and IRAK-M−/− mice and performed proteomics analysis of infarcted hearts. Functional enrichment of proteomics data indicated that IRAK-M deletion aggravated a series of pathophysiologic functions, such as acute inflammatory response, macrophage activation and mitochondrial dysfunction. S100A8/A9 acted as the central molecule in the above functions based on the protein-protein interaction network and was significantly elevated in IRAK-M−/− infarcted hearts at both the protein and mRNA levels. In conclusion, IRAK-M functioned as an essential regulator in pathophysiologic processes post-MI, exerting effects not only on controlling acute inflammatory responses but also on mediating mitochondrial respiratory function based on integrated bioinformatics analysis. SignificanceIn this study, we combined microarray datasets and a proteomics approach to explore the effect of IRAK-M on mediating biological processes and systemic molecular interactions following MI. Our data firstly showed that IRAK-M is involved in ATP synthesis and mitochondrial respiratory chain complex during MI progression. S100A8/A9 acted as the central molecule in above regulatory network and displayed a tight connection with IRAK-M. The findings provide novel evidence and clues for understanding the complex roles and molecular mechanisms of IRAK-M in the development of MI.

Itaconate inhibits ferroptosis of macrophage via Nrf2 pathways against sepsis-induced acute lung injury

Itaconate, a metabolite produced during inflammatory macrophage activation, has been extensively described to be involved in immunoregulation, oxidative stress, and lipid peroxidation. As a form of iron and lipid hydroperoxide-dependent regulated cell death, ferroptosis plays a critical role in sepsis-induced acute lung injury (ALI). However, the relationship between itaconate and ferroptosis remains unclear. This study aims to explore the regulatory role of itaconate on ferroptosis in sepsis-induced ALI. In in vivo experiments, mice were injected with LPS (10 mg/kg) for 12 h to generate experimental sepsis models. Differential gene expression analysis indicated that genes associated with ferroptosis existed significant differences after itaconate pretreatment. 4-octyl itaconate (4-OI), a cell-permeable derivative of endogenous itaconate, can significantly alleviate lung injury, increase LPS-induced levels of glutathione peroxidase 4 (GPX4) and reduce prostaglandin-endoperoxide synthase 2 (PTGS2), malonaldehyde (MDA), and lipid ROS. In vitro experiments showed that both 4-OI and ferrostatin-1 inhibited LPS-induced lipid peroxidation and injury of THP-1 macrophage. Mechanistically, we identified that 4-OI inhibited the GPX4-dependent lipid peroxidation through increased accumulation and activation of Nrf2. The silence of Nrf2 abolished the inhibition of ferroptosis from 4-OI in THP-1 cells. Additionally, the protection of 4-OI for ALI was abolished in Nrf2-knockout mice. We concluded that ferroptosis was one of the critical mechanisms contributing to sepsis-induced ALI. Itaconate is promising as a therapeutic candidate against ALI through inhibiting ferroptosis.

Fibrinogen-like protein 2 deficiency inhibits virus-induced fulminant hepatitis through abrogating inflammatory macrophage activation.

BACKGROUNDHeterogeneous macrophages play an important role in multiple liver diseases, including viral fulminant hepatitis (VFH). Fibrinogen-like protein 2 (FGL2) is expressed on macrophages and regulates VFH pathogenesis; however, the underlying mechanism remains unclear. AIMTo explore how FGL2 regulates macrophage function and subsequent liver injury during VFH. METHODSMurine hepatitis virus strain 3 (MHV-3) was used to induce VFH in FGL2-deficient (Fgl2-/-) and wild-type (WT) mice. The dynamic constitution of hepatic macrophages was examined. Adoptive transfer of Fgl2-/- or WT bone marrow-derived macrophages (BMDMs) into WT recipients with macrophages depleted prior to infection was carried out and the consequent degree of liver damage was compared. The signaling cascades that may be regulated by FGL2 were detected in macrophages.RESULTSFollowing MHV-3 infection, hepatic macrophages were largely replenished by proinflammatory monocyte-derived macrophages (MoMFs), which expressed high levels of FGL2. In Fgl2-/- mice, the number of infiltrating inflammatory MoMFs was reduced compared with that in WT mice after viral infection. Macrophage depletion ameliorated liver damage in WT mice and further alleviated liver damage in Fgl2-/- mice. Adoptive transfer of Fgl2-/- BMDMs into macrophage-removed recipients significantly reduced the degree of liver damage. Inhibition of monocyte infiltration also significantly ameliorated liver damage. Functionally, Fgl2 deletion impaired macrophage phagocytosis and the antigen presentation potential and attenuated the proinflammatory phenotype. At the molecular level, FGL2 deficiency impaired IRF3, IRF7, and p38 phosphorylation, along with NF-κB activation in BMDMs in response to viral infection. CONCLUSIONInfiltrated MoMFs represent a major source of hepatic inflammation during VFH progression, and FGL2 expression on MoMFs maintains the proinflammatory phenotype via p38-dependent positive feedback, contributing to VFH pathogenesis.

Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm.

Objective Apolipoprotein (a)/lipoprotein(a) (Lp(a)), a major carrier of oxidized phospholipids, and α7-nicotinic acetylcholine receptor (α7-nAChR) may play an important role in the development of coronary artery spasm (CAS). In CAS, the association between Lp(a) and the α7-nAChR-modulated inflammatory macrophage polarization and activation and smooth muscle cell dysfunction remains unknown. Methods We investigated the relevance of Lp(a)/α7-nAChR signaling in patient monocyte-derived macrophages and human coronary artery smooth muscle cells (HCASMCs) using expression profile correlation analyses, fluorescence-assisted cell sorting flow cytometry, immunoblotting, quantitative real-time polymerase chain reaction, and clinicopathological analyses. Results There are increased serum Lp(a) levels (3.98-fold, p = 0.011) and macrophage population (3.30-fold, p = 0.013) in patients with CAS compared with patients without CAS. Serum Lp(a) level was positively correlated with high-sensitivity C-reactive protein (r2 = 0.48, p < 0.01), IL-6 (r2 = 0.38, p = 0.03), and α7-nAChR (r2 = 0.45, p < 0.01) in patients with CAS, but not in patients without CAS. Compared with untreated or low-density lipoprotein- (LDL-) treated macrophages, Lp(a)-treated macrophages exhibited markedly enhanced α7-nAChR mRNA expression (p < 0.01) and activity (p < 0.01), in vitro and ex vivo. Lp(a) but not LDL preferentially induced CD80+ macrophage (M1) polarization and reduced the inducible nitric oxide synthase expression and the subsequent NO production. While shRNA-mediated loss of α7-nAChR function reduced the Lp(a)-induced CD80+ macrophage pool, both shRNA and anti-IL-6 receptor tocilizumab suppressed Lp(a)-upregulated α7-nAChR, p-p38 MAPK, IL-6, and RhoA-GTP protein expression levels in cultures of patient monocyte-derived macrophages and HCASMCs. Conclusions Elevated Lp(a) levels upregulate α7-nAChR/IL-6/p38 MAPK signaling in macrophages of CAS patients and HCASMC, suggesting that Lp(a)-triggered inflammation mediates CAS through α7-nAChR/p38 MAPK/IL-6/RhoA-GTP signaling induction, macrophage M1 polarization, and HCASMC activation.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models.

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1β. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

Citrulline depletion by ASS1 is required for proinflammatory macrophage activation and immune responses

Citrulline can be converted into argininosuccinate by argininosuccinate synthetase (ASS1) in the urea cycle and the citrulline-nitric oxide cycle. However, the regulation and biological function of citrulline metabolism remain obscure in the immune system. Unexpectedly, we found that macrophage citrulline declines rapidly after interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) stimulation, which is required for efficient proinflammatory signaling activation. Mechanistically, IFN-γ and/or LPS stimulation promotes signal transducers and activators of transcription 1 (STAT1)-mediated ASS1 transcription and Janus kinase2 (JAK2)-mediated phosphorylation of ASS1 at tyrosine 87, thereby leading to citrulline depletion. Reciprocally, increased citrulline directly binds to JAK2 and inhibits JAK2-STAT1 signaling. Blockage of ASS1-mediated citrulline depletion suppresses the host defense against bacterial infection invivo. We therefore define a central role for ASS1 in controlling inflammatory macrophage activation and antibacterial defense through depletion of cellular citrulline and, further, identify citrulline as an innate immune-signaling metabolite that engages a metabolic checkpoint for proinflammatory responses.

Glucocorticoids coordinate macrophage metabolism through the regulation of the tricarboxylic acid cycle

ObjectivesGlucocorticoids (GCs) are one of the most widely prescribed anti-inflammatory drugs. By acting through their cognate receptor, the glucocorticoid receptor (GR), GCs downregulate the expression of pro-inflammatory genes and upregulate the expression of anti-inflammatory genes. Metabolic pathways have recently been identified as key parts of both the inflammatory activation and anti-inflammatory polarization of macrophages, immune cells responsible for acute inflammation and tissue repair. It is currently unknown whether GCs control macrophage metabolism, and if so, to what extent metabolic regulation by GCs confers anti-inflammatory activity. MethodsUsing transcriptomic and metabolomic profiling of macrophages, we identified GC-controlled pathways involved in metabolism, especially in mitochondrial function. ResultsMetabolic analyses revealed that GCs repress glycolysis in inflammatory myeloid cells and promote tricarboxylic acid (TCA) cycle flux, promoting succinate metabolism and preventing intracellular accumulation of succinate. Inhibition of ATP synthase attenuated GC-induced transcriptional changes, likely through stalling of TCA cycle anaplerosis. We further identified a glycolytic regulatory transcription factor, HIF1α, as regulated by GCs, and as a key regulator of GC responsiveness during inflammatory challenge. ConclusionsOur findings link metabolism to gene regulation by GCs in macrophages.