Mac-1 Expression Research Articles

Inhibition of Mac-1 allows human macrophages to migrate against the direction of shear flow on ICAM-1.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

Nonsense-mediated mRNA decay of metal-binding activator MAC1 is dependent on copper levels and 3'-UTR length in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.

Mac-1 deficiency ameliorates pressure overloaded heart failure through inhibiting macrophage polarization and activation

Persistent pressure overload commonly leads to pathological cardiac hypertrophy and remodeling, ultimately leading to heart failure (HF). Cardiac remodeling is associated with the involvement of immune cells and the inflammatory response in pathogenesis. The macrophage-1 antigen (Mac-1) is specifically expressed on leukocytes and regulates their migration and polarization. Nonetheless, the involvement of Mac-1 in cardiac remodeling and HF caused by pressure overload has not been determined. The Mac-1-knockout (KO) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) for 6 weeks. Echocardiography and pressure–volume loop assessments were used to evaluate cardiac function, and cardiac remodeling and macrophage infiltration and polarization were estimated by histopathology and molecular techniques. The findings of our study demonstrated that Mac-1 expression was markedly increased in hearts subjected to TAC treatment. Moreover, compared with WT mice, Mac-1-KO mice exhibited dramatically ameliorated TAC-induced cardiac dysfunction, hypertrophy, fibrosis, oxidative stress and apoptosis. The potential positive impacts may be linked to the inhibition of macrophage infiltration and M1 polarization via reductions in NF-kB and STAT1 expression and upregulation of STAT6. In conclusion, this research reveals a new function of Mac-1 deficiency in reducing pathological cardiac remodeling and HF caused by pressure overload. Additionally, inhibiting Mac-1 could be a potential treatment option for patients with HF in a clinical setting.

Effects of chemical inhibition of histone deacetylase proteins in the growth and virulence of Macrophomina phaseolina (Tassi) Goid.

Chromatin remodeling enzymes are important “writers”, “readers” and “erasers” of the epigenetic code. These proteins are responsible for the placement, recognition, and removal of molecular marks in histone tails that trigger structural and functional changes in chromatin. This is also the case for histone deacetylases (HDACs), i.e., enzymes that remove acetyl groups from histone tails, signaling heterochromatin formation. Chromatin remodeling is necessary for cell differentiation processes in eukaryotes, and fungal pathogenesis in plants includes many adaptations to cause disease. Macrophomina phaseolina (Tassi) Goid. is a nonspecific, necrotrophic ascomycete phytopathogen that causes charcoal root disease. M. phaseolina is a frequent and highly destructive pathogen in crops such as common beans (Phaseolus vulgaris L.), particularly under both water and high temperature stresses. Here, we evaluated the effects of the classical HDAC inhibitor trichostatin A (TSA) on M. phaseolinain vitro growth and virulence. During inhibition assays, the growth of M. phaseolina in solid media, as well as the size of the microsclerotia, were reduced (p<0.05), and the colony morphology was remarkably affected. Under greenhouse experiments, treatment with TSA reduced (p<0.05) fungal virulence in common bean cv. BAT 477. Tests of LIPK, MAC1 and PMK1 gene expression during the interaction of fungi with BAT 477 revealed noticeable deregulation. Our results provide additional evidence about the role of HATs and HDACs in important biological processes of M. phaseolina.

Reshaping cardiac microenvironments by macrophage-derived extracellular vesicles-coated Pd@CeO2 heterostructures for myocardial ischemia/reperfusion injury therapy

Myocardial Ischemia/Reperfusion (MI/R) injury, a globally leading cause of mortality and disability, is commonly characterized by myriad inflammatory microenvironments, e.g. high level of reactive oxygen species (ROS), which has a high association with the consequent cell apoptosis and myocardial fibrosis. Herein, a reshaping strategy of cardiac microenvironments has been developed for relieving MI/R injury based on a biomimetic nanosystem (Pd@CeO2-M), which is composed of exterior macrophage-derived extracellular vesicles (MEVs) and encapsulated Pd@CeO2 heterostructures. Due to rapid electrons transfer on the interface, the Pd@CeO2 heterostructures exhibited outstanding ROS scavenging ability. The expression of Mac-1 and CD44 on the surface of Pd@CeO2-M contributes to adsorbing to the inflamed endothelium and efficient cellular uptake within damaged cardiac microenvironments, thus allowing for inflammation-targeting ability. In the mouse model of MI/R injury, Pd@CeO2-M accumulated and remained in the heart region over 24 hours. More importantly, Pd@CeO2-M regulated immune response and promoted anti-apoptotic and anti-inflammatory processes via upregulating the PI3K/Akt signaling pathway and inhibiting the TLR4/p38MAPK and TLR4/NF-κB signaling pathways, dissipated interstitial edema, triggered prominent angiogenesis, and ultimately improved cardiac function and ventricular remodeling. Overall, the Pd@CeO2-M heterostructures will provide an ideal paradigm of biomimetic nanomedicine confronting inflammatory diseases.

MAC-1 marks a quiescent and functionally superior HSC subset during regeneration.

Mouse hematopoietic stem cells (HSCs) have been extensively defined both molecularly and functionally at steady state, while regenerative stress induces immunophenotypical changes that limit high purity isolation and analysis. It is therefore important to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Here, we assessed the expression of macrophage-1 antigen (MAC-1) on HSCs during regeneration following transplantation and observed a transient increase in MAC-1 expression during the early reconstitution phase. Serial transplantation experiments demonstrated that reconstitution potential was highly enriched in the MAC-1+ portion of the HSC pool. Moreover, in contrast to previous reports, we found that MAC-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating MAC-1+ HSCs share molecular features with stem cells with low mitotic history. Taken together, our results suggest that MAC-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration.

Nuclear factor I-C overexpression promotes monocytic development and cell survival in acute myeloid leukemia

Nuclear factor I-C (NFIC) belongs to a family of NFI transcription factors that binds to DNA through CAATT-boxes and are involved in cellular differentiation and stem cell maintenance. Here we show NFIC protein is significantly overexpressed in 69% of acute myeloid leukemia patients. Examination of the functional consequences of NFIC overexpression in HSPCs showed that this protein promoted monocytic differentiation. Single-cell RNA sequencing analysis further demonstrated that NFIC overexpressing monocytes had increased expression of growth and survival genes. In contrast, depletion of NFIC through shRNA decreased cell growth, increased cell cycle arrest and apoptosis in AML cell lines and AML patient blasts. Further, in AML cell lines (THP-1), bulk RNA sequencing of NFIC knockdown led to downregulation of genes involved in cell survival and oncogenic signaling pathways including mixed lineage leukemia-1 (MLL-1). Lastly, we show that NFIC knockdown in an ex vivo mouse MLL::AF9 pre-leukemic stem cell model, decreased their growth and colony formation and increased expression of myeloid differentiation markers Gr1 and Mac1. Collectively, our results suggest that NFIC is an important transcription factor in myeloid differentiation as well as AML cell survival and is a potential therapeutic target in AML.

CBA strain mice as a model of spontaneous carcinogenesis

The review analyzes some parameters of CBA mice-males as model of spontaneous carcinogenesis characterizing adhesive and adaptive disorders. A weakening of the hepatocytes mutual adhesiveness force was noted already in early ontogenesis (5–10 days of postnatal development). This violation persisted and enhanced during hepatocarcinogenesis. A decrease of the β2 leukocyte integrins LFA-1 and Mac-1 expression on peripheral blood cells as well as an increase of the interleukins 6 and 10 in blood serum were determined during ontogenesis. It is significant for weakening the liver cells contact interactions (mutual adhesiveness) as well as immunity effectors and tumor cells interactions. A disbalance of the adaptive reactions and life quality important components was revealed in the CBA mice-males ontogenesis. Number of dopaminergic neurons and the neurogenesislevel in CBA micemales were decreasing. This does not contradict the dynamics of chronic stress and the aging process: an increase in the catabolic stress hormone corticosterone, a decrease in the anabolic hormone testosterone in the blood serum, a decrease in motor activity, signs of cachexia and alopecia, as well as a violation of immunological parameters.CBA mice-males with an assessment of parameters characterizing adhesive and adaptive disorders during spontaneous carcinogenesis (the hepatocytes mutual adhesiveness forсe, the expression of β2 leukocyte integrins LFA-1 and Mac-1 on peripheral blood cells, the content of interleukins 6, 10, corticosterone and testosterone in blood serum, the number of dopaminergic neurons in the midbrain during ontogenesis) as well as the frequency and size of tumours, lifespan and somatic status of animals can be used as a scientifically- and evidence-based test system to study cytostatic drugs as well as non-toxic geroprotective medications for prevention and treatment of cancer in individuals with an increased risk of malignant neoplasms developing especially hepatocellular carcinoma.

Guanxinning tablet inhibits the interaction between leukocyte integrin Mac-1 and platelet GPIbα for antithrombosis without increased bleeding risk.

Recent studies have showed that thrombosis is closely related to leucocytes involved in immunity. Interfering with the binding of leukocyte integrin Mac-1 and platelet GPIbα can inhibit thrombosis without affecting physiological coagulation. Mac-1-GPIbα is proposed as a potential safety target for antithrombotic agents. Guanxinning tablet (GXNT) is an oral Chinese patent medicine used for the treatment of angina pectoris, which contains phenolic acid active ingredients, such as salvianolic acids, ferulic acid, chlorogenic acid, caffeic acid, rosmarinic acid, tanshinol, and protocatechualdehyde. Our previous studies demonstrated that GXN exhibited significant antithrombotic effects, and clinical studies suggested that it did not increase bleeding risk. In addition, GXN exerted a significantly regulatory effect on immune inflammation. In the current study, we intended to evaluate the effects of GXN on bleeding events and explore the safety antithrombotic mechanism of GXN based on leukocyte-platelet interaction. First, we established a gastric ulcer model induced by acetic acid in rats and found that GXN not only did not increase the degree of gastrointestinal bleeding when gastric ulcer occurred, but also had a certain promoting effect on the healing of gastric ulcer. Second, in vitroexperiments showed that after pretreatment with GXN and activation by phorbol 12-myristate-13-acetate (PMA), the adhesion and aggregation of leukocytes with human platelets were reduced. It was also found that GXN reduced the expression and activation of Mac-1 in leucocytes, and inhibited platelet activation due to leukocyte engagement via Mac-1. Overall, the results suggest that GXN may be a safe antithrombotic agent, and its low bleeding risk mechanism is probably related to inhibited leukocyte-platelet aggregation and its interaction target Mac-1-GPIbα.

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

SummaryIn this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

Complement Inhibition Alleviates Cholestatic Liver Injury Through Mediating Macrophage Infiltration and Function in Mice.

Background and AimsCholestatic liver injury (CLI), which is associated with inflammatory reactions and oxidative stress, is a serious risk factor for postoperative complications. Complement system is involved in a wide range of liver disorders, including cholestasis. The present study assessed the role of complement in CLI and the therapeutic effect of the site-targeted complement inhibitor CR2-Crry in CLI.MethodsWild-type and complement gene deficient mice underwent common bile duct ligation (BDL) to induce CLI or a sham operation, followed by treatment with CR2-Crry or GdCl3. The roles of complement in CLI and the potential therapeutic effects of CR2-Crry were investigated by biochemical analysis, flow cytometry, immunohistochemistry, ELISA, and quantitative RT-PCR.ResultsC3 deficiency and CR2-Crry significantly reduced liver injuries in mice with CLI, and also markedly decreasing the numbers of neutrophils and macrophages in the liver. C3 deficiency and CR2-Crry also significantly reduced neutrophil expression of Mac-1 and liver expression of VCAM-1. More importantly, C3 deficiency and CR2-Crry significantly inhibited M1 macrophage polarization in these mice. Intravenous injection of GdCl3 inhibited macrophage infiltration and activation in the liver. However, the liver injury increased significantly. BDL significantly increased the level of lipopolysaccharide (LPS) in portal blood, but not in peripheral blood. GdCl3 significantly increased LPS in peripheral blood, suggesting that macrophages clear portal blood LPS. Oral administration of ampicillin to in GdCl3 treated mice reduced LPS levels in portal blood and alleviated liver damage. In contrast, intraperitoneal injection LPS increased portal blood LPS and reversed the protective effect of ampicillin. Interestingly, C3 deficiency did not affect the clearance of LPS.ConclusionsComplement is involved in CLI, perhaps mediating the infiltration and activation of neutrophils and macrophage M1 polarization in the liver. C3 deficiency and CR2-Crry significantly alleviated CLI. Inhibition of complement could preserve the protective function of macrophages in clearing LPS, suggesting that complement inhibition could be useful in treating CLI.

3176 – MAC-1 MARKS A QUIESCENT AND FUNCTIONALLY SUPERIOR HSC SUBSET DURING REGENERATION

Hematopoietic stem cells are capable of sustaining a lifelong production of mature blood cells as well as rapid hematopoietic regeneration upon acute stress. Mouse hematopoietic stem cells (HSCs) at steady state have been extensively defined both molecularly and functionally. When exposed to regenerative stress, however, HSCs undergo several immunophenotypical changes that limit the possibilities of isolating them at high purity. As a consequence, actively regenerating HSCs have remained less defined, and it is of great interest to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Macrophage-1 antigen (Mac-1), a cell surface marker typically expressed by myeloid cells, has been shown to be upregulated on HSCs in response to the cytotoxic drug 5-fluorouracil (5FU). Here, we assessed the expression of Mac-1 on HSCs during regeneration following transplantation and observed a transient but distinct increase in Mac-1 expression during the early reconstitution phase 4 weeks after transplantation. Remarkably, from serial transplantation experiments, we found that the functional reconstitution potential was highly enriched in this Mac-1+ portion of the HSC pool. A similar correlation between Mac-1 expression and functional HSC activity was seen following 5-FU treatment. Moreover, in contrast to previous reports, we found that Mac-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating Mac-1+ HSCs share molecular features with stem cells with low mitotic history and low historic progeny output. Taken together, our results suggest that Mac-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration. Hematopoietic stem cells are capable of sustaining a lifelong production of mature blood cells as well as rapid hematopoietic regeneration upon acute stress. Mouse hematopoietic stem cells (HSCs) at steady state have been extensively defined both molecularly and functionally. When exposed to regenerative stress, however, HSCs undergo several immunophenotypical changes that limit the possibilities of isolating them at high purity. As a consequence, actively regenerating HSCs have remained less defined, and it is of great interest to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Macrophage-1 antigen (Mac-1), a cell surface marker typically expressed by myeloid cells, has been shown to be upregulated on HSCs in response to the cytotoxic drug 5-fluorouracil (5FU). Here, we assessed the expression of Mac-1 on HSCs during regeneration following transplantation and observed a transient but distinct increase in Mac-1 expression during the early reconstitution phase 4 weeks after transplantation. Remarkably, from serial transplantation experiments, we found that the functional reconstitution potential was highly enriched in this Mac-1+ portion of the HSC pool. A similar correlation between Mac-1 expression and functional HSC activity was seen following 5-FU treatment. Moreover, in contrast to previous reports, we found that Mac-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating Mac-1+ HSCs share molecular features with stem cells with low mitotic history and low historic progeny output. Taken together, our results suggest that Mac-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration.

GPR55 Antagonist CID16020046 Protects against Atherosclerosis Development in Mice by Inhibiting Monocyte Adhesion and Mac-1 Expression.

GPR55 recognizes several lipid molecules such as lysophosphatidylinositol. GPR55 expression was reported in human monocytes. However, its role in monocyte adhesion and atherosclerosis development has not been studied. The role of GPR55 in monocyte adhesion and atherosclerosis development was investigated in human THP-1 monocytes and ApoE−/− mice using O-1602 (a potent agonist of GPR55) and CID16020046 (a specific GPR55 antagonist). O-1602 treatment significantly increased monocyte adhesion to human umbilical vein endothelial cells, and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides −750 and −503 as GPR55 responsive elements. O-1602 induction of Mac-1 was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In Apo−/− mice, administration of CID16020046 ameliorated high-fat diet-induced atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to the adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis.

Targeting S100A9 Reduces Neutrophil Recruitment, Inflammation and Lung Damage in Abdominal Sepsis.

S100A9, a pro-inflammatory alarmin, is up-regulated in inflamed tissues. However, the role of S100A9 in regulating neutrophil activation, inflammation and lung damage in sepsis is not known. Herein, we hypothesized that blocking S100A9 function may attenuate neutrophil recruitment in septic lung injury. Male C57BL/6 mice were pretreated with the S100A9 inhibitor ABR-238901 (10 mg/kg), prior to cercal ligation and puncture (CLP). Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for analysis of neutrophil infiltration as well as edema and CXC chemokine production. Blood was collected for analysis of membrane-activated complex-1 (Mac-1) expression on neutrophils as well as CXC chemokines and IL-6 in plasma. Induction of CLP markedly increased plasma levels of S100A9. ABR-238901 decreased CLP-induced neutrophil infiltration and edema formation in the lung. In addition, inhibition of S100A9 decreased the CLP-induced up-regulation of Mac-1 on neutrophils. Administration of ABR-238901 also inhibited the CLP-induced increase of CXCL-1, CXCL-2 and IL-6 in plasma and lungs. Our results suggest that S100A9 promotes neutrophil activation and pulmonary accumulation in sepsis. Targeting S100A9 function decreased formation of CXC chemokines in circulation and lungs and attenuated sepsis-induced lung damage. These novel findings suggest that S100A9 plays an important pro-inflammatory role in sepsis and could be a useful target to protect against the excessive inflammation and lung damage associated with the disease.

Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation

AbstractPlasmodium falciparum-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) not only to disrupt the barrier-permeability function of endothelial cells but also to inhibit the antiinflammatory signaling function of AT. Here we investigated the mechanisms of the proinflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration, and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation, and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of P falciparum-derived HRPII in both cellular and animal models.

Neutrophil dysfunction due to continuous mechanical shear exposure in mechanically assisted circulation in vitro.

Leukocytes play an important role in the body's immune system. The aim of this study was to assess alterations in neutrophil phenotype and function in pump-assisted circulation in vitro. Human blood was circulated for four hours in three circulatory flow loops with a CentriMag blood pump operated at a flow of 4.5 L/min at three rotational speeds (2100, 2800, and 4000rpm), against three pressure heads (75, 150, and 350mmHg), respectively. Blood samples were collected hourly for analyses of neutrophil activation state (Mac-1, CD62L, CD162), neutrophil reactive oxygen species (ROS) production, apoptosis, and neutrophil phagocytosis. Activated neutrophils indicated by both Mac-1 expression and decreased surface expression of CD62L and CD162 receptors increased with time in three loops. The highest level of neutrophil activation was observed in the loop with the highest rotational speed. Platelet-neutrophil aggregates (PNAs) progressively increased in two loops with lower rotational speeds. PNAs peaked at one hour after circulation and decreased subsequently in the loop with the highest rotational speed. Neutrophil ROS production dramatically increased at one hour after circulation and decreased subsequently in all three loops with similar levels and trends. Apoptotic neutrophils increased with time in all three loops. Neutrophil phagocytosis capacity in three loops initially elevated at one hour after circulation and decreased subsequently. Apoptosis and altered phagocytosis were dependent on rotational speed. Our study revealed that the pump-assisted circulation induced neutrophil activation, apoptosis, and functional impairment. The alterations were strongly associated with pump operating condition and duration.

Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae.

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

The Interaction between Integrin Mac-1 (αMβ2, CD11b/CD18) and Sirpα Mediates Macrophage Fusion

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. While a number of membrane proteins have been implicated in mediating cell-cell attachement during fusion, their binding partners are not yet clear. Recently, we have demonstrated that IL-4-induced fusion of mouse peritoneal macrophages depends on integrin Mac-1 (αMβ2, CD11b/CD18). Surprisingly, the genetic deficiency of ICAM-1, an established ligand of Mac-1, resulted in no decrease of macrophage fusion, suggesting the involvement of other counter-receptors for Mac-1. In the present study, we demonstrated that SIRPα, which like ICAM-1 belongs to the Ig superfamily and was previously implicated in fusion, interacts with Mac-1. Expression of Mac-1 on the surface of HEK293 cells resulted in their adhesion to recombinant ectodomain of SIRPα and this process was mediated through the αMI-domain of Mac-1. In addition, adhesion of RAW264.7 mouse macrophages to SIRPα was dependent on Mac-1. Direct interaction between the αMI-domain and SIRPα was shown by biolayer interferometry. Analyses of the interaction of αMI-domain with a peptide library spanning the ectodomain of SIRPα showed that recognition of SIRPa conforms to general principles that govern binding by Mac-1 of many of its ligands. SIRPα reportedly interacts with CD47; however, adhesion of Mac-1-expressing HEK293 cells, which express CD47, to SIRPα was not dependent on CD47 and an anti-CD47 function-blocking mAb produced only a limited inhibition of adhesion of RAW264.7 cells. Co-immunoprecipitation experiments revealed that Mac-1 forms a complex with CD47 on the surface of RAW264.7 and Mac-1-expressing HEK293 cells. Finally, co-culturing of SIPRα- and Mac-1-expressing HEK293 cells resulted in cell fusion. Collectively, these data identify SIRPα as a novel counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion. DisclosuresNo relevant conflicts of interest to declare.

LSD1 Inhibition Leads to Differentiation in Hoxa9/Meis1- but Not in MN1-Induced Acute Myeloid Leukemia

Lysine specific demethylase 1 (LSD1) represents a promising epigenetic target in the treatment of acute myeloid leukemia (AML) and inhibition of LSD1 has been demonstrated to facilitate a response of AML cells to all- trans retinoic acid (ATRA). We have previously shown that LSD1 depletion or inhibition using tranylcypromine or its derivatives induces granulocytic differentiation, reduces leukemia-initiating cell (LIC) frequency and leads to a survival benefit in Hoxa9/Meis1 (H/M)-driven AML. We have now modeled the effect of pharmacological or genetic inactivation of LSD1 in another murine leukemia model based on the retroviral overexpression of MN1.We transformed lineage-depleted bone marrow cells from conditional LSD1 knock-out (KO) mice with MN1 and induced the LSD1 KO by treatment with 4-hydroxy-tamoxifen (4-OHT). As a control we compared the results observed in this system with results from cells transformed with the combination of Hoxa9 and Meis1 (H/M). We analyzed short-term proliferation based on XTT assays and differentiation based on cell morphology as well as surface marker expression. We observed a 61% reduction in proliferation in H/M cells, while we only found 28% reduction in proliferation in MN1 cells. LSD1 KO induced marked granulocytic differentiation in H/M cells, but we did not observe any signs of granulocytic differentiation in MN1 cells. While LSD1 KO led to a reduction in the expression of c-Kit and upregulation of Ly-6G in H/M cells, c-Kit expression remained unchanged in MN1 cells. There were also no changes in Gr1 and Mac1, but MN1 cells acquired Sca1 and FcεR1. LSD1 inhibition has been shown to enhance the sensitivity of leukemic cells towards a treatment with ATRA. We therefore now compared the influence of LSD1 KO on the effect of ATRA treatment in H/M and MN1 cells. We saw a 95 % reduction in proliferation in H/M cells with LSD1 KO upon treatment with 0.1 µM of ATRA. In MN1 cells with LSD1 KO we only observed a reduction in proliferation of 28 % at this concentration.We previously found a differentiating effect of irreversible LSD1 inhibitors in H/M transformed cells. We therefore also tested different potent LSD1 inhibitors (derivatives of tranylcypromine) in MN1 cells (trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (ORY86, AW69), (1S,2R)-2-phenyl-N-(piperidin-4-ylmethyl) cyclopropanamine (AW84), GSK-LSD1). Again we observed no signs of granulocytic differentiation, but an acquisition of the surface markers Sca1 and FcεR1 without concomitant changes in the expression of c-Kit, Gr1 and Mac1. Treatment with AW69 leads to a strong reduction in LIC frequency in the H/M model system. We now investigated if pre-treatment with AW69 in vitro for 96 h also affected the ability of MN1 cells to induce leukemia in mice. In this experiment we observed no change in leukemia development upon transplantation of 20.000 cells per mouse. However while 2 of 3 mice transplanted with control treated cells developed leukemia upon transplantation of 2.000 cells per mouse, no leukemia development was seen at this cell dose with AW69-treated cells. We therefore cannot exclude a moderate effect on LIC frequency in the MN1 model.In order to test if our findings can be transferred to human AML we characterized the expression of MN1, HOXA9 and MEIS1 in 6 primary human AML cell cultures. We then treated these cells with GSK-LSD1 and assessed the expression of CD86, a known differentiation marker induced by LSD1 inhibition. In these experiments we observed a particularly strong upregulation of CD86 in samples expressing higher levels of MEIS1.In summary, our results show that while pharmacological or genetic inactivation of LSD1 potently induces differentiation in H/M-induced AML, it is not sufficient to induce differentiation in MN1-driven AML. This finding could be relevant for selecting patients for treatment with LSD1 inhibitors and warrants further molecular investigations. DisclosuresBug:Astellas: Other: Travel Funding; Janssen: Other: Travel Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Other: Travel Funding; Celgene: Honoraria, Other: Travel funding. Lübbert:Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid. Berg:Astellas: Other: Travel Funding; Riemser: Consultancy; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding; Abbvie: Other: Travel Funding.

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.