PRMT4 inhibitor TP-064 inhibits the pro-inflammatory macrophage lipopolysaccharide response in vitro and ex vivo and induces peritonitis-associated neutrophilia in vivo.

Previous in vitro studies have shown that protein arginine N-methyltransferase 4 (PRMT4) is a co-activator for an array of cellular activities, including NF-κB-regulated pro-inflammatory responses. Here we investigated the effect of PRMT4 inhibitor TP-064 treatment on macrophage inflammation in vitro and in vivo. Exposure of RAW 264.7 monocyte/macrophages to TP-064 was associated with a significant decrease in the production of pro-inflammatory cytokines upon a lipopolysaccharide challenge. Similarly, thioglycollate-elicited peritoneal cells isolated from wildtype mice treated with TP-064 showed lowered mRNA expression levels and cytokine production of pro-inflammatory mediators interleukin (IL)-1ß, IL-6, IL-12p40, and tumor necrosis factor-α in response to lipopolysaccharide exposure. However, TP-064-treated mice exhibited an ongoing pro-inflammatory peritonitis after 5 days of thioglycollate exposure, as evident from a shift in the peritoneal macrophage polarization state from an anti-inflammatory LY6ClowCD206hi to a pro-inflammatory LY6ChiCD206low phenotype. In addition, TP-064-treated mice accumulated (activated) neutrophils within the peritoneum as well as in the blood (7-fold higher; P < 0.001) and major organs such as kidney and liver, without apparent tissue toxicity. TP-064 treatment downregulated hepatic mRNA expression levels of the PRMT4 target genes glucose-6-phosphatase catalytic subunit (-50%, P < 0.05) and the cyclin-dependent kinases 2 (-50%, P < 0.05) and 4 (-30%, P < 0.05), suggesting a direct transcriptional effect of PRMT4 also in hepatocytes. In conclusion, we have shown that the PRMT4 inhibitor TP-064 induces peritonitis-associated neutrophilia in vivo and inhibits the pro-inflammatory macrophage lipopolysaccharide response in vitro and ex vivo. Our findings suggest that TP-064 can possibly be applied as therapy in NF-κB-based inflammatory diseases.

Menstrual blood-derived stromal cells modulate functional properties of mouse and human macrophages.

Menstrual blood-derived stromal cells (MenSCs) are emerging as a strong candidate for cell-based therapies due to their immunomodulatory properties. However, their direct impact on innate immune populations remains elusive. Since macrophages play a key role in the onset and development of inflammation, understanding MenSCs implication in the functional properties of these cells is required to refine their clinical effects during the treatment of inflammatory disorders. In this study, we assessed the effects that MenSCs had on the recruitment of macrophages and other innate immune cells in two mouse models of acute inflammation, a thioglycollate (TGC)-elicited peritonitis model and a monobacterial sepsis model. We found that, in the TGC model, MenSCs injection reduced the percentage of macrophages recruited to the peritoneum and promoted the generation of peritoneal immune cell aggregates. In the sepsis model, MenSCs exacerbated infection by diminishing the recruitment of macrophages and neutrophils to the site of infection and inducing defective bacterial clearance. Additional in vitro studies confirmed that co-culture with MenSCs impaired macrophage bactericidal properties, affecting bacterial killing and the production of reactive oxygen intermediates. Our findings suggest that MenSCs modulate the macrophage population and that this modulation must be taken into consideration when it comes to future clinical applications.

Disruption of hepatocyte Sialylation drives a T cell-dependent pro-inflammatory immune tone.

Through the catalysis of α2,6-linked sialylation, the enzyme ST6Gal1 is thought to play key roles in immune cell communication and homeostasis. Of particular importance, glycans with terminal α2,6-sialic acids are known to negatively regulate B cell receptor signaling and are associated with an immunosuppressive tumor microenvironment that promotes T cell anergy, suggesting that α2,6-sialic acids are a key immune inhibitory signal. Consistent with this model, mice harboring a hepatocyte-specific ablation of ST6Gal1 (H-cKO) develop a progressive and severe non-alcoholic fatty liver disease characterized by steatohepatitis. Using this H-cKO mouse, we have further discovered that loss of hepatocyte α2,6-sialylation not only increases the inflammatory state of the local tissue microenvironment, but also systemic T cell-dependent immune responses. H-cKO mice responded normally to innate and passively induced inflammation, but showed significantly increased morbidity in T cell-dependent house dust mite-antigen (HDM)-induced asthma and myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). We further discovered that H-cKO mice have a profound shift toward effector/memory T cells even among unchallenged mice, and that macrophages from both the liver and spleen expressed the inhibitory and α2,6-sialic acid-specific glycan binding molecule CD22. These findings align with previously reported pro-inflammatory changes in liver macrophages, and support a model in which the liver microenvironment sets a systemic immune tone that is regulated by tissue α2,6-sialylation and mediated by liver macrophages and systemic T cells.

Inhibition of integrin αDß2-mediated macrophage adhesion to end product of docosahexaenoic acid (DHA) oxidation prevents macrophage accumulation during inflammation.

A critical step in the development of chronic inflammatory diseases is the accumulation of proinflammatory macrophages in the extracellular matrix (ECM) of peripheral tissues. The adhesion receptor integrin αDß2 promotes the development of atherosclerosis and diabetes by supporting macrophage retention in inflamed tissue. We recently found that the end product of docosahexaenoic acid (DHA) oxidation, 2-(ω-carboxyethyl)pyrrole (CEP), serves as a ligand for αDß2 CEP adduct with ECM is generated during inflammation-mediated lipid peroxidation. The goal of this project was to identify a specific inhibitor for αDß2-CEP interaction that can prevent macrophage accumulation. Using a specially designed peptide library, Biacore-detected protein-protein interaction, and adhesion of integrin-transfected HEK 293 cells, we identified a sequence (called P5 peptide) that significantly and specifically inhibited αD-CEP binding. In the model of thioglycollate-induced peritoneal inflammation, the injection of cyclic P5 peptide reduced 3-fold the macrophage accumulation in WT mice but had no effect in αD-deficient mice. The tracking of adoptively transferred, fluorescently labeled WT and αD-/- monocytes in the model of peritoneal inflammation and in vitro two-dimensional and three-dimensional migration assays demonstrated that P5 peptide does not affect monocyte transendothelial migration or macrophage efflux from the peritoneal cavity but regulates macrophage migration through the ECM. Moreover, the injection of P5 peptide into WT mice on a high-fat diet prevents macrophage accumulation in adipose tissue in an αDß2-dependent manner. Taken together, these results demonstrate the importance of αDß2-mediated macrophage adhesion for the accumulation of infiltrating macrophages in the inflamed ECM and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes.

KCC2 Manipulation Alters Features of Migrating Interneurons in Ferret Neocortex.

KCC2 is a brain specific chloride-potassium cotransporter affecting neuronal development including migration and cellular maturation. It modulates chloride homeostasis influencing the switch of GABA from depolarizing to hyperpolarizing, which contributes to the cues that influence the termination of neuronal migration. The expression of KCC2 during migration of interneurons, therefore, correlates with the ability of these cells to respond to GABA as a stop signal. Manipulation of KCC2 in development can affect various aspects of migrating neurons, including the speed. We describe the effect of KCC2 downregulation and inhibition on features of migrating interneurons of normal ferret kits and those treated with methylazoxymethanol acetate, which increases KCC2. Treatment of organotypic cultures with Bisphenol A, an environmental toxin that alters gene expression, also downregulates KCC2 protein. In organotypic slices treated with the KCC2 antagonist VU0240551, chloride imaging shows inhibition of KCC2 via blockade of chloride flux. Time-lapse video imaging of organotypic cultures treated with either drug, shows a significant increase in the average speed, step size, and number of turns made by migrating neurons leaving the ganglionic eminence. Our findings demonstrate the harmful effect of environmental toxins on brain development and potential consequences in the pathogenesis of neurodevelopmental disorders.

Novel function of PiT1/SLC20A1 in LPS-related inflammation and wound healing.

PiT1/SLC20A1 is an inorganic phosphate transporter with additional functions including the regulation of TNFα-induced apoptosis, erythropoiesis, cell proliferation and insulin signaling. Recent data suggest a relationship between PiT1 and NF-κB-dependent inflammation: (i) Pit1 mRNA is up-regulated in the context of NF-κB pathway activation; (ii) NF-κB target gene transcription is decreased in PiT1-deficient conditions. This led us to investigate the role of PiT1 in lipopolysaccharide (LPS)-induced inflammation. MCP-1 and IL-6 concentrations were impaired in PiT1-deficient bone marrow derived macrophages (BMDMs) upon LPS stimulation. Lower MCP-1 and IL-6 serum levels were observed in Mx1-Cre; Pit1lox/lox mice dosed intraperitoneally with LPS. Lower PiT1 expression correlated with decreased in vitro wound healing and lower reactive oxygen species levels. Reduced IκB degradation and lower p65 nuclear translocation were observed in PiT1-deficient cells stimulated with LPS. Conversely, PiT1 expression was induced in vitro upon LPS stimulation. Addition of an NF-κB inhibitor abolished LPS-induced PiT1 expression. Furthermore, we showed that p65 expression activated Pit1 promoter activity. Finally, ChIP assays demonstrated that p65 directly binds to the mPit1 promoter in response to LPS. These data demonstrate a completely novel function of PiT1 in the response to LPS and provide mechanistic insights into the regulation of PiT1 expression by NF-κB.

Environmental hazard and risk assessment of thiochemicals. Application of integrated testing and intelligent assessment strategies (ITS) to fulfil the REACH requirements for aquatic toxicity.

REACH requires information on hazardous properties of substances to be generated avoiding animal testing where possible. It is the objective of the present case study with thiochemicals to extract as much information as possible from available experimental data with fish, daphnia and algae and to fill data gaps for analogues to be registered under REACH in 2018. Based on considerations of chemical similarity and common mode of action (MOA) the data gaps regarding the aquatic toxicity of the thiochemicals were largely closed by trend analysis ("category approach") and read-across within the same group, for example, thioglycolates or mercaptopropionates. Among 16 thiochemicals to be registered by 2018 there are only 2 substances with sufficient data. 36 data gaps for 14 thiochemicals were identified. Most of the required data (>60%) could be estimated by in silico methods. Only 14 tests (6 algae, 6 daphnia, 1 limit fish test and 1 acute fish test) were proposed. When the results of these tests are available it has to be discussed whether 2 further fish (limit) tests are required. For two substances (exposure-based) waiving was suggested. The relatively high toxicity of the thiochemicals is manifested in low predicted no-effect concentrations (PNECs). Only preliminary predicted environmental concentrations (PECs) could be derived for the thiochemicals for which a risk assessment has to be performed (production rate >10 t/y). The preliminary PEC/PNEC ratios indicate no risk for the aquatic compartment at the production site. PECs due to down-stream use must not exceed the estimated PNECs.

Ammonium 2,2'-thiodiacetates - Selective and environmentally safe herbicides.

2,2'-Thiodiacetic acid derivatives have a wide application potential, mainly in coordination chemistry. This research indicates that quaternary ammonium 2,2'-thiodiacetate salts may also be potent herbicidal agents used in agriculture. To provide a rationale for this statement, the toxic effect by a alkyl and aryl quaternary ammonium salts (QASs) on plant growth was investigated. The phytotoxicity of these compounds was tested against cultivated monocotyledonous (spring barley) and dicotyledonous (common radish) plants, whereas herbicidal activity was investigated in relation to popular weeds species (white goosefoot, sorrel and gallant-soldier). The results showed that aliphatic QASs possessed a low phytotoxicity to food crops and that some of them (in particular triethylammonium salt) had potent and selective herbicidal properties against common weeds, such as sorrel and gallant-soldier. However, the investigated compounds appeared to be ineffective herbicides against white goosefoot.

C-terminal truncation of IFN-γ inhibits proinflammatory macrophage responses and is deficient in autoimmune disease.

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages. In acute peritonitis this signature was absent in Mmp12 -/- mice and recapitulated in Mmp12 +/+ mice treated with a MMP12-specific inhibitor. Similarly, loss-of-MMP12 increases IFN-γ-dependent proinflammatory markers and iNOS+/MHC class II+ macrophage accumulation with worse lymphadenopathy, arthritic synovitis and lupus glomerulonephritis. In active human systemic lupus erythematosus, MMP12 levels were lower and IFN-γ higher compared to treated patients or healthy individuals. Hence, macrophage proteolytic truncation of IFN-γ attenuates classical activation of macrophages as a prelude for resolving inflammation.

Selecting the right gate to identify relevant cells for your assay: a study of thioglycollate-elicited peritoneal exudate cells in mice.

OBJECTIVE: In this study, we investigate the diversity and modulation of leukocyte populations represented in the gates defined by size and granularity at different time points of thioglycollate-induced peritonitis in mouse. RESULTS: The inflammatory cells were distributed into four regions (R1-R4) of a data plot graph defined by cell size and granularity. R1 and R2 contained agranular cells that were small in size and predominately included T (CD3+) lymphocytes along with B (B220+) lymphocytes. Macrophages (F4/80+) were the predominant cells found in the R3 region. However, these cells were present in all regions, albeit at a lower frequency in R1 and R2. Granulocytes (Gr1+) were mainly distributed in R3 and R4. The wide distribution of F4/80+ and Gr1+ cells may reflect the recruitment and activation state of the different macrophage and granulocyte populations. Based on these observations, size and granularity may contribute to an initial step in the analysis and sorting of thioglycollate-elicited peritoneal exudate cells. However, the developmental stage and cell activation state may interfere with cell segregation using size and granularity as parameters.

Thioglycolate-based task-specific ionic liquids: Metal extraction abilities vs acute algal toxicity.

We studied the extraction behavior of two thioglycolate-based ionic liquids (ILs), for heavy metals from aqueous solutions; substances of interest were methyltrioctylammonium S-hexyl thioglycolate [N1888][C6SAc] and methyltrioctylphosphonium S-hexyl thioglycolate [P1888][C6SAc]. Theses ILs previously have shown very good extraction abilities towards cadmium and copper, therefore we investigated time-dependent metal removal experiments with aqueous solutions of cobalt(II), nickel(II) and zinc(II). The highest distribution ratio (RIL/Water) was determined for zinc (RIL/Water=2000). Recovery studies for zinc after extraction were performed with different stripping agents showing a successful recycling. Additionally, the two ILs were immobilized on active charcoal, displaying great potential for solid-liquid extraction. Regarding the extraction mechanism, quantum-mechanical calculations were included, which indicate that the metal extraction depends on the stability of the metal-water cluster. Ligands (water as well as ILs) are planar coordinated in nickel complexes but showed a tetrahedral configuration for zinc. As a first estimate of the ecotoxicity of the ILs, in vivo tests toward three freshwater green algae species Tetradesmus obliquus, Desmodesmus armatus and Raphidocelis subcapitata were carried out. The EC50 values (effective concentration after 72 h) confirm high toxicity of all tested ILs to all species, displaying only small differences between the species and EC50ies.

Anti-inflammatory effects of essential oils extracted from Chamaecyparis obtusa on murine models of inflammation and RAW 264.7 cells.

Antimicrobial, antifungal and anti-inflammatory effects of essential oils extracted from Chamaecyparis obtusa (EOCO) have previously been reported. In the present study, the anti-inflammatory effects of EOCO were investigated in two murine models of inflammation: Carrageenan-induced paw edema and thioglycollate-induced peritonitis, and in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The expression levels of proinflammatory cytokines were analyzed by ELISA, the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were determined by western blotting, and nitrite concentration was measured using Griess reagent. In mice with carrageenan-induced edema, paw thickness and the expression levels of interleukin (IL)­1ß and IL-6 in paw homogenates were significantly decreased in the EOCO (5 and 10 mg/kg) group, as compared with the control group. In mice with thioglycollate-induced peritonitis, treatment with EOCO (5 and 10 mg/kg) reduced the number of total cells and suppressed tumor necrosis factor­α (TNF­α), IL­1ß and IL­6 levels in peritoneal fluid. In addition, EOCO reduced nitric oxide, TNF­α and IL­6 production, and suppressed iNOS and COX­2 expression in LPS­stimulated RAW 264.7 cells. These results suggest that EOCO may exert anti­inflammatory effects in vivo and in vitro, and that these effects may be associated with the inhibition of inflammatory mediators. Therefore, EOCO may be considered an effective therapeutic agent for the treatment of inflammatory diseases.

Molecular and cellular profiles of the resolution phase in a damage-associated molecular pattern (DAMP)-mediated peritonitis model and revelation of leukocyte persistence in peritoneal tissues.

Models of microbe-elicited peritonitis have been invaluable to identify mechanisms underlying inflammation resolution, but whether resolution mechanisms differ from an inflammatory agent to another has not been determined. Thus, we analyzed the cellular and molecular components of the resolution phase of non-microbe-induced inflammation. In thioglycollate (TG)-induced peritonitis, resolution started at 12 h (Tmax) and displayed a 22 h resolution interval (Ri). During resolution, lipoxin A4, resolvin (Rv) D1 and RvD2, protectin D1 (PD1), and maresin 1 (MaR1) were transiently produced while RvD5 was continually generated. In addition, docosahexaenoic acid (DHA)-derived mediators were produced to a higher extent than in microbial peritonitis. We also investigated leukocyte infiltration and clearance in peritoneal tissues surrounding the inflammatory site. In the omentum, resolution parameters, neutrophil apoptosis, and efferocytosis were similar to those of the peritoneal cavity. However, we noticed long-term persistence of M2-polarized macrophages and B-lymphocytes in the omentum after TG administration, whereas zymosan injection caused M1/M2-macrophage and T-lymphocyte persistence regardless of the magnitude of the inflammatory response. Our study indicates that some aspects of resolution are shaped in a stimulus-specific manner, and it ultimately argues that the tissues surrounding the inflammatory site must also be considered to address the inflammatory response globally.

Resolution of sterile inflammation: role for vitamin C.

INTRODUCTION: Macrophage reprogramming is vital for resolution of acute inflammation. Parenteral vitamin C (VitC) attenuates proinflammatory states in murine and human sepsis. However information about the mechanism by which VitC regulates resolution of inflammation is limited. METHODS: To examine whether physiological levels of VitC modulate resolution of inflammation, we used transgenic mice lacking L-gulono-γ-lactone oxidase. VitC sufficient/deficient mice were subjected to a thioglycollate-elicited peritonitis model of sterile inflammation. Some VitC deficient mice received daily parenteral VitC (200 mg/kg) for 3 or 5 days following thioglycollate infusion. Peritoneal macrophages harvested on day 3 or day 5 were examined for intracellular VitC levels, pro- and anti-inflammatory protein and lipid mediators, mitochondrial function, and response to lipopolysaccharide (LPS). The THP-1 cell line was used to determine the modulatory activities of VitC in activated human macrophages. RESULTS: VitC deficiency significantly delayed resolution of inflammation and generated an exaggerated proinflammatory response to in vitro LPS stimulation. VitC sufficiency and in vivo VitC supplementation restored macrophage phenotype and function in VitC deficient mice. VitC loading of THP-1 macrophages attenuated LPS-induced proinflammatory responses. CONCLUSION: VitC sufficiency favorably modulates macrophage function. In vivo or in vitro VitC supplementation restores macrophage phenotype and function leading to timely resolution of inflammation.

Physical and biophysical assessment of highly fluorescent, magnetic quantum dots of a wurtzite-phase manganese selenide system.

Combining fluorescence and magnetic features in a non-iron based, select type of quantum dots (QDs) can have immense value in cellular imaging, tagging and other nano-bio interface applications, including targeted drug delivery. Herein, we report on the colloidal synthesis and physical and biophysical assessment of wurtzite-type manganese selenide (MnSe) QDs in cell culture media. Aiming to provide a suitable colloidal system of biological relevance, different concentrations of reactants and ligands (e.g., thioglycolic acid, TGA) have been considered. The average size of the QDs is ∼7 nm, which exhibited a quantum yield of ∼75% as compared to rhodamine 6 G dye(®). As revealed from time-resolved photoluminescence (TR-PL) response, the near band edge emission followed a bi-exponential decay feature with characteristic times of ∼0.64 ns and 3.04 ns. At room temperature, the QDs were found to exhibit paramagnetic features with coercivity and remanence impelled by TGA concentrations. With BSA as a dispersing agent, the QDs showed an improved optical stability in Dulbecco's Modified Eagle Media(®) (DMEM) and Minimum Essential Media(®) (MEM), as compared to the Roswell Park Memorial Institute(®) (RPMI-1640) media. Finally, the cell viability of lymphocytes was found to be strongly influenced by the concentration of MnSe QDs, and had a safe limit upto 0.5 µM. With BSA inclusion in cell media, the cellular uptake of MnSe QDs was observed to be more prominent, as revealed from fluorescence imaging. The fabrication of water soluble, nontoxic MnSe QDs would open up an alternative strategy in nanobiotechnology, while preserving their luminescent and magnetic properties intact.

Inflammatory mediator profiling reveals immune properties of chemotactic gradients and macrophage mediator production inhibition during thioglycollate elicited peritoneal inflammation.

Understanding of spatiotemporal profiling of inflammatory mediators and their associations with MΦ accumulation is crucial to elucidate the complex immune properties. Here, we used murine thioglycollate elicited peritonitis to determine concentrations of 23 inflammatory mediators in peritoneal exudates and plasma before (day 0) and after (days 1 and 3) thioglycollate administration to peritoneal cavities; these mediators included TNF-α , FGF-9, IFN-γ , IP-10, RANTES, IL-1α , IL-6, IL-7, IL-10, IL-11, IL-12p70, IL-17A, lymphotactin, OSM, KC/GRO, SCF, MIP-1ß , MIP-2, TIMP-1, VEGF-A, MCP-1, MCP-3, and MCP-5. Our results showed that concentrations of most mediators in exudates and plasma reached peak levels on day 1 and were significantly reduced on day 3. Conversely, MΦ numbers started to increase on day 1 and reached peak levels on day 3. Moreover, LPS treatment in vitro significantly induced mediator productions in cell culture media and lysates from MΦ isolated on day 3. Our results also showed that on day 0, concentrations of many mediators in plasma were higher than those in exudates, whereas on day 1, the trend was reversed. Overall, the findings from thioglycollate elicited peritonitis reveal that reversible chemotactic gradients between peritoneal exudates and blood exist in basal and inflamed conditions and the inflammatory mediator production in vivo is disassociated with macrophage accumulation during inflammation resolution.

Pannexin-1 influences peritoneal cavity cell population but is not involved in NLRP3 inflammasome activation.

Pannexin-1 (Panx1) forms nonselective large channel in cell plasma membrane and has been shown to be associated with NLRP3 inflammasome activation, ATP release and phagocytes recruitment. In the current study, by manipulation of Panx1 expression in human myeloid cells and application of Panx1 deficient mice, we failed to find a correlation between Panx1 and NLRP3 inflammasome activation, although an interaction between these two proteins was evident. However, in thioglycollate induced peritonitis, Panx1 deficient mice showed much more phagocytes infiltration. Further analyses showed that mice deficient for Panx1 exhibited enlarged F4/80(low)Gr1(-)Ly6C(-)cell population in the peritonea. Our study thus reveals an important role for Panx1 in regulation of peritoneal cell population and peritonitis development.

Macrophage ADAM17 deficiency augments CD36-dependent apoptotic cell uptake and the linked anti-inflammatory phenotype.

RATIONALE: Apoptotic cell phagocytosis (efferocytosis) is mediated by specific receptors and is essential for resolution of inflammation. In chronic inflammation, apoptotic cell clearance is dysfunctional and soluble levels of several apoptotic cell receptors are elevated. Reports have identified proteolytic cleavage as a mechanism capable of releasing soluble apoptotic cell receptors, but the functional implications of their proteolysis are unclear. OBJECTIVE: To test the hypothesis that ADAM17-mediated cleavage of apoptotic cell receptors limits efferocytosis in vivo. METHODS AND RESULTS: In vivo comparison of macrophage efferocytosis in wild-type and Adam17-null hematopoietic chimeras demonstrates that ADAM17 deficiency leads to a 60% increase in efferocytosis and an enhanced anti-inflammatory phenotype in a model of peritonitis. In vitro uptake of phosphatidylserine liposomes identifies the dual-pass apoptotic cell receptor CD36 as a major contributor to enhanced efferocytosis, and CD36 surface levels are elevated on macrophages from Adam17-null mice. Further, temporal elevation of CD36 expression with inflammation may also contribute to its impact. Soluble CD36 from macrophage-conditioned media comprises 2 species based on Western blotting, and mass spectrometry identifies 3 N-terminal peptides that represent probable cleavage sites. Levels of soluble CD36 are decreased in Adam17-null conditioned media, providing evidence for involvement of ADAM17 in CD36 cleavage. Importantly, enhanced efferocytosis in vivo by macrophages lacking ADAM17 is CD36 dependent and accelerates macrophage clearance from the peritoneum, thus promoting resolution of inflammation and highlighting the impact of increased apoptotic cell uptake. CONCLUSIONS: Our studies demonstrate the importance of ADAM17-mediated proteolysis for in vivo efferocytosis regulation and suggest a possible mechanistic link between chronic inflammation and defective efferocytosis.

The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots.

Quantum dots (QDs) have many potential clinical and biological applications because of their advantages over traditional fluorescent dyes. However, the genotoxicity potential of QDs still remains unclear. In this paper, a plasmid-based system was designed to explore the genotoxic mechanism of QDs by detecting changes in DNA configuration and biological activities. The direct chemicobiological interactions between DNA and mercaptoacetic acid-coated CdSecore QDs (MAA-QDs) were investigated. After incubation with different concentrations of MAA-QDs (0.043, 0.13, 0.4, 1.2, and 3.6 µmol/L) in the dark, the DNA conversion of the covalently closed circular (CCC) DNA to the open circular (OC) DNA was significantly enhanced (from 13.9% ± 2.2% to 59.9% ± 12.8%) while the residual transformation activity of plasmid DNA was greatly decreased (from 80.7% ± 12.8% to 13.6% ± 0.8%), which indicated that the damages to the DNA structure and biological activities induced by MAA-QDs were concentration-dependent. The electrospray ionization mass spectrometry data suggested that the observed genotoxicity might be correlated with the cadmium-mercaptoacetic acid complex (Cd-MAA) that is formed in the solution of MAA-QDs. Circular dichroism spectroscopy and transformation assay results indicated that the Cd-MAA complex might interact with DNA through the groove-binding mode and prefer binding to DNA fragments with high adenine and thymine content. Furthermore, the plasmid transformation assay could be used as an effective method to evaluate the genotoxicities of nanoparticles.

Defective macrophage migration in Gαi2- but not Gαi3-deficient mice.

Various heterotrimeric G(i) proteins are considered to be involved in cell migration and effector function of immune cells. The underlying mechanisms, how they control the activation of myeloid effector cells, are not well understood. To elucidate isoform-redundant and -specific roles for Gα(i) proteins in these processes, we analyzed mice genetically deficient in Gα(i2) or Gα(i3). First, we show an altered distribution of tissue macrophages and blood monocytes in the absence of Gα(i2) but not Gα(i3). Gα(i2)-deficient but not wild-type or Gα(i3)-deficient mice exhibited reduced recruitment of macrophages in experimental models of thioglycollate-induced peritonitis and LPS-triggered lung injury. In contrast, genetic ablation of Gα(i2) had no effect on Gα(i)-dependent peritoneal cytokine production in vitro and the phagocytosis-promoting function of the Gα(i)-coupled C5a anaphylatoxin receptor by liver macrophages in vivo. Interestingly, actin rearrangement and CCL2- and C5a anaphylatoxin receptor-induced chemotaxis but not macrophage CCR2 and C5a anaphylatoxin receptor expression were reduced in the specific absence of Gα(i2). Furthermore, knockdown of Gα(i2) caused decreased cell migration and motility of RAW 264.7 cells, which was rescued by transfection of Gα(i2) but not Gα(i3). These results indicate that Gα(i2), albeit redundant to Gα(i3) in some macrophage activation processes, clearly exhibits a Gα(i) isoform-specific role in the regulation of macrophage migration.