Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and anti-inflammatory agents: key role for tnf-α

Development of chronic inflammation or autoimmunity may be related to deregulated mechanisms orchestrating successful resolution of inflammation, especially apoptosis of inflammatory cells and their subsequent clearance by macrophages (Mφ). Chronically inflamed sites are characterised by an excess of the key pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and importantly, TNF-α inhibitors, widely used in the clinical setting for the treatment of rheumatoid arthritis (RA), inflammatory bowel disease and psoriasis, significantly delay disease progression. TNF-α therefore may affect processes implicated in resolution of inflammation. Although TNF-α and pro-inflammatory bacterial products such as lipopolysaccharide (LPS) influence rates of inflammatory cell apoptosis, little is known about their effects on Mφ phagocytosis of apoptotic cells (efferocytosis). In this PhD thesis, the effects of several pro-inflammatory agents (i.e., LPS, lipoteichoic acid (LTA), peptidoglycan (PGN) and TNF-α) on efferocytosis by human blood monocytederived Mφ (MDMφ) have been investigated. LPS, LTA and PGN all inhibited MDMφ efferocytosis in a concentration- and time-dependent manner; however, LPS did not inhibit the uptake of immunoglobulin-G (IgG)-opsonized erythrocytes. Moreover, although TNF-α did inhibit efferocytosis, phagocytosis of IgG-opsonized erythrocytes was not inhibited. Furthermore, the LPS effect was attenuated by dimeric soluble human recombinant TNF receptor-1 (sTNFR1/ Fc), indicating a critical role of TNF-α. Concomitant treatments with monomeric soluble human recombinant TNF receptor-1 (sTNF-R1) or the TNF-α Converting Enzyme (TACE) inhibitor, TOPI-0, only partially reversed the inhibitory effect of LPS. Even though TNF-α release takes place within the first few hours following LPS stimulation, the LPS-induced inhibitory effect occurred only if treatment was performed for 96 hours or longer. Analysis of supernatants obtained from LPS-treated MDMφ revealed that there appears to be interplay between concentrations of TNF-α and interleukin-10 (IL-10) and that these cytokines exert opposing actions on efferocytosis. IL-10 per se increased MDMφ efferocytosis and addition of exogenous IL-10 to LPS-treated samples rescued phagocytosis. The latter effect was associated with the IL-10-induced, concentration-dependent inhibition of TNF-α release. Interestingly, when IL-10 was added to TNF-α-treated MDMφ, only slight augmentation of phagocytosis was observed. Furthermore, when IL-10-mediated effects were blocked by concomitant treatment with anti-human IL-10 receptor 1 antibody (anti-IL-10- R1Ab), the LPS inhibitory effect on phagocytosis was much greater and occurred at 24 hours after treatment. The role of IL-10 on efferocytosis was also investigated using IL-10 deficient murine bone marrow-derived Mφ (BMDMφ). IL-10 deficient BMDMφ, when compared to wild-type, were characterised by a much lower ability to phagocytose apoptotic neutrophils and this effect was independent of culture conditions (control samples and LPS or TNF-α treatments). Finally, effects of the synthetic steroid (dexamethasone) and nonsteroidal anti-inflammatory drugs (NSAID) on MDMφ phagocytosis were examined. Dexamethasone, like IL-10, augmented MDMφ efferocytosis, reversed the inhibitory effects of both LPS and TNF-α, and suppressed LPS-induced production of TNF-α. In contrast NSAID did not increase MDMφ efferocytosis per se. However, preliminary data suggest that aspirin blocks the inhibitory effect of TNF-α on phagocytosis. In summary, it has been determined that prolonged treatment with proinflammatory agents such as LPS, LTA and PGN inhibits MDMφ efferocytosis which may potentially postpone the resolution of inflammation in vivo. I have shown that TNF-α is a key mediator in this process and that IL-10 exerts an important regulatory effect on TNF-α production and consequently on efferocytosis. Furthermore, several approaches have been unveiled to successfully reverse LPS-mediated inhibition of efferocytosis by decreasing either TNF-α production or its inhibitory effect with sTNF-RI/Fc, exogenous IL- 10 or dexamethasone. These findings indicate that TNF-α and other agents which influence efferocytosis may have significance in the resolution phase of inflammation. In addition, presented findings provide important mechanistic information into the potential mode of action of anti-TNF-α agents and steroids and may help to explain their clinical success in the treatment of chronic inflammatory diseases

Seasonal leaf dimorphism in Potentilla argentea L. var. tenuiloba (Jord.) Sw. (Rosaceae)

A pattern of seasonal changes in the morphological and anatomical leaf traits is reported for Potentilla argentea L. var. tenuiloba (Jord.) Sw. of temperate-climate areas in central Poland. Leaf area, perimeter, dry mass and lamina thickness were measured in summer and autumn leaves of the same individuals. Dissection index, density and specific leaf area were calculated. Significant differences were obtained between summer and autumn leaves obtained from the same individuals. The shapes of leaves of the P. argentea plants varied in the extent of incisions between teeth and the number of teeth on the margins. Fully expanded autumn leaves were larger in weight and area than summer leaves. The autumn leaves had lower leaf mass area and density than the summer leaves. Leaves were covered by considerably more trichomes in summer than in autumn. Anatomical leaf structure also changed with the season. The summer leaves were thick, with a lower number of chloroplasts in the cells of the compact mesophyll. Autumn leaves are thinner, with loose mesophyll. Chloroplasts from the two seasonal types of leaves differ on account of starch grain and plastoglobule content. The large variations in leaf density and thickness recorded here confirm great differences in cell size and amounts of structural tissue within species. Seasonal dimorphism of leaves may result from seasonal drought or from seasonality in leaf production, leaf fall or incoming solar radiation. Within this new context of seasonal leaf dimorphism, P. argentea can still be distinguished by the absence of deeply divided leaflets on late-formed leaves. The results confirmed the presence of several morpho-anatomical leaf traits of P. argentea that allow the species to adapt to environmental seasonal conditions

The effect of EDTA and EDDS on lead uptake and localization in hydroponically grown Pisum sativum L.

Pisum sativum plants were treated for 3 days with an aqueous solution of 100 lM Pb(NO3)2 or with a mixture of lead nitrate and ethylenediaminetetraacetic acid (EDTA) or [S,S]-ethylenediaminedisuccinic acid (EDDS) at equimolar concentrations. Lead decline from the incubation media and its accumulation and localization at the morphological and ultrastructural levels as well as plant growth parameters (root growth, root and shoot dry weight) were estimated after 1 and 3 days of treatment. The tested chelators, especially EDTA, significantly diminished Pb uptake by plants as compared to the lead nitrate-treated material. Simultaneously, EDTA significantly enhanced Pb translocation from roots to shoots. In the presence of both chelates, plant growth parameters remained considerably higher than in the case of uncomplexed Pb. Considerable differences between the tested chelators were visible in Pb localization both at the morphological and ultrastructural level. In Pb?EDTA-treated roots, lead was mainly located in the apical parts, while in Pb?EDDS-exposed material Pb was evenly distributed along the whole root length. Transmission electron microscopy and EDS analysis revealed that in meristematic cells of the roots incubated in Pb?EDTA, large electron-dense lead deposits were located in vacuoles and small granules were rarely noticed in cell walls or cytoplasm, while after Pb?EDDS treatment metal deposits were restricted to the border between plasmalemma and cell wall. Such results imply different ways of transport of those complexed Pb forms.The X-ray microanalysis was performed in the Laboratory of Electron Microscopy, Nencki Institute of Experimental Biology, Warsaw, Poland at the equipment installed within the project sponsored by the EU Structural Funds: Centre of Advanced Technology BIM—Equipment purchase for the Laboratory of Biological and Medical Imaging. Conflict of interest T

CBP/p300 Bromodomain Inhibitor–I–CBP112 Declines Transcription of the Key ABC Transporters and Sensitizes Cancer Cells to Chemotherapy Drugs

The high expression of some ATP-binding cassette (ABC) transporters is linked to multidrug resistance in cancer cells. We aimed to determine if I-CBP112, which is a CBP/p300 bromodomain inhibitor, altered the vulnerability of the MDA-MB-231 cell line to chemotherapy drugs, which are used in neoadjuvant therapy in patients with triple negative breast cancer (TNBC). MDA-MB-231 cells represent TNBC, which is negative for the expression of estrogen and progesterone receptors and HER2 protein. An I-CBP112-induced decrease in the expression of all the studied ABCs in the breast, but also in the lung (A549), and hepatic (HepG2) cancer cell lines was associated with increased accumulation of doxorubicin, daunorubicin, and methotrexate inside the cells as well as with considerable cell sensitization to a wide range of chemotherapeutics. Gene promoters repressed by I-CBP112 in MDA-MB-231 cells, such as ABCC1 and ABCC10, were characterized by enhanced nucleosome acetylation and, simultaneously, by considerably lower trimethylation in the transcription-promoting form of H3K4me3. The CBP/p300 bromodomain inhibitor induced the recruitment of LSD1 to the gene promoters. The inhibition of this demethylase in the presence of I-CBP112 prevented the repression of ABCC1 and ABCC10 and, to a considerable extent, cancer cells’ sensitization to drugs. In conclusion, the CBP/p300 bromodomain inhibitor I-CBP112 can be considered as a potent anti-multidrug-resistance agent, capable of repressing key ABC transporters responsible for drug efflux in various cancer types.This research was funded by National Centre for Research and Development, grant number LIDER/22/0122/L-10/18/NCBR/2019

Characterisation of the molecular mechanism required for glucocorticoid augmentation of macrophage phagocytosis of apoptotic neutrophils

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies

Ruthenium metallodendrimer against triple-negative breast cancer in mice

Carbosilane metallodendrimers, based on the arene Ru(II) complex (CRD13) and integrated to imino-pyridine surface groups have been investigated as an anticancer agent in a mouse model with triple-negative breast cancer. The dendrimer entered into the cells efficiently, and exhibited selective toxicity for 4T1 cells. In vivo investigations proved that a local injection of CRD13 caused a reduction of tumour mass and was non-toxic. ICP analyses indicated that Ru(II) accumulated in all tested tissues with a greater content detected in the tumour.European CommissionMinisterio de Economía y CompetitividadComunidad de MadridJunta de Comunidades de Castilla-La Manch

Heterofunctionalized polyphenolic dendrimers decorated with caffeic acid: Synthesis, characterization and antioxidant activity

Dendrimers, branched polymer structures, have been widely studied as efficient drug carriers. Scientists are trying to find new dendrimer-based formulations with the properties needed for biomedical applications such as improved bioavailability, low toxicity and high transfection profiles. The unique drug delivery properties of carbosilane dendrimers have already been demonstrated. Their efficacy has been further improved by conju-gation with polyphenols, plant secondary metabolites with a wide range of biological activities, including antioxidant effects that are beneficial for human health. The present study focuses on synthesis and character-ization of two new types of carbosilane dendritic systems, one family presents one or two caffeic acid units and ammonium groups on the surface to make them water soluble. The other family has, in addition to the two mentioned functionalities, one or two polyethylene glycol (PEG) chains in the structure to increase the biocompatibility of the system. Carbosilane dendrimers with caffeic acid have low toxicity and protect eryth-rocytes against oxidative hemolysis. These dendrimers also decrease AAPH-induced ROS production in human fibroblasts.Various techniques demonstrating such antioxidant activities have been applied in the current research. The best antioxidant properties were shown for the dendrimer with two PEG-caffeic acid moieties. Further aspects of the biochemical characterization of the dendrimers are also considered and discussed.Ministerio de Ciencia e InnovaciónMinisterio de Economía y CompetitividadJunta de Comunidades de Castilla-La ManchaComunidad de MadridPolish National Agency for Academic Exchang

Copper (II) Metallodendrimers Combined with Pro-Apoptotic siRNAs as a Promising Strategy Against Breast Cancer Cells

Cancer treatment with small interfering RNA (siRNA) is one of the most promising new strategies; however, transfection systems that increase its bioavailability and ensure its delivery to the target cell are necessary. Transfection systems may be just vehicular or could contain fragments with anticancer activity that achieves a synergistic effect with siRNA. Cationic carbosilane dendrimers have proved to be powerful tools as non-viral vectors for siRNA in cancer treatment, and their activity might be potentiated by the inclusion of metallic complexes in its dendritic structure. We have herein explored the interaction between Schiff-base carbosilane copper (II) metallodendrimers, and pro-apoptotic siRNAs. The nanocomplexes formed by metallodendrimers and different siRNA have been examined for their zeta potential and size, and by transmission electron microscopy, fluorescence polarisation, circular dichroism, and electrophoresis. The internalisation of dendriplexes has been estimated by flow cytometry and confocal microscopy in a human breast cancer cell line (MCF-7), following the ability of these metallodendrimers to deliver the siRNA into the cell. Finally, in vitro cell viability experiments have indicated effective interactions between Cu (II) dendrimers and pro-apoptotic siRNAs: Mcl-1 and Bcl-2 in breast cancer cells. Combination of the first-generation derivatives with chloride counterions and with siRNA increases the anticancer activity of the dendriplex constructs and makes them a promising non-viral vector.Polish National Agency for Academic Exchange (NAWA)European CommissionMinisterio de Economía y CompetitividadComunidad de MadridJunta de Comunidades de Castilla-La Manch

In Vitro anticancer properties of copper metallodendrimers

Newly synthesized carbosilane copper dendrimers (CCD) with chloride and nitrate surface groups seem to be good candidates to be used as gene and drug carriers in anti-cancer therapy, due to their properties such as size and surface charge. Copper attached to the nanoparticles is an important element of many biological processes and recently their anti-cancer properties have been widely examined. Zeta size and potential, transmission electron microscopy (TEM), circular dichroism (CD), analysis of haemolytic activity, and fluorescence anisotropy techniques were used to characterize copper dendrimers. Additionally, their cytotoxic properties toward normal (PBMC) and cancer (1301; HL-60) cells were examined. All tested dendrimers were more cytotoxic against cancer cells in comparison with normal cells

Evaluation of dendronized gold nanoparticles as siRNAs carriers into cancer cells

Gene therapy is one of the most promising approaches for potential application in the treatment of diseases, ranging from cancer and heritable disorders to infectious diseases. Before nucleic acid molecules can reach their site of action inside target cells, they must overcome several obstacles. Thus, to fully exploit the therapeutic potential of nucleic acids, efficient delivery systems are required. We herein evaluated gold nanoparticles (AuNPs) covered with cationic carbosilane dendrons as siRNA delivery systems. Detailed analysis of formation of AuNP:siRNA complexes using circular dichroism, zeta-potential, zeta-size, electron microscopy and gel electrophoresis was performed. The stability of complexes in presence of heparin and RNase was evaluated. Internalization of AuNPs and their complexes with siRNAs into cancer cells was estimated by ultrastructure analysis and confocal microscopy. The cytotoxicity of dendrons, AuNPs and their complexes with siRNAs on 4 cancer cell lines (Caco-2, HeLa, U937 and THP-1) was estimated. We concluded that dendronization of AuNPs is a promising way to develop siRNA carriers for anticancer gene therapyUniversidad de AlcaláMinisterio de Economía y CompetitividadComunidad de MadridJunta de Comunidades de Castilla-La ManchaEuropean Commissio