Eik Hoffmann

Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells

Titanium dioxide (TiO2), also known as titanium (IV) oxide or anatase, is the naturally occurring oxide of titanium. It is also one of the most commercially used form. To date, no parameter has been set for the average ambient air concentration of TiO2 nanoparticles (NP) by any regulatory agency. Previously conducted studies had established these nanoparticles to be mainly non-cyto- and -genotoxic, although they had been found to generate free radicals both acellularly (specially through photocatalytic activity) and intracellularly. The present study determines the role of TiO2-NP (anatase, ∅ < 100 nm) using several parameters such as cyto- and genotoxicity, DNA-adduct formation and generation of free radicals following its uptake by human lung cells in vitro. For comparison, iron containing nanoparticles (hematite, Fe2O3, ∅ < 100 nm) were used. The results of this study showed that both types of NP were located in the cytosol near the nucleus. No particles were found inside the nucleus, in mitochondria or ribosomes. Human lung fibroblasts (IMR-90) were more sensitive regarding cyto- and genotoxic effects caused by the NP than human bronchial epithelial cells (BEAS-2B). In contrast to hematite NP, TiO2-NP did not induce DNA-breakage measured by the Comet-assay in both cell types. Generation of reactive oxygen species (ROS) was measured acellularly (without any photocatalytic activity) as well as intracellularly for both types of particles, however, the iron-containing NP needed special reducing conditions before pronounced radical generation. A high level of DNA adduct formation (8-OHdG) was observed in IMR-90 cells exposed to TiO2-NP, but not in cells exposed to hematite NP. Our study demonstrates different modes of action for TiO2- and Fe2O3-NP. Whereas TiO2-NP were able to generate elevated amounts of free radicals, which induced indirect genotoxicity mainly by DNA-adduct formation, Fe2O3-NP were clastogenic (induction of DNA-breakage) and required reducing conditions for radical formation.

Yolk Sac Macrophages, Fetal Liver, and Adult Monocytes Can Colonize an Empty Niche and Develop into Functional Tissue-Resident Macrophages

Tissue-resident macrophages can derive from yolk sac macrophages (YS-Macs), fetal liver monocytes (FL-MOs), or adult bone-marrow monocytes (BM-MOs). The relative capacity of these precursors to colonize a niche, self-maintain, and perform tissue-specific functions is unknown. We simultaneously transferred traceable YS-Macs, FL-MOs, and BM-MOs into the empty alveolar macrophage (AM) niche of neonatal Csf2rb(-/-) mice. All subsets produced AMs, but in competition preferential outgrowth of FL-MOs was observed, correlating with their superior granulocyte macrophage-colony stimulating factor (GM-CSF) reactivity and proliferation capacity. When transferred separately, however, all precursors efficiently colonized the alveolar niche and generated AMs that were transcriptionally almost identical, self-maintained, and durably prevented alveolar proteinosis. Mature liver, peritoneal, or colon macrophages could not efficiently colonize the empty AM niche, whereas mature AMs could. Thus, precursor origin does not affect the development of functional self-maintaining tissue-resident macrophages and the plasticity of the mononuclear phagocyte system is largest at the precursor stage.

The unfolded-protein-response sensor IRE-1α regulates the function of CD8α + dendritic cells

The role of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in homeostasis of the immune system is incompletely understood. Here we found that dendritic cells (DCs) constitutively activated the UPR sensor IRE-1α and its target, the transcription factor XBP-1, in the absence of ER stress. Loss of XBP-1 in CD11c+ cells led to defects in phenotype, ER homeostasis and antigen presentation by CD8α+ conventional DCs, yet the closely related CD11b+ DCs were unaffected. Whereas the dysregulated ER in XBP-1-deficient DCs resulted from loss of XBP-1 transcriptional activity, the phenotypic and functional defects resulted from regulated IRE-1α-dependent degradation (RIDD) of mRNAs, including those encoding CD18 integrins and components of the major histocompatibility complex (MHC) class I machinery. Thus, a precisely regulated feedback circuit involving IRE-1α and XBP-1 controls the homeostasis of CD8α+ conventional DCs.

Integrated network reconstruction, visualization and analysis using YANAsquare

BackgroundModeling of metabolic networks includes tasks such as network assembly, network overview, calculation of metabolic fluxes and testing the robustness of the network.ResultsYANAsquare provides a software framework for rapid network assembly (flexible pathway browser with local or remote operation mode), network overview (visualization routine and YANAsquare editor) and network performance analysis (calculation of flux modes as well as target and robustness tests). YANAsquare comes as an easy-to-setup program package in Java. It is fully compatible and integrates the programs YANA (translation of gene expression values into flux distributions, metabolite network dissection) and Metatool (elementary mode calculation). As application examples we set-up and model the phospholipid network in the phagosome and genome-scale metabolic maps of S.aureus, S.epidermidis and S.saprophyticus as well as test their robustness against enzyme impairment.ConclusionYANAsquare is an application software for rapid setup, visualization and analysis of small, larger and genome-scale metabolic networks.

Reactive Oxygen Species Production in the Phagosome: Impact on Antigen Presentation in Dendritic Cells

SIGNIFICANCE The NADPH oxidase 2 (NOX2) is known to play a major role in innate immunity for several decades. Phagocytic cells provide host defense by ingesting microbes and destroy them by different mechanisms, including the generation of reactive oxygen species (ROS) by NOX2, a process known as oxidative burst. The phagocytic pathway of dendritic cells (DCs), highly adapted to antigen processing, has been shown to display remarkable differences compared to other phagocytes. Contrary to macrophages and neutrophils, the main function of DC phagosomes is antigen presentation rather than pathogen killing or clearance of cell debris. RECENT ADVANCES In the last few years, it became clear that NOX2 is also involved in the establishment of adaptive immunity. Several studies support the idea of a relationship between antigen presentation and the level of antigen degradation, the latter one being regulated by the pH and ROS within phagosomes. CRITICAL ISSUES The regulation of phagosomal pH exerted by NOX2, and thereby of the efficacy of antigen cross-presentation in DCs, represents a clear illustration of how NOX2 can influence CD8(+) T lymphocyte responses. In this review, we want to put emphasis on the relationship between ROS generation and antigen processing and presentation, since there is growing evidence that the low levels of ROS generated by DCs play an important role in these processes. FUTURE DIRECTIONS In the next years, it will be interesting to unravel possible mechanisms involved and to find other possible connections between NOX family members and adaptive immune responses.

Toll-like Receptor 4 Engagement on Dendritic Cells Restrains Phago-Lysosome Fusion and Promotes Cross-Presentation of Antigens.

The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens.

Ezrin Promotes Actin Assembly at the Phagosome Membrane and Regulates Phago-Lysosomal Fusion

Phagosome maturation is defined as the process by which phagosomes fuse sequentially with endosomes and lysosomes to acquire an acidic pH and hydrolases that degrade ingested particles. While the essential role of actin cytoskeleton remodeling during particle internalization is well established, its role during the later stages of phagosome maturation remains largely unknown. We have previously shown that purified mature phagosomes assemble F‐actin at their membrane, and that the ezrin‐radixin‐moesin (ERM) proteins ezrin and moesin participate in this process. Moreover, we provided evidence that actin assembly on purified phagosomes stimulates their fusion with late endocytic compartments in vitro. In this study, we further investigated the role of ezrin in phagosome maturation. We engineered a structurally open form of ezrin and demonstrated that ezrin binds directly to the actin assembly promoting factor N‐WASP (Neural Wiskott‐Aldrich Syndrome Protein) by its FERM domain. Using a cell‐free system, we found that ezrin stimulates F‐actin assembly on purified phagosomes by recruiting the N‐WASP–Arp2/3 machinery. Accordingly, we showed that the down‐regulation of ezrin activity in macrophages by a dominant‐negative approach caused reduced F‐actin accumulation on maturing phagosomes. Furthermore, using fluorescence and electron microscopy, we found that ezrin is required for the efficient fusion between phagosomes and lysosomes. Live‐cell imaging analysis supported the notion that ezrin is necessary for the fusogenic process itself, promoting the transfer of the lysosome content into the phagosomal lumen.

Autonomous phagosomal degradation and antigen presentation in dendritic cells

Phagocytosis plays a critical role in both innate and adaptive immunity. Phagosomal fusion with late endosomes and lysosomes enhances proteolysis, causing degradation of the phagocytic content. Increased degradation participates in both innate protection against pathogens and the production of antigenic peptides for presentation to T lymphocytes during adaptive immune responses. Specific ligands present in the phagosomal cargo influence the rate of phagosome fusion with lysosomes, thereby modulating both antigen degradation and presentation. Using a combination of cell sorting techniques and single phagosome flow cytometry-based analysis, we found that opsonization with IgG accelerates antigen degradation within individual IgG-containing phagosomes, but not in other phagosomes present in the same cell and devoid of IgG. Likewise, IgG opsonization enhances antigen presentation to CD4+ T lymphocytes only when antigen and IgG are present within the same phagosome, whereas cells containing phagosomes with either antigen or IgG alone failed to present antigen efficiently. Therefore, individual phagosomes behave autonomously, in terms of both cargo degradation and antigen presentation to CD4+ T cells. Phagosomal autonomy could serve as a basis for the intracellular discrimination between self and nonself antigens, resulting in the preferential presentation of peptides derived from opsonized, nonself antigens.

Comparison of Micro- and Nanoscale Fe+3–Containing (Hematite) Particles for Their Toxicological Properties in Human Lung Cells In Vitro

The specific properties of nanoscale particles, large surface-to-mass ratios and highly reactive surfaces, have increased their commercial application in many fields. However, the same properties are also important for the interaction and bioaccumulation of the nonbiodegradable nanoscale particles in a biological system and are a cause for concern. Hematite (α-Fe₂O₃), being a mineral form of Fe(III) oxide, is one of the most used iron oxides besides magnetite. The aim of our study was the characterization and comparison of biophysical reactivity and toxicological effects of α-Fe₂O₃ nano- (d < 100 nm) and microscale (d < 5 μm) particles in human lung cells. Our study demonstrates that the surface reactivity of nanoscale α-Fe₂O₃ differs from that of microscale particles with respect to the state of agglomeration, radical formation potential, and cellular toxicity. The presence of proteins in culture medium and agglomeration were found to affect the catalytic properties of the hematite nano- and microscale particles. Both the nano- and microscale α-Fe₂O₃ particles were actively taken up by human lung cells in vitro, although they were not found in the nuclei and mitochondria. Significant genotoxic effects were only found at very high particle concentrations (> 50 μg/ml). The nanoscale particles were slightly more potent in causing cyto- and genotoxicity as compared with their microscale counterparts. Both types of particles induced intracellular generation of reactive oxygen species. This study underlines that α-Fe₂O₃ nanoscale particles trigger different toxicological reaction pathways than microscale particles. However, the immediate environment of the particles (biomolecules, physiological properties of medium) modulates their toxicity on the basis of agglomeration rather than their actual size.

Dynamin A, Myosin IB and Abp1 Couple Phagosome Maturation to F‐Actin Binding

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo‐phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)‐null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin‐dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F‐actin in vitro, and correlated this with the presence of actin‐binding and membrane‐trafficking proteins. Phagosomes isolated from myoB‐null cells showed an increased binding to F‐actin, especially late phagosomes. In contrast, early phagosomes from dymA‐null cells showed reduced binding to F‐actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F‐actin‐binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.