Tumor cell lines are often used as models for the study of nanoparticle-cell interactions. Here we demonstrate that carboxy (PS-COOH) and amino functionalized (PS-NH2) polystyrene nanoparticles of ∼100 nm in diameter are internalized by human macrophages, by undifferentiated and by PMA-differentiated monocytic THP-1 cells via diverse mechanisms. The uptake mechanisms also differed for all cell types and particles when analyzed either in buffer or in medium containing human serum. Macrophages internalized ∼4 times more PS-COOH than THP-1 cells, when analyzed in serum-containing medium. By contrast, in either medium, THP-1 cells internalized PS-NH2 more rapidly than macrophages. Using pharmacological and antisense in vitro knockdown approaches, we showed that, in the presence of serum, the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP-1 cells occurred via dynamin II-dependent endocytosis. PMA-differentiated THP-1 cells differed in their uptake mechanism from macrophages and undifferentiated THP-1 cells by internalizing the particles via macropinocytosis. In line with our in vitro data, more intravenously applied PS-COOH particles accumulated in the liver, where macrophages of the reticuloendothelial system reside. By contrast, PS-NH2 particles were preferentially targeted to tumor xenografts grown on the chorioallantoic membrane of fertilized chicken eggs. Our data show that the amount of internalized nanoparticles, the uptake kinetics, and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.

The pathogenic impact of tumor-infiltrating B cells is unresolved at present, however, some studies suggest that they may have immune regulatory potential. Here, we report that the microenvironment of various solid tumors includes B cells that express granzyme B (GrB, GZMB), where these B cells can be found adjacent to interleukin (IL)-21-secreting regulatory T cells (Treg) that contribute to immune tolerance of tumor antigens. Because Tregs and plasmacytoid dendritic cells are known to modulate T-effector cells by a GrB-dependent mechanism, we hypothesized that a similar process may operate to modulate regulatory B cells (Breg). IL-21 induced outgrowth of B cells expressing high levels of GrB, which thereby limited T-cell proliferation by a GrB-dependent degradation of the T-cell receptor z-chain. Mechanistic investigations into how IL-21 induced GrB expression in B cells to confer Breg function revealed a CD19

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH(2)) of ∼100 nm in diameter, but not carboxyl- or nonfunctionalized particles, trigger NLRP3 inflammasome activation and subsequent release of proinflammatory interleukin 1β (IL-1β) by human macrophages. PS-NH(2) induced time-dependent proton accumulation in lysosomes associated with lysosomal destabilization, release of cathepsin B, and damage of the mitochondrial membrane. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein, resulting in a conformational change of the pyrin domain of the NLRP3 protein, as was predicted by molecular modeling. Consequently, this prompted assembly of the NLRP3 inflammasome complex with recruitment and activation of caspase-1, inducing IL-1β release by cleavage of pro-IL-1β. The central role of the NLRP3 inflammasome for cytokine production was confirmed by in vitro knockdown of NLRP3 and of the adaptor protein ASC, confirming that other inflammasomes were not activated by PS-NH(2). The PS-NH(2)-mediated proinflammatory macrophage activation could be antagonized by the radical scavenger N-acetyl-L-cysteine, which prevented mitochondrial damage, caspase-1 activation, and the subsequent release of IL-1β. Our study reveals the molecular mechanism of NLRP3 inflammasome activation by amino-functionalized nanoparticles and suggests a strategy as to how such adverse effects could be antagonized.

The serine protease plasmin generated from its zymogen plasminogen is best known for its function as a key enzyme of the fibrinolytic cascade. However, beyond fibrinolysis, plasmin has a number of crucial functions in a variety of processes, including inflammation. Various cells can bind plasminogen and plasmin via plasminogen-binding sites exposing a C-terminal lysine. Plasmin, generated as a result of plasminogen activation at the cell surface, is protected from its physiological inhibitors. Apart from its ability to facilitate cell migration in tissues, plasmin is capable of triggering signaling, which depends on cellular binding via its lysine-binding sites and its proteolytic activity. Plasmin-induced signaling affects various functions of monocytes, macrophages, DCs, and others, with the list of affected cells still growing. In vitro and in vivo studies have demonstrated the ability of plasmin to stimulate the production of cytokines, ROS, and other mediators, thereby contributing to inflammation. Plasmin-induced chemotaxis of monocytes and DCs indicates that it is also a potent chemoattractant for immune cells. Therefore, excessive activation of plasmin in chronic inflammatory or autoimmune diseases might exacerbate the activation of inflammatory cells and the pathogenesis of the disease. This review focuses on the available evidence for physiological and pathophysiological roles the serine protease plasmin in inflammatory processes.

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Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB ؉ pDCs potently suppress T-cell proliferation in a GrBdependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly IntroductionPlasmacytoid dendritic cells (pDCs) represent a central link between innate and adaptive immunity and play a crucial role in viral, autoimmune, and neoplastic diseases. [1][2][3][4] One of their most prominent features is the ability of pDCs to produce and secrete large amounts of type I interferons (IFNs), thereby initiating and orchestrating antiviral immune responses. 1,5 pDCs can also function as antigen-presenting cells and stimulate effector T-cell responses. 6 However, pDC effects on T-cell subsets can include both T-cell activation (immunogenic function) 7 and induction of T-cell anergy (tolerogenic function). 7,8 pDCs therefore play an important role in fine-tuning cellular immune responses depending on the microenvironment. Several studies suggest that both the activated T cell-derived cytokine interleukin-3 (IL-3) 9 and the immunosuppressive cytokine IL-10 induce a rather tolerogenic pDC phenotype associated with suppression of T-cell responses. [10][11][12] In contrast, activation of pDCs in the presence of ligands for the pDC-characteristic Toll-like receptors (TLRs) TLR7 and TLR9 1 and for CD40 10 results in an immunogenic phenotype with such pDC triggering a proinflammatory immune response including T-cell activation and cytotoxicity. 6,13,14 Several inflammatory diseases including viral and autoimmune diseases have been found to be associated with elevated levels of extracellular granzyme B (GrB). Granzymes including GrB have been found to be locally elevated in bronchoalveolar lavage fluid from patients with chronic allergic asthma and in synovial fluid from patients with rheumatoid arthritis. 15 Infections with cytomegalovirus after renal transplantation, with the dengue fever virus or with HIV, have been associated with high serum levels of GrB. 15 Granzymes such as GrB represent a major constituent of the granules of cytotoxic cells, including cytotoxic T lymphocyte (CTL) and natural killer (NK) cells. The classical function of GrB is induction of apoptosis in target cells recognized by CTLs. 16,17 Evidence is growing that apart from its cytotoxic effec...