Induction of CIITA by IFN-γ in macrophages involves STAT1 activation by JAK and JNK.

The induction of major histocompatibility complex (MHC) class II proteins by interferon gamma (IFN-γ) in macrophages play an important role during immune responses. Here we explore the signaling pathways involved in the induction by IFN-γ of the MHC II transactivator (CIIta) required for MHC II transcriptional activation. Cyclophilin A (CypA) is required for IFN-γ-dependent induction of MHC II in macrophages, but not when it is mediated by GM-CSF. The effect of CypA appears to be specific because it does not affect the expression of other molecules or genes triggered by IFN-γ, such as FcγR, NOS2, Lmp2, and Tap1. We found that CypA inhibition blocked the IFN-γ-induced expression of CIIta at the transcriptional level in two phases. In an early phase, during the first 2 h of IFN-γ treatment, STAT1 is phosphorylated at Tyrosine 701 and Serine 727, residues required for the induction of the transcription factor IRF1. In a later phase, STAT1 phosphorylation and JNK activation are required to trigger CIIta expression. CypA is needed for STAT1 phosphorylation in this last phase and to bind the CIIta promoter. Our findings demonstrate that STAT1 is required in a two-step induction of CIIta, once again highlighting the significance of cross talk between signaling pathways in macrophages.

Carbon Nanotubes as Optical Sensors in Biomedicine.

Single-walled carbon nanotubes (SWCNTs) have become potential candidates for a wide range of medical applications including sensing, imaging, and drug delivery. Their photophysical properties (i.e., the capacity to emit in the near-infrared), excellent photostability, and fluorescence, which is highly sensitive to the local environment, make SWCNTs promising optical probes in biomedicine. In this Perspective, we discuss the existing strategies for and challenges of using carbon nanotubes for medical diagnosis based on intracellular sensing as well as discuss also their biocompatibility and degradability. Finally, we highlight the potential improvements of this nanotechnology and future directions in the field of carbon nanotubes for biomedical applications.

It takes two to tango: Understanding the interactions between engineered nanomaterials and the immune system.

The immune system represents our primary defense system against foreign intrusion, including pathogens as well as particles. In order to understand the potential toxicity of engineered nanomaterials of ever increasing sophistication, it is necessary to understand the sophistication of the immune system with its multiple, specialized cell types and soluble mediators. Moreover, it is important to consider not only material-intrinsic properties of the pristine nanomaterial, but also the acquired, context-dependent 'identity' of a nanomaterial in a living system resulting from the adsorption of biomolecules on its surface. The immune system has evolved to recognize a vast array of microbes through so-called pattern recognition; we discuss in the present review whether engineered nanomaterials with or without a corona of biomolecules could also be sensed as 'pathogens' by immune-competent cells.

Extracellular entrapment and degradation of single-walled carbon nanotubes.

Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials.

Macrophage clearance of neutrophil extracellular traps is a silent process.

Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, in recent years, excessive NET formation has been implicated in several pathological conditions. Indeed, NETs that are not removed from tissues or from the circulation might serve to trigger autoimmune responses. We observed that physiological amounts of DNase I do not suffice to completely degrade NETs in vitro, suggesting that additional mechanisms are required for the removal of these extracellular structures. We show in this article that human monocyte-derived macrophages are able to engulf NETs in a cytochalasin D-dependent manner, indicating that this is an active, endocytic process. Furthermore, preprocessing of NETs by DNase I facilitated their clearance by macrophages. In addition, both recombinant C1q and endogenous C1q derived from human serum were found to opsonize NETs, and this facilitated NET clearance. Upon internalization, NETs were apparently degraded in lysosomes, as treatment with chloroquine led to accumulation of extranuclear DNA in human monocyte-derived macrophages. Finally, uptake of NETs alone did not induce proinflammatory cytokine secretion, whereas LPS-induced production of IL-1ß, IL-6, and TNF-α was promoted by the uptake of NETs. In summary, we show that macrophages are capable of clearance of NETs and that this occurs in an immunologically silent manner.

CREB and AP-1 activation regulates MKP-1 induction by LPS or M-CSF and their kinetics correlate with macrophage activation versus proliferation.

MAPK phosphatase-1 (MKP-1) is a protein phosphatase that plays a crucial role in innate immunity. This phosphatase inactivates ERK1/2, which are involved in two opposite functional activities of the macrophage, namely proliferation and activation. Here we found that although macrophage proliferation and activation induce MKP-1 with different kinetics, gene expression is mediated by the proximal promoter sequences localized between -380 and -180 bp. Mutagenesis experiments of the proximal element determined that CRE/AP-1 is required for LPS- or M-CSF-induced activation of the MKP-1 gene. Moreover, the results from gel shift analysis and chromatin immunoprecipitation indicated that c-Jun and CREB bind to the CRE/AP-1 box. The distinct kinetics shown by M-CSF and LPS correlates with the induction of JNK and c-jun, as well as the requirement for Raf-1. The signal transduction pathways that activate the induction of MKP-1 correlate kinetically with induction by M-CSF and LPS.

Homogeneous conjugation of peptides onto gold nanoparticles enhances macrophage response.

Murine bone marrow macrophages were able to recognize gold nanoparticle peptide conjugates, while peptides or nanoparticles alone were not recognized. Consequently, in the presence of conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6, as well as nitric oxide synthase (NOS2) were induced. Furthermore, macrophage activation by gold nanoparticles conjugated to different peptides appeared to be rather independent of peptide length and polarity, but dependent on peptide pattern at the nanoparticle surface. Correspondingly, the biochemical type of response also depended on the type of conjugated peptide and could be correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical nanoparticle conjugate design to either activate the immune system or hide from it in order to reach their targets before being removed by phagocytes.

Peptides conjugated to gold nanoparticles induce macrophage activation.

Macrophages that react against pathogenic organisms can also be activated with artificial nanometric units consisting of gold nanoparticles (Au NPs) with a peptide coating. Using bone marrow-derived macrophages, here we show that these cells have the capacity to recognize Au NPs once conjugated to two biomedically relevant peptides, the amyloid growth inhibitory peptide (AGIP) and the sweet arrow peptide (SAP), while they do not recognize peptides or NPs alone. The recognition of these conjugates by macrophages is mediated by a pattern recognition receptor, the TLR-4. Consequently, pro-inflammatory cytokines such as TNF-alpha, IL-1 beta and IL-6, as well as nitric oxide synthase were induced and macrophage proliferation was stopped when exposed to the peptide-conjugated Au NPs. Contamination by lipopolysaccharide in our experimental system was excluded. Furthermore, macrophage activation appeared to be independent of peptide length and polarity. As a result of macrophage activation, conjugated Au NPs were internalized and processed. These results open up a new avenue in the world of adjuvants and illustrate the basic requirements for the design of NP conjugates that efficiently reach their target.

NMR structural studies of the ItchWW3 domain reveal that phosphorylation at T30 inhibits the interaction with PPxY-containing ligands.

In this work, we study the role of phosphorylation as a regulatory mechanism for the interaction between the E3 ubiquitin ligase ItchWW3 domain and two PPxY motifs of one of its targets, the Epstein-Barr virus latent membrane protein 2A. Whereas ligand phosphorylation only diminishes binding, domain phosphorylation at residue T30 abrogates it. We show that two ItchWW domains can be phosphorylated at this position, using CK2 and PKA kinases and/or with stimulated T lymphocyte lysates. To better understand the regulation process, we determined the NMR structures of the ItchWW3-PPxY complex and of the phosphoT30-ItchWW3 variant. The peptide binds the domain using both XP and tyrosine grooves. A hydrogen bond from T30 to the ligand is also detected. This hydrogen-bond formation is precluded in the variant, explaining the inhibition upon phosphorylation. Our results suggest that phosphorylation at position 30 in ItchWW domains can be a mechanism to inhibit target recognition in vivo.

JNK1 Is required for the induction of Mkp1 expression in macrophages during proliferation and lipopolysaccharide-dependent activation.

Macrophages proliferate in the presence of their growth factor, macrophage colony-stimulating factor (M-CSF), in a process that is dependent on early and short ERK activation. Lipopolysaccharide (LPS) induces macrophage activation, stops proliferation, and delays ERK phosphorylation, thereby triggering an inflammatory response. Proliferating or activating responses are balanced by the kinetics of ERK phosphorylation, the inactivation of which correlates with Mkp1 induction. Here we show that the transcriptional induction of this phosphatase by M-CSF or LPS depends on JNK but not on the other MAPKs, ERK and p38. The lack of Mkp1 induction caused by JNK inhibition prolonged ERK-1/2 and p38 phosphorylation. The two JNK genes, jnk1 and jnk2, are constitutively expressed in macrophages. However, only the JNK1 isoform was phosphorylated and, as determined in single knock-out mice, was necessary for Mkp1 induction by M-CSF or LPS. JNK1 was also required for pro-inflammatory cytokine biosynthesis (tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6) and LPS-induced NO production. This requirement is independent of Mkp1 expression, as shown in Mkp1 knock-out mice. Our results demonstrate a critical role for JNK1 in the regulation of Mkp1 induction and in LPS-dependent macrophage activation.

Cyclophilin A is required for M-CSF-dependent macrophage proliferation.

The immunosuppressor sanglifehrin A (SfA) is a member of a family of immunophilin cyclophilin A-binding molecules and does not inhibit calcineurin activity. Sanglifehrin A inhibits M-CSF-dependent macrophage proliferation by arresting the G1 phase of the cell cycle but does not affect cell viability. This immunosuppressor exerts its action on proliferation by inactivating cyclin-dependent kinase 2 (Cdk2) activity. Moreover, c-myc expression is also repressed. In the early steps of M-CSF signaling, SfA inhibits the phosphorylation of Raf-1 and the external regulated kinases (ERK)1/2 and mitogen-activated protein kinase phosphatase-1, which are required for proliferation. The effects of SfA are not related to a block of the proteosome activity. These data show that immunophilin contributes to M-CSF-dependent proliferation through activation of the Raf-1/MEK/ERK pathway and the regulation of Cdk activities, which is required for cell cycle progression.

Macrophage-colony-stimulating factor-induced proliferation and lipopolysaccharide-dependent activation of macrophages requires Raf-1 phosphorylation to induce mitogen kinase phosphatase-1 expression.

Macrophages are key regulators of immune responses. In the absence of an activating signal, murine bone marrow-derived macrophages undergo proliferation in response to their specific growth factor, namely M-CSF. The addition of bacterial LPS results in macrophage growth arrest and their engagement in a proinflammatory response. Although participation of ERKs is required for both macrophage proliferation and activation, ERK phosphorylation follows a more delayed pattern in response to activating agents. In primary macrophages, mitogen kinase phosphatase-1 (MKP-1) is a key regulator of the time course of MAPK activity. Here we showed that MKP-1 expression is dependent on Raf-1 activation. The time course of Raf-1 activation correlated with that of ERK-1/2. However, whereas ERK phosphorylation in response to M-CSF is Raf-1 dependent, in response to LPS, an alternative pathway directs the activation of these kinases. Inhibition of Raf-1 activity increased the expression of cyclin-dependent kinase inhibitors and growth arrest. In contrast, no effect was observed in the expression of proinflammatory cytokines and inducible NO synthase following LPS stimulation. The data reported here reveal new insights into how signaling determines opposing macrophage functions.