Histones, DNA, and Citrullination Promote Neutrophil Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4.

Neutrophil extracellular traps (NETs) promote atherosclerosis by inducing proinflammatory cytokines, but the underlying mechanism remains unknown. NET DNA is immunogenic, but given the cytotoxicity of NET histones, it is unclear how it activates cells without killing them. Here, we show that histones, DNA, citrullination, and fragmentation synergize to drive inflammation below the histone cytotoxicity threshold. At low concentrations, nucleosomes induce cytokines, but high concentrations kill cells before cytokines are produced. The synergy between histones and DNA is critical for sub-lethal signaling and relies on distinct roles for histones and DNA. Histones bind and activate TLR4, whereas DNA recruits TLR4 to histone-containing endosomes. Citrullination is dispensable for NETosis but potentiates histone-mediated signaling. Consistently, chromatin blockade or PAD4 deficiency reduces atherosclerosis. Inflammation is also reduced in infected mice expressing GFP-tagged histones that block TLR4 binding. Thus, chromatin promotes inflammation in sterile disease and infection via synergistic mechanisms that use signals with distinct functions.

Transcriptional profiling unveils type I and II interferon networks in blood and tissues across diseases.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4+ effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease.

Reactive Oxygen Species Localization Programs Inflammation to Clear Microbes of Different Size.

How the number of immune cells recruited to sites of infection is determined and adjusted to differences in the cellular stoichiometry between host and pathogen is unknown. Here, we have uncovered a role for reactive oxygen species (ROS) as sensors of microbe size. By sensing the differential localization of ROS generated in response to microbes of different size, neutrophils tuned their interleukin (IL)-1ß expression via the selective oxidation of NF-κB, in order to implement distinct inflammatory programs. Small microbes triggered ROS intracellularly, suppressing IL-1ß expression to limit neutrophil recruitment as each phagocyte eliminated numerous pathogens. In contrast, large microbes triggered ROS extracellularly, amplifying IL-1ß expression to recruit numerous neutrophils forming cooperative clusters. Defects in ROS-mediated microbe size sensing resulted in large neutrophil infiltrates and clusters in response to small microbes that contribute to inflammatory disease. These findings highlight the impact of ROS localization on signal transduction.

Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis.

Secretion of the cytokine interleukin-1ß (IL-1ß) by macrophages, a major driver of pathogenesis in atherosclerosis, requires two steps: Priming signals promote transcription of immature IL-1ß, and then endogenous "danger" signals activate innate immune signaling complexes called inflammasomes to process IL-1ß for secretion. Although cholesterol crystals are known to act as danger signals in atherosclerosis, what primes IL-1ß transcription remains elusive. Using a murine model of atherosclerosis, we found that cholesterol crystals acted both as priming and danger signals for IL-1ß production. Cholesterol crystals triggered neutrophils to release neutrophil extracellular traps (NETs). NETs primed macrophages for cytokine release, activating T helper 17 (TH17) cells that amplify immune cell recruitment in atherosclerotic plaques. Therefore, danger signals may drive sterile inflammation, such as that seen in atherosclerosis, through their interactions with neutrophils.

Oxidation matters: the ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation.

During innate immune responses the delicate balance of protein synthesis, quality control and degradation is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins. The regulated degradation of ubiquitin-tagged proteins by the ubiquitin proteasome system (UPS) represents one major pathway for the maintenance of cellular proteostasis and regulatory processes under these conditions. In addition, MHC class I antigen presentation is strictly dependent on an appropriate peptide supply by the UPS to efficiently prime CD8(+) T cells and to initiate an adaptive immune response. We here discuss recent efforts in defining the link between innate immune mechanisms like cytokine and ROS production, the induction of an efficient adaptive immune response and the specific involvement of the UPS therein. Cytokines and/or infections induce translation and the production of free radicals, which in turn confer oxidative damage to nascent as well as folded proteins. In parallel, the same signaling cascades are able to accelerate the protein turnover by the concomitantly induced ubiquitin conjugation and degradation of such damaged polypeptides by immunoproteasomes. The ability of immunoproteasomes to efficiently degrade such oxidant-damaged ubiquitylated proteins protects cells from accumulating toxic ubiquitin-rich aggregates. At the same time, this innate immune mechanism facilitates a sufficient peptide supply for MHC class I antigen presentation and connects it to initiation of adaptive immunity.

Synaptic transmission and short-term plasticity at the calyx of Held synapse revealed by multielectrode array recordings.

We assessed the potential of using multielectrode arrays (MEAs) to investigate several physiological properties of the calyx of Held synapse in the medial nucleus of the trapezoid body of gerbil. Due to the large size of the synapse, it became widely employed in studies on synaptic mechanisms. Electrical stimulation at the midline evoked a characteristic compound signal consisting of a presynaptic volley (C(1)) and a postsynaptic response (C(2)). The C(1) was blocked by tetrodotoxin, whilst the C(2) was blocked by perfusion of low Ca(2+) external solution, or the AMPA-R antagonists CNQX, and GYKI52466. NMDA-R blocker D-AP5, partially inhibited the postsynaptic response at P12, but showed no effect in P30 animals. The inhibitory effects of GABA or glycine on postsynaptic responses were reciprocal with regard to animal's maturity: GABA caused a pronounced reduction of C(2) amplitude in P20-22 animals, while glycine showed a stronger inhibition in P27-28 animals. Low-frequency super-threshold stimulation of the afferents induced facilitation of the postsynaptic C(2) amplitudes and only minor changes in temporal characteristics of the signals. At stimulation frequencies >200 Hz, however, significant depression occurs accompanied by increases in transmission delay and in the width of the postsynaptic response. This study suggests MEAs as a useful tool to study calyx of Held synapse by simultaneous recordings of pre- and postsynaptic elements of synaptically interconnected neurons in the auditory brainstem. Moreover, MEAs enable convenient analysis of activity-dependent depression and modulation of neuronal activity by glycine and GABA at later developmental stages not accessible to patch recordings.