NF-κB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression.

Epidemiological studies indicate that overweight and obesity are associated with increased cancer risk. To study how obesity augments cancer risk and development, we focused on hepatocellular carcinoma (HCC), the common form of liver cancer whose occurrence and progression are the most strongly affected by obesity among all cancers. We now demonstrate that either dietary or genetic obesity is a potent bona fide liver tumor promoter in mice. Obesity-promoted HCC development was dependent on enhanced production of the tumor-promoting cytokines IL-6 and TNF, which cause hepatic inflammation and activation of the oncogenic transcription factor STAT3. The chronic inflammatory response caused by obesity and enhanced production of IL-6 and TNF may also increase the risk of other cancers.

Anthrax toxin induces macrophage death by p38 MAPK inhibition but leads to inflammasome activation via ATP leakage.

Detection of microbial constituents by membrane associated and cytoplasmic pattern recognition receptors is the essence of innate immunity, leading to activation of protective host responses. However, it is still unclear how immune cells specifically respond to pathogenic bacteria. Using virulent and nonvirulent strains of Bacillus anthracis, we have shown that secretion of ATP by infected macrophages and the sequential activation of the P2X7 purinergic receptor and nucleotide binding oligomerization domain (NOD)-like receptors are critical for IL-1-dependent host protection from virulent B. anthracis. Importantly, lethal toxin produced by virulent B. anthracis blocked activation of protein kinases, p38 MAPK and AKT, resulting in opening of a connexin ATP release channel and induction of macrophage death. Prevention of cell death or ATP release through constitutive p38 or AKT activation interfered with inflammasome activation and IL-1ß production, thereby compromising antimicrobial immunity.

Loss of CMAH during Human Evolution Primed the Monocyte-Macrophage Lineage toward a More Inflammatory and Phagocytic State.

Humans and chimpanzees are more sensitive to endotoxin than are mice or monkeys, but any underlying differences in inflammatory physiology have not been fully described or understood. We studied innate immune responses in Cmah-/- mice, emulating human loss of the gene encoding production of Neu5Gc, a major cell surface sialic acid. CMP-N-acetylneuraminic acid hydroxylase (CMAH) loss occurred ∼2-3 million years ago, after the common ancestor of humans and chimpanzees, perhaps contributing to speciation of the genus HomoCmah-/- mice manifested a decreased survival in endotoxemia following bacterial LPS injection. Macrophages from Cmah-/- mice secreted more inflammatory cytokines with LPS stimulation and showed more phagocytic activity. Macrophages and whole blood from Cmah-/- mice also killed bacteria more effectively. Metabolic reintroduction of Neu5Gc into Cmah-/- macrophages suppressed these differences. Cmah-/- mice also showed enhanced bacterial clearance during sublethal lung infection. Although monocytes and monocyte-derived macrophages from humans and chimpanzees exhibited marginal differences in LPS responses, human monocyte-derived macrophages killed Escherichia coli and ingested E. coli BioParticles better. Metabolic reintroduction of Neu5Gc into human macrophages suppressed these differences. Although multiple mechanisms are likely involved, one cause is altered expression of C/EBPß, a transcription factor affecting macrophage function. Loss of Neu5Gc in Homo likely had complex effects on immunity, providing greater capabilities to clear sublethal bacterial challenges, possibly at the cost of endotoxic shock risk. This trade-off may have provided a selective advantage when Homo transitioned to butchery using stone tools. The findings may also explain why the Cmah-/- state alters severity in mouse models of human disease.

Tamm-Horsfall glycoprotein engages human Siglec-9 to modulate neutrophil activation in the urinary tract.

Urinary tract infections are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic Escherichia coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared with wild-type mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia.

OBJECTIVES: The Gram-negative bacillus Stenotrophomonas maltophilia (SM) is an emerging MDR opportunistic pathogen. Recent studies identify a potentially relevant activity of azithromycin against Gram-negative bacteria overlooked in standard bacteriological testing. We investigated azithromycin activity against SM in testing conditions incorporating mammalian tissue culture medium and host defence factors. METHODS: MIC testing, chequerboard assays, time-kill assays and fluorescence microscopy were performed for azithromycin, the cationic peptide antibiotic colistin and the human defence peptide cathelicidin LL-37 alone or in combination in cation-adjusted Mueller-Hinton broth or mammalian tissue culture media. Azithromycin sensitization of SM to host immune clearance was tested in a human neutrophil killing assay and a murine pneumonia model. RESULTS: We observed potent bactericidal activity of azithromycin against SM in mammalian tissue culture medium absent in bacteriological medium. Colistin and LL-37 strongly potentiated azithromycin killing of SM by increasing drug entry. Additionally, azithromycin sensitized SM to neutrophil killing and increased SM clearance in the murine pneumonia model. CONCLUSIONS: Despite lack of activity in standard MIC testing, azithromycin synergizes with cationic peptide antibiotics to kill SM in medium mimicking tissue fluid conditions. Azithromycin, alone or in combination with colistin, merits further exploration in therapy of drug-resistant SM infections.

Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages.

Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1ß (IL-1ß). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1ß signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B Streptococcus (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1ß release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1ß release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, Streptococcus pneumoniae, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating cis- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1ß secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1ß production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a SIGLEC5/14 fusion polymorphism, and we found increased IL-1ß expression in primary macrophages from SIGLEC14+/+ individuals compared to those with the SIGLEC14-/+ and SIGLEC14-/- genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation.

Proton-pump inhibitors do not influence clinical outcomes in patients with Staphylococcus aureus bacteremia.

BACKGROUND: Proton-pump inhibitors (PPIs) are commonly used in clinical practice for gastric acid suppression. However, these agents have also been associated with certain negative clinical outcomes. We evaluated the real-world effects of incident PPI use on clinical outcomes in patients with Staphylococcus aureus bacteremia. METHODS: This retrospective cohort study included patients admitted to Veterans Affairs hospitals with positive S. aureus blood cultures collected between 2002 and 2013 that received appropriate antibiotics within 48 hours of culture collection. Clinical outcomes among three PPI exposure groups, each compared to nonusers, were assessed with propensity-score-matched Cox proportional-hazard regression models: pretreated PPI users initiating therapy in the 30 days prior to culture and either (a) continuing PPI therapy after culture, or (b) not continuing after culture, and (c) de novo users initiating at culture. RESULTS: Clinical outcomes, including inpatient mortality, intensive care discharge, 30-day mortality, 30-day readmission, and 30-day Clostridium difficile infection (CDI) were similar among PPI users and nonusers. Though length of stay was longer in pretreated, continuing PPI users [time-to-discharge hazard ratio (HR) 0.78, 95% confidence interval (CI) 0.65-0.93], 14-day mortality was significantly lower than in nonusers (HR 0.66, 95% CI 0.50-0.87). CONCLUSIONS: In our large national cohort study, PPIs were not associated with an increased risk of negative clinical outcomes, including mortality and CDI, in patients with S. aureus bacteremia.

Characterization of Freestream Disturbances in Conventional Hypersonic Wind Tunnels.

While low disturbance ("quiet") hypersonic wind tunnels are believed to provide more reliable extrapolation of boundary layer transition behavior from ground to flight, the presently available quiet facilities are limited to Mach 6, moderate Reynolds numbers, low freestream enthalpy, and subscale models. As a result, only conventional ("noisy") wind tunnels can reproduce both Reynolds numbers and enthalpies of hypersonic flight configurations, and must therefore be used for flight vehicle test and evaluation involving high Mach number, high enthalpy, and larger models. This article outlines the recent progress and achievements in the characterization of tunnel noise that have resulted from the coordinated effort within the AVT-240 specialists group on hypersonic boundary layer transition prediction. New Direct Numerical Simulation datasets elucidate the physics of noise generation inside the turbulent nozzle wall boundary layer, characterize the spatiotemporal structure of the freestream noise, and account for the propagation and transfer of the freestream disturbances to a pitot-mounted sensor. The new experimental measurements cover a range of conventional wind tunnels with different sizes and Mach numbers from 6 to 14 and extend the database of freestream fluctuations within the spectral range of boundary layer instability waves over commonly tested models. Prospects for applying the computational and measurement datasets for developing mechanism-based transition prediction models are discussed.

CO2 Laser in Neurosurgery: A Hands-on Experience.

Since its first use in 1963 by Engr. Kumar Naranbhai Patel, carbon dioxide (CO2) laser has evolved to attain a vital role in surgical neurology. The utilisation of laser is increasing in neurosurgery, especially minimally invasive and functional procedures. The laser serves three important functions in surgical procedures: photo-coagulation, photo-vaporisation, and photo-activation. Due to its favorable properties, CO2 laser is now recommended for many neurosurgical procedures. The development of low profile, and flexible transfer media has facilitated the use of laser in micro-neurosurgical procedures. CO2 laser has gained popularity as a unique cutting tool. The characteristic features of CO2 laser cause photo-vaporisation more than photo-coagulation. This allows to achieve a contact-free, relatively bloodless excision of many highly vascular lesions, especially helpful in critical anatomical areas. In this report, we share our first hands-on experience with flexible CO2 laser at Prince Sultan Military Medical City, Riyadh. The CO2 laser was utilised to achieve intracranial resection of a huge, markedly vascular glomus jugulare lesion. There was a significant reduction in blood loss with laser use, when compared with surgical resection utilising bipolar cautery, suction and cavitron ultrasonic aspirator. Postoperative recovery was also rapid and uneventful due to relatively less edema at surgical bed. We would like to favour the use of CO2 laser, especially when dealing with complex lesions with increased vascularity located in close relationship with vital neural and vascular structures in an attempt to minimise damage to intact tissue.

Group A streptococcal M protein activates the NLRP3 inflammasome.

Group A Streptococcus (GAS) is among the top ten causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections, including necrotizing fasciitis and toxic shock syndrome. Here, we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine interleukin-1ß (IL-1ß) and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K+ efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1ß activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1ß release could aggravate tissue injury.

Siglec-5 and Siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group B Streptococcus.

Group B Streptococcus (GBS) causes invasive infections in human newborns. We recently showed that the GBS ß-protein attenuates innate immune responses by binding to sialic acid-binding immunoglobulin-like lectin 5 (Siglec-5), an inhibitory receptor on phagocytes. Interestingly, neutrophils and monocytes also express Siglec-14, which has a ligand-binding domain almost identical to Siglec-5 but signals via an activating motif, raising the possibility that these are paired Siglec receptors that balance immune responses to pathogens. Here we show that ß-protein-expressing GBS binds to both Siglec-5 and Siglec-14 on neutrophils and that the latter engagement counteracts pathogen-induced host immune suppression by activating p38 mitogen-activated protein kinase (MAPK) and AKT signaling pathways. Siglec-14 is absent from some humans because of a SIGLEC14-null polymorphism, and homozygous SIGLEC14-null neutrophils are more susceptible to GBS immune subversion. Finally, we report an unexpected human-specific expression of Siglec-5 and Siglec-14 on amniotic epithelium, the site of initial contact of invading GBS with the fetus. GBS amnion immune activation was likewise influenced by the SIGLEC14-null polymorphism. We provide initial evidence that the polymorphism could influence the risk of prematurity among human fetuses of mothers colonized with GBS. This first functionally proven example of a paired receptor system in the Siglec family has multiple implications for regulation of host immunity.