IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation.

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

RIPK3 Promotes Mefv Expression and Pyrin Inflammasome Activation via Modulation of mTOR Signaling.

Mutations in MEFV, the gene encoding pyrin in humans, are associated with the autoinflammatory disorder familial Mediterranean fever. Pyrin is an innate sensor that assembles into an inflammasome complex in response to Rho-modifying toxins, including Clostridium difficile toxins A and B. Cell death pathways have been shown to intersect with and modulate inflammasome activation, thereby affecting host defense. Using bone marrow-derived macrophages and a murine model of peritonitis, we show in this study that receptor-interacting protein kinase (RIPK) 3 impacts pyrin inflammasome activation independent of its role in necroptosis. RIPK3 was instead required for transcriptional upregulation of Mefv through negative control of the mechanistic target of rapamycin (mTOR) pathway and independent of alterations in MAPK and NF-κB signaling. RIPK3 did not affect pyrin dephosphorylation associated with inflammasome activation. We further demonstrate that inhibition of mTOR was sufficient to promote Mefv expression and pyrin inflammasome activation, highlighting the cross-talk between the mTOR pathway and regulation of the pyrin inflammasome. Our study reveals a novel interaction between molecules involved in cell death and the mTOR pathway to regulate the pyrin inflammasome, which can be harnessed for therapeutic interventions.

Direct Detection of Surface Species Formed on Iridium Electrocatalysts during the Oxygen Evolution Reaction.

The effect of surface orientations on the formation of iridium oxide species during the oxygen evolution reaction (OER) remains yet unknown. Herein, we use a needle-shaped iridium atom probe specimen as a nanosized working electrode to ascertain the role of the surface orientations in the formation of oxide species during OER. At the beginning of electrolysis, the top 2-3 nm of (024), (026), (113), and (115) planes are covered by IrO-OH, which activates all surfaces towards OER. A thick subsurface oxide layer consisting of sub-stoichiometric Ir-O species is formed on the open (024) planes as OER proceeds. Such metastable Ir-O species are thought to provide an additional contribution to the OER activity. Overall, this study sheds light on the importance of the morphological effects of iridium electrocatalysts for OER. It also provides an innovative approach that can directly reveal surface species on electrocatalysts at atomic scale.

Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida.

The DNA sensor absent in melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1ß and IL-18 and pyroptosis. AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida-infected bone marrow-derived macrophages. Furthermore, caspase-1 activation was compromised in bone marrow-derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome.

Integrity Monitoring of Multimodal Perception System for Vehicle Localization.

Autonomous driving systems tightly rely on the quality of the data from sensors for tasks such as localization and navigation. In this work, we present an integrity monitoring framework that can assess the quality of multimodal data from exteroceptive sensors. The proposed multisource coherence-based integrity assessment framework is capable of handling highway as well as complex semi-urban and urban scenarios. To achieve such generalization and scalability, we employ a semantic-grid data representation, which can efficiently represent the surroundings of the vehicle. The proposed method is used to evaluate the integrity of sources in several scenarios, and the integrity markers generated are used for identifying and quantifying unreliable data. A particular focus is given to real-world complex scenarios obtained from publicly available datasets where integrity localization requirements are of high importance. Those scenarios are examined to evaluate the performance of the framework and to provide proof-of-concept. We also establish the importance of the proposed integrity assessment framework in context-based localization applications for autonomous vehicles. The proposed method applies the integrity assessment concepts in the field of aviation to ground vehicles and provides the Protection Level markers (Horizontal, Lateral, Longitudinal) for perception systems used for vehicle localization.

Curcumin Reduces the Motility of Salmonella enterica Serovar Typhimurium by Binding to the Flagella, Thereby Leading to Flagellar Fragility and Shedding.

UNLABELLED: One of the important virulence properties of the pathogen is its ability to travel to a favorable environment, cross the viscous mucus barrier (intestinal barrier for enteric pathogens), and reach the epithelia to initiate pathogenesis with the help of an appendage, like flagella. Nonetheless, flagella can act as an "Achilles heel," revealing the pathogen's presence to the host through the stimulation of innate and adaptive immune responses. We assessed whether curcumin, a dietary polyphenol, could alter the motility of Salmonella, a foodborne pathogen. It reduced the motility of Salmonella enterica serovar Typhimurium by shortening the length of the flagellar filament (from ∼8 µm to ∼5 µm) and decreasing its density (4 or 5 flagella/bacterium instead of 8 or 9 flagella/bacterium). Upon curcumin treatment, the percentage of flagellated bacteria declined from ∼84% to 59%. However, no change was detected in the expression of the flagellin gene and protein. A fluorescence binding assay demonstrated binding of curcumin to the flagellar filament. This might make the filament fragile, breaking it into smaller fragments. Computational analysis predicted the binding of curcumin, its analogues, and its degraded products to a flagellin molecule at an interface between domains D1 and D2. Site-directed mutagenesis and a fluorescence binding assay confirmed the binding of curcumin to flagellin at residues ASN120, ASP123, ASN163, SER164, ASN173, and GLN175. IMPORTANCE: This work, to our knowledge the first report of its kind, examines how curcumin targets flagellar density and affects the pathogenesis of bacteria. We found that curcumin does not affect any of the flagellar synthesis genes. Instead, it binds to the flagellum and makes it fragile. It increases the torsional stress on the flagellar filament that then breaks, leaving fewer flagella around the bacteria. Flagella, which are crucial ligands for Toll-like receptor 5, are some of the most important appendages of Salmonella Curcumin is an important component of turmeric, which is a major spice used in Asian cooking. The loss of flagella can, in turn, change the pathogenesis of bacteria, making them more robust and fit in the host.

Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification.

Intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) manipulate their host cells through the interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organized in pathogenicity islands. The virulence-associated genomic stretch of STM3117-3120 has structural features of pathogenicity islands and is present exclusively in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117-encoded lactoylglutathione lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Δlgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Δlgl, which was also reflected in the form of oxidative DNA damage and upregulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant-containing vacuoles (MCVs). The perturbation in the pH of the MCV milieu and bacterial cytosol enhances the Salmonella pathogenicity island 2 translocation in Δlgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation of Δlgl-containing vacuoles were affected as these non-phagocytic cells maintain less acidic vacuoles compared to those in macrophages. Remarkably, ectopic expression of Toll-like receptors 2 and 4 on epithelial cells partially restored the survival of Δlgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affected the virulence of the bacteria.

Comparison of continuous transmuscular quadratus lumborum block and continuous psoas compartment block for posterior total hip arthroplasty: A randomised controlled trial.

Background and Aims: Analgesia for hip surgery involves cutaneous anaesthesia at the site of the skin incision and the anterior hip capsule. This study aimed to compare continuous ultrasound (US)-guided transmuscular quadratus lumborum block (TQLB) with psoas compartment block (PCB) for analgesia in patients undergoing total hip arthroplasty (THA) under general anaesthesia (GA). Methods: This randomised, observer-blinded trial included 18-70-year-old American Society of Anesthesiologists physical status I-III patients undergoing THA under GA with either US-guided continuous TQLB or PCB. Primary objectives included a visual analogue scale (VAS; 0-100 mm) at rest and mobilisation at 6 h postoperatively (analysed by intention to treat and per protocol) using a non-inferiority margin of 20 mm. Secondary objectives included VAS at other time points, 24-h fentanyl consumption (analysed using the Wilcoxon rank-sum test), sensory dermatomes anaesthetised, motor weakness 30 min after block, and haemodynamic response to skin incision (analysed using the Chi-squared or Fisher's exact test). A P value less than 0.05 was considered statistically significant. Results: VAS (0-100 mm) score at 6 h on rest was 25.34 ± 14.25 and 27.3 ± 9.6, mean difference (MD) was 1.9 [-3.3, 7.1] and at movement was 35.1 ± 23.0 and 38.6 ± 17.0, MD was 3.5 [-5.2, 12.2], in the PCB (n = 29) and QLB (n = 30) groups, respectively (i.e. less than the non-inferiority margin). However, VAS (rest) at 1, 12, and 24 h postoperatively and median (IQR) 24-h fentanyl consumption was significantly higher in the QLB group (1212.5 [300-2345] µg] when compared to the PCB group (635 [100-1645] µg; P = 0.0004). Conclusion: Though statistically, continuous QLB was non-inferior to continuous PCB for pain at rest and mobilisation at 6-hours postoperatively, a higher 24-hour perioperative fentanyl consumption and VAS show that QLB was clinically inferior to PCB.

Guanylate binding proteins facilitate caspase-11-dependent pyroptosis in response to type 3 secretion system-negative Pseudomonas aeruginosa.

Detection of bacterial ligands is a pre-requisite for inflammasome activation. During Pseudomonas aeruginosa infection, flagellin which is secreted through the T3SS is detected by the NLRC4 inflammasome. Activation of the NLRC4 inflammasome is believed to contribute to high IL-1ß production and pathogenicity in cystic fibrosis patients with chronic P. aeruginosa infection. Interestingly, the majority of P. aeruginosa isolated from cystic fibrosis patients with chronic airway infection are non-motile and T3SS-negative, suggesting that yet un-characterized inflammasome pathways regulate IL-1ß production in cystic fibrosis patients. Here we demonstrate the role of guanylate-binding proteins (GBPs) in regulating bacterial proliferation and inflammasome activation in response to T3SS-negative P. aeruginosa. Bacterial ligands liberated by the action of GBP2 and IRGB10 activate caspase-11 and regulate non-canonical NLRP3 inflammasome activation and IL-1ß release. Overall, our results reveal the role of caspase-11 in inhibiting bacterial proliferation and promoting IL-1ß secretion during T3SS-negative P. aeruginosa infection. This study suggests that non canonical inflammasomes might have co-evolved to detect Gram-negative bacterial pathogens that have evolved to bypass detection by canonical NLRs.

Epithelial Cell Damage Activates Bactericidal/Permeability Increasing-Protein (BPI) Expression in Intestinal Epithelium.

As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP) that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI) is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn's Disease, Ulcerative colitis, and Infectious enteritis's. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin). Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium and Shigella flexneri) whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC) did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.

Of Men Not Mice: Bactericidal/Permeability-Increasing Protein Expressed in Human Macrophages Acts as a Phagocytic Receptor and Modulates Entry and Replication of Gram-Negative Bacteria.

Macrophages as immune cells prevent the spreading of pathogens by means of active phagocytosis and killing. We report here the presence of an antimicrobial protein, bactericidal/permeability-increasing protein (BPI) in human macrophages, which actively participates in engulfment and killing of Gram-negative pathogens. Our studies revealed increased expression of BPI in human macrophages during bacterial infection and upon stimulation with various pathogen-associated molecular patterns, viz., LPS and flagellin. Furthermore, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand, Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages.

Halobacterial nano vesicles displaying murine bactericidal permeability-increasing protein rescue mice from lethal endotoxic shock.

Bactericidal/permeability-increasing protein (BPI) had been shown to possess anti-inflammatory and endotoxin neutralizing activity by interacting with LPS of Gram-negative bacteria. The current study examines the feasibility of using murine BPI (mBPI) expressed on halophilic Archaeal gas vesicle nanoparticles (GVNPs) for the treatment of endotoxemia in high-risk patients, using a murine model of D-galactosamine-induced endotoxic shock. Halobacterium sp. NRC-1was used to express the N-terminal 199 amino acid residues of mBPI fused to the GVNP GvpC protein, and bound to the surface of the haloarchaeal GVNPs. Our results indicate that delivery of mBPIN-GVNPs increase the survival rate of mice challenged with lethal concentrations of lipopolysaccharide (LPS) and D-galactosamine. Additionally, the mBPIN-GVNP-treated mice displayed reduced symptoms of inflammation, including inflammatory anemia, recruitment of neutrophils, liver apoptosis as well as increased pro-inflammatory serum cytokine levels.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria.

Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases.

Bactericidal/permeability increasing protein: a multifaceted protein with functions beyond LPS neutralization.

Bactericidal permeability increasing protein (BPI), a 55-60 kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.