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Graphical abstract, Highlights • Lipoteichoic acid (LTA) reduced the levels of the antioxidant enzymes in H9c2 cells. • LTA from Streptococcus sanguinis induced ROS production in H9c2 cells. • Low antioxidant enzymes levels and ROS production could be related to infective endocarditis (IE). • Antioxidants could protect against oxidative stress induced by LTA in IE., Infective endocarditis (IE) is an illness where the heart is invaded by bacteria, like Streptococcal and Staphylococcal species that contain lipoteichoic acid (LTA) related to an essential role in this disease. This study is the first in evaluating antioxidant enzyme levels in embryonic cardiomyocyte cell line (H9c2) induced by LTA from Streptococcus sanguinis. LTA increased reactive oxygen species (ROS) and reduced the levels of the antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD)-1 and catalase (CAT) but did not affect glutathione content. At the highest LTA concentration (15 μg/ml), SOD-1 and CAT levels did not change, and this effect was related to the induction of mRNA levels of Nrf2 induced by LTA. These results suggest that low antioxidant enzyme levels and ROS production could be related to IE.

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition., Journal of Biological Chemistry, 296, ISSN:0021-9258, ISSN:1083-351X

Graphical abstract, Highlights • Monocyte test assays were poorly efficient for the detection of little bacterial contamination in icodextrin batches. • TLR2- and TLR4-transfected cell lines were appropriate for detecting PGN and LPS contaminations. • Mutanolysin treatment of icodextrins enhanced the response of TLR2-transfected cells to contaminating PGN. • Using TLR-transfected cell lines is a valuable approach for selecting icodextrin batches for peritoneal dialysis solutions., Icodextrin is a starch derivative used for preparing solutions of peritoneal dialysis. Unfortunately, peptidoglycans (PGN) and lipopolysaccharides (LPS) have been reported to contaminate certain icodextrin batches and to contribute to the development of sterile peritonitis. The decision of selecting or rejecting icodextrin batches is however difficult, because of limitations in the detection of these bacterial contaminants. Besides monocyte activation tests of cytokine release, a number of bio-assays using stably TLR-transfected cell lines have been developed. Here, we compared the efficacy of TLR2- and TLR4-transfected cells to detect bacterial contamination with the responses of monocytes exposed to the same icodextrin samples. In contrast to monocyte models of cytokine release, we found that TLR2- and TLR4-transfected cell lines are highly sensitive to detect little PGN and LPS contaminations in the presence of icodextrin. With the intent to increase PGN reactivity, mutanolysin was used to generate soluble fragments in icodextrin samples. We found that such an enzymatic treatment led to an enhanced response of TLR2-transfected cells, even though parental icodextrin samples were poorly reactive. Altogether, these findings indicate that the use of TLR2- and TLR4-transfected cell lines is a valuable approach for helping to the decision of selecting icodextrin batches for peritoneal dialysis.

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Early recognition of the pathogen and subsequent innate immune response play a vital role in disease susceptibility and outcome. Genetic variations in innate immune genes can alter the immune response and influence susceptibility and outcome of meningitis disease. Here we conducted a sequencing study of coding regions from 46 innate immune genes in 435 pneumococcal meningitis patients and 416 controls, to determine the role of genetic variation on pneumococcal meningitis susceptibility and disease outcome. Strongest signals for susceptibility were rs56078309 CXCL1 (p = 4.8e − 04) and rs2008521 in CARD8 (p = 6.1e − 04). For meningitis outcome the rs2067085 in NOD2 (p = 5.1e − 04) and rs4251552 of IRAK4 were the strongest associations with unfavorable outcome (p = 6.7e − 04). Haplotype analysis showed a haplotype block, determined by IRAK4 rs4251552, significantly associated with unfavorable outcome (p = 0.004). Cytokine measurements from cerebrospinal fluid showed that with the IRAK4 rs4251552 G risk allele had higher levels of IL-6 compared to individuals with A/A genotype (p = 0.04). We show that genetic variation within exons and flanking regions of 46 innate immunity genes does not yield significant association with pneumococcal meningitis. The strongest identified signal IRAK4 does imply a potential role of genetic variation in pneumococcal meningitis., Highlights • Exome variations of 46 innate immune genes show no association with pneumococcal meningitis susceptibility and outcome. • Variations that have previously been associated with invasive pneumococcal disease or meningitis could not be replicated. • Highest signals found were variations in CXCL1 and CARD8 for susceptibility and NOD2 and IRAK4 for outcome. • The IRAK4 rs4251552 variant shows a trend where patients with a G-allele have higher IL-6 levels in their liquor. The pneumococcus is the most common causative pathogen of bacterial meningitis. We investigated the association of genetic variation within the coding sequences and flanking intronic regions of 46 innate immunity genes. These genes are responsible for our first line of defense during invading pathogens. In a cohort of 435 pneumococcal meningitis patients and 416 controls we found that variation within CXCL1 and CARD8 were the strongest signals associated with pneumococcal meningitis susceptibility. For disease severity we identified variations in NOD2 and IRAK4 genes to be associated with outcome. These findings can help in the understanding and treatment of pneumococcal meningitis.

ApoE is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the antibacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models. The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy, and CD spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxin components such as lipopolysaccharide (LPS) and lipid A, yielding similar increases in the apoE α-helical content. Moreover, high-molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with lipid A. Together, our results demonstrate the ability of apoE to kill Gram-negative bacteria, interact with their endotoxins, which leads to the structural changes in apoE and the formation of aggregate-like complexes., Graphical abstract

The severe infection is becoming a significant health problem which threaten the lives of patients and the safety and economy of society. In the way of finding new strategy, antimicrobial peptides (AMPs) - an important part of host defense family, emerged with tremendous potential. Up to date, huge numbers of AMPs has been investigated from both natural and synthetic sources showing not only the ability to kill microbial pathogens but also propose other benefits such as wound healing, anti-tumor, immune modulation. In this review, we describe the involvements of AMPs in biological systems and discuss the opportunity in developing AMPs for clinical applications. In the detail, their properties in antibacterial activity is followed by their application in some infection diseases and cancer. The key discussions are the approaches to improve biological activities of AMPs either by modifying chemical structure or incorporating into delivery systems. The new applications and perspectives for the future of AMPs would open the new era of their development., Graphical abstract Unlabelled Image

CD5L (CD5 molecule-like) is a secreted glycoprotein that participates in host response to bacterial infection. CD5L influences the monocyte inflammatory response to the bacterial surface molecules lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by inhibiting TNF secretion. Here we studied the intracellular events that lead to macrophage TNF inhibition by human CD5L. To accomplish this goal, we performed functional analyses with human monocytic THP1 macrophages, as well as with peripheral blood monocytes. Inhibition of phosphatidylinositol 3-kinase (PtdIns3K) reversed the inhibitory effect of CD5L on TNF secretion. Among the various PtdIns3K isoforms, our results indicated that CD5L activates PtdIns3K (whose catalytic subunit is termed PIK3C3), a key modulator involved in autophagy. Further analysis revealed a concomitant enhancement of autophagy markers such as cellular LC3-II content, increased LC3 puncta, as well as LC3-LysoTracker Red colocalization. Moreover, electron microscopy showed an increased presence of cytosolic autophagosomes in THP1 macrophages overexpressing CD5L. Besides preventing TNF secretion, CD5L also inhibited IL1B and enhanced IL10 secretion. This macrophage anti-inflammatory pattern of CD5L was reverted upon silencing of autophagy protein ATG7 by siRNA transfection. Additional siRNA experiments in THP1 macrophages indicated that the induction of autophagy mechanisms by CD5L was achieved through cell-surface scavenger receptor CD36, a multiligand receptor expressed in a wide variety of cell types. Our data represent the first evidence that CD36 is involved in autophagy and point to a significant contribution of the CD5L-CD36 axis to the induction of macrophage autophagy.

Background & Aims Neutrophils are important immune effectors required for sterile and non-sterile inflammatory responses. However, neutrophils are associated with pathology in drug-induced liver injury, acute alcoholic liver disease, and ischemia-reperfusion injury. An understanding of the complex mechanisms that control neutrophil recruitment to the injured liver is desirable for developing strategies aimed at limiting neutrophil-mediated cellular damage. Methods Wt, tlr2−/−, tlr4−/−, and s100a9−/− mice were administered CCl4 either acutely (8, 24, 48, or 72 h) or chronically (8 weeks) and livers investigated by histological (IHC for neutrophils, fibrogenesis, proliferation, and chemotactic proteins) or molecular approaches (qRT-PCR for neutrophil chemoattractant chemokines and cytokines as well as pro-fibrogenic genes). Results Mice lacking TLR2 or S100A9 failed to recruit neutrophils to the injured liver and had a defective hepatic induction of the neutrophil chemokine CXCL-2. Hierarchy between TLR2 and S100A9 proved to be complex. While induction of S100A9 was dependent on TLR2 in isolated neutrophils, there was a more complicated two-way signalling cross-talk between TLR2 and S100A9 in whole liver. However, wound-healing and regenerative responses of the liver were unaffected in these genetic backgrounds as well as in wild type mice, in which neutrophils were depleted by infusion of Ly-6G antibody. Conclusions We have identified TLR2 and S100A8/S100A9 as key regulators of hepatic CXCL-2 expression and neutrophil recruitment. This novel TLR2-S100A9-CXCL-2 pathway may be of use in development of new strategies for selectively manipulating neutrophils in liver disease without impairing normal wound healing and regenerative responses.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that results in significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics, and infection, and a critical current target for the development of new therapeutic and prophylactic interventions. Staphylococcus aureus is an AD-associated pathogen producing virulence factors that induce skin barrier disruption in vivo and contribute to AD pathogenesis. We show, using immortalized and primary keratinocytes, that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment and tight junction damage. V8-induced integrity damage was inhibited by an IL-1β-mediated mechanism, independent of effects on claudin-1. Induction of keratinocyte expression of the antimicrobial/host defense peptide human β-defensin 2 (hBD2) was found to be the mechanism underpinning this protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was protective. This modulatory property of hBD2, unrelated to antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential.