Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may ...become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane ...of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBPchr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.
Caspase‐11 non‐canonical inflammasome recognizes intracellular lipopolysaccharide and activates caspase‐1, resulting in pyroptosis and secretion of interleukin‐1β and interleukin‐18 in macrophages during inflammatory responses.
The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a ...barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.
Abstract In this systematic review we analyzed studies that assessed serum concentrations of lipopolysaccharide (LPS) and/or lipopolysacharide-binding protein (LBP) in diabetic patients compared with ...healthy people. Articles were selected using PubMed and Scopus. Search terms used were endotoxemia, endotoxins, LPS, LBP, diabetes mellitus (DM), type 1 (T1DM), type 2 (T2DM), insulin resistance, humans, epidemiologic studies, population-based, survey, representative, cross-sectional, case–control studies, observational, and clinical trials. Two authors independently extracted articles using predefined data fields, including study quality indicators. There was a great variability in the estimates of metabolic endotoxemia among the studies. Most of the studies observed higher LPS or LBP concentrations in diabetic subjects than in healthy controls. T1DM and T2DM subjects presented higher mean fasting LPS of 235.7% and 66.4% compared with non-diabetic subjects, respectively. Advanced complications (e.g. macroalbuminuria) and disease onset exacerbate endotoxemia. Antidiabetic medications decrease fasting LPS concentrations. Among these medications, rosiglitazone and insulin present higher and lower effects, respectively, compared with other treatments. T1DM and T2DM seem to increase metabolic endotoxemia. However, some confounders such as diet, age, medication, smoking and obesity influence both diabetes and endotoxemia manifestation. A better understanding of the interaction of these factors is still needed.
The hypothalamus receives serotonergic projections from the raphe nucleus in a sex-specific manner. During systemic inflammation, hypothalamic levels of serotonin (5-hydroxytryptamine 5-HT) decrease ...in male rats. The present study evaluated the involvement of endothelin-1 (ET-1) in the febrile response, hypolocomotion, and changes in hypothalamic 5-HT levels during systemic inflammation in male and female rats. An intraperitoneal injection of lipopolysaccharide (LPS) induced a febrile response and hypolocomotion in both male and female rats. However, although LPS reduced hypothalamic levels of 5-HT and its metabolite 5-hydroxyindol acetic acid (5-HIAA) in male rats, it increased these levels in female rats. An intracerebroventricular injection of the endothelin-B receptor antagonist BQ788 significantly reduced LPS-induced fever and hypolocomotion and changes in hypothalamic 5-HT and 5-HIAA levels in both male and female rats. The i.c.v. administration of ET-1 induced a significant fever and hypolocomotion, but reduced the hypothalamic levels of 5-HT and 5-HIAA in both males and females. These results suggest an important sexual dimorphism during systemic inflammation regarding the release of 5-HT in the hypothalamus. Moreover, ET-1 arises as an important mediator involved in the changes in hypothalamic 5-HT levels in both male and female rats.
•Hypothalamic serotonin levels are reduced in male rats during systemic inflammation.•However, in females, hypothalamic serotonin levels are increased after lipopolysaccharide injection.•The febrile response and hypolocomotion are similar in both in male and female rats.•Endothelin-1, through ETB receptors, seems to be involved in the changes in hypothalamic serotonin levels.
Background. The relationships between soluble CD 14 (sCD14), endotoxin (lipopolysaccharide LPS), and progression of atherosclerosis have not been defined in human immunodeficiency virus (HIV) ...infection. Methods. We retrospectively assessed serum sCD14 and LPS levels of 91 subjects in a prospective 3-year study of carotid artery intima-media thickness (CIMT) (AIDS Clinical Trials Group ACTG 5078), where subjects were enrolled as risk factor-controlled triads of HIV-uninfected (n = 36) and HIV-infected individuals with (n = 29) or without (n = 26) protease inhibitor (PI)-based therapy for ≥2 years. The primary end point was the yearly rate of change of CIMT (∆CIMT). Results. In multivariate analysis of the HIV-infected subjects, each 1 μg/mL above the mean of baseline serum sCD14 corresponded to an additional 1.52 μm/y (95% confidence interval, .07-2.98; P= .04) in the ∆CIMT. Every 100 pg/mL above the mean of baseline serum LPS corresponded to an additional 0.49 μm/y (95% confidence interval, .18–. 81; P =. 003) in the ∆CIMT. However, in univariate analysis in the HIV-uninfected group sCD14 (P = .33) and LPS (P = .27) levels were not associated with higher ∆CIMT. HIV infection and PI therapy were not associated with baseline serum LPS and sCD14 levels (P > .1). Conclusions. Our data are among the first to suggest that serum biomarkers of microbial translocation (LPS) and macrophage activation (sCD14) predict subclinical atherosclerosis progression in HIV-infected persons.
The aim of this study was to identify miRNAs that regulate AKI and develop their applications as diagnostic biomarkers and therapeutic agents. First, kidney tissues from two different AKI mouse ...models, namely, AKI induced by the administration of lipopolysaccharide (LPS) causing sepsis (LPS-AKI mice) and AKI induced by renal ischemia-reperfusion injury (IRI-AKI mice), were exhaustively screened for their changes of miRNA expression compared with that of control mice by microarray analysis followed by quantitative RT-PCR. The initial profiling newly identified miRNA-5100, whose expression levels significantly decreased in kidneys in both LPS-AKI mice and IRI-AKI mice. Next, the administration of miRNA-5100-mimic conjugated with a nonviral vector, polyethylenimine nanoparticles (PEI-NPs), via the tail vein significantly induced miRNA-5100 overexpression in the kidney and prevented the development of IRI-AKI mice by inhibiting several apoptosis pathways in vivo. Furthermore, serum levels of miRNA-5100 in patients with AKI were identified as significantly lower than those of healthy subjects. ROC analysis showed that the serum expression level of miRNA-5100 can identify AKI (cut-off value 0.14, AUC 0.96, sensitivity 1.00, specificity 0.833, p<0.05). These results suggest that miRNA-5100 regulates AKI and may be useful as a novel diagnostic biomarker and therapeutic target for AKI.
In crustaceans, anti-lipopolysaccharide factors (ALFs) are important immune effectors that have sequence diversity and exhibit broad antimicrobial activities. In this study, we characterized a novel ...ALF homolog SpALF6 from mud crab Scylla paramamosain and its variant SpALF6-V, which was generated by mutations of two amino acids (H46 to R and A110 to P) due to the presence of two single nucleotide polymorphisms (SNPs). SpALF6 was an anionic peptide with isoelectric point (pI) 6.79, whereas SpALF6-V was a cationic protein with pI 7.98. These two proteins shared a common lipopolysaccharide (LPS)-binding domain (LBD) with pI 6.05. SpALF6 was expressed mainly in hemocytes and up-regulated by Vibrio parahaemolyticus or Staphylococcus aureus challenge, indicating that SpALF6 may participate in the antibacterial immune responses. To investigate the likely functional differences between SpALF6 and SpALF6-V and elucidate the underlying mechanisms, a single amino acid mutant SpALF6-M (from H46 to R, outside but very close to LBD), which had the same pI as SpALF6-V, was harvested by a fusion PCR. Then, both SpALF6 and SpALF6-M were overexpressed and purified to test antimicrobial activity and binding activity to microbial cells or polysaccharides. SpALF6-M exhibited more potent antimicrobial and cell-binding activity on Gram-positive bacteria and fungi than SpALF6. Furthermore, SpALF6-M possessed stronger lipoteichoic acid (LTA)-binding activity than SpALF6, demonstrating that this particular positively charged amino acid outside but close to LBD contributed to the increase in SpALF6-M antibacterial activity. In addition, SpALF6 LBD peptide and its biotin-labeled form were synthesized in this study. Results showed that this anionic LBD peptide itself did not exhibit any significant antimicrobial activity against 10 kinds of microorganisms but it possessed strong binding activity to LPS, LTA, and peptidoglycan. These findings suggested that this anionic LBD was still an important active center and required collaboration with some particular positively charged amino acids outside LBD to exhibit antibacterial activity. Thus, SpALF6-M antimicrobial activity was increased by the mutation of H46 to R instead of A110 to P, which did not change the protein charge, suggesting that SpALF6-V may have more potent antimicrobial activity than SpALF6 and play more important roles in antibacterial immunity. This study provided a new insight into the mechanisms of how ALF amino acid sequence diversity resulted in their functional divergence.
•We characterized a novel ALF homolog SpALF6 and its variant SpALF6-V.•SpALF6-M having the same pI as SpALF6-V was produced by mutation of H46 in SpALF6 to R.•SpALF6-M possessed stronger antimicrobial and LTA-binding activities than SpALF6.•The anionic LBD of SpALF6 did not exhibit any antimicrobial activity.•The SNP locus of SpALF6-V can be a selective marker of disease resistance in breeding.
Benproperine (BNP) is a nonnarcotic antitussive drug that is used to treat bronchitis. In the present study, we examined the anti-inflammatory effects of BNP in vitro and in vivo. BNP was found to ...reduce the secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, in lipopolysaccharide (LPS)-treated RAW264.7 monocyte/macrophage-lineage cells in vitro. As IL-6 is a biomarker for sepsis and has been suggested to exacerbate symptoms, we used an animal model to determine whether BNP reduces IL-6 levels in vivo and improves sepsis symptoms. Notably, BNP reduced IL-6 levels in the lungs of LPS-treated mice and improved LPS-induced hypothermia, one of the symptoms of sepsis. BNP reduced the mortality of septic mice administered a lethal dose of LPS. To reveal the mechanisms underlying the anti-inflammatory function of BNP, we assessed intracellular signaling in LPS-treated RAW264.7 cells. BNP induced the phosphorylation of protein kinase B (Akt) in RAW264.7 cells with/without LPS treatment. Wortmannin, an inhibitor of phosphoinositide 3-kinase reduced the phosphorylation levels of Akt. Wortmannin also obstructed the reduction of IL-6 secretion caused by BNP. Altogether, BNP was found to exhibit an anti-inflammatory function via Akt signaling. Therefore, BNP could be a drug candidate for inflammatory diseases, including sepsis.
Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16−, intermediate ...CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14++CD16+ monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14++CD16+ monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14++CD16+ monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. After CD14++CD16+ monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.
PDF
