Bacterial Ligands Research Articles

TLR5 Activation Exacerbates Airway Inflammation in Asthma.

Innate immune activation through exposure to indoor and outdoor pollutants is emerging as an important determinant of asthma severity. For example, household levels of the bacterial product lipopolysaccharide (LPS) are associated with increased asthma severity. We hypothesized that activation of the innate immune receptor TLR5 by its bacterial ligand flagellin will exacerbate airway inflammation and asthma symptoms. We determined the effect of flagellin co-exposure with ovalbumin in a murine model of allergic asthma. We evaluated the presence of flagellin activity in house dust of asthma patients. Finally, we analyzed the association of a dominant-negative polymorphism in TLR5 (rs5744168) with asthma symptoms in patients with asthma. We showed that bacterial flagellin can be found in the house dust of patients with asthma and that this bacterial product exacerbates allergic airway inflammation in an allergen-specific mouse model of asthma. Furthermore, a dominant-negative genetic polymorphism in TLR5, the receptor for flagellin, is associated with decreased symptoms in patients with asthma. Together, our results reveal a novel genetic protective factor (TLR5 deficiency) and a novel environmental pollutant (microbial flagellin) that influence asthma severity. (Clinical trials NCT01688986 and NCT01087307).

A new role for host annexin A2 in establishing bacterial adhesion to vascular endothelial cells: lines of evidence from atomic force microscopy and an in vivo study

Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacterium that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence is lacking in the field of bacterial infections of ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. We conclude that the endothelial surface ANXA2 plays an important role in initiating pathogen–host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface.

Microbial Danger Signals Control Transcriptional Induction of Distinct MHC Class I L Lineage Genes in Atlantic Salmon.

Antigen processing and presentation by major histocompatibility complex (MHC) molecules is a cornerstone in vertebrate immunity. Like mammals, teleosts possess both classical MHC class I and multiple families of divergent MHC class I genes. However, while certain mammalian MHC class I-like molecules have proven to be integral in immune regulation against a broad array of pathogens, the biological relevance of the different MHC class I lineages in fish remains elusive. This work focuses on MHC class I L lineage genes and reveals unique regulatory patterns of six genes (Sasa-lia, Sasa-lda, Sasa-lca, Sasa-lga, Sasa-lha, and Sasa-lfa) in antimicrobial immunity of Atlantic salmon (Salmo salar L.). Using two separate in vivo challenge models with different kinetics and immune pathologies combined with in vitro stimulation using viral and bacterial TLR ligands, we show that de novo synthesis of different L lineage genes is distinctly regulated in response to various microbial stimuli. Prior to the onset of classical MHC class I gene expression, lia was rapidly and systemically induced in vivo by the single-stranded (ss) RNA virus salmonid alpha virus 3 (SAV3) but not in response to the intracellular bacterium Piscirickettsia salmonis. In contrast, lga expression was upregulated in response to both viral and bacterial stimuli. A role for distinct MHC class I L-lineage genes in anti-microbial immunity in salmon was further substantiated by a marked upregulation of lia and lga gene expression in response to type I IFNa stimulation in vitro. Comparably, lha showed no transcriptional induction in response to IFNa stimulation but was strongly induced in response to a variety of viral and bacterial TLR ligands. In sharp contrast, lda showed no response to viral or bacterial challenge. Similarly, induction of lca, which is predominantly expressed in primary and secondary lymphoid tissues, was marginal with the exception of a strong and transient upregulation in pancreas following SAV3 challenge Together, these findings suggest that certain Atlantic salmon MHC class I L lineage genes play important and divergent roles in early anti-microbial response and that their regulation, in response to different activation signals, represents a system for selectively promoting the expression of distinct non-classical MHC class I genes in response to different types of immune challenges.

Mucosal-associated invariant T cells and disease.

Mucosal-associated invariant T (MAIT) cells are unique innate-like T cells that bridge innate and adaptive immunity. They are activated by conserved bacterial ligands derived from vitamin B biosynthesis and have important roles in defence against bacterial and viral infections. However, they can also have various deleterious and protective functions in autoimmune, inflammatory and metabolic diseases. MAIT cell involvement in a large spectrum of pathological conditions makes them attractive targets for potential therapeutic approaches.

Active and passive biosorption of Pb(II)using live and dead biomass of marine bacterium Bacillus xiamenensis PbRPSD202: Kinetics and isotherm studies

A highly lead(II) resistant (up to 2200 mg/l) bacterium PbRPSD202 was selected among 210 lead resistant bacteria isolated from marine environment of Paradeep Port, Odisha for possible biosoption of toxic Pb (II) ions from metals polluted environments. The bacterium was identified as Bacillus xiamenensis following the phenotypic as well as 16S rRNA gene sequence analysis. In addition to Pb(II), it also showed resistance towards other heavy metals like Cd(II), Cr(VI), As(III), Cu(II), Ni(II) and Zn(II). Batch biosorption of Pb(II) using both live and dead biomass of this strain was investigated under different operational parametric conditions such as pH, temperature, NaCl concentration, shaking speed, treatment time, biomass concentration and initial Pb(II) concentration. The maximum Pb(II) uptake of 216.75 and 207.4 mg/g biomass was obtained with live and dead biomass, respectively, at the optimum condition (4% w/v NaCl, pH 6.0, 35 °C, 140 rpm and 1 g/l biosorbent dose). Both active as well as passive Pb(II) bio-sorption process showed best fit with the pseudo-second-order kinetic model. The sorption mechanism was favoured with Langmuir isotherm model indicating monolayer type adsorption. FTIR and FESEM-EDX analysis further ensured the possible interactions of Pb(II) with bacterial cell surface ligands like hydroxyl, carbonyl, carboxyl and amine groups during surface adsorption. TEM analysis revealed the intracellular accumulation of lead ions. This investigation highlights the potential application of this bacterium for bioremediation of lead(II) from the multiple metals contaminated saline environment through biosorption.

Improving our mechanistic understanding of the indirect effects of CMV infection in transplant recipients.

Cytomegalovirus (CMV) is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal, and viral infections. However, the pathogenesis of this phenomenon is poorly understood. We determined whether inflammatory responses to different Toll-like receptor (TLR) ligands are blunted during CMV infection in solid-organ transplant (SOT) patients. Peripheral blood mononuclear cells from 38 SOT patients with and without CMV were incubated in the presence of various viral, fungal, and bacterial TLR ligands. Cytokines were measured in the supernatant by multiplex enzyme-linked immunosorbent assay. Patients had blunted cytokine responses to bacterial, fungal, and viral ligands during CMV infection when compared to the absence of CMV infection. This was independent of viral load, clinical presentation of CMV infection or immunosuppression, supporting the clinical observation in SOT recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. Moreover, in the absence of CMV infection, patients with subsequent CMV infection had lower cytokines in response to TLR ligands compared to those without subsequent CMV infection, suggesting that inherent differences in patients not directly related to CMV also contribute to this increased susceptibility. In summary, these data provide novel ex vivo evidence to support indirect effects of CMV.

STAT2 dependent Type I Interferon response promotes dysbiosis and luminal expansion of the enteric pathogen Salmonella Typhimurium.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2-/- mice, we observed decreased hypoxia in the gut tissue of Stat2-/- mice. Neutrophil numbers were similar in wild-type and Stat2-/- mice, yet Stat2-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium.

Strong selection of the TLR2 coding region among the Lagomorpha suggests an evolutionary history that differs from other mammals.

Toll-like receptors (TLRs) are one of the first lines of defense against pathogens and are crucial for triggering an appropriate immune response. Among TLRs, TLR2 is functional in all vertebrates and has high ability in detecting bacterial and viral pathogen ligands. The mammals' phylogenetic tree of TLR2 showed longer branches for the Lagomorpha clade, raising the hypothesis that lagomorphs experienced an acceleration of the mutation rate. This hypothesis was confirmed by (i) Tajima's test of neutrality that revealed different evolutionary rates between lagomorphs and the remaining mammals with lagomorphs presenting higher nucleotide diversity; (ii) genetic distances were similar among lagomorphs and between lagomorphs and other mammals; and (iii) branch models reinforced the existence of an acceleration of the mutation rate in lagomorphs. These results suggest that the lagomorph TLR2 has been strongly involved in pathogen recognition, which probably caused a host-pathogen arms race that led to the observed acceleration of the mutation rate.

PorA-Loop4 derived peptides of Neisseria meningitidis cause a G1 cell cycle arrest through the Akt signalling pathway in human brain microvascular endothelial cells

Neisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.

P126 MULTIPLE SIGNALING PATHWAY DEFECTS IN THE INTESTINAL STEM CELL NICHE LIKELY TRIGGER CD-LIKE ILEITIS IN SAMP1/YITFC MICE

Intestinal epithelial barrier dysfunction is considered as a risk factor in the pathogenesis of Crohn’s disease (CD), and since there is no FDA approved therapy available, there is a need for identifying a relevant model to study and test therapies to correct this defect. Previously, we demonstrated that inflammatory environment of CD affects properties of intestinal stem cell (ISC) niche, ablating its ability to form enteroids (EnOs) in our mouse model for CD-like ileitis (SAMP/YitFc, SAMP) (doi.org/10.1016/S0016-5085(17)32213-8). However, EnOs from SAMP mice even prior to disease onset (5-wks, SAMP-5) harbor impaired features, such as smaller size (2.6 ± 0.4 vs 3.9 ± 0.2 fold to ctrl at day 1, P=0.003), number (0.6 ± 0.1 fold to ctrl, P=0.000), reduced viability (63.7%±7.5 vs ctrl P=0.003) and rudimental branching complexity [cyst-like structures (42.7%±2.3 vs 11%±1.2)], compared to controls (ctrls). These EnOs produce exacerbated expression of genes involved in epithelial secretory cell functions when challenged with purified bacterial ligands, such as LPS [higher Muc2 (10.8 ± 3.2 vs 2.5 ± 1.8 P=0.00), Lyz (11.0 ± 5.2 vs 1.1 ± 0.4 P=0.00), Reg3γ (6.7 ± 9.9 vs 1.1 ± 0.5 P=0.04), Crp (7.4 ± 5.4 vs 1.3 ± 0.6 P=0.05), and Defa1 (4006.0 ± 1472.5 vs 2.1 ± 0.7 P=0.00) mRNA vs AKR EnOs]. To further investigate this phenotype, we generated the first high-resolution transcriptional profile of SAMP ISC niche by laser capture microdissection and RNA sequencing. We found that signaling pathways involved in the cellular energy machinery were dramatically reduced in inflamed SAMP (25-wks, SAMP-25) ISC niche compared to that of inflammation-free mice [mitochondrial energy metabolism (P ≤ 0.00, Padj ≤ 0.002, normalized enrichment score NES ≤ -2.4); oxidative phosphorylation (P ≤ 0.00 Padj ≤ 0.001 NES ≤ -2.5)]. Fibrosis (P ≤ 0.04 Padj≤ 0.09 NES ≤ 1.3) and UPR (P ≤ 0.01 Padj ≤ 0.03 NES ≤ 1.5) pathways were enriched in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Bile acid metabolism (P ≤ 0.00, Padj ≤ 0.002 NES ≤ -1.8), ROS (P ≤ 0.001 Padj ≤ 0.003 NES ≤ -1.8), Wnt3a (P ≤ 0.001 Padj ≤ 0.01 NES ≤ -1.8), and Notch1 (P ≤ 0.001 Padj ≤ 0.02 NES ≤ -1.8) pathways were decrease in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Collectively, these data suggest that multiple signaling pathways contribute to ISC niche defect in SAMP mice, and that many key biological functions are already altered prior to ileitis onset and may be responsible for disease initiation. Finally, the evidence that altered gene expression is maintained in ex vivo expanded EnOs indicates that these cultures recapitulate reactive epithelial changes specific of experimental CD. This aspect is of paramount importance since it emphasizes the physiologically relevant value of EnOs generated from SAMP mice, rather than those originated from healthy individuals, to mimic CD-like ileitis.

P126 MULTIPLE SIGNALING PATHWAY DEFECTS IN THE INTESTINAL STEM CELL NICHE LIKELY TRIGGER CD-LIKE ILEITIS IN SAMP1/YITFC MICE

Intestinal epithelial barrier dysfunction is considered as a risk factor in the pathogenesis of Crohn’s disease (CD), and since there is no FDA approved therapy available, there is a need for identifying a relevant model to study and test therapies to correct this defect. Previously, we demonstrated that inflammatory environment of CD affects properties of intestinal stem cell (ISC) niche, ablating its ability to form enteroids (EnOs) in our mouse model for CD-like ileitis (SAMP/YitFc, SAMP) (doi.org/10.1016/S0016-5085(17)32213-8). However, EnOs from SAMP mice even prior to disease onset (5-wks, SAMP-5) harbor impaired features, such as smaller size (2.6 ± 0.4 vs 3.9 ± 0.2 fold to ctrl at day 1, P=0.003), number (0.6 ± 0.1 fold to ctrl, P=0.000), reduced viability (63.7%±7.5 vs ctrl P=0.003) and rudimental branching complexity [cyst-like structures (42.7%±2.3 vs 11%±1.2)], compared to controls (ctrls). These EnOs produce exacerbated expression of genes involved in epithelial secretory cell functions when challenged with purified bacterial ligands, such as LPS [higher Muc2 (10.8 ± 3.2 vs 2.5 ± 1.8 P=0.00), Lyz (11.0 ± 5.2 vs 1.1 ± 0.4 P=0.00), Reg3γ (6.7 ± 9.9 vs 1.1 ± 0.5 P=0.04), Crp (7.4 ± 5.4 vs 1.3 ± 0.6 P=0.05), and Defa1 (4006.0 ± 1472.5 vs 2.1 ± 0.7 P=0.00) mRNA vs AKR EnOs]. To further investigate this phenotype, we generated the first high-resolution transcriptional profile of SAMP ISC niche by laser capture microdissection and RNA sequencing. We found that signaling pathways involved in the cellular energy machinery were dramatically reduced in inflamed SAMP (25-wks, SAMP-25) ISC niche compared to that of inflammation-free mice [mitochondrial energy metabolism (P ≤ 0.00, Padj ≤ 0.002, normalized enrichment score NES ≤ -2.4); oxidative phosphorylation (P ≤ 0.00 Padj ≤ 0.001 NES ≤ -2.5)]. Fibrosis (P ≤ 0.04 Padj≤ 0.09 NES ≤ 1.3) and UPR (P ≤ 0.01 Padj ≤ 0.03 NES ≤ 1.5) pathways were enriched in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Bile acid metabolism (P ≤ 0.00, Padj ≤ 0.002 NES ≤ -1.8), ROS (P ≤ 0.001 Padj ≤ 0.003 NES ≤ -1.8), Wnt3a (P ≤ 0.001 Padj ≤ 0.01 NES ≤ -1.8), and Notch1 (P ≤ 0.001 Padj ≤ 0.02 NES ≤ -1.8) pathways were decrease in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Collectively, these data suggest that multiple signaling pathways contribute to ISC niche defect in SAMP mice, and that many key biological functions are already altered prior to ileitis onset and may be responsible for disease initiation. Finally, the evidence that altered gene expression is maintained in ex vivo expanded EnOs indicates that these cultures recapitulate reactive epithelial changes specific of experimental CD. This aspect is of paramount importance since it emphasizes the physiologically relevant value of EnOs generated from SAMP mice, rather than those originated from healthy individuals, to mimic CD-like ileitis.

Insights into the substrate binding specificity of quorum-quenching acylase PvdQ

Quorum sensing is a cell to cell signaling mechanism that enables them to coordinate their behaviors in a density-dependent manner mediated by small diffusible signaling molecules, which can control the virulence and biofilm gene expression in many Gram-negative and positive bacteria. N-acyl homoserine lactone acylase PvdQ from human opportunistic pathogen Pseudomonas aeruginosa is a quorum-quenching enzyme that can hydrolyze the amide bond of the quorum signaling N-acyl homoserine lactones (AHLs) thereby degrading the signaling molecules, turning off the biofilm phenotype and resulting in a reduction of bacterial virulence. Previous studies demonstrated that PvdQ has different preferences for N-acyl substrates with different acyl chain lengths and substituents. However, the substrate binding specificity determinants of the quorum-quenching enzyme PvdQ with the different bacterial ligands are unknown and unintuitive. Further, elucidation of these determinants can lead to mutants with efficiency and broader substrate promiscuity. To investigate this question, a computational study was carried out combining multiple molecular docking methods, molecular dynamics simulations, residue interaction network analysis, and binding free energy calculations. The main findings are: firstly, the results from pKa predictions support that the pKa of the N-terminus of Serβ1 was depressed due to the surrounding residues. Multiple molecular docking studies provide useful information about the detailed binding modes and binding affinities. Secondly, 300 ns molecular dynamics simulations were carried out to analyze the overall molecular motions of substrate-bound and substrate-free PvdQ. The specific interactions between the active site of PvdQ and different ligands revealed the determinants for the preference among the ligands. A systematic comparison and analysis of the protein dynamic fingerprint of each complex demonstrated that binding of the most favorable ligand, C12-homoserine lactone (C12-HSL), reduced the global motions of the complex and maintained the correct arrangement of the catalytic site. Further, the residue interaction network analysis of each system illustrated that there are more communication contacts and pathways between the residues in the C12-HSL complex as compared to complexes with the other ligands. The binding of the C12-HSL ligand facilitates structural communication between the two knobs and the active site. While the binding of the other ligands tend to impair specific communication pathways between the two knobs and the active site, and lead to a catalytically inefficient state. Finally, simulation results from free energy landscape and binding free energy analysis revealed that the C12-HSL ligand has the lowest binding free energy and greater stability than the less favored ligands. Each of the following residues: Serβ1, Hisβ23, Pheβ24, Metβ30, Pheβ32, Leuβ50, Asnβ57, Thrβ69, Valβ70, Trpβ162, Trpβ186, Asnβ269, Argβ297 and Leuα146, play different roles in substrate binding specificity. This is the first computational study that provides molecular information for structure-dynamic-function relationships of PvdQ with different ligands and demonstrates determinants of bacterial substrate binding specificity.

Quantifying Human Innate Cytokine and Chemokine Responses Ex Vivo via Pattern Recognition Receptor Stimulation.

Linkages between human innate immune capacity, the environment in which we live, and the development of clinical tolerance versus a spectrum of disease phenotypes are a major focus of inflammatory disease research. While extensive epidemiologic evidence indicates key roles for the microbiome and other environmental factors, the underlying mechanisms that explain how these stimuli lead to a given clinical phenotype remain speculative. Here we review strategies for characterizing human cytokine production ex vivo in response to innate immune receptor stimulation with defined ligands. Human cytokine and chemokine biomarker data provides a tool to test hypotheses on the relationship between innate immune capacity in vivo and expression of current or future clinical phenotypes. The most important limitations of experimental strategies that have been used to date are reviewed. Detailed experimental protocols are provided for characterization of pattern recognition receptor (PRR)-driven stimulation with a panel of bacterial (TLR4, TLR5) and viral (TLR3, TLR7/8, RIG-I/MDA5) ligands to assess the role played by human pro-inflammatory, anti-inflammatory, Th1-like, and Th2-like responses. The importance of characterizing human innate immune phenotypes extends beyond discovery-based research to development of improved strategies for prevention or inhibition of chronic inflammatory diseases, improved design of immunization programs, and more effective cancer immunotherapy.

A New Role for Host Annexin A2 in Establishing Bacterial Adhesion to Vascular Endothelial Cell: Lines of Evidence from in vivo Study and Micro Force Spectroscopy

Background: Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacteria that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence are completely lacking in the field of bacterial infections of ECs. Methods: In the present study, we employ a novel, anatomically-based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. Findings: We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. Interpretation: The endothelial surface ANXA2 plays an important role in initiating pathogen-host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface. Funding: This work was supported by NIH (B.G.) and Natural Science Foundation of China (F.L.). Declaration of Interest: The authors declare that they have no conflicts of interest. Ethical Approval: All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB).

Role of TLR4 in the gut-brain axis in Parkinson’s disease: a translational study from men to mice

ObjectiveRecent evidence suggesting an important role of gut-derived inflammation in brain disorders has opened up new directions to explore the possible role of the gut-brain axis in neurodegenerative diseases. Given...

1532. Increased Risk of Bacterial, Fungal and Other Viral Infections During CMV Infection: Decreased Cytokine Production in Response to Toll-Like Receptor Ligands

BackgroundIn the solid-organ transplant (SOT) setting, CMV is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal and viral infections. However, the pathogenesis of this phenomenon is poorly understood. The aim of our study was to determine whether inflammatory responses to different Toll-like receptor ligands are blunted during CMV infection in SOT patients.MethodsCMV D+/R− SOT patients had blood drawn at the end of CMV prophylaxis and then weekly after onset of CMV viremia. PBMCs were extracted and incubated for 24 hours in the presence of bacterial (LPS), fungal (Zymosan [ZYM]), and viral (Resiquimod [R848]) ligands. Proinflammatory (IL1β), Th1 (IFNγ), Th2 (IL4), immunoregulatory (IL10), and chemotactic (MCP1) cytokines were measured in the supernatant by multiplex ELISA.ResultsThirty-eight SOT patients were followed for at least 9 months. Patients who developed subsequent CMV infection had lower cytokines in response to bacterial, fungal and viral ligands (LPS, ZYM, and R848) at the end of prophylaxis compared with those with no CMV infection. These results were independent of immunosuppression and peripheral blood cell counts. Specifically, these trends were significantly different with respect to IFNγ, IL1β, and IL10 production in response to LPS (P = 0.003, 0.003, and 0.039, respectively), R848 (P < 0.001, 0.039, and <0.001, respectively) and ZYM (P = 0.039, 0.003, and 0.003, respectively), as well as for MCP1 in response to R848 or ZYM (P = 0.039 for both). In the cohort with CMV infection, cytokine responses to TLR ligands were even lower during the acute CMV infection when compared with the end of prophylaxis, although this was significant only for IL10 production after R848 stimulation (P = 0.034). There was no influence of CMV viral load or duration of viremia on cytokine levels.ConclusionResponse to non-CMV antigens during CMV infection was blunted supporting the clinical observation in transplant recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. However, inherent differences in patients that are neither directly related to CMV nor to their net level of immunosuppression also contribute to this increased susceptibility, as cytokine levels at the end of prophylaxis were lower among patients with compared with those without subsequent CMV infection.Disclosures All authors: No reported disclosures.

The Possibility of an Infectious Etiology of Alzheimer Disease

Over the past three decades, there has been constant postulation regarding the infectious etiology of Alzheimer disease (AD), which in turn suggests the vital role of various infectious agents in AD-associated inflammatory pathways. Recent findings indicate anti-microbial properties of Aβ, and suggest that Aβ production and deposition in AD might be induced by infectious agents. Several types of spirochetes have been associated to dementia, cortical atrophy, and pathological and biological hallmarks of AD. A significant association between AD spirochetes and other pathogens like HSV-1 and Chlamydia pneumonia has now become well established. In neurons infected by HSV-1 showed Aβ and hyperphosphorylated Tau accumulation. The expression of pro-inflammatory molecules have been found to be enhanced by specific bacterial ligands, and viral and bacterial DNA and RNA, thus activating the immune system. Aβ has now been established as anti-microbial peptide capable of inducing pore formation, thus justifying their infection-mediated accumulation. Thus, a proper combination of anti-inflammatory, anti-viral, and antibiotic therapeutics might potentially prevent the progression of AD. Here, we discussed the potential role of bacterial, fungi, and viral infections in AD causation and progression, and the potential-associated therapies to counter the AD condition.

Mutual Regulation of TLR/NLR and CEACAM1 in the Intestinal Microvasculature: Implications for IBD Pathogenesis and Therapy.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) displays multiple activities, among which pathogen binding and angiogenesis are particularly prominent. These same functions are also exerted by Toll- and NOD-like receptors (TLRs and NLRs), which are critical mediators of innate immune responses. We investigated whether a functional inter-relationship exists between CEACAM1 and TLRs and NLRs and its potential impact on induction of intestinal angiogenesis. This hypothesis was tested using human intestinal microvascular endothelial cells, a unique cell population exposed to microbial products under physiological and pathological conditions. The results show that activation of TLR2/4, TLR4, NOD1, and NOD2 by specific bacterial ligands selectively and differentially upregulates the levels of cellular and soluble CEACAM1 produced by intestinal microvascular endothelial cells. The results also show that CEACAM1 regulates the migration, transmigration, and tube formation of these endothelial cells and mediates vessel sprouting induced by specific TLR and NLR bacterial ligands. Combined, these results demonstrate a close and reciprocal regulatory interaction between CEACAM1 and bacterial products in mediating multiple functions essential to new vessel formation in the gut mucosa. A coordinated and reciprocal interaction of CEACAM1 and microbiota-derived factors is necessary to optimize angiogenesis in the gut mucosa. This suggests that a coordination of endogenous and exogenous innate immune responses is necessary to promote intestinal angiogenesis under physiological and inflammatory conditions such as inflammatory bowel disease.

Mucosal-associated invariant and γδ T cell subsets respond to initial Mycobacterium tuberculosis infection.

Innate immune responses that control early Mtb infection are poorly understood, but understanding these responses may inform vaccination and immunotherapy strategies. Innate T cells that respond to conserved bacterial ligands such as mucosal-associated invariant T (MAIT) and γδ T cells are prime candidates to mediate these early innate responses but have not been examined in subjects who have been recently exposed to Mtb. We recruited a cohort living in the same household with an active tuberculosis (TB) case and examined the abundance and functional phenotypes of 3 innate T cell populations reactive to M. tuberculosis: γδ T, invariant NK T (iNKT), and MAIT cells. Both MAIT and γδ T cells from subjects with Mtb exposure display ex vivo phenotypes consistent with recent activation. However, both MAIT and γδ T cell subsets have distinct response profiles, with CD4+ MAIT and γδ T cells accumulating after infection. Examination of exposed but uninfected contacts demonstrates that resistance to initial infection is accompanied by robust MAIT cell CD25 expression and granzyme B production coupled with a depressed CD69 and IFNγ response. Finally, we demonstrate that MAIT cell abundance and function correlate with the abundance of specific gut microbes, suggesting that responses to initial infection may be modulated by the intestinal microbiome.

Cationic lipids as one-component vaccine adjuvants: A promising alternative to alum

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1β secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants.