Immunogenecity of Modified Alkane Polymers Is Mediated through TLR1/2 Activation

Background: With the advancement of biomedical technology, artificial materials have been developed to replace diseased, damaged or nonfunctional body parts. Among such materials, ultra high molecular weight alkane or modified alkyl polymers have been extensively used in heart valves, stents, pacemakers, ear implants, as well as total joint replacement devices. Although much research has been undertaken to design the most non-reactive biologically inert polyethylene derivatives, strong inflammatory responses followed by rejection and failure of the implant have been noted. Methodology/Principal Findings: Purification of the alkane polymers from the site of inflammation revealed extensive ‘‘in vivo’ ’ oxidation as detected by fourier transformed infra-red spectroscopy. Herein, we report the novel observation that oxidized alkane polymers induced activation of TLR1/2 pathway as determined by ligand dependent changes in intrinsic tyrosine fluorescence intensity and NF-kB luciferase gene assays. Oxidized polymers were very effective in activating dendritic cells and inducing secretion of pro-inflammatory cytokines. Molecular docking of the oxidized alkanes designated ligand specificity and polymeric conformations fitting into the TLR1/2 binding grooves

Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses

Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life

Vibrio cholerae Proteome-Wide Screen for Immunostimulatory Proteins Identifies Phosphatidylserine Decarboxylase as a Novel Toll-Like Receptor 4 Agonist

Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called “EPSIA”, Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFα and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced ∼40% and ∼15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant

The synthetic bacterial lipopeptide Pam3CSK4 modulates respiratory syncytial virus infection independent of TLR activation

Respiratory syncytial virus (RSV) is an important cause of acute respiratory disease in infants, immunocompromised subjects and the elderly. However, it is unclear why most primary RSV infections are associated with relatively mild symptoms, whereas some result in severe lower respiratory tract infections and bronchiolitis. Since RSV hospitalization has been associated with respiratory bacterial co-infections, we have tested if bacterial Toll-like receptor (TLR) agonists influence RSVA2- GFP infection in human primary cells or cell lines. The synthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), the prototype ligand for the heterodimeric TLR1/TLR2 complex, enhanced RSV infection in primary epithelial, myeloid and lymphoid cells. Surprisingly, enhancement was optimal when lipopeptides and virus were added simultaneously, whereas addition of Pam3CSK4 immediately after infection had no effect. We have identified two structurally related lipopeptides without TLR-signaling capacity that also modulate RSV infection, whereas Pam3CSK4-reminiscent TLR1/2 agonists did not, and conclude that modulation of infection is independent of TLR activation. A similar TLR-independent enhancement of infection could also be demonstrated for wild-type RSV strains, and for HIV-1, measles virus and human metapneumovirus. We show that the effect of Pam3CSK4 is primarily mediated by enhanced binding of RSV to its target cells. The Npalmitoylated cystein

Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.</p> <p>Results</p> <p>The study presents association between TLR gene mutations and increased susceptibility to <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further <it>in silico </it>analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.</p> <p>Conclusion</p> <p>The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^thamino acid position in LRR motif (TLR1–LRR10) and 4^thresidue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.</p

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections

Lung epithelium as a sentinel and effector system in pneumonia – molecular mechanisms of pathogen recognition and signal transduction

Pneumonia, a common disease caused by a great diversity of infectious agents is responsible for enormous morbidity and mortality worldwide. The bronchial and lung epithelium comprises a large surface between host and environment and is attacked as a primary target during lung infection. Besides acting as a mechanical barrier, recent evidence suggests that the lung epithelium functions as an important sentinel system against pathogens. Equipped with transmembranous and cytosolic pathogen-sensing pattern recognition receptors the epithelium detects invading pathogens. A complex signalling results in epithelial cell activation, which essentially participates in initiation and orchestration of the subsequent innate and adaptive immune response. In this review we summarize recent progress in research focussing on molecular mechanisms of pathogen detection, host cell signal transduction, and subsequent activation of lung epithelial cells by pathogens and their virulence factors and point to open questions. The analysis of lung epithelial function in the host response in pneumonia may pave the way to the development of innovative highly needed therapeutics in pneumonia in addition to antibiotics

Gram-positive bacterial lipoglycans based on a glycosylated diacylglycerol lipid anchor are microbe-associated molecular patterns recognized by TLR2

Innate immune recognition is the first line of host defense against invading microorganisms. It is a based on the detection, by pattern recognition receptors (PRRs), of invariant molecular signatures that are unique to microorganisms. TLR2 is a PRR that plays a major role in the detection of Gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. These microbe-associated molecular patterns (MAMPs) display a structure based on a lipid anchor, being either an acylated cysteine, a glycosylated diacylglycerol or a mannosyl-phosphatidylinositol respectively, and having in common a diacylglyceryl moiety. A fourth class of macroamphiphile, namely lipoglycans, whose lipid anchor is made, as for lipoteichoic acids, of a glycosylated diacylglycerol unit rather than a mannosyl-phosphatidylinositol, is found in Gram-positive bacteria and produced by certain Actinobacteria, including Micrococcus luteus, Stomatococcus mucilaginosus and Corynebacterium glutamicum. We report here that these alternative lipoglycans are also recognized by TLR2 and that they stimulate TLR2-dependant cytokine production, including IL-8, TNF-α and IL-6, and cell surface co-stimulatory molecule CD40 expression by a human macrophage cell line. However, they differ by their co-receptor requirement and the magnitude of the innate immune response they elicit. M. luteus and S. mucilaginosus lipoglycans require TLR1 for recognition by TLR2 and induce stronger responses than C. glutamicum lipoglycan, sensing of which by TLR2 is dependent on TLR6. These results expand the repertoire of MAMPs recognized by TLR2 to lipoglycans based on a glycosylated diacylglycerol lipid anchor and reinforce the paradigm that macroamphiphiles based on such an anchor, including lipoteichoic acids and alternative lipoglycans, induce TLR2-dependant innate immune responses