Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.     

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.     

Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes

Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.     

Profiling Carbohydrate-Receptor Interaction with Recombinant Innate Immunity Receptor-Fc Fusion Proteins

The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity. However, due to the complexity of polysaccharides and their various sources from medicinal fungi, quantitative analysis of medicinal polysaccharide extracts with regard to their functions represents a major challenge. To profile carbohydrate-immune receptor interactions, the extracellular domains of 17 receptors were cloned as Fc-fusion proteins, such that their interactions with immobilized polysaccharides could be probed in an enzyme-linked immunosorbent assay. The results show that several innate immune receptors, including Dectin-1, DC-SIGN, Langerin, Kupffer cell receptor, macrophage mannose receptor, TLR2, and TLR4, interact with the polysaccharide extracts from G. lucidum (GLPS). This analysis revealed distinct polysaccharide profiles from different sources of medicinal fungi, and the innate immune receptor-based enzyme-linked immunosorbent assay described here can serve as a high-throughput profiling method for the characterization and quality control of medicinal polysaccharides. It also provides a means to dissect the molecular mechanism of medicinal polysaccharide-induced immunomodulation events.     

Endotoxin,Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 μM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.Neisseria meningitidis (meningococci) is a gram-negative, aerobic diplococci that is an obligate human pathogen. Infections caused by N. meningitidis are an important cause of morbidity and mortality worldwide. Meningococci colonize the nasopharyngeal mucosa of approximately 10% of healthy individuals but can cross epithelial and endothelial cell barriers and enter the bloodstream, causing septicemia, with mortality rates of 20 to 50% (4). Meningitis occurs when bacteria transverse the blood cerebrospinal fluid, causing a fatal outcome in 15 to 20% of infected patients. Bacterial adherence is initially mediated by type IV pili with host cell receptors. PilT is the molecular motor responsible for pili retraction, which mediates a tight interaction. An important virulence factor of N. meningitidis is the endotoxin lipooligosaccharide (LOS), which is located in the bacterial outer cell membrane. Meningococcal LOS is composed of a conserved inner core of membrane-associated lipid A (16) to which variable α- and β-chains attach (13).As one of many first lines of defense against invading pathogens like Neisseria bacteria, epithelial cells produce antimicrobial peptides (AMPs). These peptides are effector molecules for the innate immune response, with both direct antimicrobial activity and a broad spectrum of immunomodulatory functions (18, 22). LL-37 is the single known human cathelicidin and is expressed in various immune cells as well as in epithelial cells of inflamed skin, mouth, tongue, esophagus, and lungs. It has been shown that LL-37 interacts with bacterial membranes through both electrostatic and hydrophobic effects. It remains unknown whether LL-37 ultimately kills bacteria by formation of torroidal pores as described by Henzler Wildman et al. (11) or by detergent-like disintegration of the membrane via the carpet model as described by Shai (24), but increasing membrane permeability, osmotic swelling, and loss of the vital proton gradient are important characteristics of the killing process (21). Membrane interactions of LL-37 (and other AMPs) appear to be highly selective for the negative surface charge on prokaryotic membranes. However, it has been shown by Tzeng et al. (28) that meningococci regulate AMP attack via mechanisms that include lipid A modification and an efflux pump. LL-37 toxicity for eukaryotic cells remains low, probably because eukaryotic cell membranes do not have a negative net charge (31).In order to further investigate the bactericidal activity of LL-37, various Neisseria strains were examined for their susceptibility to LL-37. Our results show that LL-37 exhibits potent killing activity against N. meningitidis, whereas adhesion to host cells, LOS, and the capsule was found to contribute to resistance to LL-37. Neisseria bacteria can respond to sublethal doses of LL-37 to increase capsule production.     

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P相似文献   

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.     

Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5

Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.     

In vitro and in vivo evaluation of intrinsic immunogenicity of reporter and insulin gene therapy plasmids

BACKGROUND: Plasmid DNA vectors offer the potential of safe gene therapy avoiding viral vector-mediated toxicity and immunogenicity. As plasmid DNA is bacterial in origin, presence of bacterial lipopolysaccharide (LPS) or unmethylated CpG dinucleotides may stimulate host innate immunity. METHODS: Primary cultures of mouse and rat dendritic cells were established and incubated with bacterial lipopolysaccharide; immunostimulatory CpG oligodeoxynucleotide; control GpC oligodeoxynucleotide; and a range of (pVR1012) plasmids encoding transgenes with increasing CpG content (wild-type and mutant human preproinsulin; non-mammalian eukaryotic eGFP reporter gene; and bacterial beta-galactosidase reporter gene). IL-12 secretion was assayed to determine in vitro plasmid immunogenicity. Local inflammatory response following intramuscular injection of these plasmids, with or without a non-ionic carrier SP1017, was characterised in vivo. RESULTS: Dose-responsive LPS and CpG stimulation of IL-12 secretion from dendritic cells was demonstrated. All plasmids induced significant IL-12 secretion in comparison to control unstimulated cells. The beta-galactosidase plasmid had highest CpG content and induced significantly higher IL-12 secretion than constructs containing a eukaryotic transgene. Injection of rat muscle with the beta-galactosidase construct induced greater inflammatory response than human preproinsulin constructs. This was further enhanced by SP1017. At 2 days post-injection, monocyte/macrophage injection site infiltration predominated with CD8-positive lymphocytes predominating at 7 days. There was no evidence of transgene expression in infiltrating immune cells. CONCLUSIONS: Dendritic cell immunostimulation may be employed as an in vitro bioassay of innate immune response to plasmid DNA vectors during evaluation for clinical gene therapy.     

Time course and mechanistic analysis of human umbilical cord perivascular cell mitigation of lipopolysaccharide-induced systemic and neurological inflammation

Background aimsBecause of their potent immunomodulatory and anti-inflammatory properties, mesenchymal stromal cells are a major focus in the field of stem cell therapy. However, the precise mechanisms underlying this are not entirely understood. Human umbilical cord perivascular cells (HUCPVCs) are a promising cell therapy candidate. This study was designed to evaluate the time course and mechanisms by which HUCPVCs mitigate lipopolysaccharide (LPS)-induced systemic and neurological inflammation in immunocompetent mice. To explore the underlying mechanisms, the authors investigated the biodistribution and fate of HUCPVCs.MethodsMale C57BL/6 mice were randomly allocated to four groups: control, LPS, HUCPVCs or LPS + HUCPVCs. Quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and cytokine arrays were used to assess changes in pro-inflammatory mediators systemically and in the brain. Depressive-like behavioral changes were evaluated via a forced swim test. Quantum dot (qDot) labeling and immunohistochemistry were used to assess the biodistribution and fate of HUCPVCs and interactions with recipient innate immune cells.ResultsA single intravenously delivered dose of HUCPVCs significantly reduced the systemic inflammation induced by LPS within the first 24 h after administration. HUCPVC treatment abrogated the upregulated expression of pro-inflammatory genes in the hippocampus and cortex and attenuated depressive-like behavior induced by LPS treatment. Biodistribution analysis revealed that HUCPVC-derived qDots rapidly accumulated in the lungs and demonstrated limited in vivo persistence. Furthermore, qDot signals were associated with major recipient innate immune cells and promoted a shift in macrophages toward a regulatory phenotype in the lungs.ConclusionsOverall, this study demonstrates that HUCPVCs can successfully reduce systemic and neurological inflammation induced by LPS within the first 24 h after administration. Biodistribution and fate analyses suggest a critical role for the innate immune system in the HUCPVC-based immunomodulation mechanism.     

Galleria mellonella apolipophorin III – an apolipoprotein with anti-Legionella pneumophila activity

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.     

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag⁺/Cl^? colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔH_ITC upon interacting with free Ag⁺ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag⁺ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag⁺/Cl^? colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag⁺/Cl^? colloids. Biomineralization of Ag⁺/Cl^? colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.     

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.     

Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kappaB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kappaB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to na?ve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kappaB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.     

Fatty Acid Binding Proteins FABP9 and FABP10 Participate in Antibacterial Responses in Chinese Mitten Crab,Eriocheir sinensis

Invertebrates rely solely on the innate immune system for defense against pathogens and other stimuli. Fatty acid binding proteins (FABP), members of the lipid binding proteins superfamily, play a crucial role in fatty acid transport and lipid metabolism and are also involved in gene expression induced by fatty acids. In the vertebrate immune system, FABP is involved in inflammation regulated by fatty acids through its interaction with peroxidase proliferator activate receptors (PPARs). However, the immune functions of FABP in invertebrates are not well characterized. For this reason, we investigated the immune functionality of two fatty acid binding proteins, Es-FABP9 and Es-FABP10, following lipopolysaccharide (LPS) challenge in the Chinese mitten crab (Eriocheir sinensis). An obvious variation in the expression of Es-FABP9 and Es-FABP10 mRNA in E. sinensis was observed in hepatopancreas, gills, and hemocytes post-LPS challenge. Recombinant proteins rEs-FABP9 and rEs-FABP10 exhibited distinct bacterial binding activity and bacterial agglutination activity against Escherichia coli and Staphylococcus aureus. Furthermore, bacterial growth inhibition assays demonstrated that rEs-FABP9 responds positively to the growth inhibition of Vibrio parahaemolyticuss and S. aureus, while rEs-FABP10 responds positively to the growth inhibition of Aeromonas hydrophila and Bacillus subtilis. Coating of agarose beads with recombinant rEs-FABP9 and rEs-FABP10 dramatically enhanced encapsulation of the beads by crab hemocytes in vitro. In conclusion, the data presented here demonstrate the participation of these two lipid metabolism-related proteins in the innate immune system of E. sinensis.     

IL-1R-associated kinase 4 is required for lipopolysaccharide-induced activation of APC

The bacterial product LPS is a critical stimulus for the host immune system in the response against the corresponding bacterial infection. LPS provides an activation stimulus for macrophages and a maturation signal for dendritic cells to set up innate and adaptive immune responses, respectively. The signaling cascade of myeloid differentiation factor 88-->IL-1R-associated kinase (IRAK)-->TNFR-associated factor 6 has been implicated in mediating LPS signaling. In this report, we studied the function of IRAK-4 in various LPS-induced signals. We found that IRAK-4-deficient cells were severely impaired in producing some IFN-regulated genes as well as inflammatory cytokines in response to LPS. Among the critical downstream signaling pathways induced by LPS, NF-kappaB activation but not IFN regulatory factor 3 or STAT1 activation was defective in cells lacking IRAK-4. IRAK-4 was also required for the proper maturation of dendritic cells by LPS stimulation, particularly in terms of cytokine production and the ability to stimulate Th cell differentiation. Our results demonstrate that IRAK-4 is critical for the LPS-induced activations of APCs.     

Immature human dendritic cells infected with Leishmania infantum are resistant to NK-mediated cytolysis but are efficiently recognized by NKT cells

Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-gamma. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania.     

Prenylation: From bacteria to eukaryotes

For their protection from host cell immune defense, intracellular pathogens of eukaryotic cells developed a variety of mechanisms, including secretion systems III and IV which can inject bacterial effectors directly into eukaryotic cells. These effectors may function inside the host cell and may be posttranslationally modified by host cell machinery. Recently, prenylation was added to the list of possible posttranslational modifications of bacterial proteins. In this work we describe the current state of the knowledge about the prenylation of eukaryotic and prokaryotic proteins and prenylation inhibitors. The bioinformatics analyses suggest the possibility of prenylation for a number of Francisella genus proteins.     

The effect of composition of the core region of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 lipopolysaccharides on the surface properties of cells

The pH dependences of electrokinetic potentials (EKP) of the cells of two Escherichia coli K-12 strains (D21 and JM 103) with known lipopolysaccharide (LPS) core composition have been determined by the method of microelectrophoresis. At pH 4.6–5.2, the negative surface charge of the cells with Re core LPS was reliably higher. It was shown that the interaction of bacteria with lysozyme results in a decrease of optical density of suspensions due to higher sensitivity of the cells with complete LPS core to hypotonic shock. LPS release from bacterial cell wall depended also on bacterial LPS core composition and increased with LPS core extension. Electrokinetic measurements and the study of the interaction of cells with lysozyme suggest that higher negative surface charge of E. coli JM 103 cells (Re type LPS) is associated with higher quantity and density of LPS packing in the cell wall as compared with the cells of E. coli D21 (Ra type LPS).     

Mechanical Stretch Inhibits Lipopolysaccharide-induced Keratinocyte-derived Chemokine and Tissue Factor Expression While Increasing Procoagulant Activity in Murine Lung Epithelial Cells

Previous studies have shown that the innate immune stimulant LPS augments mechanical ventilation-induced pulmonary coagulation and inflammation. Whether these effects are mediated by alveolar epithelial cells is unclear. The alveolar epithelium is a key regulator of the innate immune reaction to pathogens and can modulate both intra-alveolar inflammation and coagulation through up-regulation of proinflammatory cytokines and tissue factor (TF), the principal initiator of the extrinsic coagulation pathway. We hypothesized that cyclic mechanical stretch (MS) potentiates LPS-mediated alveolar epithelial cell (MLE-12) expression of the chemokine keratinocyte-derived cytokine (KC) and TF. Contrary to our hypothesis, MS significantly decreased LPS-induced KC and TF mRNA and protein expression. Investigation into potential mechanisms showed that stretch significantly reduced LPS-induced surface expression of TLR4 that was not a result of increased degradation. Decreased cell surface TLR4 expression was concomitant with reduced LPS-mediated NF-κB activation. Immunofluorescence staining showed that cyclic MS markedly altered LPS-induced organization of actin filaments. In contrast to expression, MS significantly increased LPS-induced cell surface TF activity independent of calcium signaling. These findings suggest that cyclic MS of lung epithelial cells down-regulates LPS-mediated inflammatory and procoagulant expression by modulating actin organization and reducing cell surface TLR4 expression and signaling. However, because LPS-induced surface TF activity was enhanced by stretch, these data demonstrate differential pathways regulating TF expression and activity. Ultimately, loss of LPS responsiveness in the epithelium induced by MS could result in increased susceptibility of the lung to bacterial infections in the setting of mechanical ventilation.