A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

Pseudomonas aeruginosa Quorum Sensing Molecule N-(3-Oxododecanoyl)-L-Homoserine-Lactone Induces HLA-G Expression in Human Immune Cells.

HLA-G is a nonclassical class I human leukocyte antigen (HLA) involved in mechanisms of immune tolerance. The objective of this study was to determine whether N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL), a quorum sensing molecule produced by Pseudomonas aeruginosa, could modify HLA-G expression to control the host immune response. We evaluated the ability of 3O-C12-HSL to induce HLA-G expression in primary immune cells, monocytes (U937 and THP1), and T-cell lines (Jurkat) in vitro and analyzed the cellular pathway responsible for HLA-G expression. We studied the HLA-G promoter with a luciferase assay and interleukin-10 (IL-10) and p38/CREB signaling with enzyme-linked immunosorbent assay and immunofluorescence, respectively. We observed that 3O-C12-HSL is able to induce HLA-G expression in human monocytes and T cells. We showed that the induction of HLA-G by 3O-C12-HSL is p38/CREB and IL-10 dependent. 3O-C12-HSL treatment is able to arrest only the U937 cell cycle, possibly due to the peculiar expression of the ILT2 receptor in the U937 cell line. Our observations suggest HLA-G as a mechanism to create a protected niche for the bacterial reservoir, similar to the role of HLA-G molecules during viral infections.

High-sensitivity monoclonal antibodies specific for homoserine lactones protect mice from lethal Pseudomonas aeruginosa infections.

A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity.

Among numerous molecules found in the gut ecosystem, quorum sensing (QS) molecules represent an overlooked part that warrants highlighting. QS relies on the release of small molecules (auto-inducers) by bacteria that accumulate in the environment depending on bacterial cell density. These molecules not only are sensed by the microbial community but also interact with host cells and contribute to gut homeostasis. It therefore appears entirely appropriate to highlight the role of these molecules on the immune system in dysbiosis-associated inflammatory conditions where the bacterial populations are imbalanced. Here, we intent to focus on one of the most studied QS molecule family, namely, the type I auto-inducers represented by N-acyl-homoserine lactones (AHL). First described in pathogens such as Pseudomonas aeruginosa, these molecules have also been found in commensals and have been recently described within the complex microbial communities of the mammalian intestinal tract. In this mini-review, we will expound on this emergent field of research. We will first recall evidence on AHL structure, synthesis, receptors, and functions regarding interbacterial communication. Then, we will discuss their interactions with the host and particularly with agents of the innate and adaptive gut mucosa immunity. This will reveal how this new set of molecules, driven by microbial imbalance, can interact with inflammation pathways and could be a potential target in inflammatory bowel disease (IBD). The discovery of the general impact of these compounds on the detection of the bacterial quorum and on the dynamic and immune responses of eukaryotic cells opens up a new field of pathophysiology.

Influence of Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone on mast cells.

Quorum sensing system is a cell-to-cell communication system that plays a pivotal role in virulence expression in bacteria. Recent advances have demonstrated that the Pseudomonas aeruginosa quorum sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC(12)-HSL), exerts effects on mammalian cells and modulates host immune response. Mast cells (MCs) are strategically located in the tissues that are constantly exposed to external stimulus. Therefore, it is very much possible that 3OC(12)-HSL may interact with MCs. Little is known, however, about specific effects of 3OC(12)-HSL on MCs. To address this, we investigated the influence of 3OC(12)-HSL on cell viability, apoptosis, intracellular calcium and cytokine release in MCs. We found that at high concentrations (100 microM), 3OC(12)-HSL inhibited proliferation and induced apoptosis in P815. The 3OC(12)-HSL treatment significantly increased intracellular calcium release in both P815 and HMC-1. We also observed that 3OC(12)-HSL-induced histamine release and degranulation in HMC-1 cells. Furthermore, 3OC(12)-HSL-induced IL-6 production at lower concentrations (6.25-12.5 microM) but steadily reduced IL-6 production at high concentration (50-100 muM). These data demonstrate that P. aeruginosa 3OC(12)-HSL affects MCs function.

The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone.

The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acyl-homoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl-homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl-homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl-homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems.

Significant immunomodulatory effects of Pseudomonas aeruginosa quorum-sensing signal molecules: possible link in human sepsis.

Pathogenic bacteria use quorum-sensing signal molecules to co-ordinate the expression of virulence genes. Animal-based studies have demonstrated the immunomodulatory effects of quorum-sensing signal molecules. In the present study, we have examined the impact of these molecules on normal human immune function in vitro and compared this with immune changes in patients with sepsis where quorum-sensing signal molecules were detected in the sera of patients. Quorum-sensing signal molecules inhibited normal dendritic cell and T-cell activation and proliferation, and down-regulated the expression of co-stimulatory molecules on dendritic cells; in MLDCRs (mixed lymphocyte dendritic cell reactions), secretion of IL (interleukin)-4 and IL-10 was enhanced, but TNF-alpha (tumour necrosis factor-alpha), IFN-gamma (interferon-gamma) and IL-6 was reduced. Quorum-sensing signal molecules induced apoptosis in dendritic cells and CD4(+) cells, but not CD8(+) cells. Dendritic cells from patients with sepsis were depleted and ex vivo showed defective expression of co-stimulatory molecules and dysfunctional stimulation of allogeneic T-lymphocytes. Enhanced apoptosis of dendritic cells and differential CD4(+) Th1/Th2 (T-helper 1/2) cell apoptotic rate, and modified Th1/Th2 cell cytokine profiles in MLDCRs were also demonstrated in patients with sepsis. The pattern of immunological changes in patients with sepsis mirrors the effects of quorum-sensing signal molecules on responses of immune cells from normal individuals in vitro, suggesting that quorum-sensing signal molecules should be investigated further as a cause of immune dysfunction in sepsis.

Generation of High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones.

A number of bacteria use a class of chemical compounds called acyl-homoserine lactones (AHLs) as quorum sensing (QS) signals to coordinate their behavior at the population level, including pathogens like Pseudomonas aeruginosa. Blocking QS using antibodies is an attractive strategy for infection control as this process takes a central role in P. aeruginosa infections. Here the methods involved in the generation of high sensitivity anti-QS monoclonal antibodies from an immunized sheep phage display antibody library are described. A panel of AHL compounds conjugated to carrier proteins are used for sheep immunization and a phage display antibody library is constructed using the immune repertoire of sheep as a source of antibody genes. High sensitivity single chain antibody fragments (scFv) are isolated from the library using "smart selection strategies" and reformatted into single chain antibodies (scAbs). The resultant monoclonal antibodies: (1) recognize HSL compounds at low nanomolar concentrations; (2) have the potential to reduce virulence gene expression in P. aeruginosa; and (3) offer protection in a nematode model of infection.

Immunization with 3-oxododecanoyl-L-homoserine lactone-protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection.

Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-L-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL-carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL-BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3 x 10(6) c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-alpha in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.

Stereospecificity in allergic contact dermatitis to simple substituted methylene lactone derivatives.

The enantiomers of beta,gamma-dimethyl- and beta-methyl-alpha-methylene-gamma-butyrolactones have been synthesized stereospecifically from glutamic acid and beta-hydroxy isobutyric acid, respectively. Guinea pigs have been sensitized (Freund complete adjuvant technique) and tested to them. Both enantiomers of beta-methyl lactone as well as (+)-beta,gamma-dimethyl lactone induced enantiospecific allergic contact dermatitis (ACD); in turn, (-)-beta,gamma-dimethyl lactone showed no specificity. An interpretation is proposed.

Allergic contact dermatitis to tulips: an example of enantiospecificity.

Allergic contact dermatitis (ACD), an immunological reaction of the skin resulting from contact with reactive compounds occurring in plants was shown to the enantiospecific (animals sensitized to a compound do not react to its nonsuperimposable mirror image). Thus, when guinea pigs were experimentally sensitized to (+)-tulipalin B (a compound present in tulip bulbs) they did not react to its enantiomer, (-)-tulipalin B. This was also true for (+)- and (-)-beta-hydroxy-gamma-methyl-alpha-methylene-gamma-butyrolactones.

Allergic contact dermatitis to laurel (Laurus nobilis L.): isolation and identification of haptens.

Two methods, using methylenelactone dimethylamino adducts, were used to remove selectively alpha-methylene gamma-butyrolactones from Laurus nobilis L. extracts. Isolated lactones were identified and "treated" extracts recovered. Two guinea-pig groups were sensitized to crude extracts and "treated" extracts, respectively, and tested with primary sensitizer and with different lactones. Only the first group showed strong skin reactions to crude extracts and to the lactones. Treated extracts were shown to be anallergic.

Molecular recognition in allergic contact dermatitis. The concept of double-headed haptens.

The sensitizing capacity of double-headed haptens containing the pyrocatechol and alpha-methylene-gamma-butyrolactone groups reveals that only the pyrocatechol end is recognized as a sensitizer. Prior sensitization to the double-headed hapten provides protection against (induces immune tolerance to) further sensitization to the lactone hapten.

ZCH-2B8a, an antibody targeting actin-binding protein coronin-1a, is a potential therapeutic agent for B-lineage malignancies.

BACKGROUND: Monoclonal antibody (mAb)-based targeted therapy is one of the most promising strategies to cure cancers. MAb ZCH-2B8a (2B8a) was a novel antibody generated in our laboratory, which presented potential to be a therapeutic agent for hematologic malignancies. METHODS: We investigated the reactivity profile of 2B8a mAb, identified the targeting antigen by proteomic and genetic approaches and evaluated its potential to exert tumor cell killing. RESULTS: 2B8a antigen was strictly expressed on lymph tissues and hematopoietic cells (mainly leukocytes), and was highly expressed on B-lineage leukemia cell lines and acute lymphoblastic leukemia (ALL) cells from patients. 2B8a antibody was quickly internalized into the target cells once binding to the antigen, but was capable of killing tumor cells through complement dependent cytotoxicity. To identify the 2B8a antigen, the proteins of Raji cells were immunoprecipitated with 2B8a antibody and analyzed by mass spectrometry, which indicated that coronin-1a was a potential candidate. Then, coronin-1a gene was cloned from Raji cells, inserted into plasmid pcDNA3.1 (+), and transfected into CHO cells. The intracellular 2B8a antigen level was significantly increased in the coronin-1a transfectant cell line. CONCLUSION: 2B8a mAb is a novel antibody targeting coronin-1a, which has the potential to be a therapeutic agent for B-lineage malignancies.

Enterodiol and enterolactone modulate the immune response by acting on nuclear factor-kappaB (NF-kappaB) signaling.

Lignan-rich whole-grain cereals, beans, berries, and nuts show protective effects against a variety of chronic diseases, including cancer. Lignans are converted by intestinal microflora to enterolactone (EL) and its oxidation product enterodiol (ED). To investigate the immunomodulatory effect of EL and ED in human cells, peripheral blood lymphocytes were treated with increasing physiologically relevant concentrations of EL and ED (0-1000 microM) and stimulated with lipopolysaccharide (LPS) and anti-CD3 plus anti-CD28 monoclonal antibodies. A dose-related inhibition of cell proliferation and cytokine production was observed, with EL being the most active. Molecular investigations in THP-1 cells showed that both EL and ED prevented inhibitory-kappaB (I-kappaB) degradation and nuclear factor-kappaB (NF-kappaB) activation, which in turn resulted in decreased tumor necrosis factor-alpha (TNF-alpha) production. EL and ED were also able to pass the intestinal barrier and modulate cytokine production. The findings of the present study reveal potential mechanisms that could explain some in vivo beneficial effects of lignans.

Bacterial quorum sensing: a new target for anti-infective immunotherapy.

BACKGROUND: Cell-to-cell communication via exchange of small molecules, 'autoinducers', is a widespread phenomenon among Gram-negative and -positive bacteria. This intercellular signaling that synchronizes population-wide gene expression in a cell-density-dependent manner is termed 'quorum sensing' (QS). The discovery that Gram-negative bacteria employ non-peptide structures, N-acyl homoserine lactones, to globally regulate production of secondary metabolites and proteins, initiated a new area of research. Subsequently, other quorum-sensing systems and small signaling molecules were identified. With the emergence of antibiotic-resistant bacteria, most prominently methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, new approaches for combating infections are needed. Inhibition of QS results in attenuation of virulence rather than direct killing of microbes. OBJECTIVE: We highlight current trends in preventing bacterial infections using quorum-quenching strategies. METHODS: We mainly focus on P. aeruginosa and S. aureus and their QS systems as targets for intervention. RESULTS/CONCLUSION: New research strongly suggests that QS systems represent attractive targets for discovery of novel anti-infective agents, including immunotherapeutic strategies.

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion.

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.

The quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) enhances the host defence by activating human polymorphonuclear neutrophils (PMN).

The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.