Birgit Prior

Induction of Neutrophil Chemotaxis by the Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

ABSTRACT Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

Tasting Pseudomonas aeruginosa Biofilms: Human Neutrophils Express the Bitter Receptor T2R38 as Sensor for the Quorum Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone.

Bacteria communicate with one another via specialized signaling molecules, known as quorum sensing molecules or autoinducers. The Pseudomonas aeruginosa-derived quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (AHL-12), however, also activates mammalian cells. As shown previously, AHL-12-induced chemotaxis, up-regulated CD11b expression, and enhanced phagocytosis of polymorphonuclear neutrophils. Circumstantial evidence concurred with a receptor for AHL-12, which has been elusive so far. We now investigated the bitter receptor T2R38 as a potential candidate. Although identified as a taste receptor, extragustatory cells express T2R38, for example, epithelial cells in the lung. We now detected T2R38 in peripheral blood neutrophils, monocytes, and lymphocytes. T2R38 is not only found on the cell membrane but also intracellular. In neutrophils, T2R38 was located in vesicles with characteristics of lipid droplets, and super-resolution microscopy showed a co-localization with the lipid droplet membrane. Neutrophils take up AHL-12, and it co-localized with T2R38 as seen by laser scan microscopy. Binding of AHL-12 to T2R28 was confirmed by pull-down assays using biotin-coupled AHL-12 as bait. A commercially available antibody to T2R38 inhibited binding of AHL-12 to neutrophils, and this antibody by itself stimulated neutrophils, similarly to AHL-12. In conclusion, our data provide evidence for expression of functional T2R38 on neutrophils, and are compatible with the notion that T2R38 is the receptor for AHL-12.

Immune defense against S. epidermidis biofilms: components of the extracellular polymeric substance activate distinct bactericidal mechanisms of phagocytic cells

Bacteria, organized in biofilms, are a common cause of relapsing or persistent infections and the ultimate cause of implant-associated osteomyelitis. Bacterial biofilms initiate a prominent local inflammatory response with infiltration of polymorphonuclear neutrophils (PMN), the main protagonists of the local innate host defense against bacteria. In our previous work we found that PMN recognize and adhere to biofilms, and that phagocytosis and degranulation of bactericidal substances, such as lactoferrin, were initiated. In contrast to the situation with planktonic bacteria, opsonization of biofilms with immunoglobulin and complement was not required for PMN activation, suggesting that biofilms contain signaling components for PMN. In the present study we identified in the bacteria-free extracellular substance of Staphylococcus epidermidis biofilms protein fractions that activated PMN in vitro.

T lymphocytes in acute bacterial infection: increased prevalence of CD11b+ cells in the peripheral blood and recruitment to the infected site

T‐cell activation, particularly of CD8+ cells, is invariably associated with viral infections. We now provide evidence for the activation of T cells in patients with localized bacterial soft tissue infections. During acute disease we detected in the peripheral blood of these patients, small though conspicuous populations of CD4+ CD28+ CD11b+ and CD8+ CD28+ CD11b+ cells, indicative of an expansion of effector T cells. Moreover, we identified CD4+ and CD8+ cells at the infected site, in addition to highly activated polymorphonuclear neutrophils (PMN). In keeping with their role as first‐line defence, PMN were preponderant, but T cells amounted to 20% of the infiltrated cells. The majority of the infiltrated T cells expressed CXCR6, a homing receptor for non‐lymphoid tissue. The infiltrated T cells produced interferon‐γ (IFN‐γ), while the peripheral blood cells obtained at the same time did not. In conclusion, in response to localized bacterial infections, T cells are activated and recruited to the infected site. We propose that these T cells, e.g. by producing IFN‐γ, enhance the efficiency of the infiltrated phagocytic cells, particularly of the PMN, thereby supporting the local host defence.

Activation of Neutrophils by the Extracellular Polymeric Substance of S.Epidermidis Biofilms is Mediated by The Bacterial Heat Shock Protein Groel

In patients with implant-associated osteomyelitis, formation of bacterial biofilms on osteosynthesis materials or endoprosthetic devices is considered the “common cause of persistent infection”. As we showed previously for Staphylococcus aureus or S. epidermidis implant infection, activation of the local host response with infiltration of phagocytic cells and release of bactericidal and potentially cytotoxic entities, as well as production of proinflammatory and osteolysis-inducing cytokines contributes greatly to the persistence of the infection and the ensuing tissue damage with bone degradation. In this study we addressed the question, how phagocytic cells, particularly neutrophils, recognise bacterial biofilms. We found that the protein fraction of the biofilm extracellular substance (EPS) activated neutrophils: up-regulation of defence-relevant functions, among others increased surface expression of the adhesion proteins CD11b and CD66b was seen, as was production of oxygen-radicals. Subsequently, we identified the bacterial heat-shock protein GroEL as a likely candidate: GroEL is present in the EPS; depletion of GroEL from the EPS reduced neutrophil activation, culture of neutrophils with recombinant GroEL up-regulated CD11b and CD66b surface expression, and induced oxygen radical production. According to the literature GroEL and its human homologue heat shock protein (HSP)60 may bind to different surface receptors, including toll-like receptor (TLR)4 and scavenger receptors. Under our experimental conditions, the TLR4 pathway appeared to be crucial for the EPS-induced up-regulation of CD11b and CD66b, but not for induction of oxygen-radical production; suggesting involvement of additional receptors. In conclusion, we identified within the bacterial biofilm the bacterial heat-shock protein GroEL as an activator of the local innate immune response.

Human polymorphonuclear neutrophils express RANK and are activated by its ligand, RANKL

The receptor activator of NF‐κB (RANK) is especially well studied in the context of bone remodeling, and RANK and its ligand, RANKL, are key molecules in the induction of bone resorbing osteoclasts. We now report that polymorphonuclear neutrophils (PMNs) contain preformed RANK, stored in secretory vesicles and in specific granules. Upon stimulation of PMNs in vitro, RANK was translocated to the cell membrane. In patients with persistent bacterial infections, RANK surface expression was enhanced compared with that of healthy individuals. The functional activity of RANK was assessed by determining migration of PMNs toward RANKL. A time‐ and dose‐dependent migration was seen, leading to the conclusion that RANK on PMNs is functional. We presume that regulated RANK expression contributes to the fine tuning of PMN migration, for example, on and through inflamed endothelium that is known to express RANKL.

The Pseudomonas quinolone signal (PQS) stimulates chemotaxis of polymorphonuclear neutrophils.

Cross-talk between bacteria and mammalian cells is increasingly recognized as an important factor, especially during chronic infections. In particular, the interaction of extracellular bacterial signaling molecules with cells of the innate immune response is of special interest. In this context, we investigated whether the Pseudomonas quinolone signal (PQS) which is a quorum sensing molecule produced by bacteria and participates in biofilm formation and virulence has any influence on polymorphonuclear neutrophils (PMN), the cells of the “first line defense” against bacterial infections. We found that PQS did not enhance the bactericidal activity of PMN and did not induce apoptosis at concentrations up to 100 μM. However, PQS stimulated chemotaxis of PMN in doses of 10-100 μM. This PQS-dependent chemotaxis could be inhibited with SB203580 which blocks MAPkinase p38, suggesting a signaling pathway similar to AHL-12 induction. Using bacterial cell culture supernantants of Pseudomonas aeruginosa wild-type cells and a PQS-deficient mutant strain support the in vivo relevance of these findings. Since PQS is produced in the early phase of biofilm formation, PMN infiltration could be timely enough to eradicate bacteria before biofilm formation is completed, which confers the bacteria with a relative resistance to host defense mechanisms.

Activation of T Lymphocytes in Response to Persistent Bacterial Infection: Induction of CD11b and of Toll-Like Receptors on T Cells.

T cell activation is invariably associated with virus infections, but activation of T cells is also noted, for example, in patients with persistent bacterial infections with intracellular pathogens or localised bacterial biofilms. The latter is characterised by a destructive inflammatory process. Massive infiltration of leukocytes, predominantly of polymorphonuclear neutrophils (PMNs) and of T lymphocytes, is seen. While PMN influx into sites of bacterial infection is in line with their role as “first-line defence” a role of T cells in bacterial infection has not yet been delineated. We now found evidence for activation and expansion of peripheral blood T cells and an upregulation of Toll-like receptors 1, 2, and 4 on small portions of T cells. T cells recovered from the infected site were terminally differentiated and produced interferon gamma, a cytokine known to enhance functions of phagocytic cells, leading to the conclusion that infiltrated T cells support the local immuner defence.

Galectin-3 inhibits the chemotaxis of human polymorphonuclear neutrophils in vitro.

In the recent years, the participation of the animal lectin galectin (gal)-3 in inflammation and in host defence mechanisms was extensively studied. In vivo studies implied - among others - a role of gal-3 in the recruitment of polymorphonuclear neutrophils (PMN) to sites of bacterial infection. In that context, we asked the question whether gal-3 was chemotactic for PMN. Functional assays revealed that gal-3 was not chemotactic for PMN, but that it inhibited the spontaneous migration and the chemotaxis of PMN towards complement C5a, interleukin (IL)-8, or ATP. Moreover, gal-3 inhibited the shape change and the actin polymerisation of PMN that occurs in response to C5a or IL-8. By use of FITC-labelled gal-3, we found that it attached rapidly to the PMN membrane in a lactose-sensitive manner. In response to gal-3 the MAP kinase p38 was phosphorylated. This kinase is crucial for the migration of PMN towards end-target chemokines, such as C5a, and is activated in response to C5a or IL-8. When PMN were preincubated with gal-3, the C5a-induced p38 phosphorylation was transiently enhanced, but eventually down-modulated. We conclude that by interfering with the chemokine-induced p38 phosphorylation gal-3 inhibits chemotaxis of PMN.

Neutrophil-derived MRP-14 is up-regulated in infectious osteomyelitis and stimulates osteoclast generation

Bone infections of patients with joint replacement by endoprosthesis (so called “periprosthetic joint infection”) pose a severe problem in the field of orthopedic surgery. The diagnosis is often difficult, and treatment is, in most cases, complicated and prolonged. Patients often require an implant exchange surgery, as the persistent infection and the accompanying inflammation lead to tissue damage with bone degradation and consequently, to a loosening of the implant. To gain insight into the local inflammatory process, expression of the proinflammatory cytokine MRP‐14, a major content of neutrophils, and its link to subsequent bone degradation was evaluated. We found MRP‐14 prominently expressed in the affected tissue of patients with implant‐associated infection, in close association with the chemokine CXCL8 and a dense infiltrate of neutrophils and macrophages. In addition, the number of MRP‐14‐positive cells correlated with the presence of bone‐resorbing osteoclasts. MRP‐14 plasma concentrations were significantly higher in patients with implant‐associated infection compared with patients with sterile inflammation or healthy individuals, advocating MRP‐14 as a novel diagnostic marker. A further biologic activity of MRP‐14 was detected: rMRP‐14 directly induced the differentiation of monocytes to osteoclasts, thus linking the inflammatory response in implant infections with osteoclast generation, bone degradation, and implant loosening.