The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

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title: Lyme disease: A tick-borne spirochetosis
abstract: A treponema-like spirochete was detected in and isolated from adult Ixodes dammini, the incriminated tick vector of Lyme disease. Causally related to the spirochetes may be long-lasting cutaneous lesions that appeared on New Zealand White rabbits 10 to 12 weeks after infected ticks fed on them. Samples of serum from patients with Lyme disease were shown by indirect immunofluorescence to contain antibodies to this agent. It is suggested that the newly discovered spirochete is involved in the etiology of Lyme disease.

title: Lyme disease.
abstract: Within the last decade, Lyme borreliosis has emerged as a complex new infection whose distribution is worldwide. The disorder is caused by a recently recognized spirochete, B. burgdorferi, transmitted by ticks of the I. ricinus complex. Certain species of mice are critical in the life cycle of the spirochete, and deer appear to be crucial to the tick. Although the disorder's basic outlines are similar everywhere, there are regional variations in the causative spirochete, animal hosts, and clinical manifestations of the illness. In the United States, Lyme disease commonly begins in summer with a characteristic skin lesion, erythema migrans, accompanied by flu-like or meningitis-like symptoms. Weeks or months later, the patients may have neurologic or cardiac abnormalities, migratory musculoskeletal pain, or arthritis, and more than a year after onset, some patients have chronic joint, skin, or neurologic abnormalities. After the first several weeks of infection, almost all patients have a positive antibody response to the spirochete, and serologic determinations are currently the most practical laboratory aid in diagnosis. Treatment with appropriate antibiotics is usually curative, but longer courses of therapy are often needed later in the illness, and some patients may not respond.

title: Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis.
abstract: Borrelia burgdorferi sensu lato has been subdivided into three genospecies: B. burgdorferi sensu stricto, B. garinii, and B. burgdorferi group VS461. Sixty-eight isolates cultured from patients and 26 strains from ticks were characterized with use of SDS-PAGE, western blotting, and rRNA gene restriction analysis. Fifty-seven of 58 strains obtained from the skin of 70 patients who had erythema migrams or acrodermatitis chronica atrophicans were of group VS461, whereas the genotype of the remaining strain was unidentifiable. Of 10 strains cultured from CSF (n = 3) and skin (n = 7) of 20 patients with extracutaneous symptoms of Lyme borreliosis, nine were B. garinii and one was B. burgdorferi sensu stricto. Of these 20 patients, 17 had neuroborreliosis, one had arthritis and carditis, one had myalgia, and one had erythema and arthralgia. All 26 isolates from ticks were of group VS461. In conclusion, infections due to group VS461 and B. garinii are associated with cutaneous and extracutaneous symptoms, respectively. Our findings suggest that B. burgdorferi genotypes have different pathogenic potentials.

title: Lyme disease.
abstract: Within the last decade, Lyme borreliosis has emerged as a complex new infection whose distribution is worldwide. The disorder is caused by a recently recognized spirochete, B. burgdorferi, transmitted by ticks of the I. ricinus complex. Certain species of mice are critical in the life cycle of the spirochete, and deer appear to be crucial to the tick. Although the disorder's basic outlines are similar everywhere, there are regional variations in the causative spirochete, animal hosts, and clinical manifestations of the illness. In the United States, Lyme disease commonly begins in summer with a characteristic skin lesion, erythema migrans, accompanied by flu-like or meningitis-like symptoms. Weeks or months later, the patients may have neurologic or cardiac abnormalities, migratory musculoskeletal pain, or arthritis, and more than a year after onset, some patients have chronic joint, skin, or neurologic abnormalities. After the first several weeks of infection, almost all patients have a positive antibody response to the spirochete, and serologic determinations are currently the most practical laboratory aid in diagnosis. Treatment with appropriate antibiotics is usually curative, but longer courses of therapy are often needed later in the illness, and some patients may not respond.

title: Tick-host-pathogen interactions in Lyme borreliosis.
abstract: Borrelia burgdorferi, the spirochetal agent of Lyme borreliosis, is predominantly transmitted by Ixodes ticks. Spirochetes have developed many strategies to adapt to the different environments that are present in the arthropod vector and the vertebrate host. This review focuses on B. burgdorferi genes that are preferentially expressed in the tick and the vertebrate host, and describes how selected gene products facilitate spirochete survival throughout the enzootic life cycle. Interestingly, B. burgdorferi also enhances expression of specific Ixodes scapularis genes, such as TROSPA and salp15. The importance of these genes and their products for B. burgdorferi survival within the tick, and during the transmission process, will also be reviewed. Moreover, we discuss how such vector molecules could be used to develop vector-antigen-based vaccines to prevent the transmission of B. burgdorferi and, potentially, other arthropod-borne microbes.

title: Fibrinolysis and host response in bacterial infections.
abstract: The plasminogen activation system is part of the fibrinolysis which is tightly regulated and protected against dysfunction by various activators and inhibitors. However, microorganisms including bacteria, fungi and also parasites have been proven to interact in a specific manner with components of the fibrinolytic pathways. Pathogenic bacteria are capable to subvert the function of proteases, activators or inhibitors for their own benefits including dissemination within the host and evasion of host inflammatory immune response. Here, we provide a state of the art overview of the divers strategies employed by bacteria to interact with components of the fibrinolytic system and to exploit the system for invasion. Moreover, the role of factors of the fibrinolytic cascade in inflammatory host response due to different bacterial infections will be presented.

title: Plasminogen Is Required for Efficient Dissemination of B. burgdorferi in Ticks and for Enhancement of Spirochetemia in Mice
abstract: The role of the host plasminogen activation system in transmission of and invasion by Borrelia burgdorferi, the tick-borne spirochetal agent of Lyme disease, was investigated using plasminogen (Plg)-knockout mice. PLG was not detected in spirochetes from unfed ticks, but binding occurred as ticks fed on the host's blood. Plasminogen activators were derived from the host blood meal. PLG was required for efficient dissemination of B. burgdorferi within the tick and for enhancement of spirochetemia in mice but was not critical for transmission and infection. These results provide evidence for a bacterium using a vertebrate protease to disseminate in an invertebrate vector and underscores the interplay among vector, pathogen, and host in promoting the life cycle and disease.

title: Binding of human plasminogen and urokinase-type plasminogen activator to the Lyme disease spirochete, Borrelia burgdorferi.
abstract: Many bacteria that spread in the skin produce enzymes that digest extracellular matrix components. Borrelia burgdorferi spreads from a skin inoculation site to form the characteristic erythema migrans skin lesion. It was determined that B. burgdorferi does not produce collagenase, elastase, hyaluronidase, or other enzymes that digest extracellular matrix components. However, B. burgdorferi bound human plasmin, plasminogen (Pgn), and urokinase-type plasminogen activator (uPA). When spirochetes were sequentially incubated with Pgn and uPA, bioactive plasmin was generated on the surface of B. burgdorferi. B. burgdorferi did not produce an endogenous Pgn activator. Fluorochrome-conjugated uPA and Pgn colocalized to the terminus of the spirochete. In a mouse model, uPA-treated B. burgdorferi were more infectious than control spirochetes. Binding of host uPA and Pgn to form a bioactive extracellular matrix protease on B. burgdorferi represents a mechanism that could facilitate dissemination and localization of spirochetes to sites of vascular injury.

title: Binding of human urokinase type plasminogen activator and plasminogen to Borrelia species.
abstract: Borrelia burgdorferi binds human urokinase plasminogen activator (uPA), which cleaves plasminogen (Pgn) to plasmin. The ability of other Borrelia species to bind uPA, Pgn, or both was investigated. Borrelia coriacae, Borrelia garinii, Borrelia parkeri, Borrelia anserina, and Borrelia turicatae were compared with infectious and noninfectious B. burgdorferi isolates. All Borrelia species lacked endogenous proteases capable of digesting casein, but all species bound human uPA and Pgn, generating Pgn-dependent proteolytic activity. There were no significant differences in the amount of plasmin, Pgn, or uPA bound per spirochete of the different species. On unfixed borreliae, fluorochrome-conjugated uPA bound to all species. Early binding was at the terminus of B. burgdorferi, whereas diffuse binding was observed on B. coriacae, B. garinii, B. parkeri, and B. turicatae. These studies demonstrate that binding of human uPA and Pgn to borreliae occurs on multiple species with apparent differences in surface distribution.

title: Reciprocal upregulation of urokinase plasminogen activator and its inhibitor, PAI‐2, by Borrelia burgdorferi affects bacterial penetration and host‐inflammatory response
abstract: The mammalian plasminogen activation system (PAS) is a complex system involved in multiple physiological and pathological processes. Borrelia burgdorferi interacts with certain components of the PAS. Here we further investigate this interaction to determine its effect on bacterial dissemination and host cell migration in vitro. We show that stimulation of monocytic cells with B. burgdorferi induces the transient production and secretion of urokinase plasminogen activator (uPA), shortly followed by its physiological inhibitor, plasminogen activator inhibitor‐2 (PAI‐2). Mono Mac 6 (MM6) cells as well as peripheral blood monocytes enhanced transmigration of B. burgdorferi across a barrier coated with fibronectin mediated by uPA. Moreover, the induction of PAI‐2 or the addition of recombinant PAI‐2 did not have a significant effect on the uPA‐potentiated transmigration of B. burgdorferi. In contrast, the induction of PAI‐2 by B. burgdorferi resulted in significantly diminished invasion by monocytic cells across a reconstituted basement membrane (matrigel), which could be partially restored by treatment with purified uPA. These results show that the PAS plays a twofold role in the pathogenesis of B. burgdorferi infection, both by enhancing bacterial dissemination and by diminishing host‐cell inflammatory migration.

title: Inhibition of Borrelia burgdorferi-bound fibrinolytic enzymes by alpha2-antiplasmin, PAI-1 and PAI-2.
abstract: Lyme disease is caused by Borrelia burgdorferi. Human plasminogen and urokinase-type plasminogen activator bind to the surface of the spirochete where plasmin is generated. We have suggested that bound urokinase and plasminogen are utilized by the organism to disseminate. We tested whether the physiological inhibitors of urokinase, plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2), could regulate the activity of spirochete-bound urokinase. The k(ass) of PAI-1 and PAI-2 for bound urokinase were 1.3 x 10(6) M(-1)s(-1) and 6.9 x 10(4)M(-1)s(-1), respectively, whereas the k(ass) for free urokinase were 7.2 x 10(6) M(-1)s(-1) and 5.3 x 10(5) M(-1)s(-1), respectively. Plasmin associated with the spirochete was not inhibited by alpha2-antiplasmin. These results suggest that PAI-1, PAI-2 and alpha2 antiplasmin would not be efficient regulators of fibrinolytic protease activity on the Borrelial surface and would not pose a barrier to utilization of these enzymes for dissemination in the human host.

title: Borrelia burgdorferi and Other Bacterial Products Induce Expression and Release of the Urokinase Receptor (CD87)1
abstract: The urokinase-type plasminogen activator receptor (uPAR, CD87) is a highly glycosylated 55- to 60-kDa protein anchored to the cell membrane through a glycosylphosphatidylinositol moiety that promotes the acquisition of plasmin on the surface of cells and subsequent cell movement and migration by binding urokinase-type plasminogen activator. uPAR also occurs in a soluble form in body fluids and tumor extracts, and both membrane and soluble uPAR are overexpressed in patients with tumors. uPAR may be a factor in inflammatory disorders as well. We investigated whether Borrelia burgdorferi could stimulate up-regulation of cell membrane uPAR in vitro. B. burgdorferi, purified native outer surface protein A, and a synthetic outer surface protein A hexalipopeptide stimulated human monocytes to up-regulate membrane uPAR as measured by immunofluorescence/FACS and Western blot. The presence of soluble uPAR in culture supernatants, measured by Ag capture ELISA, was also observed. LPS from Salmonella typhimurium and lipotechoic acid from Streptococcus pyogenes also induced the up-regulation of both membrane and soluble uPAR protein by monocytes. Up-regulation of uPAR was induced by conditioned medium from B. burgdorferi/monocyte cocultures. The up-regulation of uPAR by B. burgdorferi was concomitant with an increase in uPAR mRNA, indicating that synthesis was de novo. The expression and release of uPAR in response to B. burgdorferi and other bacterial components suggests a role in the pathogenesis of Lyme disease as well as in other bacterial infections.

title: The Urokinase Receptor Can Be Induced by Borrelia burgdorferi through Receptors of the Innate Immune System
abstract: Monocytic cells exposed to Borrelia burgdorferi, through unknown receptors, overexpress the urokinase receptor (uPAR), a key mediator of the plasminogen activation system. We show that combined blockade of CD14 and TLR2 causes a significant inhibition of B. burgdorferi-induced uPAR in Mono Mac 6 (MM6) cells. Other pattern recognition receptors tested (CD11b/CD18, the mannose receptor, and the N-formyl-methionyl-leucyl-phenylalanine receptor) did not have demonstrated roles in B. burgdorferi-mediated uPAR induction. We dissected the result for CD14 andTLR2 by investigating the singular contributions of each. Independent functional blockade of CD14 or TLR2 failed to inhibit B. burgdorferi-mediated uPAR induction. 1,25-Dihydroxyvitamin D3 differentiation of MM6 cells increased CD14 expression 12-fold but did not augment B. burgdorferi-mediated uPAR expression. Peritoneal exudate macrophages (PEM) from CD14- or TLR2-deficient mice were not defective in B. burgdorferi-mediated synthesis of uPAR mRNA and protein. Increased uPAR mRNA or protein or both were apparent in PEM from transgenic and control mice, even at a ratio of one Borrelia spirochete per cell. We conclude that signaling for the uPAR response, as mediated by B. burgdorferi, proceeds with CD14 and TLR2 as partial contributors. That part under control of CD14 and TLR2 represents a new link between the host plasminogen activation and innate immunity systems.

title: uPA and uPAR in fibrinolysis, immunity and pathology.
abstract: The urokinase plasminogen activator (uPA) and its receptor (uPAR) exert pleiotropic functions over the course of both physiological and pathological processes. Data generated in deficient mice have underlined how uPA not only has a key role in fibrinolysis but also modulates the development of protective immunity. By contrast, uPAR anchors uPA at the cell surface and, in addition to favoring extracellular matrix degradation, regulates cell migration, cell adhesion and cell proliferation, thus influencing the development of inflammatory and immune responses. Here, we review the expression and function of uPA and uPAR, and correlate their role in fibrinolysis with immune, infectious and proliferative disorders. This Review is the second in a series on i nteractions between haemostasis and inflammation .

title: uPAR: a versatile signalling orchestrator
abstract: The plasminogen system has been implicated in clot lysis, wound healing, tissue regeneration, cancer and many other processes that affect health and disease. The urokinase receptor uPAR was originally thought to assist the directional invasion of migrating cells, but it is now becoming increasingly evident that this proteinase receptor elicits a plethora of cellular responses that include cellular adhesion, differentiation, proliferation and migration in a non-proteolytic fashion.

title: The soluble D2D388-274 fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion
abstract: We have previously shown that chymotrypsin-cleaved soluble uPAR (D2D388-274) elicits migration of monocytic cells through interaction with FPRL-1, a G protein-coupled receptor that is homologous to the fMLP receptor. Here, we report that D2D388-274 also modulates the ability of monocytes to migrate in response to other chemokines. Pretreatment of monocytes with increasing amounts of D2D388-274 prevents cell migration in response to MCP-1, RANTES and fMLP. We demonstrate that D2D388-274 does not inhibit MCP-1 receptor binding, elicit CCR2 internalization and prevent MCP-1-induced intracellular Ca2+ increase. Thus, CCR2 receptor desensitization cannot account for D2D388-274-mediated inhibition of MCP-1-induced cell migration. Rather, we show that pretreatment of monocytes with D2D388-274 dramatically decreases chemokine-induced integrin-dependent rapid cell adhesion by interacting with FPRL-1. Together, our results indicate that chemokine-dependent cell migration can be regulated not only by homologous and heterologous receptor desensitization, but also by inhibition of integrin-dependent cell adhesion, an important step in cell transmigration.

title: The urokinase receptor is required for human monocyte chemotaxis in vitro.
abstract: Mononuclear phagocytes (Mphi) produce urokinase-type plasminogen activator (uPA) and also express a specific cell-surface receptor for urokinase, uPAR. The concomitant expression of these proteins provides a mechanism by which Mphi can degrade extracellular matrix proteins during directed cell migration. In this study, we sought to determine if uPAR plays a role in Mphi chemotaxis that is distinct from its role in matrix proteolysis. Exposing adherent monocytes to a chemotactic gradient causes plasma membrane uPAR to localize strongly to the leading edge of cell migration. Adherence alone or exposure to FMLP had no effect on uPAR expression. Using Boyden chamber chemotaxis assays, we demonstrate that treating mononuclear cells with an anti-uPAR mAb (either as an intact mAb or F[ab']2) ablates chemotaxis induced by FMLP and monocyte chemotactic peptide-1 (P < 0.001). Inactivating the catalytic activity of uPAR-bound uPA had no effect on chemotaxis. Similarly, blocking uPAR expression with an antisense oligonucleotide to uPAR completely ablates chemotaxis, but blocking uPA expression with an antisense oligonucleotide to uPA has a minimal effect. We therefore demonstrate that expression and unimpeded function of uPAR plays an obligate role in M phi chemotaxis by mechanisms that are largely independent of its ligand, uPA. Combined with its known role in mediating pericellular proteolysis, these observations demonstrate that uPAR is essential for both locomotion and traversing tissue barriers during M phi migration.

title: Function of the urokinase receptor (CD87) in neutrophil Chemotaxis
abstract: During recruitment, leukocytes respond to chemotaxins and traverse matrix barriers. Urokinase‐type plasminogen activator (uPA), bound to its receptor (uPAR; CD87) facilitates plasmin formation, which promotes matrix proteolysis. Polymorphonuclear leukocytes (PMNs) are critical to the inflammatory response and express both uPA and CD87. To determine whether uPA and CD87 are required for PMN Chemotaxis, PMNs were pretreated with an anti‐CD87 monoclonal antibody (mAb), a neutralizing anti‐uPA mAb, or uPA. PMN Chemotaxis was profoundly suppressed by the anti‐CD87 mAb but was unaffected by anti‐uPA mAb or uPA. The role CD87 plays in Chemotaxis may be related to its ability to associate with CR3. CD87/CR3 coupling can be disrupted by specific saccharides. The same saccharides that disrupt CD87/CR3 coupling (NADC, D‐mannose, and mannoside) inhibit PMN Chemotaxis. We conclude that CD87 plays a crucial role in PMN Chemotaxis in vitro that is independent of uPA enzyme activity but may be related to the ability of CD87 to interact with CR3.

title: Urokinase‐deficient and urokinase receptor‐deficient mice have impaired neutrophil antimicrobial activation in vitro
abstract: Leukocytes express both urokinase‐type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). We have shown that neutrophil recruitment to the lung during P. aeruginosa pneumonia is impaired in uPAR‐deficient (uPAR−/−) mice but is normal in uPA−/− mice. However, both uPA−/− mice and uPAR−/− mice have impaired lung clearance of P. aeruginosa compared with wild‐type (WT) mice. To determine the role of uPA and uPAR in antibacterial host defense, we compared neutrophil bacterial‐phagocytosis, respiratory burst, and degranulation among uPA−/−, uPAR−/−, and WT mice. Nutrophil phagocytosis was significantly diminished comparing uPA−/− and uPAR−/− mice with WT mice at all time points. The generation of superoxide by both uPA−/− and uPAR−/− neutrophils was about half of that seen in WT neutrophils. Degranulation of azurophilic granules was significantly diminished in uPA−/− neutrophils compared with either uPAR−/− or WT neutrophils. By contrast, agonist‐stimulated release of specific granules was not diminished in either uPA−/− or uPAR−/− mice compared with WT. We conclude that the uPA/uPAR system modulates several of the crucial steps in neutrophil activation that result in bacterial killing and effective innate host defense.

title: Urokinase-type plasminogen activator receptor plays a role in neutrophil migration during lipopolysaccharide-induced peritoneal inflammation but not during Escherichia coli-induced peritonitis.
abstract: Urokinase-type plasminogen activator receptor (uPAR) is expressed on many different cells, including leukocytes. uPAR has been implicated to play a role in neutrophil migration to sites of inflammation.



To determine the role that uPAR plays in neutrophil recruitment in response to bacterial products or intact bacteria, uPAR gene-deficient (uPAR(-/-)) and wild-type (wt) mice were injected intraperitoneally with either Escherichia coli or lipopolysaccharide (LPS) derived from this bacterium.



uPAR(-/-) mice demonstrated a decreased LPS-induced neutrophil migration into peritoneal lavage fluid, whereas the chemokine and cytokine response was unaltered. In contrast, during E. coli-induced peritonitis, uPAR(-/-) mice had a normal neutrophil migration into the primary site of infection. The unaltered neutrophil trafficking in uPAR(-/-) mice during bacterial infection was corroborated by histological assessment of liver and lung tissue and myeloperoxidase levels in tissue homogenates. uPAR(-/-) mice displayed slightly but significantly lower bacterial loads in the peritoneal cavity, together with a decreased dissemination to the circulation early during the infection.



These data suggest that uPAR, in part, mediates neutrophil migration into the peritoneal cavity on local instillation of LPS but that this function of uPAR can be compensated for during peritonitis caused by intact E. coli.

title: The urokinase plasminogen activator receptor is crucially involved in host defense during acute pyelonephritis.
abstract: The urokinase plasminogen activator receptor (uPAR) is expressed at the cell surface of inflammatory cells and plays an important role in neutrophil migration. To investigate the in vivo role of uPAR during urinary tract infection, acute pyelonephritis was induced in uPAR-/- and wild-type (WT) mice by intravesical inoculation with 1 x 10(9) colony-forming units (CFU) of uropathogenic Escherichia coli. Mice were killed after 24 and 48 h, after which bacterial outgrowth and cytokine levels in kidney homogenates were determined. Influx of neutrophils was quantified by myeloperoxidase-enzyme-linked immunosorbent assay. uPAR-/- kidneys had significantly higher numbers of E. coli CFU, accompanied by higher levels of interleukin-1beta (IL-1beta), IL-6, keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha). However, the number of infiltrating neutrophils was similar in uPAR-/- and WT mice at both time points, suggesting that uPAR-/- neutrophils have a lower ability to eliminate E. coli. To further investigate this, neutrophil oxidative burst and phagocytosis was measured. The generation of reactive oxygen species upon stimulation with E. coli was not diminished in uPAR-/- neutrophils compared with WT. Interestingly, uPAR-/- neutrophils displayed significantly impaired phagocytosis of E. coli organisms compared with WT neutrophils. We conclude that uPAR is crucially involved in host defense through phagocytosis during E. coli induced acute pyelonephritis.

title: Urokinase Receptor-Deficient Mice Have Impaired Neutrophil Recruitment in Response to Pulmonary Pseudomonas aeruginosa Infection1
abstract: Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). Evidence in vitro has implicated uPAR as a modulator of β2 integrin function, particularly CR3 (CD11b/CD18, Mac-1). Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a β2 integrin-dependent mechanism. We demonstrate that mice deficient in uPAR (uPAR−/−) have profoundly diminished neutrophil recruitment in response to P. aeruginosa pneumonia compared with wild-type (WT) mice. The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA−/− mice is indistinguishable from recruitment in WT mice. uPAR−/− mice have impaired clearance of P. aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology. WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR−/− neutrophils is unaffected. We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P. aeruginosa pneumonia and that this requirement is independent of uPA. Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P. aeruginosa. This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.

title: Urokinase Receptor Is Necessary for Adequate Host Defense Against Pneumococcal Pneumonia1
abstract: Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the β2 integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for β2 integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a β2 integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR−/−), uPA-deficient (uPA−/−), and wild-type (Wt) mice were intranasally inoculated with 105 CFU S. pneumoniae. uPAR−/− mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA−/− mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a β2 integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.

title: Structure, Function and Expression on Blood and Bone Marrow Cells of the Urokinase‐Type Plasminogen Activator Receptor, uPAR
abstract: Several important functions have been assigned to the receptor for urokinase‐type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro‐uPA, to the plasma membrane where trace amounts of plasmin will initiate a series of events referred to as “reciprocal zymogen activation” where plasmin converts pro‐uPA to the active enzyme, uPA, which in turn converts plasma membrane‐associated plasminogen to plasmin. This is an efficient machinery to generate broad‐spectrum proteolytic activity which is spatially restricted to the plasma membrane, since plasmin that diffuses away from the plasma membrane is rapidly inactivated by circulating inhibitors (i.e., α2‐antiplasmin). The system is controlled by a series of plasminogen activator inhibitors (PAIs), most importantly PAI‐1 and PAI‐2, providing means of temporally restricting the process of plasminogen activation.

title: Increasing the Recruitment of Neutrophils to the Site of Infection Dramatically Attenuates Borrelia burgdorferi Infectivity1
abstract: Borrelia burgdorferi infection causes an initial skin lesion called erythema migrans (EM) in human Lyme disease and in models of monkey and rabbit borreliosis. EM results from the inflammatory response triggered by spirochete replication and likely develops to contain the initial infection but allows bacterial dissemination to occur. The essential lack of neutrophil involvement in EM histopathology prompted us to examine the consequence of increasing their recruitment in the inflammatory response to the Lyme disease agent. B. burgdorferi was modified genetically to constitutively express and secrete the chemokine KC, a neutrophil chemoattractant. After inoculation into the dermis of the murine host, control spirochetes induced an infiltration of macrophages, neutrophils, and basophils within 6 h; however, the recruited neutrophils and basophils were quickly substituted by eosinophils, and the inflammatory response became macrophage dominant by 16 h. Such a response failed to contain the initial infection and allowed the spirochetes to disseminate. In contrast, B. burgdorferi with KC secretion induced an intensive neutrophil infiltration at the inoculation site, and as a result, the host’s ability to control the initial infection was greatly enhanced. Taken together, this study suggests that the failure of sufficient neutrophil recruitment and activation during the initial inflammatory response may allow B. burgdorferi to effectively colonize the mammalian host.

title: Sonic hedgehog induces transcription-independent cytoskeletal rearrangement and migration regulated by arachidonate metabolites.
abstract: Sonic hedgehog (Shh) is a morphogen pivotal for development and tissue maintenance. Biological effects of Shh are mediated through a pathway that involves binding to patched1 (Ptch1), thereby releasing Smoothened (Smo) from inhibition resulting in the activation of Gli transcription factors, which mediate the induction of Shh target genes. Here, we describe a novel signal transduction pathway for Shh, which is transcription/translation-independent, SuFu insensitive, and consequently independent of Gli-mediated induction of transcription. Through this alternative pathway Shh, transduced via Smo, induced altered cell morphology together with lamellipodia formation. Migration assays demonstrate that this cytoskeletal rearrangement mediates the migratory response to Shh. This Shh-induced, Smo mediated migration utilizes and requires the metabolism of arachidonic acid through the 5-lipoxygenase pathway. These data provide a link between a seemingly novel Gli-independent Hh signaling pathway and the leukotriene metabolism, and might explain the developmental abnormalities observed in both patients with defective leukotriene metabolism as well as in rodent models of defective Rho family GTPase signaling.

title: Protease-activated receptor-4 inhibition protects from multiorgan failure in a murine model of systemic inflammation.
abstract: Coagulation proteases may act as cell signaling molecules via protease-activated receptor (PAR) cleavage, subsequently affecting cellular and inflammatory responses. Activation of PARs in the setting of systemic inflammation and disseminated intravascular coagulation (DIC) might thus exacerbate the inflammatory response contributing to tissue and organ damage. To investigate the role of PAR-4 in these processes, we subjected mice to a model of systemic inflammation and DIC (Shwartzman reaction) in the absence or presence of a cell-penetrating pepducin antagonist of PAR-4 (P4pal-10). P4pal-10 dose-dependently diminished the severity of endotoxemia and preserved liver, kidney, as well as lung function. Moreover, systemic inflammation and local (neutrophilic) inflammatory responses were attenuated. In vitro migration assays and P4pal-10 treatment in neutropenic mice suggest an essential role for neutrophils in PAR-4-mediated pathology. P4pal-10 treatment of thrombocytopenic mice excluded the involvement of platelets in this phenomenon. These results uncover an important role for PAR-4 in the Shwartzman reaction and suggest that inhibition of PAR-4 signaling in neutrophils could be protective in systemic inflammation and DIC.

title: Carditis in Lyme disease susceptible and resistant strains of laboratory mice infected with Borrelia burgdorferi.
abstract: The clinical and pathologic evolution of cardiac Lyme disease was evaluated in four-week-old susceptible C3H/He (C3H) and resistant C57Bl/6 (B6) mice on days 3, 6, 10, 15, 30, 60, and 90 after intradermal inoculation with Borrelia burgdorferi strain N40. Culture, DNA polymerase chain reaction, in situ nucleic acid hybridization, immunoperoxidase histochemical analysis, and silver stain were used to detect spirochetes. Spirochetes were first detected by culture on day 6 in two of four C3H mice. The hearts of all mice of both genotypes were culture positive by day 10 and infection persisted through day 90. The spirochetes had a predilection for connective tissue in the heart base, especially around the aorta, epicardium of the upper ventricles and atria, myocardial interstitium, and endocardium. Carditis was first detectable on day 10, reaching a maximum severity on day 15, then resolved, except for persistence of periaortic lymphoplasmacytic infiltrates through day 90 in C3H mice and through day 60 in B6 mice. The C3H mice developed more severe disease than the B6 mice, and this was associated with the earlier appearance, greater numbers, and later clearance of spirochetes in C3H mice. Electrocardiographs of infected and control mice revealed bradycardia and tachycardia in many C3H mice, but in very few B6 mice. Serum creatinine phosphokinase levels did not become elevated at any interval.

title: β2 Integrins Control the Severity of Murine Lyme Carditis
abstract: Infection of C57BL/6 (B6) mice with the Lyme disease spirochete Borrelia burgdorferi can result in development of arthritis and carditis. B. burgdorferi induces expression of β2/CD18 integrins, adhesion molecules that mediate the firm adhesion of leukocytes to the endothelium necessary for cellular extravasation during inflammation. The important role of β2/CD18 integrins during extravasation suggests that these molecules play a role in the development of Lyme arthritis and carditis. The dependency of these inflammatory processes on the β2 integrins was investigated in CD18 hypomorph mice, which express low levels of CD18. The results indicate that CD18 deficiency did not abrogate development of Lyme arthritis or carditis. Moreover, it resulted in increased severity of Lyme carditis. B. burgdorferi-infected CD18 hypomorph mice showed an increased macrophage infiltration of the heart, while they produced lower levels of borreliacidal anti-B. burgdorferi antibodies compared to wild-type mice. In accordance with these results, we demonstrate that dendritic cells from CD18 hypomorph mice secrete higher levels of monocyte/macrophage chemoattractant protein 1 (MCP-1/CCL2) in response to B. burgdorferi. Similarly, we show by real-time PCR that B. burgdorferi-infected hearts from CD18 hypomorph mice express increased levels of MCP-1 RNA compared to wild-type mice. Overall, our results indicate that β2 integrin deficiency does not abrogate B. burgdorferi-induced inflammation; rather, it results in increased recruitment of macrophages into the B. burgdorferi-infected heart, likely due to the increased expression of MCP-1 in this tissue. Thus, β2 integrins may play a regulatory role in B. burgdorferi-induced inflammation beyond mediating adhesion of leukocytes to the endothelium.

title: Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis.
abstract: Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.

title: Distinct Characteristics of Resistance toBorrelia burgdorferi-Induced Arthritis in C57BL/6N Mice
abstract: Studies of mice infected with Borrelia burgdorferi have indicated that the severity of arthritis is influenced by the genetic composition of the host: the C3H mouse develops severe arthritis while BALB/c and C57BL/6 mice develop mild arthritis. In this study, the effects of increasing infectious dose on the severity of arthritis were determined in these three mouse strains. C3H/He mice developed severe arthritis at all infectious doses, with 100% infection requiring 200 spirochetes. In BALB/cAnN mice, arthritis severity was dependent on infectious dose; symptoms were mild with infection by 200 B. burgdorferi and progressively more severe with increasing infectious dose. Infection of BALB/cAnN mice with 2 × 104B. burgdorferi resulted in arthritis with severity identical to that in C3H/He mice. Spirochete levels in rear ankle joints of C3H/HeJ and C3H/HeN mice were relatively high, as detected by PCR, and did not increase with infectious dose. Spirochete levels in joints from BALB/cAnN mice increased with increasing infectious dose to levels found in severely arthritic C3H/He mice. Thus, resistance to severe arthritis in BALB/cAnN mice was conditional: it could be overcome by high infectious dose and the arthritis became severe when high levels of B. burgdorferi were present in joints. A unique response to increasing infectious dose was seen in C57BL/6N mice, which displayed mild to moderate arthritis at all doses of B. burgdorferi tested, up to 2 × 105. At all infectious doses, the levels of spirochetes in ankle joints of C57BL/6N mice were high, equivalent to those found in the severely arthritic C3H/He mice. The arthritis observed in infected (C57BL/6N × C3H/HeN)F1 mice was of severity intermediate between those of the two parental strains. The finding that resistance to severe arthritis in C57BL/6N mice could not be overcome by high infectious doses and was independent of spirochete levels in joints suggested that it was mediated by a distinct mechanism from that operating in BALB/cAnN mice.

title: Borrelia burgdorferi inhibits human neutrophil functions.
abstract: Outer surface proteins OspA and OspB are among the most prominent Borrelia burgdorferi surface molecules. We constructed OspAB and OspA complementation mutants of B. burgdorferi Osp-less strain B313 and investigated the role of these surface proteins in the interactions of B. burgdorferi, human neutrophils and the complement system. We found that (1) OspB inhibits the phagocytosis and oxidative burst of human neutrophils at low serum concentrations, whereas OspA induces the oxidative burst in neutrophils; (2) OspB may have an inhibiting role in serum sensitivity and complement activation; (3) all studied strains inhibit the chemotaxis of human neutrophils specifically towards fMLP but not towards C5a, regardless of their Osp expression. These results suggest that although OspA and OspB are co-ordinately transcribed, they differ in their effects on human neutrophil functions. Our findings suggest that B. burgdorferi exploits a wide variety of immune evasion mechanisms, besides previously documented complement resistance, to survive in the vertebrate host.

title: Differential expression of cytokine mRNA in skin specimens from patients with erythema migrans or acrodermatitis chronica atrophicans.
abstract: Erythema migrans, the characteristic skin manifestation of acute Lyme borreliosis, is a self-limited lesion. In contrast, acrodermatitis chronica atrophicans, the typical cutaneous manifestation of late Lyme borreliosis, is a chronic skin condition. In an effort to understand pathogenic factors that lead to different outcomes in dermatoborrelioses, skin biopsy samples from 42 patients with erythema migrans and 27 patients with acrodermatitis chronica atrophicans were analyzed for mRNA expression of five pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interferon-gamma, and interleukin-2) and two anti-inflammatory cytokines (interleukin-4 and interleukin-10) by in situ hybridization with cytokine-specific riboprobes. Among the 27 patients who had erythema migrans alone with no associated signs or symptoms, the major cytokines expressed in perivascular infiltrates of T cells and macrophages were the pro-inflammatory cytokine interferon-gamma and the anti-inflammatory cytokine interleukin-10. In the 15 erythema migrans patients who had associated signs and symptoms, including headache, elevated temperature, arthralgias, myalgias, or fatigue, a larger number of macrophages and greater expression of macrophage-derived pro-inflammatory cytokines, tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6, were also found. In comparison, infiltrates of T cells and macrophages in the skin lesions of acrodermatitis chronica atrophicans patients had very little or no interferon-gamma expression. Instead, they usually expressed only the pro-inflammatory cytokine tumor necrosis factor alpha and the anti-inflammatory cytokine interleukin-4. Thus, the activation of pro-inflammatory cytokines in erythema migrans lesions, particularly interferon-gamma, seems to be important in the control of the spirochetal infection. In contrast, the restricted pattern of cytokine expression in acrodermatitis chronica atrophicans, including the lack of interferon-gamma, may be less effective in spirochetal killing, resulting in the chronicity of this skin lesion.

title: Phagocytosis of Borrelia burgdorferi, the Lyme Disease Spirochete, Potentiates Innate Immune Activation and Induces Apoptosis in Human Monocytes
abstract: We have previously demonstrated that phagocytosed Borrelia burgdorferi induces activation programs in human peripheral blood mononuclear cells that differ qualitatively and quantitatively from those evoked by equivalent lipoprotein-rich lysates. Here we report that ingested B. burgdorferi induces significantly greater transcription of proinflammatory cytokine genes than do lysates and that live B. burgdorferi, but not B. burgdorferi lysate, is avidly internalized by monocytes, where the bacteria are completely degraded within phagolysosomes. In the course of these experiments, we discovered that live B. burgdorferi also induced a dose-dependent decrease in monocytes but not a decrease in dendritic cells or T cells and that the monocyte population displayed morphological and biochemical hallmarks of apoptosis. Particularly noteworthy was the finding that apoptotic changes occurred predominantly in monocytes that had internalized spirochetes. Abrogation of phagocytosis with cytochalasin D prevented the death response. Heat-killed B. burgdorferi, which was internalized as well as live organisms, induced a similar degree of apoptosis of monocytes but markedly less cytokine production. Surprisingly, opsonophagocytosis of Treponema pallidum did not elicit a discernible cell death response. Our combined results demonstrate that B. burgdorferi confined to phagolysosomes is a potent inducer of cytosolic signals that result in (i) production of NF-κB-dependent cytokines, (ii) assembly of the inflammasome and activation of caspase-1, and (iii) induction of programmed cell death. We propose that inflammation and apoptosis represent mutually reinforcing components of the immunologic arsenal that the host mobilizes to defend itself against infection with Lyme disease spirochetes.

title: Distinct Roles for MyD88 and Toll-Like Receptors 2, 5, and 9 in Phagocytosis of Borrelia burgdorferi and Cytokine Induction
abstract: The contribution of Toll-like receptors (TLRs) to phagocytosis of Borrelia burgdorferi has not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88−/− mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalize B. burgdorferi. Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis of B. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88−/− BMDM but only partially affected in TLR2−/− BMDM or knockdown of TLR5 and unaffected in TLR9−/− BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88−/− BMDM. Addition of cytochalasin D to TLR2−/− BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88−/− BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake of B. burgdorferi and that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5.

title: The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor.
abstract: The spirochete Borrelia burgdorferi is the causative agent of Lyme borreliosis (Lyme disease) and is transmitted to mammalian hosts by tick vectors. In humans, the bacteria induce a complex disease, which involves the skin, joints, heart, and nervous system. However, the pathogenic principles of this multisystem illness are far from being understood. To disseminate from the site of the tick bite and invade multiple organ sites, spirochetes have to penetrate normal tissue barriers, such as vascular basement membranes and other organized extracellular matrices. Substantial evidence from other invasive bacterial infections suggest that spirochetes may use endogenous or host-derived enzymes--in particular, proteinases--for this purpose. Here we show that B. burgdorferi binds human plasmin(ogen)--mainly via its outer cell surface lipoprotein A. Binding of plasminogen to spirochetal receptor leads to an accelerated formation of active plasmin in the presence of host-derived plasminogen activator. The cell-surface-associated plasmin cannot be regulated by the serum inhibitor alpha 2-antiplasmin and degrades high-molecular-weight glycoproteins, such as fibronectin. It is suggested that the acquisition of host-derived proteinase plasmin(ogen) contributes to the pathogenicity of B. burgdorferi.

title: Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain Barrier
abstract: Neurological manifestations of Lyme disease in humans are attributed in part to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system (CNS) by Borrelia burgdorferi. However, how the spirochetes cross the BBB remains an unresolved issue. We examined the traversal of B. burgdorferi across the human BBB and systemic endothelial cell barriers using in vitro model systems constructed of human brain microvascular endothelial cells (BMEC) and EA.hy 926, a human umbilical vein endothelial cell (HUVEC) line grown on Costar Transwell inserts. These studies showed that B. burgdorferi differentially crosses human BMEC and HUVEC and that the human BMEC form a barrier to traversal. During the transmigration by the spirochetes, it was found that the integrity of the endothelial cell monolayers was maintained, as assessed by transendothelial electrical resistance measurements at the end of the experimental period, and that B. burgdorferi appeared to bind human BMEC by their tips near or at cell borders, suggesting a paracellular route of transmigration. Importantly, traversal of B. burgdorferi across human BMEC induces the expression of plasminogen activators, plasminogen activator receptors, and matrix metalloproteinases. Thus, the fibrinolytic system linked by an activation cascade may lead to focal and transient degradation of tight junction proteins that allows B. burgdorferi to invade the CNS.

title: Binding of human plasminogen to Borrelia burgdorferi
abstract: We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in the binding of Glu- or Lys-plasminogen to B. burgdorferi. Binding of plasminogen was similar in low-passage (infectious) and high-passage (noninfectious) isolates of B. burgdorferi. Plasminogen bound to the surface of B. burgdorferi could be converted into plasmin by a human urokinase-type plasminogen activator. 125I-labeled plasminogen ligand blots of borrelial membrane proteins demonstrated two prominent binding proteins at approximately 70 and approximately 30 kDa. By Western blot (immunoblot), the 30-kDa protein was found to be outer surface protein A (Osp A) of B. burgdorferi. 125I-labeled plasminogen binding to both the 70-kDa protein and Osp A was inhibited by approximately 90% with a 1,000-fold excess of unlabeled plasminogen. By scanning densitometry, the 70-kDa band bound > 10 time more 125I-labeled plasminogen than did Osp A. An Osp A-deficient mutant of B. burgdorferi and wild-type B. burgdorferi bound equal amounts of 125I-labeled plasminogen. Ligand blots of membrane proteins from an Osp A-deficient mutant showed association of 125I-labeled plasminogen at only the 70-kDa protein. Two-dimensional gel electrophoresis showed that the 70-kDa protein had a pI of approximately 5.3, clearly separable from Osp A. The association of host plasmin(ogen) with borrelial surface proteins provides a mechanism by which B. burgdorferi can digest extracellular matrix and disseminate.

title: The plasminogen activation system enhances brain and heart invasion in murine relapsing fever borreliosis.
abstract: The role of the plasminogen activation system (PAS) was investigated during the course of infection of a relapsing fever Borrelia species in plasminogen-deficient (plg -/-) and control (plg +/+ and plg +/-) mice. Subcutaneous inoculation of 10(4) spirochetes resulted in a peak spirochetemia five days after infection with 20-23 x 10(6) organisms per milliliter of whole blood in all mice, indicating that the PAS had no effect on the development of this phase of the infection. Anemia, thrombocytopenia, hepatitis, carditis, and splenomegaly were noted in all mice during and immediately after peak spirochetemia. Fibrin deposition in organs was noted in plg -/- mice but not in controls during these stages. Significantly greater spirochetal DNA burdens were consistently observed in the hearts and brains of control mice 28-30 days after infection, as determined by PCR amplification of this organism's flagellin gene (flaB), followed by quantitative densitometry. Furthermore, the decreased spirochetal load in brains of plg -/- mice was associated with a significant decrease in the degree of inflammation of the leptomeninges in these mice. These findings indicate a role for the PAS in heart and brain invasion by relapsing fever Borrelia, resulting in organ injury.

title: Reference gene selection for real-time RT-PCR in regenerating mouse livers.
abstract: The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.

title: IL-1 can act as number one.
abstract: Both interleukin-1 receptor (IL-1R) and toll-like receptor 2 (TLR2) signal via the adapter molecule MyD88. However, in response to a cutaneous infection challenge, the downstream signaling events diverge and IL-1R, but not TLR2, proves to be essential for host defense.

title: MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus.
abstract: MyD88 is an important signaling adaptor for both TLR and IL-1R family members. Here, we evaluated the role of TLR2/MyD88 and IL-1R/MyD88 signaling in host defense against S. aureus by using a cutaneous infection model in conjunction with bioluminescent bacteria. We found that lesions of S. aureus-infected MyD88- and IL-1R-deficient mice were substantially larger with higher bacterial counts compared with wild-type mice. In contrast, TLR2-deficient mice had lesions that were only moderately larger with minimally higher bacterial counts. In addition, MyD88- and IL-1R- but not TLR2-deficient mice had severely decreased recruitment of neutrophils to the site of infection. This neutrophil recruitment was not dependent upon IL-1R/MyD88 signaling by recruited bone marrow-derived cells, suggesting that resident skin cells utilize IL-1R/MyD88 signaling to promote neutrophil recruitment.

title: Cytokines in murine lyme carditis: Th1 cytokine expression follows expression of proinflammatory cytokines in a susceptible mouse strain.
abstract: The cardiac infiltrate seen in murine Lyme carditis is composed predominantly of macrophages, but small numbers of T cells are also present. To identify the cytokines present in cardiac lesions from susceptible mice, semiquantitative polymerase chain reaction was done on cardiac tissue from mice infected with Borrelia burgdorferi. The temporal expression of proinflammatory and T cell-derived cytokines was characterized in cardiac tissue at days 0, 3, 7, 14, 21, and 42 after infection with B. burgdorferi. Early in the course of infection, up-regulation of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha was detected. The Th1 cytokine interferon-gamma appeared after the expression of the proinflammatory cytokines and remained elevated throughout the study. Interleukin-4 was not detectable at any time in cardiac lesions. These data are the first to identify cytokines expressed at the lesional level in murine Lyme carditis and to demonstrate a Th1 pattern of cytokine expression in this lesion.

title: Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist.
abstract: Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis.

title: Murine Lyme disease: no evidence for active immune down-regulation in resolving or subclinical infection.
abstract: Macrophages in vitro rapidly ingest and kill Borrelia burgdorferi, yet some spirochetes in vivo may survive in the host and lead to complications of Lyme disease. One strategy for such survival may be the down-regulation of the immune system. To test this, we evaluated the degree of macrophage activation in a site of active disease-the heart-by examining cytokine expression in murine macrophages from control and B. burgdorferi-infected animals. Using double-label immunofluorescent staining in situ, we showed that infiltrating macrophages in infected hearts produce interleukin (IL)-1. By semiquantitative reverse transcription-polymerase chain reaction analysis, increased levels of mRNA were measured for the proinflammatory cytokines IL-1, tumor necrosis factor-alpha, and IL-12 during peak and resolving disease. No increases in the anti-inflammatory cytokines IL-10 and transforming growth factor-beta were detected. In an infected site without active disease--the peritoneal cavity--no increases in levels of proinflammatory or anti-inflammatory cytokines were detected in local macrophages. Thus, there is no evidence of pressure toward the down-regulation of inflammatory activity in the regions tested.

title: Pattern of Proinflammatory Cytokine Induction in RAW264.7 Mouse Macrophages Is Identical for Virulent and Attenuated Borrelia burgdorferi1
abstract: Lyme disease pathogenesis results from a complex interaction between Borrelia burgdorferi and the host immune system. The intensity and nature of the inflammatory response of host immune cells to B. burgdorferi may be a determining factor in disease progression. Gene array analysis was used to examine the expression of genes encoding cytokines, chemokines, and related factors in the joint tissue of infected C3H/HeJ mice and in a murine macrophage-like cell line in response to a disseminating or attenuated clinical isolate of B. burgdorferi. Both isolates elicited a robust proinflammatory response in RAW264.7 cells characterized by an increase in transcript levels of genes encoding CC and CXC chemokines, proinflammatory cytokines, and TNF superfamily members. Transcription of genes encoding IL-1β, IL-6, MCP-1, MIP-1α, CXCR4, and TLR2 induced in RAW264.7 cells by either live or heat-killed spirochetes did not differ significantly at any time point over a 24-h period, nor was there a difference in the protein levels of IL-10, TNF-α, IL-6, and IL-12p70 in culture supernatants. Thus, induction of host macrophage expression of proinflammatory mediators by host macrophages does not contribute to the differential pathogenicity of different B. burgdorferi strains.

title: Salp15 Binding to DC-SIGN Inhibits Cytokine Expression by Impairing both Nucleosome Remodeling and mRNA Stabilization
abstract: Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC) functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi–induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK)-dependent decrease of IL-6 and TNF-α mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor–induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.

title: Generation and characterization of urokinase receptor-deficient mice.
abstract: Mice homozygously deficient for the urokinase-type plasminogen activator (u-PA) receptor (u-PAR-1-) were generated by homologous recombination in D3, embryonic stem cells. The genomic sequences comprising exon 2 through 5 of the u-PAR gene were replaced by the neomycin resistance gene, resulting in inactivation of both u-PAR splice variants. The inactivated u-PAR allele was transmitted via mendelian inheritance, and fertility. Inactivation of u-PAR was confirmed by the absence of binding of rabbit anti-murine u-PAR or of an aminoterminal fragment of murine u-PA (mu-PA.1-48) to u-PAR-1- embryonic fibroblasts and macrophages. u-PAR-1- mice displayed normal lysis of a murine plasma clot injected via the jugular vein. Invasion of macrophages into the peritoneal cavity after thioglycollate stimulation was similar in u-PAR-1- and u-PAR-1- mice. u-PAR-1- peritoneal macrophages had a threefold decreased initial rate of u-PA-mediated plasminogen activation in vitro but degraded extracellular matrix proteins in vitro as efficiently as u-PAR-1- macrophages.

title: Correlation between plasmid content and infectivity in Borrelia burgdorferi.
abstract: Infectivity-associated plasmids were identified in Borrelia burgdorferi B31 by using PCR to detect each of the plasmids in a panel of 19 clonal isolates. The clones exhibited high-, low-, and intermediate-infectivity phenotypes based on their frequency of isolation from needle-inoculated C3H/HeN mice. Presence or absence of 21 of the 22 plasmids was determined in each of the clones by using PCR primers specific for regions unique to each plasmid, as identified in the recently available genome sequence. Southern blot hybridization results were used to confirm the PCR results in some cases. Plasmid lp25 exhibited a direct correlation with infectivity in that it was consistently present in all clones of high or intermediate infectivity and was absent in all low-infectivity clones. lp28-1, containing the vmp-like sequence locus, also correlated with infectivity; all clones that lacked lp28-1 but contained lp25 had an intermediate infectivity phenotype, in which infection was primarily restricted to the joints. Plasmids cp9, cp32-3, lp21, lp28-2, lp28-4, and lp56 apparently are not required for infection in this model, because clones lacking these plasmids exhibited a high-infectivity phenotype. Plasmids cp26, cp32-1, cp32-2 and/or cp32-7, cp32-4, cp32-6, cp32-8, cp32-9, lp17, lp28-3, lp36, lp38, and lp54 were consistently present in all clones examined. On the basis of these results, lp25 and lp28-1 appear to encode virulence factors important in the pathogenesis of B. burgdorferi B31.

title: Long-term protection of mice from Lyme disease by vaccination with OspA
abstract: Mice vaccinated with recombinant outer surface protein A (OspA) have been shown to be protected from infection with Borrelia burgdorferi, the agent of Lyme disease, when sacrificed 14 days after challenge with an intradermal inoculum of the spirochete. To determine whether infection was not merely delayed and that protection was long-lasting, we sacrificed vaccinated mice 60, 120, and 180 days after challenge; and to determine whether vaccinated mice retained their immune state over long periods, we challenged mice with B. burgdorferi 60, 90, 120, and 150 days after vaccination. The results of both groups of experiments show that the mice remained free from infection and disease and extend the usefulness of OspA as a vaccine candidate for Lyme borreliosis.

title: CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis.
abstract: Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mphi) recruitment into M. tuberculosis-infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44(-/-)) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-gamma were reduced in the lungs of infected CD44(-/-) mice. The production of IFN-gamma by splenocytes of CD44(-/-) mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mphi-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44(-/-) Mphis. This suggests that CD44 is a receptor on Mphis for binding of M. tuberculosis. CD44(-/-) mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mphi binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mphi recruitment, and protective immunity against pulmonary tuberculosis.

title: Gene-expression profiles in murine melioidosis.
abstract: Melioidosis, caused by the bacterium Burkholderia pseudomallei, is a septicemic illness, often associated with pneumonia and bacterial dissemination to distant sites. Recently we reported the inflammatory mRNA profile in blood leukocytes during human melioidosis. Knowledge of the inflammatory gene expression profile in the pulmonary compartment after infection with B. pseudomallei, however, is highly limited. We therefore aimed to characterize the inflammatory mRNA profile in the pulmonary and systemic compartment during murine melioidosis. By using a newly developed mouse specific Multiplex-Ligation-dependent-Probe-Amplification (MLPA) assay we determined the expression profile of 33 genes encoding inflammatory proteins in lung tissue, leukocytes in bronchoalveolar-lavage-fluid (BALF) and blood leukocytes in mice before and at several time points after intranasal infection with B. pseudomallei. Relative to naïve mice, mice intranasally infected with B. pseudomallei showed increased transcription of a whole array of genes involved in inflammation, Toll-like receptor-signaling, coagulation, fibrinolysis, cell adhesion, tissue repair and homeostasis in the lung, BALF and blood compartment. Notably, many inflammatory genes were shown to be differentially expressed during the course of infection. These data provide new information on compartmentalized inflammatory gene-expression profiles after infection with B. pseudomallei, increasing our insights into the extent of inflammation activation in the pulmonary and systemic compartment during melioidosis.

title: The Noxa/Mcl-1 axis regulates susceptibility to apoptosis under glucose limitation in dividing T cells.
abstract: Throughout lymphocyte development, cellular persistence and expansion are tightly regulated by survival and apoptosis. Within the Bcl-2 family, distinct apoptogenic BH3-only members like Bid, Bim, and Puma appear to function in specific cell death pathways. We found that naive human T cells after mitogenic activation, apart from expected protective Bcl-2 members, also rapidly upregulate the BH3-only protein Noxa in a p53-independent fashion. The specific role of Noxa became apparent during glucose limitation and involves interaction with the labile Bcl-2 homolog Mcl-1. Knockdown of Noxa or Mcl-1 results in protection or susceptibility, respectively, to apoptosis induced by glucose deprivation. Declining Mcl-1 levels and apoptosis induction are inversely correlated to Noxa levels and prevented by readdition of glucose. We propose that the Noxa/Mcl-1 axis is an apoptosis rheostat in dividing cells, in a selective pathway that functions to restrain lymphocyte expansion and can be triggered by glucose deprivation.

title: Impaired arterial neointima formation in mice with disruption of the plasminogen gene.
abstract: To define the role of plasminogen (Plg) in the smooth muscle cell response after arterial wall injury, neointima formation was evaluated after electric injury of the femoral artery in plasminogen-deficient (Plg-/-) mice. The injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and induced transient platelet-rich mural thrombosis. In wild-type (Plg+/+) mice, vascular wound healing was characterized by lysis of the thrombus, transient infiltration of inflammatory cells, and progressive removal of necrotic debris and thrombosis. Topographic analysis revealed repopulation of the media and accumulation in the neointima of smooth muscle cells originating from the noninjured borders, which progressed into the necrotic center. In Plg-/- mice, wound healing was significantly impaired with delayed removal of necrotic debris, reduced leucocyte infiltration and smooth muscle cell accumulation, and decreased neointima formation. Smooth muscle cells accumulated at the uninjured borders, but failed to migrate into the necrotic center. Proliferation of smooth muscle cells was not affected by Plg deficiency. Evans blue staining revealed no genotypic differences in reendothelialization. Thus, Plg plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting cellular migration.

title: Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)
abstract: Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.  34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.  Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

title: The Mannose Receptor Mediates Dengue Virus Infection of Macrophages
abstract: Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell–produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.

title: Oxidized Phospholipids Inhibit Phagocytosis and Impair Outcome in Gram-Negative Sepsis In Vivo1
abstract: Oxidized phospholipids that are generated during inflammation exert anti-inflammatory properties and prevent death during murine endotoxemia. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) inhibits the interaction of LPS with LPS-binding protein and CD14. In this study, we determined the functional properties of OxPAPC and potential interference with CD14 during abdominal sepsis caused by Escherichia coli. Administration of OxPAPC rendered mice highly susceptible to E. coli peritonitis, as indicated by an accelerated mortality and enhanced bacterial outgrowth and dissemination. CD14−/− mice also displayed increased mortality and bacterial outgrowth and OxPAPC did not further impair host defense in these animals. The mechanisms by which OxPAPC and CD14 deficiency impaired the immune response differed: whereas CD14−/− mice demonstrated a strongly reduced recruitment of phagocytes to the site of the infection, OxPAPC did not influence the influx of inflammatory cells but strongly diminished the phagocytosing capacity of neutrophils and macrophages by a CD14-independent mechanism. Furthermore, OxPAPC potently inhibited uptake of fluorospheres as well as receptor-mediated endocytosis and fluid-phase pinocytosis. These data suggest that oxidized phospholipids such as produced during inflammatory reactions may contribute to mortality during Gram-negative sepsis in vivo via impairment of the phagocytic properties of professional phagocytes.

title: TLR2-Dependent MyD88 Signaling Contributes to Early Host Defense in Murine Enterococcus faecium Peritonitis
abstract: The incidence of infections with Enterococcus faecium is increasing worldwide. TLRs have been implicated in the recognition of pathogens and the initiation of an adequate innate immune response. We here sought to determine the roles of MyD88, the common adaptor protein involved in TLR signaling, TLR2, TLR4, and CD14 in host defense against E. faecium peritonitis. MyD88 knockout (KO) mice demonstrated an impaired early response to E. faecium peritonitis, as reflected by higher bacterial loads in peritoneal fluid and liver accompanied by a markedly attenuated neutrophil influx into the abdominal cavity. In vitro, not only MyD88 KO macrophages but also TLR2 KO and CD14 KO macrophages displayed a reduced responsiveness to E. faecium. In accordance, transfection of TLR2 rendered human embryonic kidney 293 cells responsive to E. faecium, which was enhanced by cotransfection of CD14. TLR2 KO mice showed higher bacterial loads in peritoneal fluid after in vivo infection with E. faecium and a diminished influx of neutrophils, whereas CD14 KO mice had an unaltered host response. E. faecium phagocytosis and killing were not affected by MyD88, TLR2, or CD14 deficiency. TLR4 did not play a role in the immune response to E. faecium in vitro or in vivo. These data suggest that MyD88 contributes to the effective clearance of E. faecium during peritonitis at least in part via TLR2 and by facilitating neutrophil recruitment to the site of the infection.

title: A rapid and simple microfluorometric phagocytosis assay.
abstract: A microfluorometric method for phagocytosis study has been developed using fluorescein conjugated Escherichia coli K-12 particles. This technique is based on the uptake of fluorescent particles and quenching of extracellular fluorescence at the end of the assay. A murine macrophage cell line, J774, was used as a phagocyte model. The cells were harvested from tissue culture flasks and adjusted to 1 x 10(6) cells/ml. They were then dispensed into a 96-well tissue culture plate, 100 microliters/well, and incubated at 37 degrees C in 5% CO2 for 1 h to allow cells to adhere to the bottom of the wells. The culture medium was aspirated and 100 microliters of fluorescent E. coli particles suspended in Hanks' buffer were added. The plates were further incubated for various time periods. Buffer solution in the wells was removed by aspiration. Extracellular fluorescence was then quenched by adding 100 microliters of trypan blue (250 micrograms/ml, pH 4.4). The dye was removed after 1 min. The intensity of fluorescence associated with intracellular fluorescent particles was measured directly in the wells using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid and objective measurement of phagocytosis activity. Using a cultured cell line and a 96-well microtiter plate format, this assay can facilitate the screening of a large number of various biological and pharmacological substances for their modulating effects on phagocytosis.

title: Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils
abstract: BackgroundSpecies of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry.ResultsIn this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain.ConclusionThese results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.