Christopher J. Day

MCP-1 Is Induced by Receptor Activator of Nuclear Factor-κB Ligand, Promotes Human Osteoclast Fusion, and Rescues Granulocyte Macrophage Colony-stimulating Factor Suppression of Osteoclast Formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-κB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.

MCP-1-induced Human Osteoclast-like Cells Are Tartrate-resistant Acid Phosphatase, NFATc1, and Calcitonin Receptor-positive but Require Receptor Activator of NFκB Ligand for Bone Resorption

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFκB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP+/CTR+ multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP+/CTR+ multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP+/CTR+ multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP+/CTR+ multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.

Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1

Staphylococcus aureus causes diseases ranging from superficial wound infections to more invasive manifestations like osteomyelitis and endocarditis. The evasion of host phagocytes recruited to the site of infection is essential to the success of S. aureus as a pathogen. A single S. aureus strain can produce up to five different bicomponent pore-forming leukotoxins that lyse immune cells by forming pores in the cellular plasma membrane. Although these leukotoxins have been considered redundant due to their cytotoxic activity toward human neutrophils, each toxin displays varied species and cell-type specificities. This suggests that cellular factors may influence which cells each toxin targets. Here we describe the identification of CD11b, the α subunit of the αM/β2 integrin (CD11b/CD18), macrophage-1 antigen, or complement receptor 3, as a cellular receptor for leukocidin A/B (LukAB), an important toxin that contributes to S. aureus killing of human neutrophils. We demonstrate that CD11b renders human neutrophils susceptible to LukAB-mediated killing by purified LukAB as well as during S. aureus infection ex vivo. LukAB directly interacts with human CD11b by binding to the I domain, a property that determines the species specificity exhibited by this toxin. Identification of a LukAB cellular target has broad implications for the use of animal models to study the role of LukAB in S. aureus pathogenesis, explains the toxin’s tropism toward human neutrophils and other phagocytes, and provides a cellular therapeutic target to block the effect of LukAB toward human neutrophils.

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.

Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP‐1 and RANTES

Chemokines MCP‐1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP‐1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1α is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1α was able to overcome the potent inhibition of GM‐CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6‐ and 49‐fold, P = 4.0 × 10−7 and 4.0 × 10−8, respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP‐1, RANTES and MIP1α. Unexpectedly, treatment with MCP‐1 in the absence of RANKL resulted in 458‐fold induction of CCR4 (P = 1.0 × 10−10), while RANTES treatment resulted in twofold repression (P = 1.0 × 10−4). Since CCR2b and CCR4 are MCP‐1 receptors, these data support the existence of an MCP‐1 autocrine loop in human osteoclasts differentiated using RANKL. J. Cell. Biochem.

Differential carbohydrate recognition by Campylobacter jejuni strain 11168: influences of temperature and growth conditions.

The pathogenic clinical strain NCTC11168 was the first Campylobacter jejuni strain to be sequenced and has been a widely used laboratory model for studying C. jejuni pathogenesis. However, continuous passaging of C. jejuni NCTC11168 has been shown to dramatically affect its colonisation potential. Glycan array analysis was performed on C. jejuni NCTC11168 using the frequently passaged, non-colonising, genome sequenced (11168-GS) and the infrequently passaged, original, virulent (11168-O) isolates grown or maintained under various conditions. Glycan structures recognised and bound by C. jejuni included terminal mannose, N-acetylneuraminic acid, galactose and fucose. Significantly, it was found that only when challenged with normal oxygen at room temperature did 11168-O consistently bind to sialic acid or terminal mannose structures, while 11168-GS bound these structures regardless of growth/maintenance conditions. Further, binding of un-capped galactose and fucosylated structures was significantly reduced when C. jejuni was maintained at 25°C under atmospheric oxygen conditions. These binding differences identified through glycan array analysis were confirmed by the ability of specific lectins to competitively inhibit the adherence of C. jejuni to a Caco-2 intestinal cell line. Our data suggests that the binding of mannose and/or N-acetylneuraminic acid may provide the initial interactions important for colonisation following environmental exposure.

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni.

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

Significance The pneumococcus accounts for 25% of deaths in children under 5 y of age in developing countries. One of the most important virulence factors expressed by this pathogen is the pore-forming toxin, pneumolysin (Ply), an example of a Gram-positive cholesterol-dependent cytolysin (CDC). We show that Ply interacts with the Lewis histo-blood group antigen sialyl LewisX and that blocking this interaction can protect RBCs from lysis. We also identify glycan receptors on RBCs for the CDC streptolysin O from group A streptococcus. Our study supports the emerging paradigm shift that CDCs have cellular receptors other than cholesterol that define target cell tropism. The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Gene array identification of osteoclast genes: Differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) stimulates the formation of multinucleate osteoclast‐like cells. Treatment with M‐CSF alone results in the formation of macrophage‐like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R∝), transcription factors (nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPα)), and ribosomal proteins (60S L17 and 40S S20). Real‐time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c‐Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate‐resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30‐fold. Constant exposure of M‐CSF‐ and RANKL‐treated cells to GM‐CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.

Characterisation of a Multi-ligand Binding Chemoreceptor CcmL (Tlp3) of Campylobacter jejuni

Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.