Susu M. Zughaier

Differential Induction of the Toll-Like Receptor 4-MyD88-Dependent and -Independent Signaling Pathways by Endotoxins

ABSTRACT The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), and the MyD88-independent molecules beta interferon (IFN-β), nitric oxide, and IFN-γ-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-α, IL-1β, and MIP-3α, but significantly less IFN-β, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-β, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-α and MIP-3α in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-α release but did not influence nitric oxide release. IFN-β polyclonal antibody and IFN-α/β receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

Cationic Antimicrobial Peptide Resistance in Neisseria meningitidis

Cationic antimicrobial peptides (CAMPs) are important components of the innate host defense system against microbial infections and microbial products. However, the human pathogen Neisseria meningitidis is intrinsically highly resistant to CAMPs, such as polymyxin B (PxB) (MIC > or = 512 microg/ml). To ascertain the mechanisms by which meningococci resist PxB, mutants that displayed increased sensitivity (> or =4-fold) to PxB were identified from a library of mariner transposon mutants generated in a meningococcal strain, NMB. Surprisingly, more than half of the initial PxB-sensitive mutants had insertions within the mtrCDE operon, which encodes proteins forming a multidrug efflux pump. Additional PxB-sensitive mariner mutants were identified from a second round of transposon mutagenesis performed in an mtr efflux pump-deficient background. Further, a mutation in lptA, the phosphoethanolamine (PEA) transferase responsible for modification of the lipid A head groups, was identified to cause the highest sensitivity to PxB. Mutations within the mtrD or lptA genes also increased meningococcal susceptibility to two structurally unrelated CAMPs, human LL-37 and protegrin-1. Consistently, PxB neutralized inflammatory responses elicited by the lptA mutant lipooligosaccharide more efficiently than those induced by wild-type lipooligosaccharide. mariner mutants with increased resistance to PxB were also identified in NMB background and found to contain insertions within the pilMNOPQ operon involved in pilin biogenesis. Taken together, these data indicated that meningococci utilize multiple mechanisms including the action of the MtrC-MtrD-MtrE efflux pump and lipid A modification as well as the type IV pilin secretion system to modulate levels of CAMP resistance. The modification of meningococcal lipid A head groups with PEA also prevents neutralization of the biological effects of endotoxin by CAMP.

Incidence of macrolide resistance in Streptococcus pneumoniae after introduction of the pneumococcal conjugate vaccine: population-based assessment

BACKGROUND The prevalence of macrolide resistance in Streptococcus pneumoniae has risen in recent years after the introduction of new macrolides and their increased use. We assessed emergence of macrolide-resistant invasive S pneumoniae disease in Atlanta, GA, USA, before and after the licensing, in February 2000, of the heptavalent pneumococcal conjugate vaccine for young children. METHODS Prospective population-based surveillance was used to obtain pneumococcal isolates and demographic data from patients with invasive pneumococcal disease. We calculated cumulative incidence rates for invasive pneumococcal disease for 1994-2002 using population estimates and census data from the US Census Bureau. FINDINGS The incidence of invasive pneumococcal disease in Atlanta fell from 30.2 per 100,000 population (mean annual incidence 1994-99) to 13.1 per 100,000 in 2002 (p<0.0001). Striking reductions were seen in children younger than 2 years (82% decrease) and in those 2-4 years (71% decrease), age-groups targeted to receive pneumococcal conjugate vaccine. Significant declines were also noted in adults aged 20-39 (54%), 40-64 (25%), and 65 years and older (39%). Macrolide resistance in invasive S pneumoniae disease in Atlanta, after increasing steadily from 4.5 per 100,000 in 1994 to 9.3 per 100,000 in 1999, fell to 2.9 per 100,000 by 2002. Reductions in disease caused by mefE-mediated and erm-mediated macrolide-resistant isolates of conjugate-vaccine serotypes 6B, 9V, 19F, and 23F, and the vaccine-associated serotype 6A were also recorded. INTERPRETATION Vaccines can be a powerful strategy for reducing antibiotic resistance in a community.

Neisseria meningitidis Lipooligosaccharide Structure-Dependent Activation of the Macrophage CD14/Toll-Like Receptor 4 Pathway

ABSTRACT Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS α or β chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-d-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an ∼10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.

Antimicrobial peptides and endotoxin inhibit cytokine and nitric oxide release but amplify respiratory burst response in human and murine macrophages

Antimicrobial peptides (AMPs), in addition to their antibacterial properties, are also chemotactic and signalling molecules that connect the innate and adaptive immune responses. The role of AMP [α defensins, LL‐37, a cathepsin G‐derived peptide (CG117‐136), protegrins (PG‐1), polymyxin B (PMX) and LLP1] in modulating the respiratory burst response in human and murine macrophages in the presence of bacterial endotoxin [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] was investigated. AMP were found to neutralize endotoxin induction of nitric oxide and TNFα release in macrophages in a dose‐dependent manner. In contrast, macrophages primed overnight with AMP and LOS or LPS significantly enhanced reactive oxygen species (ROS) release compared with cells primed with endotoxin or AMP alone, while no responses were seen in unprimed cells. This enhanced ROS release by macrophages was seen in all cell lines including those obtained from C3H/HeJ (TLR4–/–) mice. Similar effects were also seen when AMP and endotoxin were added directly with zymosan to trigger phagocytosis and the respiratory burst in unprimed RAW 264.7 and C3H/HeJ macrophages. Amplification of ROS release was also demonstrated in a cell‐free system of xanthine and xanthine oxidase. Although AMP inhibited cytokine and nitric oxide induction by endotoxin in a TLR4‐dependent manner, AMP and endotoxin amplified ROS release in a TLR4‐independent manner possibly by exerting a prolonged catalytic effect on the ROS generating enzymes such as the NADPH‐oxidase complex.

Impact of vitamin D supplementation on markers of inflammation in adults with cystic fibrosis hospitalized for a pulmonary exacerbation

Patients with cystic fibrosis (CF) suffer from chronic lung infection and inflammation leading to respiratory failure. Vitamin D deficiency is common in patients with CF, and correction of vitamin D deficiency may improve innate immunity and reduce inflammation in patients with CF. We conducted a double-blinded, placebo-controlled, randomized clinical trial of high-dose vitamin D to assess the impact of vitamin D therapy on antimicrobial peptide concentrations and markers of inflammation. We randomized 30 adults with CF hospitalized with a pulmonary exacerbation to 250 000 IU of cholecalciferol or placebo, and evaluated changes in plasma concentrations of inflammatory markers and the antimicrobial peptide LL-37 at baseline and 12 weeks post intervention. In the vitamin D group, there was a 50.4% reduction in tumor necrosis factor-α (TNF-α) at 12 weeks (P<0.01), and there was a trend for a 64.5% reduction in interleukin-6 (IL-6) (P=0.09). There were no significant changes in IL-1β, IL-8, IL-10, IL-18BP and NGAL (neutrophil gelatinase-associated lipocalin). We conclude that a large bolus dose of vitamin D is associated with reductions in two inflammatory cytokines, IL-6 and TNF-α. This study supports the concept that vitamin D may help regulate inflammation in CF, and that further research is needed to elucidate the potential mechanisms involved and the impact on clinical outcomes.

Induction of Reactive Oxygen Species-mediated Autophagy by a Novel Microtubule-modulating Agent

Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G1 population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents.

High-dose cholecalciferol reduces parathyroid hormone in patients with early chronic kidney disease: a pilot, randomized, double-blind, placebo-controlled trial

BACKGROUND Vitamin D deficiency contributes to secondary hyperparathyroidism, which occurs early in chronic kidney disease (CKD). OBJECTIVES We aimed to determine whether high-dose cholecalciferol supplementation for 1 y in early CKD is sufficient to maintain optimal vitamin D status (serum 25-hydroxyvitamin D [25(OH)D] concentration ≥30 ng/mL) and decrease serum parathyroid hormone (PTH). A secondary aim was to determine the effect of cholecalciferol on blood pressure and serum fibroblast growth factor-23 (FGF23). DESIGN This was a double-blind, randomized, placebo-controlled trial. Forty-six subjects with early CKD (stages 2-3) were supplemented with oral cholecalciferol (vitamin D group; 50,000 IU/wk for 12 wk followed by 50,000 IU every other week for 40 wk) or a matching placebo for 1 y. RESULTS By 12 wk, serum 25(OH)D increased in the vitamin D group only [baseline (mean ± SD): 26.7 ± 6.8 to 42.8 ± 16.9 ng/mL; P < 0.05] and remained elevated at 1 y (group-by-time interaction: P < 0.001). PTH decreased from baseline only in the vitamin D group (baseline: 89.1 ± 49.3 to 70.1 ± 24.8 pg/mL; P = 0.01) at 12 wk, but values were not significantly different from baseline at 1 y (75.4 ± 29.5 pg/mL; P = 0.16; group-by-time interaction: P = 0.09). Group differences were more pronounced in participants with secondary hyperparathyroidism (group-by-time interaction: P = 0.004). Blood pressure and FGF23 did not change in either group. CONCLUSIONS After 1 y, this oral cholecalciferol regimen was safe and sufficient to maintain serum 25(OH)D concentrations and prevent vitamin D insufficiency in early CKD. Furthermore, serum PTH improved after cholecalciferol treatment, particularly in patients who had secondary hyperparathyroidism.

The role of vitamin D in regulating the iron-hepcidin-ferroportin axis in monocytes

Chronic kidney disease affects 40% of adults aged 65 and older. Anemia of CKD is present in 30% of patients with CKD and is associated with increased cardiovascular risk, decreased quality of life, and increased mortality. Hepcidin-25 (hepcidin), the key iron regulating hormone, prevents iron egress from macrophages and thus prevents normal recycling of the iron needed to support erythropoiesis. Hepcidin levels are increased in adults and children with CKD. Vitamin D insufficiency is highly prevalent in CKD and is associated with erythropoietin hyporesponsiveness. Recently, hepcidin levels were found to be inversely correlated with vitamin D status in CKD. The aim of this study was to investigate the role of vitamin D in the regulation of hepcidin expression in vitro and in vivo. This study reports that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, is associated with decreased hepcidin and increased ferroportin expression in lipopolysaccharide (LPS) stimulated THP-1 cells. 1,25(OH)2D3 also resulted in a dose-dependent decrease in pro-hepcidin cytokines, IL-6 and IL-1β, release in vitro. Further, we show that high-dose vitamin D therapy impacts systemic hepcidin levels in subjects with early stage CKD. These data suggest that improvement in vitamin D status is associated with lower systemic concentrations of hepcidin in subjects with CKD. In conclusion, vitamin D regulates the hepcidin-ferroportin axis in macrophages which may facilitate iron egress. Improvement in vitamin D status in patients with CKD may reduce systemic hepcidin levels and may ameliorate anemia of CKD.

Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence of Neisseria meningitidis

ABSTRACT Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.