Tom Sprong

NOD2 mediates anti-inflammatory signals induced by TLR2 ligands: implications for Crohn's disease.

Mutations of the NOD2 gene have been associated with an increased susceptibility to Crohns disease, but the pathogenetic mechanisms mediated by NOD2 remain elusive. In the present study, we demonstrate that the 3020insC frameshift‐mutation in the NOD2 gene associated with Crohns disease results in defective release of IL‐10 from blood mononuclear cells after stimulation with the Toll‐like receptor (TLR)2 ligands, peptidoglycan and Pam3Cys‐KKKK, but not with bacterial LPS, a TLR4 ligand. The potential pathophysiological significance of this finding in patients with Crohns disease and who are homozygous for this NOD2 mutation was substantiated by the finding of decreased anti‐inflammatory cytokine release when cells from these patients were stimulated with different species of Bacteroides, an enteric microorganism implicated in the pathogenesis of Crohns disease. In conclusion, defective NOD2 function results in a pro‐inflammatory cytokine bias after stimulation of mononuclear cells with TLR2 stimuli, and this could contribute to the overwhelming inflammation seen in Crohns disease.

Chronic Q fever: review of the literature and a proposal of new diagnostic criteria.

A review was performed to determine clinical aspects and diagnostic tools for chronic Q fever. We present a Dutch guideline based on literature and clinical experience with chronic Q fever patients in The Netherlands so far. In this guideline diagnosis is categorized as proven, possible or probable chronic infection based on serology, PCR, clinical symptoms, risk factors and diagnostic imaging.

Vascular Endothelial Growth Factor Is Increased During The First 48 Hours Of Human Septic Shock And Correlates With Vascular Permeability

Meningococcal septic shock is an important cause of morbidity and mortality in children and young adults worldwide and is the prototypical gram-negative septic shock. One of the key factors in the development of shock is increased microvascular permeability. Vascular endothelial growth factor (VEGF) is a central factor in angiogenesis and is an important mediator of vascular permeability. Thirteen patients with meningococcal infection (eight presenting with shock) were investigated in the early phase of invasive meningococcal disease. Cytokines, complement activation, and VEGF plasma concentrations were measured during the first 48 h on the pediatric intensive care unit. Increased cytokine concentrations and activation of the complement system were observed. VEGF plasma concentrations were increased (median 193 pg/mL, range 71-1082) and were highest in the presence of shock (208 pg/mL, 169-1082) compared with patients presenting without shock (92 pg/mL range 71-299). VEGF concentration at admission correlated with the severity of disease (pediatric risk of mortality score, R = 0.90 [Spearman], P = 0.0001) and the amount of fluids administered within the first 24 h (R = 0.90, P < 0.0001). In all patients, a decrease in VEGF was associated with a decrease in fluid intake during t = 24 to 48 h. The results suggest that apart from correlation with IL-1β, −10, −12, and complement activation, microvascular permeability in sepsis is also closely linked to the plasma concentration of VEGF. The role of VEGF in sepsis-associated increased microvascular permeability needs further exploration and may represent a new therapeutic target.

Bartonella quintana Lipopolysaccharide Is a Natural Antagonist of Toll-Like Receptor 4

ABSTRACT Bartonella quintana is a gram-negative microorganism that causes trench fever and chronic bacteremia. B. quintana lipopolysaccharide (LPS) was unable to induce the production of proinflammatory cytokines in human monocytes. Interestingly, B. quintana LPS is a potent antagonist of Toll-like receptor 4 (TLR4), as it inhibited both mRNA transcription and the release of tumor necrosis factor alpha, interleukin 1β (IL-1β), and IL-6 by Escherichia coli LPS in human monocytes, at ratios ranging from 1,000:1 to 10:1 (B. quintana LPS to E. coli LPS). Likewise, B. quintana LPS blocked the interaction of E. coli LPS with TLR4 in transfected cell lines. The extent of the inhibitory effect of B. quintana LPS was demonstrated in microarray studies, which showed downregulation of practically all genes induced by LPS in monocytes. Because of the role of TLR4 in inflammation, B. quintana LPS may prove useful as a potent anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation.

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response

To determine the relative contribution of lipopolysaccharide (LPS) and non‐LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild‐type serogroup B meningococci (strain H44/76), and LPS‐deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral‐blood mononuclear cells with wild‐type and LPS‐deficient meningococci showed that non‐LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β production and virtually all interferon (IFN)‐γ production. Based on tricine sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of LPS in proteinase K‐treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine‐inducing capacity of isolated and purified LPS and LPS‐containing meningococci. At concentrations of >107 bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non‐LPS components was additive to that by LPS. Experiments with mice showed that non‐LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non‐LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF‐α, IL‐1β, and IFN‐γ production.

Pentraxin 3 and C-reactive protein in severe meningococcal disease.

The long pentraxin 3 (PTX3) is an important element of the innate immune system and has potential as a diagnostic tool in inflammatory conditions. We studied PTX3 in patients admitted to an intensive care unit with severe meningococcal disease and compared it with the short pentraxin C-reactive protein (CRP). Twenty-six patients with meningococcal disease were studied, 17 patients presented with meningococcal septic shock (shock group), and 9 patients presented with meningococcal meningitis or bacteremia (no-shock group). Pentraxin 3 and CRP were measured by enzyme-linked immunosorbent assay. High plasma concentrations of PTX3 (median, 579 &mgr;g/L) were seen at admission in patients with meningococcal disease. Concentrations were significantly higher in patients with shock compared with patients without shock (medians, 801 and 256 &mgr;g/L, respectively; P = 0.006). In contrast, CRP at admission was lower in the shock group as compared with the no-shock group (medians, 58 and 165 mg/L, respectively; P = 0.008). High PTX3 and low CRP concentration at admission discriminated between presence and absence of shock (area under the receiver operating characteristic curve, 0.85; P = 0.007 for PTX3 and area under the receiver operating characteristic curve, 0.84; P = 0.01 for CRP). PTX3 did not correlate with disease severity (pediatric risk of mortality) and days spent in the intensive care unit. PTX3 at admission and PTX3 peak concentration both showed a negative correlation with plasma fibrinogen concentrations. C-reactive protein concentration at admission correlated negatively with disease severity. In conclusion, PTX3 was an early indicator of shock in patients with severe meningococcal disease that followed a pattern of induction distinct from CRP.

Complement activation and complement-dependent inflammation by Neisseria meningitidis are independent of lipopolysaccharide

ABSTRACT Fulminant meningococcal sepsis has been termed the prototypical lipopolysaccharide (LPS)-mediated gram-negative septic shock. Systemic inflammation by activated complement and cytokines is important in the pathogenesis of this disease. We investigated the involvement of meningococcal LPS in complement activation, complement-dependent inflammatory effects, and cytokine or chemokine production. Whole blood anticoagulated with lepirudin was stimulated with wild-type Neisseria meningitidis H44/76 (LPS+), LPS-deficient N. meningitidis H44/76lpxA (LPS−), or purified meningococcal LPS (NmLPS) at concentrations that were relevant to meningococcal sepsis. Complement activation products, chemokines, and cytokines were measured by enzyme-linked immunosorbent assays, and granulocyte CR3 (CD11b/CD18) upregulation and oxidative burst were measured by flow cytometry. The LPS+ and LPS−N. meningitidis strains both activated complement effectively and to comparable extents. Purified NmLPS, used at a concentration matched to the amount present in whole bacteria, did not induce any complement activation. Both CR3 upregulation and oxidative burst were also induced, independent of LPS. Interleukin-1β (IL-1β), tumor necrosis factor alpha, and macrophage inflammatory protein 1α production was predominantly dependent on LPS, in contrast to IL-8 production, which was also markedly induced by the LPS− meningococci. In this whole blood model of meningococcal sepsis, complement activation and the immediate complement-dependent inflammatory effects of CR3 upregulation and oxidative burst occurred independent of LPS.

Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways

Despite the differences in the molecular structure between lipopolysaccharides (LPS) isolated from Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium, the potential differences in their biological effects in vivo have not been investigated. In the present study, TNF and LT double knock‐out (TNF−/−LT−/−) mice were almost as susceptible as TNF+/+LT+/+ controls to S. typhimurium LPS, but they were significantly more resistant to lethal endotoxemia induced by E. coli or K. pneumoniae LPS. The effect was not due to endotoxin‐associated proteins. In the knock‐out mice, this difference in lethality was accompanied by decreased interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) production after challenge with E. coli LPS, whereas after S. typhimurium LPS more IL‐1 and IFN‐γ were produced. In contrast, more IL‐10 was produced after challenge of mice with E. coli LPS than with S. typhymurium LPS. The hypothesis that a combination of pro‐inflammatory cytokines is responsible for the mortality after S. typhimurium LPS was suggested by experiments in mice deficient in IL‐1β‐converting enzyme (ICE−/− mice). ICE‐/‐mice, lacking mature IL‐1β and IL‐18, but also defective in IFN‐γ and TNF production, were completely protected against both E. coli and S. typhimurium LPS. Experiments in Toll‐like receptor (TLR)‐4 defective mice suggested that the difference is not due to differential activation of TLR4. In conclusion, TNF and LT play a central role in the lethality due to E. coli LPS, whereas the lethal effects of S. typhimurium LPS are mediated through mechanisms also involving other cytokines such as IFN‐γ, IL‐1 and IL‐18.

A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease

Macrophage migration inhibitory factor (MIF) is an abundantly expressed proinflammatory cytokine playing a critical role in innate immunity and sepsis and other inflammatory diseases. We examined whether functional MIF gene polymorphisms (–794 CATT5–8 microsatellite and –173 G/C SNP) were associated with the occurrence and outcome of meningococcal disease in children. The CATT5 allele was associated with the probability of death predicted by the Pediatric Index of Mortality 2 (P=0.001), which increased in correlation with the CATT5 copy number (P=0.04). The CATT5 allele, but not the —173 G/C alleles, was also associated with the actual mortality from meningoccal sepsis [OR 2.72 (1.2‐6.4), P=0.02]. A family‐based association test (i.e., transmission disequilibrium test) performed in 240 trios with 1 afflicted offspring indicated that CATT5 was a protective allele (P=0.02) for the occurrence of meningococcal disease. At baseline and after stimulation with Neisseria meningitidis in THP‐1 monocytic cells or in a whole‐blood assay, CATT5 was found to be a low‐expression MIF allele (P= 0.005 and P=0.04 for transcriptional activity; P=0.09 and P=0.09 for MIF production). Taken together, these data suggest that polymorphisms of the MIF gene affecting MIF expression are associated with the occurrence, severity, and outcome of meningococcal disease in children.—Renner, P., Roger, T., Bochud, P.‐Y., Sprong, T., Sweep, F. C. G. J., Bochud, M., Faust, S. N., Haralambous, E., Betts, H., Chanson, A.‐L., Reymond, M. K., Mermel, E., Erard, V., van Deuren, M., Read, R. C., Levin, M., Calandra, T. A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease. FASEB J. 26, 907–916 (2012). www.fasebj.org

Complement plays a central role in Candida albicans-induced cytokine production by human PBMCs

In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans‐induced host proinflammatory cytokine production via C5a‐C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a‐C5aR signaling pathway, either with anti‐C5a antagonist antibodies or with the C5aR antagonist W‐54001, C. albicans‐induced IL‐6 and IL‐1β levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans‐induced IL‐6 and IL‐1β production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti‐Candida host defense in humans.