Innate Immunity recent issues

Invited review: Profiling structural elements of short-chain lipopolysaccharide of non-typeable Haemophilus influenzae

Schweda, E. K.H., Twelkmeyer, B., Jianjun Li, — 2008-07-31

Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. A characteristic feature of H. influenzae LPS is the extensive intra- and inter-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behaviour of the bacterium. The chemical composition of non-typeable Haemophilus influenzae (NTHi) LPS is highly diverse. It contains a number of different monosaccharides (Neu5Ac, L-glycero-D-manno heptose, D-glycero -D-manno heptose, Kdo, D-Glc, D-Gal, D-GlcNAc, D-GalNAc) and non-carbohydrate substituents. Prominent non-carbohydrate components are O-acetyl groups, glycine and phosphates. We now know that sialic acid (N-acetylneuraminic acid or Neu5Ac) and certain oligosaccharide extensions are important in the pathogenesis of NTHi; however, the biological implications for many of the various features are still unknown. Electrospray ionization mass spectrometry in combination with separation techniques like CE and HPLC is an indispensable tool in profiling glycoform populations in heterogeneous LPS samples. Mass spectrometry is characterized by its extreme sensitivity. Trace amounts of glycoforms expressing important virulence determinants can be detected and characterized on minute amounts of material. The present review focuses on LPS structures and mass spectrometric methods which enable us to profile these in complex mixtures.

Flagellin and lipopolysaccharide up-regulation of IL-6 and CXCLi2 gene expression in chicken heterophils is mediated by ERK1/2-dependent activation of AP-1 and NF-{kappa}B signaling pathways

Kogut, M. H., Genovese, K. J., Haiqi He, , Kaiser, P. — 2008-07-31

The Toll-like receptor agonists, flagellin (FLG) and lipopolysaccharide (LPS), stimulate chicken heterophils to induce the expression and secretion of pro-inflammatory cytokines by a mechanism involving the triggering of differential MEK-ERK signaling cascades. However, the translocation and activation of transcription factors potentially involved in the control of cytokine gene expression remains unknown. Herein, we examined the effects of FLG and LPS on the activation of the transcription factors NF-B and AP-1 and their role in regulating heterophil activation leading to cytokine gene expression. Treatment of heterophils with either FLG or LPS induced a significant increase in DNA binding by the NF-B family members p50, c-Rel, and RelB. Likewise, FLG and LPS induced a significant increase in DNA binding by the AP-1 family members c-Jun and JunD. The activation of both NF-B and AP-1 was inhibited following treatment of heterophils with specific inhibitors of ERK1/2 (U0126 and PD098059), NF-B (Bay 11-7086 and the cell-permeable NF-B peptide, SN50), and AP-1 (Tanshinone IIA). Likewise, the up-regulation of gene expression of the pro-inflammatory cytokine, IL-6, and the inflammatory chemokine, CXCLi2, were inhibited when heterophils were treated with the same specific inhibitors. Taken together these data demonstrate that FLG and LPS stimulate the up-regulation of expression of IL-6 and CXCLi2 through an ERK1/2-dependent activation of both NF-B and AP-1.

Host species-specific usage of the TLR4-LPS receptor complex

Lizundia, R., Sauter, K.-S., Taylor, G., Werling, D. — 2008-07-31

Recognition of LPS depends on the interaction of at least three molecules forming the LPS-receptor complex. The most important ones, CD14, MD2 and Toll-like receptor (TLR) 4 share a high degree of homology between species. In the present study, we investigated the importance of species-specific restriction on the recognition of LPS using stably transfected HEK293 cell lines expressing either human or bovine LPS-receptor complex components. Species-specific MD2 appeared to confer LPS recognition, whereas species-specific CD14 only appeared to play a minor role. In addition to the recognition of LPS, there is evidence that the fusion (F) protein of respiratory syncytial virus (RSV), which is the most common viral respiratory pathogen during infancy world-wide, interacts with TLR4, and plays an important role in the initiation of the innate immune response. Our findings suggest that human and bovine RSV may activate human and bovine TLR4 receptors, respectively, in the presence of both MD2 and CD14. However, no clear role for the RSV F protein of either human or bovine RSV alone in stimulating TLR4-dependent NF-B activation was observed.

Purified human plasma glycosaminoglycans reduced NF-{kappa}B activation, pro-inflammatory cytokine production and apoptosis in LPS-treated chondrocytes

2008-07-31

Introduction: There have been several cases reporting a significant increase in chondroitin sulphate plasma levels in patients with different types of disease, such as systemic lupus erythematosus, rheumatoid arthritis, and liver disease. At present, the precise role of chondroitin sulphate molecules in blood is unclear. Previous investigations have shown that the addition of purified human plasma glycosaminoglycans (GAGs), containing a high percentage of chondroitin-4-sulphate (C4S) was able to inhibit lipid peroxidation and to protect cells from reactive oxygen species damage, suggesting antioxidant activity. Starting from these reports, the aim of this study was to evaluate the effectiveness of GAG structures purified from normal human plasma in reducing inflammation using a model of lipopolysaccharide (LPS)-induced increase of pro-inflammatory cytokines in mouse articular chondrocyte cultures.

Results: Chondrocyte stimulation with LPS (50 µg/ml) for 24 h enhanced gene expression of tumor necrosis factor alpha (TNF-), interleukin-1 beta (IL-1β), interleukin 6 (IL-6), interferon gamma (IFN-), inducible nitric oxide synthase (iNOS) and increases in their related protein levels, as well as NF-B activation, IB phosphorylation and apoptosis evaluated by the increase in caspase-3 expression and its related protein amount. LPS treatment also generated a high amount of nitric oxide (NO). The addition of different doses of purified human GAGs to LPSstimulated chondrocytes reduced inflammatory cytokines and iNOS both at mRNA and protein levels, blocked NF-B activation and cytoplasmic IB phosphorylation, limited cell death by inhibiting apoptosis, and reduced NO concentrations.

Conclusions: These results further support the hypothesis that plasma GAGs may function as immunomodulators and their increased release and degradation could be a biological response acting to modulate inflammation during disease.

Lipopolysaccharide associates with pro-atherogenic lipoproteins in periodontitis patients

2008-07-31

Introduction: Periodontitis patients are known to suffer from endotoxemia, which may be among the major risk factors for atherosclerosis. In health, lipopolysaccharide (LPS) is mainly carried with high density lipoprotein (HDL) particles. Shift of LPS toward lipoproteins with lower densities may result in less effective endotoxin scavenging. Our aim was to determine plasma LPS activity and lipoprotein-distribution before and after treatment in periodontitis patients.

Patients and Methods: Very low and intermediate density (VLDL-IDL), low density (LDL), HDL₂, HDL₃, and lipoprotein-deficient plasma (LPDP) were isolated by sequential ultracentrifugation. Patients included 34 subjects aged 53.5 ± 8.3 years, before and 6 months after periodontal treatment.

Results: The mean LPS distribution decreased among lipoprotein classes as follows: VLDL-IDL 41.3 ± 12.1%, LPDP 25.0 ± 7.0%, HDL₃ 13.1 ± 5.2%, LDL 11.5 ± 3.7%, and HDL₂ 9.2 ± 2.8%. Plasma and VLDL-IDL-associated LPS correlated positively, and LDL- and HDL-associated LPS negatively with clinical periodontal parameters and plasma cytokine concentrations. Mean plasma LPS activity increased after periodontal treatment from 44.0 ± 17.0 to 55.7 ± 24.2 EU/ml (P = 0.006). No significant changes were found in LPS lipoprotein distribution and lipoprotein compositions after the treatment.

Conclusions: Endotoxemia increases with severity of periodontitis. In periodontitis, LPS associates preferentially with the pro-atherogenic VLDL-IDL fraction. Periodontal treatment has only minor effects on plasma LPS activity or distribution, which reflects persistence of the disease.

Kupffer cell products and interleukin 1? directly promote VLDL secretion and apoB mRNA up-regulation in rodent hepatocytes

Bartolome, N., Arteta, B., Martinez, M. J., Chico, Y., Ochoa, B. — 2008-07-31

Plasma VLDL accumulation in Gram-negative sepsis is partly ascribed to an increased hepatic VLDL production driven by pro-inflammatory cytokines. We previously showed that hepatocytes of the Kupffer cell (KC)-rich periportal area are major contributors to enhanced VLDL production in lipopolysaccharide (LPS)-injected rats. However, it remains to be established whether KC generated products directly affect the number (apoB) and composition of secreted VLDL. Using rat primary cells, we show here that hepatocytes respond to stimulation by soluble mediators released by LPS-stimulated Kupffer cells with enhanced secretion of apoB and triglycerides in phospholipid-rich VLDL particles. Unstimulated KC products also augmented the secretion of normal VLDL, doubling apoB mRNA abundance. IL-1β treatment resulted in concentration-dependent increases of hepatocyte apoB mRNA and protein secretion, increases that were greater, but not additive, when combined with IL-6 and TNF-. Lipid secretion and MTP mRNA levels were unaffected by cytokines. In summary: (i) enhanced secretion of phospholipid-rich VLDL particles is a net hepatocyte response to LPS-stimulated KC products, which gives a clue about the local role of Kupffer cells in septic dyslipidemia induction; and (ii) pro-inflammatory cytokines act redundantly to enhance apoB secretion involving translational apoB up-regulation, but other humoral components or KC mediators are necessary to accomplish increased lipid association.

Review: Chemical and biological features of Burkholderia cepacia complex lipopolysaccharides

De Soyza, A., Silipo, A., Lanzetta, R., Govan, J. R., Molinaro, A. — 2008-06-18

The Burkholderia cepacia complex comprises 10 closely related Gram-negative organisms all of which appear capable of causing disease in humans. These organisms appear of particular relevance to patients with cystic fibrosis. Lipopolysaccharide (LPS) is an important virulence determinant in Gram-negative pathogens. In this review, we highlight important data within the field commenting on LPS/lipid A structure-to-function relationships and cytokine induction capacity of Burkholderia strains studied so far.

Evidence for infection, inflammation and shock in sudden infant death: parallels between a neonatal rat model of sudden death and infants who died of sudden infant death syndrome

Blood-Siegfried, J., Rambaud, C., Nyska, A., Germolec, D. R. — 2008-06-18

This study compared pathological findings from a neonatal rat model of sudden death with those from 40 sudden infant death syndrome (SIDS) infants collected at autopsy. In the rat model, influenza A virus was administered intranasally on postnatal day 10, and on day 12 a sublethal, intraperitoneal dose of Escherichia coli endotoxin; mortality was 80%. Tissue samples from the animals and infants were fixed in formaldehyde, embedded in paraffin, and sections stained with hematoxylin and eosin. Tissues from the SIDS specimens were additionally cultured for bacteria and viruses; post-mortem blood samples were evaluated for signs of inflammation. All sections were examined by a pediatric forensic pathologist familiar with SIDS pathology. Comparisons between the rat model and the human SIDS cases revealed that both exhibited gross and microscopic pathology related to organ shock, possibly associated with the presence of endotoxin. Uncompensated shock appeared to be a likely factor that caused death in both infants and rat pups. Response to a shock-inducing event might have played an important role in the events leading to death. The similarities between the neonatal rats and the human cases indicate that further research with the model might elucidate additional aspects of SIDS pathology.

The effect of systemic iNOS inhibition during human endotoxemia on the development of tolerance to different TLR-stimuli

Draisma, A., Dorresteijn, M., Pickkers, P., van der Hoeven, H. — 2008-06-18

The phenomenon of repeated exposure to endotoxin resulting in diminished release of pro-inflammatory cytokines is called endotoxin tolerance, in which there is a putative role for nitric oxide (NO). We investigated the effect of selective inducible NO-synthase (iNOS) inhibition during experimental human endotoxemia on the development of tolerance to various Toll-like receptor (TLR) agonists ex vivo. Volunteers received 2 ng/kg Escherichia coli endotoxin in the absence (n = 7) or presence (n = 7) of the selective iNOS inhibitor aminoguanidine (bolus 5 mM followed by a continuous infusion of 1.5 mmol/h). At 0, 2 and 4 h, blood samples were drawn for ex vivo stimulation with different TLR agonists. Experimental endotoxemia did not induce tolerance to TLR-2 and TLR-7 stimulation. In TLR-3, TLR-4 and TLR-5 stimulated whole blood, pro- and anti-inflammatory cytokine release was attenuated at 4 h, indicating that endotoxin-induced tolerance is not confined to subsequent TLR-4 stimulation alone. Aminoguanidine-treated subjects also developed tolerance to TLR-4 stimulation. In contrast, tolerance to TLR-3 stimulation did not occur for IL-10, and tolerance in TLR-5 stimulated blood did not develop for both pro- and anti-inflammatory cytokines. The role of NO in the development of tolerance is different for the various TLRs stimulated and pro- and anti-inflammatory cytokines measured.

(+)-Catechin inhibits tumour angiogenesis and regulates the production of nitric oxide and TNF-{alpha} in LPS-stimulated macrophages

Guruvayoorappan, C., Kuttan, G. — 2008-06-18

The anti-angiogenic activity of (+)-catechin as well as its regulatory effect on the production of nitric oxide and TNF were studied using in vivo and in vitro models. In vivo angiogenic activity was studied using B16F-10 melanoma cell-induced capillary formation in C57BL/6 mice. Administration of (+)-catechin significantly inhibited (36.09%) the number of tumour-directed capillaries induced by injecting B16F-10 melanoma cells on the ventral side of C57BL/6 mice. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1β, IL-6, TNF-, GM-CSF and the direct endothelial cell proliferating agent, VEGF. Administration of (+)-catechin could differentially regulate elevation of these cytokines. The differential elevation is further evidenced by the increased production of IL-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the B16F-10 injected, (+)-catechin-treated animals. In vitro L929 bioassay revealed the inhibition of TNF- production by (+)-catechin treatment. In the rat aortic ring assay, (+)-catechin inhibited the microvessel outgrowth at non-toxic concentrations. (+)-Catechin at non-toxic concentrations (5—25 µg/ml) showed significant inhibition in the proliferation, migration and tube formation of endothelial cells, which are the key events in the process of angiogenesis. (+)-Catechin also showed inhibitory effect on VEGF mRNA levels in B16F-10 melanoma cells. (+)-Catechin inhibited the production of NO and TNF- in LPS-stimulated primary macrophages. Taken together, these results demonstrate that (+)-catechin inhibits tumour-specific angiogenesis by regulating the production of pro- and anti-angiogenic factors such as pro-inflammatory cytokines, nitric oxide, VEGF, IL-2 and TIMP-1. These results also suggest that (+)-catechin could significantly inhibit nitrite and TNF- production in LPS-stimulated macrophages.

Expression profiling and binding properties of fibrinogen-related proteins (FREPs), plasma proteins from the schistosome snail host Biomphalaria glabrata

Zhang, S.-M., Yong Zeng, , Loker, E. S. — 2008-06-18

A growing body of evidence suggests an important role for fibrinogen-like proteins in innate immunity in both vertebrates and invertebrates. It has been shown that fibrinogen-related proteins (FREPs), plasma proteins present in the freshwater snail Biomphalaria glabrata, the intermediate host for the human blood fluke Schistosoma mansoni, are diverse and involved in snail innate defense responses. To gain further insight into the functions of FREPs, recombinant FREP proteins (rFREPs) were produced in Escherichia coli and antibodies (Abs) were raised against the corresponding rFREPs. We first show that most FREP proteins exist in their native conformation in snail hemolymph as multimeric proteins. Western blot analyses reveal that expression of multiple FREPs including FREP4 in plasma from M line and BS-90 snails, which are susceptible and resistant to S. mansoni infection, respectively, is up-regulated significantly after infection with the trematode Echinostoma paraensei. Moreover, our assays demonstrate that FREPs are able to bind E. paraensei sporocysts and their secretory/excretory products (SEPs), and a variety of microbes (Gram-positive and Gram-negative bacteria and yeast). Furthermore, this binding capability shows evidence of specificity with respect to pathogen type; for example, 65—75-kDa FREPs (mainly FREP4) bind to E. paraensei sporocysts and their SEPs whereas 95-kDa and 125-kDa FREPs bind the microbes assayed. Our results suggest that FREPs can recognize a wide range of pathogens, from prokaryotes to eukaryotes, and different categories of FREPs seem to exhibit functional specialization with respect to the pathogen encountered.

Hydrogen peroxide induces the production of tumor necrosis factor-{alpha} in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

Nakao, N., Kurokawa, T., Nonami, T., Tumurkhuu, G., Koide, N., Yokochi, T. — 2008-06-18

The effect of hydrogen peroxide (H₂O₂) on production of tumor necrosis factor (TNF)- was examined in RAW 264.7 murine macrophage cells. H₂O₂ led to production of TNF- up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H₂O₂ induced TNF- production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H₂O₂induced TNF- production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H₂O₂ induced the phosphorylation of p38 and SAPK. Further, H₂O₂ significantly augmented the AP-1 activity, but not nuclear factor (NF)-B activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H₂O₂-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H₂O₂-induced TNF production. H₂O₂ significantly enhanced lipopolysaccharide (LPS)-induced TNF- production. Therefore, H₂ O₂ was suggested to induce TNF- production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.

Invited review: Vascular cells contribute to atherosclerosis by cytokine- and innate-immunity-related inflammatory mechanisms

Loppnow, H., Werdan, K., Buerke, M. — 2008-04-07

Cardiovascular diseases are the human diseases with the highest death rate and atherosclerosis is one of the major underlying causes of cardiovascular diseases. Inflammatory and innate immune mechanisms, employing monocytes, innate receptors, innate cytokines, or chemokines are suggested to be involved in atherogenesis. Among the inflammatory pathways the cytokines are central players. Plasma levels of cytokines and related proteins, such as CRP, have been investigated in cardiovascular patients, tissue mRNA expression was analyzed and correlations to vascular diseases established. Consistent with these findings the generation of cytokine-deficient animals has provided direct evidence for a role of cytokines in atherosclerosis. In vitro cell culture experiments further support the suggestion that cytokines and other innate mechanisms contribute to atherogenesis. Among the initiation pathways of atherogenesis are innate mechanisms, such as toll-like-receptors (TLRs), including the endotoxin receptor TLR4. On the other hand, innate cytokines, such as IL-1 or TNF, or even autoimmune triggers may activate the cells. Cytokines potently activate multiple functions relevant to maintain or spoil homeostasis within the vessel wall. Vascular cells, not least smooth muscle cells, can actively contribute to the inflammatory cytokine-dependent network in the blood vessel wall by: (i) production of cytokines; (ii) response to these potent cell activators; and (iii) cytokine-mediated interaction with invading cells, such as monocytes, T-cells, or mast cells. Activation of these pathways results in accumulation of cells and increased LDL- and ECM-deposition which may serve as an `immunovascular memory' resulting in an ever-growing response to subsequent invasions. Thus, vascular cells may potently contribute to the inflammatory pathways involved in development and acceleration of atherosclerosis.

Removal of immune-stimulatory components from surfaces by plasma discharges

Hasiwa, M., Kylian, O., Hartung, T., Rossi, F. — 2008-04-07

Immune-stimulating microbiological components like lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan bound onto surfaces lead to severe problems when brought in contact with the organism via surgical instruments or implants. We have shown, in recent studies, that it is possible to detect different immune-stimulating components directly on the surface, via an indirect detection method, using human whole-blood and the monocyte reaction to measure the inflammatory mediator release (IL-1β) by ELISA. With regard to the inactivation of pyrogenic substances, we present a method based on the application of a low-pressure microwave plasma discharge working at low temperatures. We found a fast (10 s to a few minutes) removal rate of the immune-stimulating competence for LPS, LTA and zymosan. To mimic the bacterial cell-wall, LPS in combination with muramyl dipeptide was employed and the decreasing rate of the inflammatory signal did not differ from pure LPS.

Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1{beta}, TNF-{alpha} and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS

Diya Zhang, , Lili Chen, , Shenglai Li, , Zhiyuan Gu, , Jie Yan, — 2008-04-07

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P < 0.05) suppressed by anti-TLR2 antibody or JNK inhibitor, and the phosphorylation level of JNK was significantly increased (P < 0.05). These results indicate that TLR2—JNK is the main signaling pathway of P. gingivalis LPS-induced cytokine production, while the cytokine induction by E. coli LPS was mainly via TLR4—NF-B and TLR4—p38MAPK. This suggests that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in THP-1 cells, and that the TLR2—JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.

Interleukin-10 inhibits tumor necrosis factor-{alpha} production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

2008-04-07

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)- production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF- production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-B, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF- production via reduced MyD88 expression.

Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-{alpha}), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis

2008-04-07

The role of TLRs and MyD88 in the maintenance of gut integrity in response to dextran sodium sulfate (DSS)-induced colitis was demonstrated recently and led to the conclusion that the innate immune response to luminal commensal flora provides necessary signals that facilitate epithelial repair and permits a return to homeostasis after colonic injury. In this report, we demonstrate that a deficit in a single neutrophil chemokine, CXCL1/KC, also results in a greatly exaggerated response to DSS. Mice with a targeted mutation in the gene that encodes this chemokine responded to 2.5% DSS in their drinking water with significant weight loss, bloody stools, and a complete loss of gut integrity in the proximal and distal colon, accompanied by a predominantly mononuclear infiltrate, with few detectable neutrophils. In contrast, CXCL1/KC^{— /—} and wild-type C57BL/6J mice provided water showed no signs of inflammation and, at this concentration of DSS, wild-type mice showed only minimal histopathology, but significantly more infiltrating neutrophils. This finding implies that neutrophil infiltration induced by CXCL1/KC is an essential component of the intestinal response to inflammatory stimuli as well as the ability of the intestine to restore mucosal barrier integrity.

Editorial: From JER to INI

Holst, O. — 2008-02-19

Review: Innate immunity to tropical theileriosis

Ahmed, J. S., Glass, E. J., Salih, D. A., Seitzer, U. — 2008-02-19

The intracellular protozoan parasite Theileria annulata causes a severe, and often fatal, disease of pure and cross-bred cattle in tropical and subtropical countries. The present review refers to the importance of innate immunity as far as it is known to date in this infectious disease. Specifically, macrophages and the mediators produced by these cells are outlined. In addition, the latest findings concerning cattle breed differences in susceptibility to T. annulata infection in relation to macrophage activation are discussed.

Folimycin (concanamycin A) inhibits LPS-induced nitric oxide production and reduces surface localization of TLR4 in murine macrophages

Eswarappa, S. M., Basu, N., Joy, O., Chakravortty, D. — 2008-02-19

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and signals through a receptor complex which consists of TLR4, MD-2 and CD14. LPS signaling in macrophages induces the production of many pro-inflammatory molecules, including nitric oxide (NO). In this study, we have shown that folimycin, a macrolide antibiotic and a specific inhibitor of vacuolar ATPase (V-ATPase), inhibits LPS-induced NO production, but not TNF production, in murine elicited peritoneal macrophages. However, folimycin did not affect interferon- induced NO production. LPS-induced iNOS mRNA and protein expression and NF-B activation were also inhibited by folimycin. Interestingly, folimycin-treated cells showed reduced surface expression of TLR4 molecules and dilated Golgi apparatus. These findings suggest that folimycin, by inhibiting V-ATPases, alters intra-Golgi pH, which in turn causes defective processing and reduced surface expression of TLR4 reducing the strength of LPS signaling in murine macrophages.

Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus

2008-02-19

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone -D-GalpA-(1->4)-β-D-DAG-(1->6)--D-DAG-(1->1)--D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[⁵-12:1(3-OH)]] and 12:0[3-O(⁵-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3OH) and 12:0[3,6-(OH)₂] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF- inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.

Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins

2008-02-19

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure -chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.

Clearance of bacterial lipopolysaccharides and lipid A by the liver and the role of arginino-succinate synthase

Satoh, M., Ando, S., Shinoda, T., Yamazaki, M. — 2008-02-19

The liver is thought to be involved in the systemic clearance and detoxification of lipopolysaccharide (LPS). Argininosuccinate synthase (AS), a liver cytosolic urea cycle enzyme, has been found to bind to and inactivate LPS and lipid A. To elucidate the participation of AS in the clearance of LPS by liver and hepatocytes, we investigated the correlation between AS content and the removal of lipid A and LPS in vivo and in vitro, tracing levels of biological activity. A hepatotoxic model in which mice were injected with CCl₄ revealed a significant reduction in lipid A clearance along with liver failure on day 1; total body clearance was changed to 0.534 ml/min from 1.42 ml/min. AS content in liver concomitantly decreased to about half and AS leaked to blood at about 6 µg/ml. Total body clearance of i.v. injected AS was estimated at 0.083 ml/min, which predicted about 24-h leakage of AS after CCl₄ injection. The treatment also reduced the clearance of R-type LPSs to a lesser degree the larger its polysaccharide portion. S-type LPS, which has a large O-antigen polysaccharide, exhibited enhancement of clearance on CCl₄ treatment. When pretreated in vitro with AS and injected into normal mice, lipid A and R-type LPS showed a similar pattern of clearance of residual activities to the untreated forms, but S-type LPS exhibited enhancement of clearance. Comparison between different strains of mice revealed a correlation of AS content in liver and lipid A clearance, where the higher AS strain C3H/He mice showed a more rapid clearance than the lower AS strains C57BL/6 and BALB/c. Primary spheroid cultures of hepatocytes treated with 0.1 µM dexamethasone and 1 µM glucagon showed about a 2-fold increase in AS amount and a more rapid clearance of LPS from culture medium than untreated cells. These results suggest that AS in hepatocytes may be involved in the process of lipid A and LPS clearance and the extracellular leakage of AS may also participate in the systemic detoxification.