The local transcriptional response was studied in different locations of lungs from pigs experimentally infected with the respiratory pathogen Actinobacillus pleuropneumoniae serotype 5B, using porcine cDNA microarrays. This infection gives rise to well-demarcated infection loci in the lung, characterized by necrotic and haemorrhagic lesions. Lung tissue was sampled from necrotic areas, from visually unaffected areas and from areas bordering on necrotic areas. Expression pattern of these areas from infected pigs was compared to healthy lung tissue from un-infected pigs. Transcription of selected genes important in the innate defence response were further analysed by quantitative real-time reverse-transcriptase PCR. A clear correlation was observed between the number of differentially expressed genes as well as the magnitude of their induction and the sampling location in the infected lung, with the highest number of differentially expressed genes, and the most highly induced genes found in necrotic areas. Interestingly, a group of differentially regulated genes was represented in all three areas, comprising genes encoding cytokines, acute phase proteins, and factors related to regulation of apoptosis and the complement system. Interferon- was down-regulated in both necrotic and bordering areas. Evidence of neutrophil recruitment was seen by the up-regulation of chemotactic factors for neutrophils. In conclusion, we found subsets of genes expressed at different levels in the three selected areas of the infected lung as compared to the control group. Thus it is demonstrated that an infection with clearly defined infected loci leads to a rapid disseminated intra-organ response in neighbouring seemingly unaffected tissue areas of the infected organ. Within the lung, we found a clear division of induced genes as, in unaffected areas a large part of differently expressed genes were involved in systemic reactions to infections, while differently expressed genes in necrotic areas were mainly concerned with homeostasis regulation.

Muramyldipeptide (MDP), the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2). Muramyldipeptide enhances the activities of lipopolysaccharide (LPS), but the mechanism underlying this effect is unclear. Here, we obtained evidence that intravenously injected MDP augments LPS-induced hypothermia in wild-type mice, but not in mice deficient in interleukin (IL)-1/β and/or tumor-necrosis factor (TNF)-. Muramyldipeptide also: (i) increased pro-IL-1β in tissues, but did not increase IL-1β in serum (since caspase-1 was not activated by MDP); (ii) down-regulated the expression of suppressor of cytokine signaling 1 (SOCS1; a negative-feedback regulator of LPS-induced signaling); and (iii) augmented the LPS-induced production of TNF-, IL-12 p40, and interferon (IFN)-. Moreover, by performing in vivo and in vitro experiments, we obtained evidence that macrophages were involved in these effects of MDP. These results suggest that two different mechanisms may underlie the augmenting effect of MDP: namely, stimulation of pro-IL-1β production by, and down-regulation of SOCS1 in, macrophages. We consider that this work may help to elucidate the pathogenesis of mixed bacterial infections, including septic shock and multiple organ dysfunction syndrome (MODS).

Abscess formation associated with secondary peritonitis causes severe morbidity and can be fatal. Formation of abscesses requires the presence of CD4⁺ T-cells. Zwitterionic polysaccharides (ZPSs) represent a novel class of immunomodulatory bacterial antigens that stimulate CD4⁺ T-cells in a major histocompatibility complex (MHC) class II-dependent manner. The capsular polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 possesses a zwitterionic charge with free amino groups and promotes T-cell-dependent abscess formation in an experimental mouse model. So far, nothing is known about the function of Interleukin (IL)-6 in intraperitoneal abscess formation. Here, we demonstrate that macrophages and dendritic cells (DCs), the most prevalent professional antigen-presenting cells involved in the formation of abscesses, secrete Interleukin (IL)-6 and are incorporated in the abscess capsule. Sp1 inhibits apoptosis of CD4⁺ T-cells and causes IL-17 expression by CD4⁺ T-cells in an IL-6-dependent manner. Abrogation of the Sp1-induced pleiotropic effects of IL-6 in IL-6-deficient mice and mice treated with an IL-6-specific neutralizing antibody results in significant inhibition of abscess formation. The data delineate the essential role of IL-6 in the linkage of innate and adaptive immunity in polysaccharide-mediated abscess formation.

Human odontoblasts trigger immune response s to oral bacteria that invade dental tissues during the caries process. To date, their ability to regulate the expression of the nucleotide-binding domain leucine-rich repeat containing receptor NOD2 when challenged by Gram-positive bacteria is unknown. In this study, we investigated NOD2 expression in healthy and inflamed human dental pulps challenged by bacteria, and in cultured odontoblast-like cells stimulated with lipoteichoic acid (LTA), a Toll-like receptor (TLR) 2 agonist which is specific for Gram-positive bacteria. We found that NOD2 gene expression was significantly up-regulated in pulps with acute inflammation compared to healthy ones. In vitro, LTA augmented NOD2 gene expression and protein level in odontoblast-like cells. The increase was more pronounced in odontoblast-like cells compared to dental pulp fibroblasts. Blocking experiments in odontoblast-like cells with anti-TLR2 antibody strongly reduced the NOD2 gene expression increase, whereas stimulation with the synthetic TLR2 ligand Pam₂CSK₄ confirmed NOD2 gene up-regulation following TLR2 engagement. These data suggest that NOD2 up-regulation is part of the odontoblast immune response to Gram-positive bacteria and might be important in protecting human dental pulp from the deleterious effects of cariogenic pathogens.

Cystic fibrosis is an autosomal recessive disorder and it is characterised by chronic bacterial airway infection which leads to progressive lung deterioration, sometimes with fatal outcome. Burkholderia multivorans and Burkholderia cenocepacia are the species responsible for most of the infections of cystic fibrosis patients. Lipopolysaccharide endotoxins (LPSs) are among the foremost factors of pathogenesis of Gram-negative infection and, in particular, lipid A is the endotoxic portion of LPS responsible for eliciting host innate immune response. In this work, the complete primary structure of the lipid A from B. multivorans C1576 has been defined and, further, its pro-inflammatory activity in a cystic fibrosis airways model is shown. The structure of B. multivorans lipid A was attained by chemical, mass spectrometry and nuclear magnetic resonance analyses whereas its biological activity was assessed on the intestinal epithelial cell line CACO-2 cells, on the airway epithelial IB3-1 cells, carrying the F508/W1282X CFTR mutation and on an ex vivo model of culture explants of nasal polyps.

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli Rc chemotype (J5) mutant lipopolysaccharide (J5dLPS)–group B meningococcal outer membrane protein (OMP) complex vaccine protected animals from lethal sepsis. The protection from sepsis is believed to be dependent on high levels of antibodies against the core glycolipid (CGL), a region of LPS that is rather conserved among Enterobacteriaceae. The addition of unmethylated deoxycytidyl-deoxyguanosine dinucleotide (CpG)-containing oligodeoxynucleotides (ODN) was used as an immuno-adjuvant to improve antibody responses. In preparation for a Phase I human trial, we elucidated potential contributions by which the sepsis vaccine (J5dLPS–OMP) and CpG ODN might enhance the antibody response and provide evidence that the generation of immune responses is Toll-like receptor (TLR) dependent. Toll-like receptor 2, TLR4, and TLR9 were each essential for generating robust cytokine and antibody responses. The signature cytokine of dendritic cells, interleukin-12, was one of the cytokines that demonstrated synergy with the optimal TLR ligand/engagement combination. We conclude that the involvement of multiple TLRs upon immunization was critical for the generation of optimal antibody responses. These observations provide further evidence for the inclusion of innate immune-based adjuvants during the development of next-generation vaccines.

Platelet factor 4 (CXCL4), a member of the CXC chemokine subfamily released in high amounts by activated platelets, has been identified as a monocyte survival factor that induces monocyte differentiation into macrophages. Although CXCL4 has been shown to have biological effects unique to chemokines, nothing is known about the role of CXCL4-derived human macrophages or CXCL4 in human immunodeficiency virus (HIV) disease. In this study, CXCL4-derived macrophages are compared with macrophage-colony stimulating factor (M-CSF)-derived macrophages for their ability to support HIV-1 replication. We show that CXCL4-derived macrophages can be infected with macrophage-tropic HIV-1 that uses either CC-chemokine receptor 5 (CCR5) or CXC-chemokine receptor 4 (CXCR4) as a co-receptor for viral entry. We also find that M-CSF and the chemokines, monocyte chemoattractant protein 1 (MCP-1; CCL2) and macrophage-inflammatory-protein-1-alpha (MIP-1; CCL3) are produced upon R5- and X4-tropic HIV-1 replication in both M-CSF- and CXCL4-derived human macrophages. In addition, CXCL4 added to M-CSF-derived macrophages after virus adsorption and maintained throughout the infection enhances HIV-1 replication. We thus propose a novel role for CXCL4 in HIV disease.

Yersinia pseudotuberculosis serotyping scheme contains 21 serotypes based on the distribution of about 30 different O-factors within the species. The chemical structures of LPSs and the genetic basis of their biosynthesis has been determined for a number of Y. pseudotuberculosis strains representing different serotypes; thus, an overall picture of the relationship between genetics and structures is emerging. In this work, we have performed a structural and genetic analysis of the Y. pseudotuberculosis serotype O:15 O-specific polysaccharide. Our results showed that the set-up of the Y. pseudotuberculosis O:15 gene cluster is a hybrid between those of Y. pseudotuberculosis serotypes O:1b and O:5a, possibly due to a single recombination event. The hybrid nature could also be seen in the structure of the O-specific polysaccharide repeating unit pentasaccharide. It contains a tetrameric backbone identical to that of O:5a while the branching paratofuranose residue is identical to that of O:1b.

Lactoferrin is a non-heme iron-binding glycoprotein, produced by mucosal epithelial cells and granulocytes in most mammalian species. It is involved in regulation of immune responses, possesses anti-oxidant, anti-carcinogenic, anti-inflammatory properties, and provides protection against various microbial infections. In addition, lactoferrin has been implicated in protection against the development of insult-induced systemic inflammatory response syndrome (SIRS) and its progression into septic conditions in vivo. Here we show a potential mechanism by which lactoferrin lessens oxidative insult at the cellular and tissue levels after lipopolysaccharide (LPS) exposure. Lactoferrin pretreatment of cells decreased LPS-mediated oxidative insults in a dose-dependent manner. Lipopolysaccharide-induced oxidative burst was found to be of mitochondrial origin, and release of reactive oxygen species (ROS) was localized to the respiratory complex III. Importantly, lactoferrin nearly abolished LPS-induced increases in mitochondrial ROS generation and the accumulation of oxidative damage in the DNA. In vivo, pretreatment of experimental animals with lactoferrin significantly (P < 0.05) lowered LPS-induced mitochondrial dysfunction as shown by both decreased release of H₂O₂ and DNA damage in the mitochondria. In contrast, deferoxamine, an iron chelating compound, provided only partial protection in LPS-treated animals. Together, these data suggest that lactoferrin protects against oxidative insult at the mitochondrial level, and indicate a potential utility of lactoferrin in prevention and treatment of SIRS.

Knowledge on gene expression in the liver during respiratory infections is limited although it is well-established that this organ is an important site of synthesis of several systemic innate immune components as response to infections. In the present study, the early transcriptional hepatic response of genes associated with innate immune responses was studied in pigs 14–18 h after intranasal inoculation with Actinobacillus pleuropneumoniae, using innate immune focused microarrays and quantitative real-time PCR (qPCR). The microarray analysis of liver tissue established that 51 genes were differentially expressed. A large group of these genes encoded proteins involved in the acute phase response, including serum amyloid A, C-reactive protein, fibrinogen, haptoglobin and tumor necrosis factor- the expression of which were all found to be up-regulated and glutathione S-transferase, transthyretin, transferrin and albumin which were down-regulated. Additional genes associated with innate immune responses were investigated using qPCR; genes encoding interleukin-(IL-)1, IL-6, IL-8, lipopolysaccharide binding protein, lactotransferrin, and PigMAP were up-regulated and interferon 1, ₁-acid glycoprotein, mannan-binding lectin A, surfactant protein D, and surfactant protein A1 were down-regulated in the liver of infected animals. Down-regulation of ₁-acid glycoprotein during infection has not been described previously in any species. These results confirm that the liver plays an important role in initiating and orchestrating the innate immune response to A. pleuropneumoniae infection.

Glucuronidation is an important metabolic process of detoxification in all vertebrates. The reaction is catalyzed by a multigene family of UDP-glucuronosyltransferases (UGTs) able to convert many xenobiotics and endobiotics (hydrophobic substances) to inactive, water-soluble glucuronides. The UGTs play a protective role, facilitating the elimination of potentially toxic metabolites via urine, bile and feces; therefore, impairment of UGTs may have important toxicological consequences. The regulation of UGTs during bacterial infection or inflammation is not well described. In this study, we investigated the in vitro effect of lipopolysaccharide (LPS) on the expression of the UGT1A6 isoform in human colon carcinoma Caco-2 cells. Results demonstrated a significant down-regulation of UGT1A6 expression, both in terms of mRNA and protein levels, and a reduced UGT activity after LPS exposure of cell cultures, suggesting a role for endotoxins on UGT regulation mechanisms.

The aim of this work was to establish and characterize a cell culture model for lipopolysaccharide (LPS)-induced activation of human endothelial cells. Monocytic THP-1 cells were stimulated for 4 h with 10 ng/ml LPS from Pseudomonas aeruginosa in media containing 10% human plasma. Culture supernatants containing LPS and factors secreted by THP-1 in response to stimulation were applied to human umbilical vein endothelial cells (HUVECs). Nuclear factor-B (NF-B) activity, expression of adhesion molecules, and cytokine secretion were quantified. In addition, the effect of adsorptive removal of tumour necrosis factor- (TNF-) from the THP-1 culture supernatant on HUVEC activation was assessed. After 4 h of stimulation, THP-1 cells secreted various mediators including TNF- (854 ± 472 pg/ml), interleukin (IL)-8 (2069 ± 710 pg/ml), IL-18 (305 ± 124 pg/ml), IL-10 (14 ± 5 pg/ml), and IL-1β (24 ± 11 pg/ml). Stimulated HUVECs showed significantly increased NF-B activity and secreted high amounts of IL-6 and IL-8. Additionally, adhesion molecules ICAM-1 and E-selectin were increased both in the culture supernatant and at the cell surface. Removal of TNF- from the THP-1 culture supernatant prior to HUVEC stimulation resulted in a decrease in NF-B activity, expression of adhesion molecules, as well as IL-6 secretion. The cell culture model established in this study permits the monitoring of LPS-induced endothelial activation, which plays a central role in sepsis and may serve to assess the effect of mediator modulation by methods such as extracorporeal blood purification.

After binding to lipopolysaccharide (LPS)-binding protein, LPS is transferred to CD14 and then to the MD2–Toll-like receptor 4 complex, which results in the progression of sepsis. We investigated how 3,4-methylenedioxyphenol (sesamol), an inexpensive natural product in sesame seeds, affects the binding of LPS and LPS-binding protein and the release of pro-inflammatory cytokines. Sesamol: (i) dose-dependently inhibited LPS from binding to LPS binding protein; (ii) significantly decreased the release of tumor necrosis factor- and interleukin-1β in LPS-challenged peritoneal macrophages in medium and in the serum of LPS-challenged rats; and (iii) significantly reduced the mortality rate in mice given a lethal dose of LPS. We hypothesize that sesamol blocks LPS from binding to LPS-binding protein and inhibits the release of pro-inflammatory cytokines, both of which are associated with a decrease of mortality in endotoxemia.

To determine whether growth of bacteria in biofilms triggers a specific immune response, we compared cytokine induction in human monocytes and mouse macrophages by planktonic and biofilm bacteria. We compared Pseudomonas aeruginosa and Staphylococcus aureus, two bacteria often colonizing the airways of cystic fibrosis patients. Planktonic and biofilm S. aureus induced equivalent amounts of cytokine in human monocytes. In contrast, biofilm-forming P. aeruginosa induced a higher production of tumor necrosis factor and interleukin-6 than their planktonic counterpart, both for clinical isolates and laboratory strains. This increased cytokine production was partly dependent on phagocytosis. In contrast, no difference in cytokine induction was observed with mouse macrophages. We investigated the structures of the lipopolysaccharides (LPSs) of these Gram-negative bacteria in biofilm and planktonic cultures of P. aeruginosa. Switch between the two life-styles was shown to cause several reversible LPS structure modifications affecting the lipid A and polysaccharide moieties of both clinical isolates and laboratory strains. In addition, LPS isolated from biofilm-grown bacteria induced slightly more inflammatory cytokines than that extracted from its planktonic counterpart. Our results, therefore, show that P. aeruginosa biofilm LPS undergoes structural modifications that only partially contribute to an increased inflammatory response from human monocytes.

We investigated the immunopharmacological aspects of innate immune responses via Toll-like receptors (TLRs), NOD1 and NOD2, in terms of induction of the histamine-forming enzyme, histidine decarboxylase (HDC), activity in mice. Intravenous injection of TLR4-agonistic synthetic lipid A definitely induced HDC activity in the liver, spleen, and lungs, especially the lungs, in mice, where maximum activity was induced about 3 h after the injection of lipid A. The TLR2/6 agonistic synthetic diacyl-type lipopeptide FSL-1 and TLR3-agonistic poly I:C were also effective in inducing HDC, while the NOD2-agonistic synthetic muramyldipeptide (MDP) and NOD1-agonistic synthetic FK156 and FK565 exhibited only weak activities in this respect. Mice primed with intravenous injection of NOD1 or NOD2 agonists produced higher HDC activity following the 4–6 h later intravenous challenge with the above TLR agonists. Among the priming agents, FK565 exhibited the strongest activity, and it was effective via various administration routes – intraperitoneal, subcutaneous, intramuscular, as well as intravenous injection; furthermore, oral (gastric) administration was effective, although it needed a dose 10 times higher than that required for other administration routes. These findings suggest that HDC is induced in association with TLRs and NOD1/2, and that the newly formed histamine by the induced HDC might play important roles in the regulation of inflammatory and immune responses in various organs.

Hyaluronan is a biological polysaccharide that may exist in different degrees of polymerization. Several investigations reported that low molecular mass hyaluronan may have pro-inflammatory activity, while high molecular mass hyaluronan can exert beneficial effects. Starting from these data, the aim of this study was to investigate the effect of hyaluronan of different molecular mass in mouse articular chondrocyte cultures stimulated with lipopolysaccharide (LPS). Inflammation was induced in chondrocytes by acute treatment with 2.0 µg/ml LPS. High levels of tumour necrosis factor (TNF)-, interleukin (IL)-1β, IL-6, interferon (IFN)- and iNOS gene expression and their related proteins were found in chondrocytes 24 h after treatment with LPS. High concentrations of NO, NF-B activation, IB phosphorylation and apoptosis, evaluated by the increase in caspase-3 expression and its related protein amount were also produced by LPS stimulation. In contrast, LPS reduced aggrecan and collagen type II (Col2A) expression and their protein production. The treatment of chondrocytes with hyaluronan of different molecular mass produced the following effects: (i) low molecular mass hyaluronan exerted a slight inflammatory effect in untreated chondrocytes, while in LPS-treated chondrocytes it enhanced cytokine production and decreased aggrecan and Col2A compared with cells treated with LPS alone; (ii) no effect was exerted on LPS-induced apoptosis and NO production; (iii) medium molecular mass hyaluronan did not exert any inflammatory/anti-inflammatory activity in LPS-untreated/treated cells and failed to reduce apoptosis; and (iv) high molecular mass hyaluronan had no inflammatory effect in LPS-untreated cells while it was able to reduce all the detrimental effects stimulated by LPS treatment. These data confirm the multifactorial role played by hyaluronan and suggest, in particular, that hyaluronan may modulate inflammation during pathologies by its different degrees of polymerization.

C1-Inhibitor (C1-INH) is an important biological inhibitor, regulating several protein cascade systems. Recent research has shown that the molecule exhibits properties not dependent on its protease inhibition activity. Serum and whole blood from pigs and humans were pre-incubated with C1-INH, iC1-INH or the complement inhibitors SPICE or compstatin. Whole, live Escherichia coli were then added for further incubation. Complement activation, a range of cytokines, chemokines and growth factors, as well as the leukocyte activation markers wCD11R3 (pig) and CD11b (human) were measured. Both C1-INH and iC1-INH dose-dependently and significantly (P < 0.05) reduced a range of E. coli-induced pro-inflammatory cytokines and chemokines in porcine and human whole blood, as well as growth factors in human whole blood. Differences between the two forms of C1-INH and between the two species were modest. Most of these anti-inflammatory effects could not be explained by complement inhibition, as specific complement inhibitors had minor effect on several of the mediators. C1-Inhibitor had no inhibitory effect on E. coli-induced complement activation, while iC1-INH enhanced complement activation. The presented data indicate that C1-INH has broad anti-inflammatory effects in E. coli-induced inflammation in pig and human whole blood. These effects are largely independent of the protease inhibition activity.

A widely applied method to study the activation of the innate immune system is in vitro stimulation of whole blood using lipopolysaccharide (LPS). However, it is unclear if in vitro cytokine production relates to in vivo cytokine levels elicited during experimental endotoxemia or sepsis. To determine the correlation between in vitro cytokine production and the in vivo inflammatory response, blood was obtained from 15 healthy volunteers for in vitro incubation with Escherichia coli LPS, immediately followed by experimental E. coli endotoxemia. Correlations of in vitro and peak in vivo cytokine concentrations were determined using Pearson correlation coefficient. In stimulated whole blood, tumor necrosis factor (TNF)-, Interleukin (IL)-1β, IL-6, IL-10 and interferon (IFN)- were induced to 279 ± 53, 392 ± 64, 5312 ± 624, 83 ± 20 and 343 ± 85 pg/ml, respectively, whereas in vivo cytokine induction led to cytokine levels of 603 ± 123, 11 ± 1, 4999 ± 1228, 167 ± 25 and 194 ± 40 pg/ml, respectively. Correlation coefficients between the in vitro and in vivo cytokine concentrations were for TNF-, IL-1β, IL-6, IL-10 and IFN- -0.10 (P = 0.7), 0.09 (P = 0.8), 0.36 (P = 0.2), 0.19 (P = 0.5) and 0.40 (P = 0.1), respectively. Comparison between in vitro and in vivo stimulation with LPS shows no correlation between the amount of cytokines produced. In vitro cytokine production, therefore, does not predict the in vivo inflammatory response.

Mycobacterium avium subsp. paratuberculosis adapts to the environment via the regulation of genes affecting its envelope’s composition. Bacteria grown in milk (in vivo conditions) presented differences in the cell wall-associated lipids and in the expression of genes involved in lipid metabolism (FadE8, FadE6 and MAP1420) and host cell invasion (MAP1203, LprL). A different lipid profile was also observed in the envelope of intracellular bacteria after 1 h of infection. Intracellular bacteria showed up-regulation of a LuxR regulator which controls the envelope’s composition by up-regulation of FadE8, MAP1420, MAP1203 and LprL and by down-regulation of pks12, mmpL2 and MAP2594. A LuxR-overexpressing strain with a lipid-deficient envelope phenotype, infected epithelial cells more efficiently than the wild-type bacteria; however, it was not more resistant than the wild-type strain to the action of bactericidal proteins. Here we show that LuxR regulates virulence determinants and is involved in mycobacteria adaptation to the host.

Growing evidence shows that C1q modulates the growth and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because C1q regulates both innate and acquired immune responses, we postulated that C1q modulates the transition from monocytes to DCs, i.e. the interface between innate and acquired immunity. Human peripheral blood monocytes cultured with soluble C1q and DC growth factors (granulocyte-macrophage colony-stimulating factor + Interleukin-4) failed to down-regulate monocyte-associated (CD14, CD16) and up-regulate DC-associated (CD83, CD86) markers. Impaired DC differentiation was not due to apoptosis; further analysis revealed the development of CD14^hiCD11c^hiCD16 ^+/- cells that have previously been associated with both innate and acquired immunity. Monocyte–DC precursors expressed gC1qR, the receptor for globular heads of C1q, from the outset, while cC1qR, the receptor for the collagen tails of C1q, was expressed at low levels. Notably, the binding pattern of monoclonal antibodies specific to the globular heads of C1q indicated that C1q is bound to monocytes via globular heads, presumably through gC1qR. Moreover, gC1qR levels decreased, while cC1qR levels were dramatically amplified as monocytes differentiated into immature DC. Thus, specific C1q/C1q receptor (R) interactions may control the transition from the monocyte state (innate immunity) toward the professional antigen-presenting cell state (adaptive immunity).

Macrophage inflammatory protein-3/C-C chemokine ligand 20 (MIP-3/CCL20) is an antimicrobial peptide that plays an important role in innate immunity. In addition to direct microbicidal effects, MIP-3/CCL20 also exhibits cytokine-like functions that are critical during dendritic cell activation. The aim of the present study was to investigate further which signaling pathways are involved in the MIP-3/CCL20 mRNA expression in response to whole-cell Porphyromonas gingivalis. Primary gingival epithelial cells (GECs) and the immortalized oral keratinocyte cell-line OKF6/TERT-2 were stimulated with whole-cell P. gingivalis. Prior to stimulation, GECs and OKF6/TERT-2 cells were pretreated with specific inhibitors for nuclear-factor-B (NF-B), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and phosphatidylinositol-3-kinase (PI3K). In GECs and OKF6/TERT-2 cells, activation of NF-B was examined after exposure to P. gingivalis. The gene expression of MIP-3/CCL20 was significantly induced in response to P. gingivalis (P ≤ 0.05) compared to unstimulated control cells. This induction was specifically blocked when cells were pre-incubated with inhibitors for NF-B, MAPK, and PLC (P ≤ 0.05), but not for PI3K. These results demonstrate that P. gingivalis induces the MIP-3/CCL20 mRNA in a NF-B-, PLC-, and MAPK-dependent manner.

The innate immune response provides a critical first-line defense against Mycobacterium tuberculosis, an intracellular pathogen that represents a major health threat world-wide. A synthetic lipopeptide (LP) mimicking the lipid moiety of the cell-wall associated 19-kDa lipoprotein from M. tuberculosis has recently been assigned an important role in the induction of an antibacterial immune response in host macrophages. Here, we present experimental data on the biological activities and the biophysical mechanisms underlying cell activation by synthetic 19-kDa M. tuberculosis-derived lipopeptide (Mtb-LP). Investigation of the geometry of the LP (i.e. the molecular conformation and supramolecular aggregate structure) and the preference for membrane intercalation provide an explanation for the biological activities of the mycobacterial LP. Cell activation by low concentrations of Mtb-LP was enhanced by the lipopolysaccharide–binding protein and CD14. However, surprisingly, we found that activation of human macrophages to induce pro- as well as anti-inflammatory mediators (tumor necrosis factor(TNF)-, Interleukin(IL)-6, IL-8, and IL-10) in response to the Mtb-LP is strongly reduced in the presence of serum. This observation could be confirmed for the immune response of murine macrophages which showed a strongly enhanced TNF- release in the absence of serum, suggesting that the molecular mechanisms of immune recognition of the Mtb-LP are tailored to the ambient conditions of the lung.

The intestinal epithelium maintains a state of controlled inflammation despite continuous contact with Gram-negative commensal bacteria and lipopolysaccharide (LPS) on its luminal surface. Recognition of LPS by the Toll-like receptor (TLR) 4/MD-2 complex results in pro-inflammatory gene expression and cytokine secretion in intestinal epithelial cells (IECs). We have shown that IECs express low levels of MD-2 and TLR4 and are poorly responsive to LPS. In this study, we did a comprehensive analysis to understand the immune-mediated and epigenetic mechanisms by which IECs down-regulate MD-2 expression. Expression of MD-2 and TLR4 mRNA was examined in human inflammatory bowel disease and intestinal epithelial cell lines (T84, HT-29, Caco-2). Nuclear factor-B transcriptional activation was used as a measure of LPS responsiveness. Intestinal epithelial cells in patients with inflammatory bowel disease exhibited increased expression of MD-2 and TLR4 mRNA. Lipopolysaccharide responsiveness in IECs was polarized to the basolateral membrane. Bisulfite sequencing of the MD-2 promoter demonstrated methylation of CpG dinucleotides. Inhibition of methylation by 5-azacytidine and histone de-actylation by trichostatin A, two forms of epigenetic silencing, resulted in increased mRNA expression of MD-2 in IECs. These results demonstrate various molecular mechanisms by which IECs down-regulate MD-2 and, thereby, protect against dysregulated inflammation to commensal bacteria in the intestinal lumen.

Integrins are a large family of adhesion receptors that are known to be key signaling molecules in both physiological and pathological processes. Previous studies have demonstrated that the expression of integrin receptors in the pulmonary epithelium can change under various pathological conditions, such as injury, inflammation, or malignant transformation. We hypothesize that integrin expression can be altered by stimulation of lung epithelial cells with an opportunistic bacterial pathogen Pseudomonas aeruginosa. Using the A549 adenocarcinoma cell line that expressed a low level of several integrin subunits we have demonstrated that P. aeruginosa infection in vitro caused a rapid up-regulation of 5, v, β1, and β4 integrins at both the mRNA and protein level. Neither heat-killed P. aeruginosa strain PAK nor its live isogenic mutants lacking pili or lipopolysaccharide (LPS) core oligosaccharide showed any effect on integrin expression in A549 cells as compared to the use of the wild-type PAK strain. These results establish that up-regulation of integrin expression is dependent on the internalization of live bacteria possessing intact pili and LPS. Gene silencing of integrin-linked kinase in A549 cells caused a significant decrease in the release of pro-inflammatory cytokines in response to P. aeruginosa stimulation. Although further studies are warranted towards understanding the precise role of integrin receptors in prominent inflammation caused by P. aeruginosa, our findings suggest a possibility of using specific integrin inhibitors for therapy of pulmonary inflammatory conditions caused by pathogenic micro-organisms.

Animal studies have shown that previous exposure to lipopolysaccharide (LPS) can limit ischemia-reperfusion injury. We tested whether pretreatment with LPS also protects against ischemia-reperfusion injury in humans in vivo. Fourteen volunteers received bolus injections of incremental dosages of LPS on 5 consecutive days (LPS group). Before the first and 1 day after the last LPS administration, the forearm circulation of the non-dominant arm was occluded for 10 min, with concomitant intermittent handgripping to induce transient ischemia. After reperfusion, 0.1 mg of ^99mTc-labeled annexin A5 (400 MBq) was injected intravenously to detect phosphatidylserine expression as an early marker of ischemia-reperfusion injury. Similarly, the control group (n = 10) underwent the ischemic exercise twice, but without pretreatment with LPS. Annexin A5 targeting was expressed as the percentage difference in radioactivity in the thenar muscle between both hands. Endotoxin tolerance developed during 5 consecutive days of LPS administration. Annexin A5 targeting was 12.1 ± 2.2% and 10.4 ± 2.1% before LPS treatment at 1 h and 4 h after reperfusion, compared to 12.2 ± 2.4% and 8.9 ± 2.1% at 1 h and 4 h after reperfusion on day 5 (P = 1.0 and 0.6, respectively). Also, no significant changes in annexin A5 targeting were found in the control group. So, in this model, LPS-tolerance does not protect against ischemia-reperfusion injury in humans in vivo.

Hepatic steatosis is a risk factor for the progression of non-alcoholic fatty liver disease. The role of pro-inflammatory interleukin (IL)-6 in hepatic steatosis etiology is controversial. We investigated in vivo and in primary hepatocyte cultures whether IL-6 has a modulator role in liver and mitochondria lipid composition and cell death in a choline-deficient (CD) diet rat model of hepatic steatosis. Dietary choline deficiency increased triglycerides and cholesterol, and reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the membrane integrity marker PC:PE ratio in liver. Choline-deficient diet enhanced systemic IL-6, and IL-6 receptor expression and cell death vulnerability in hepatocytes. Derangement of the mitochondrial electron transport chain and of its phospholipid environment was found in CD rat liver mitochondria, which exhibited elevated concentrations of triglycerides, cardiolipin and PC and elevated PC:PE ratio. The cell treatment with IL-6, but not PC, eliminated much of the CD-promoted lipid imbalance in mitochondria but not tumor-necrosis factor (TNF)--induced cell death. However, PC supplementation prevented the TNF--induced DNA fragmentation, cytochrome-c release and caspase-3 activity in control and CD hepatocytes. In conclusion, IL-6 ameliorated the mitochondria lipid disturbance in hepatocytes isolated from steatotic animals. Furthermore, PC is identified as a new survival agent that reverses several TNF--inducible responses that are likely to promote steatosis and necrosis.

Pig farmers and cigarette smokers are continuously exposed to pathogen-associated molecular patterns (PAMPs) have an increased prevalence of respiratory disorders, such as chronic bronchitis and chronic obstructive pulmonary decease (COPD). We hypothesized that markers of innate immunity, T-helper (Th) cell cytokine profile and acute responses to pro-inflammatory stimuli differ between smokers and farmers, who are exposed to organic material on a daily basis and healthy non-exposed subjects. Eleven non-smoking pig farmers, 12 non-farming smokers and 12 controls underwent bronchial lipopolysaccharide (LPS) challenge and exposure in a pig barn during 3 h on separate days. Toll-like receptor 2 (TLR2), TLR4 and CD14 on blood monocytes and neutrophils and intracellular cytokine profile of Th cells were assessed before and 7 h after exposures. The same outcomes were analysed on peripheral blood and purified neutrophils from farmers and controls after stimulation ex vivo with dust from a pig barn and LPS. Circulating neutrophils and IL-13 and IL-4 producing Th cells were increased in smokers and farmers and TLR2 expression on blood monocytes was decreased in farmers compared with controls and smokers. After in vivo exposure, altered TLR expression was only observed in controls and the ex vivo stimulations showed an attenuated response in farmers compared to the control group. The inflammatory systemic response to pro-inflammatory stimuli is altered in farmers and smokers probably because of adaptive mechanisms arising from chronic exposure to organic material. This increased proportion of Th2 cells and reduced TLR2 expression may have health-related implications and may be related to the increased prevalence of respiratory disorders observed in these groups.

Porphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly involved in the development of chronic periodontitis. It displays a significant amount of lipid A structural heterogeneity, containing both tetra- (LPS_1435/1449) and penta-acylated (LPS₁₆₉₀) lipid A structures with opposing effects on E-selectin expression in human endothelial cells. Little is known about how these two isoforms of P. gingivalis LPS could differentially affect host innate immune responses in human gingival epithelia. The present study compares the modulatory effects of P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ on the expression of human β-defensins (hBDs) in the reconstituted human gingival epithelium, and examines the involvements of a panel of pattern recognition receptors in the modulatory effects concerned. It is shown that hBD-1, hBD-2 and hBD-3 mRNAs are significantly up-regulated by P. gingivalis LPS₁₆₉₀, but down-regulated by P. gingivalis LPS_1435/1449. Toll-like receptor (TLR) 2 and CD14 mRNAs are also differentially regulated, and the modulation of hBD-2 expression may be through the co-operation of both TLR2 and TLR4. This study suggests that P. gingivalis LPS with different lipid A structures could differentially modulate host innate immune responses in human gingival epithelia, which may be a hitherto undescribed novel pathogenic mechanism of P. gingivalis in periodontal pathogenesis.

Pathogenic leptospires evoke severe diseases in humans but only cause mild chronic or asymptomatic infection in many host animals. The reasons for this diversity of infection remain unclear. Here, we demonstrated that Leptospira interrogans serovar Lai strain Lai had a similar ability to adhere to and enter primary and immortal (THP-1 and J774A.1) macrophages from human and mouse, but its intracellular fate in human macrophages differed markedly from that in mouse. The leptospires resided within membrane-bound vacuoles in the murine macrophages, but occurred free in the cytosol of human macrophages, with no surrounding vesicular membrane. Most leptospires in murine macrophages co-localized with the late-endosomal/lysosomal marker LAMP-1 and then were killed by lysosomal hydrolases, while most leptospires in human macrophages did not co-localize with this marker and survived. Enumeration of colony-forming units plus quantitative fluorimetry showed that in human, but not in murine, macrophages, the amounts of leptospires increased with incubation time. The infected human macrophages differed from mouse macrophages by displaying gradually enhanced apoptosis, in parallel with the increase in number of leptospires. These data strongly suggest that the outcome for intracellular leptospires depends on differences among host macrophages, which may account for some of the differences in the severity of leptospirosis in humans and animals.

This study investigated the influence of the cholinergic system on neuro-inflammation using nicotinic and muscarinic receptor agonists and antagonists. Intracerebroventricular (ICV) injection of lipopolysaccharide (LPS, 50 µg) was used to induce neuro-inflammation in rats and estimations of pro-inflammatory cytokines, 7 nicotinic acetylcholine receptor (nAChR) mRNA expression were done in striatum, cerebral cortex, hippocampus and hypothalamus at 24 h after LPS injection. Nicotine (0.2, 0.4 and 0.8 mg/kg, i.p.) or oxotremorine (0.2, 0.4 and 0.8 mg/kg, i.p.) were administered 2 h prior to sacrifice. We found that only nicotine was able to block the pro-inflammatory cytokines induced by LPS whereas, oxotremorine was found ineffective. Methyllycaconitine (MLA; 1.25, 2.5 and 5 mg/kg, i.p.), an 7 nAChR antagonist or dihydro-β-erythroidine (DHβE; 1.25, 2.5 and 5 mg/kg, i.p.), an 4β2 nAChR antagonist, was given 20 min prior to nicotine in LPS-treated rats. Methyllycaconitine antagonized the anti-inflammatory effect of nicotine whereas DHβE showed no effect demonstrating that 7 nAChR is responsible for attenuation of LPS-induced pro-inflammatory cytokines. This study suggests that the inhibitory role of the central cholinergic system on neuro-inflammation is mediated via 7 nicotinic acetylcholine receptor and muscarinic receptors are not involved.

Protease-activated receptors (PARs) are G-protein-coupled receptors with an active role in host defense. The two most highly expressed members of the PAR family in gingival epithelial cells (GECs) are PAR1 and PAR2. The major virulence factors of periodontal pathogen Porphyromonas gingivalis are its proteases which can activate PAR2. However, little is known about the function of PARs in GECs when they are activated by their endogenous agonist enzymes. The purpose of this study was to characterize how the expression of innate immune markers is modulated when PAR1 and PAR2 are activated by their agonist enzymes, thrombin and trypsin, respectively. Here, we report that activation of PAR1 and PAR2 induces cell proliferation at low concentration. Activation of PAR via proteolytic activity of thrombin and trypsin induces expression of CXCL5/ENA-78 and CCL20/MIP3 in a concentration-dependent manner. Induction of CXCL5 via PAR1 was inhibited in the presence of PAR1 cleavage blocking antibodies and by PAR1 siRNA. The induction of CXCL5 and CCL20 via PAR2 was inhibited by PAR2 siRNA. These findings indicate an active role in innate immune responses by PAR1 and PAR2 in GECs. Modulation of innate immunity by PARs may contribute to co-ordinated and balanced immunosurveillance in GECs.

The structural prerequisites for lipopolysaccharide (LPS) and its partial structures for the activation of the Limulus clotting cascade (Limulus amebocyte lysate [LAL] test) are described and compared with the corresponding requirements for the activation of human immune cells such as mononuclear cells. A necessary, but not sufficient, structural motif for this is the presence of the 4'-phosphate-diglucosamine backbone recognition structure (‘epitope’) in lipid A. High activity is only expressed by assemblies of endotoxins, but this is largely independent of the type of supramolecular aggregate structure. A particular conformation of the epitope within the lipid A assembly must be present, which is influenced by addition of further saccharide units to the lipid A moiety, but also reacts slightly to the acylation pattern. In contrast, the cytokine production of human immune cells induced by LPS sensitively depends on the type of its aggregate structure. In the case of a hexa-acylated bisphosphorylated lipid A structure, high activity is only observed with cubic inverted aggregates. Furthermore, addition of antimicrobial agents (such as polymyxin B) leads to a nearly complete inhibition of cytokine production, whereas the reduction in the Limulus assay is much lower. These data are important since a reliable determination of endotoxin concentrations, in particular with respect to its ability to elicit severe infections, is of high interest.