Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

Nontransformed, GM-CSF-dependent macrophage lines are a unique model to study tissue macrophage functions.

Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

Immune response to Propionibacterium acnes in patients with sarcoidosis--in vivo and in vitro.

BACKGROUND: Propionibacterium acnes was found in lungs and lymph nodes of patients with sarcoidosis and may induce hypersensitivity type granuloma formation. Data regarding the immune response to P. acnes of European sarcoid patients are scarce. METHODS: We assessed the total IgG and IgA amount and specific antibodies to P. acnes and to Staphylococcus aureus, serving as a control, in BAL fluid of 64 patients with sarcoidosis and of 21 healthy volunteers. In a subcohort of sarcoid patients and controls, TNF-α and GM-CSF production of BAL cells stimulated with heat-killed P. acnes were measured. RESULTS: In sarcoid patients, the total IgG and IgA levels in BAL fluid were significantly elevated compared to healthy volunteers. IgG and IgA titres against P. acnes and S. aureus were increased in sarcoid patients, yet based on the total amount of antibodies, only antibodies directed against P. acnes were relatively and significantly increased. Furthermore, BAL cells of sarcoid patients produced significantly more TNF-α and GM-CSF upon stimulation with heat-killed P. acnes compared to controls. CONCLUSIONS: Patients with sarcoidosis had elevated levels of specific antibodies against P. acnes which suggest contact with this bacterium in the past. Furthermore, BAL cells of sarcoid patients produced inflammatory cytokines (TNF-α and GM-CSF) upon stimulation with P. acnes indicating potential involvement of this pathogen in the pathogenesis of sarcoidosis in some patients.

Bacterial RNA mediates activation of caspase-1 and IL-1ß release independently of TLRs 3, 7, 9 and TRIF but is dependent on UNC93B.

Recognition of foreign nucleic acids is important for the induction of an innate immune response against invading pathogens. Although the pathways involved in sensing bacterial DNA and viral RNA are now well established, only limited knowledge is available on mechanisms underlying recognition of bacterial RNA. It has been reported that intracellular delivery of Escherichia coli RNA activates the Nlrp3 inflammasome, but whether this is a general property of bacterial RNA remains unclear as are the pathways involved in pro-IL-1ß induction and caspase-1 activation by bacterial RNA. In this study, we report that bacterial RNA from both Gram-positive and Gram-negative bacteria induces activation of caspase-1 and secretion of IL-1ß by murine dendritic cells and bone-marrow derived macrophages. Stimulation was independent of the presence of 5'-triphosphate termini and occurred with whole RNA preparations from bacteria but not from eukaryotes. Induction of pro-IL-1ß as well as the priming for caspase-1 activation by bacterial RNA was dependent on UNC93B, an endoplasmic reticulum protein essential for delivery of TLRs to the endosome, whereas the established nucleic acid sensing endosomal TLRs 3, 7, and 9 were dispensable. Additionally, caspase-1 activation and IL-1ß production by transfected bacterial RNA were absent in MyD88-deficient cells but independent of TRIF. Thus, our data indicate the presence of a yet unidentified intracellular nucleic acid receptor involved in bacterial RNA-induced inflammasome activation and release of IL-1ß.

Absence of TRIF signaling in lipopolysaccharide-stimulated murine mast cells.

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-ß (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-ß production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-ß responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αß responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.

Innate, antigen-independent role for T cells in the activation of the immune system by Propionibacterium acnes.

Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL-12-mediated IFN-gamma production. We show that P. acnes elicits IL-12p40 and p35 mRNA expression in macrophages, and IFN-gamma mRNA in liver CD4(+) T cells and NK cells. After priming with P. acnes, CD4(+) T cells serve as the major IFN-gamma mRNA source. In the absence of CD4(+) T cells, CD8(+) T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN-gamma to induce the P. acnes-driven immune effects. Moreover, in the absence of alpha beta T cells, gamma delta T cells also enable the development of strongly enhanced TNF-alpha and IFN-gamma responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T-cell types, independent of their antigen specificity, exert NK-cell-like functions, which contribute decisively to the activation of the innate immune system.

MyD88/TLR9 mediated immunopathology and gut microbiota dynamics in a novel murine model of intestinal graft-versus-host disease.

BACKGROUND: The bacterial microflora aggravates graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation, but the underlying mechanisms of manifestations of intestinal GvHD (iGvHD) in the gut remain poorly understood. AIM: To analyse the gut flora composition and the impact of bacterial sensing via Toll-like receptors (TLRs) in iGvHD. METHODS: By mimicking clinical low-intensity conditioning regimens used in humans, a novel irradiation independent, treosulfan and cyclophosphamide-based murine allogeneic transplantation model was established. A global survey of the intestinal microflora by cultural and molecular methods was performed, the intestinal immunopathology in TLR-deficient recipient mice with iGvHD investigated and finally, the impact of anti-TLR9 treatment on iGvHD development assessed. RESULTS: The inflammatory responses in iGvHD were accompanied by gut flora shifts towards enterobacteria, enterococci and Bacteroides/Prevotella spp. Analysis of iGvHD in MyD88(-/-), TRIF(-/-), TLR2/4(-/-), and TLR9(-/-) recipient mice showed that bacterial sensing via TLRs was essential for iGvHD development. Acute iGvHD was characterised by increasing numbers of apoptotic cells, proliferating cells, T cells and neutrophils within the colon. These responses were significantly reduced in MyD88(-/-), TLR2/4(-/-), TRIF(-/-) and TLR9(-/-) mice, as compared with wild-type controls. However, TRIF(-/-) and TLR2/4(-/-) mice were not protected from mortality, whereas TLR9(-/-) mice displayed increased survival rates. The important role of TLR9-mediated immunopathology was independently confirmed by significantly reduced macroscopic disease symptoms and colonic apoptosis as well as by reduced T-cell and neutrophil numbers within the colon after treatment with a synthetic inhibitory oligonucleotide. CONCLUSIONS: These results emphasise the critical role of gut microbiota, innate immunity and TLR9 in iGvHD and highlight anti-TLR9 strategies as novel therapeutic options.

Conventional bone marrow-derived dendritic cells contribute to toll-like receptor-independent production of alpha/beta interferon in response to inactivated parapoxvirus ovis.

Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus Parapoxvirus. It displays only limited homology with orthopoxviruses and has some molecular features such as an unusual high GC content distinct from orthopoxviruses. Inactivated PPVO (iPPVO) displays strong immunostimulatory capacities mediating antiviral activity in vivo. The role of dendritic cells (DC) and the pattern recognition receptors and signaling requirements responsible for immunostimulation by iPPVO are unknown. We demonstrate here that bone marrow-derived plasmacytoid DC (BM-pDC) and bone marrow-derived conventional DC (BM-cDC) secrete alpha/beta interferon (IFN-alpha/beta) in response to iPPVO. Furthermore, iPPVO induces tumor necrosis factor alpha (TNF-alpha) and interleukin-12/23p40 (IL-12/23p40) release and major histocompatibility complex class II (MHC-II), MHC-I, and CD86 upregulation by bone marrow-derived DC (BMDC). After engulfment, iPPVO is located in endosomal compartments and in the cytosol of BMDC. iPPVO elicits IFN-alpha/beta by Toll-like receptor (TLR)-independent pathways in BM-cDC, since IFN-alpha/beta release does not require myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-alpha release and enhanced expression of MHC-I and CD86 but not of MHC-II by BMDC chiefly requires MyD88 but not TLR2 or TLR4. Induction of IFN-alpha by iPPVO in BM-cDC occurred in the absence of IFN regulatory factor 3 (IRF3) but required the presence of IRF7, whereas iPPVO-triggered IFN-beta production required the presence of either IRF7 or IRF3. These results provide the first evidence that iPPVO mediates its immunostimulatory properties by TLR-independent and TLR-dependent pathways and demonstrate an important role of cDC for IFN-alpha/beta production.

Key role of splenic myeloid DCs in the IFN-alphabeta response to adenoviruses in vivo.

The early systemic production of interferon (IFN)-alphabeta is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-alphabeta response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-alphabeta mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-alphabeta production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-alphabeta response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-alphabeta and IL-6 in vivo by distinct pathways and confirm that IFN-alphabeta positively regulates the IL-6 response. Finally, by measuring TNF-alpha responses to LPS in Ad-infected wild type and IFN-alphabetaR(-/-) mice, we show that IFN-alphabeta is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-alphabeta response, which is responsible for the bulk of IFN-alphabeta production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-alphabeta induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.

MyD88/IL-18-dependent pathways rather than TLRs control early parasitaemia in non-lethal Plasmodium yoelii infection.

Plasmodium falciparum GPI contributes to malaria pathology by inducing cytokine release. It has been shown to be recognized through TLR2 and to a lesser extent TLR4 in vitro. However, previous findings on the role of TLRs in parasite clearance or pathology in vivo are conflicting. Thus, we analyzed the impact of TLR-signalling on protection using the P. yoelii infection model. Deficiency of single TLRs as well as triple TLR2/4/9-deficiency had no impact on parasitaemia. In contrast, mice deficient for the adaptor protein MyD88 were more susceptible to P. yoelii infection in that they exhibited an increased parasitaemia in the early phase of the infection and a higher lethality. This phenotype was caused mainly by impaired IL-18 signalling since parasitaemia in IL-18-deficient mice was also increased at early time points during P. yoelii infection compared to wild-type control mice. However, no lethality was observed in IL-18-deficient mice. Since parasitaemia in IL-1R-deficient mice was also slightly increased during P. yoelii infection, impaired IL-1R signalling contributed to the increased susceptibility of MyD88-deficient mice to a lesser extent. These findings correlated with a reduced IFN-gamma production in MyD88- and IL-18-deficient mice, but not in TLR2/4/9-deficient mice. We conclude that mainly IL-18/MyD88-dependent signalling but not TLR2/4/9-signalling is important for early parasite control in our model.

Lipopolysaccharide sensing an important factor in the innate immune response to Gram-negative bacterial infections: benefits and hazards of LPS hypersensitivity.

In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.

IFN-y enhances T(H)1 polarisation of monocyte-derived dendritic cells matured with clinical-grade cytokines using serum-free conditions.

Using serum-free conditions, human monocyte-derived dendritic cells (MoDCs) tend to mature insufficiently in a T(H)1-polarizing direction under approved and standardized clinical conditions. However, for the initiation of an efficient tumour antigen-specific cytotoxic T-cell response, the induction of a distinct T(H)1 response is favourable. Therefore, to improve T(H)1 polarisation, the influence of interferon-gamma (IFN-gamma) on the maturation of MoDCs was investigated with clinical-grade cytokines or lipopolysaccharide (LPS) in serum-free medium focusing on the viability, phenotypic characteristics, cytokine profile and restimulating capacities. As in previous research, we confirmed that in respect of viability and phenotypic characteristics, cytokine cocktails consisting of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6 and prostaglandin (PG) E2, mature MoDCs most efficiently. However, these cytokine-matured MoDCs secreted relatively high levels of IL-10 and only low levels of IL-12p70. Remarkably, if IFN-gamma was added, significantly lower levels of IL-10 concomitant with higher levels of IL-12p70 could be detected. Pretreatment with IFN-gamma did not improve the phenotypic characteristics nor the T(H)1 polarisation of MoDCs. Nevertheless, MoDCs matured with clinical-grade cytokines and IFN-gamma could be re-stimulated most effectively with IFN-gamma. In conclusion, our work demonstrates that addition of INF-gamma to clinical-grade cytokine cocktails readily matures MoDCs and enhances their T(H)1 polarisation efficiently under serum-free conditions.

Bacterial coinfection restrains antiviral CD8 T-cell response via LPS-induced inhibitory NK cells.

Infection of specific pathogen-free mice with lymphocytic choriomeningitis virus (LCMV) is a widely used model to study antiviral T-cell immunity. Infections in the real world, however, are often accompanied by coinfections with unrelated pathogens. Here we show that in mice, systemic coinfection with E. coli suppresses the LCMV-specific cytotoxic T-lymphocyte (CTL) response and virus elimination in a NK cell- and TLR2/4-dependent manner. Soluble TLR4 ligand LPS also induces NK cell-mediated negative CTL regulation during LCMV infection. NK cells in LPS-treated mice suppress clonal expansion of LCMV-specific CTLs by a NKG2D- or NCR1-independent but perforin-dependent mechanism. These results suggest a TLR4-mediated immunoregulatory role of NK cells during viral-bacterial coinfections.

New advances in the development of sarcoidosis models: a synopsis of a symposium sponsored by the Foundation for Sarcoidosis Research.

Sarcoidosis is a complex disease with variable phenotypes that will require a multisystem approach to understand pathophysiology. One of the most challenging problems in sarcoidosis research is the absence of valid and widely accepted experimental models that accurately simulate human disease. The Foundation of Sarcoidosis Research (FSR) has funded five projects for the development of novel experimental models for sarcoidosis, presented and discussed in a workshop organized during the European Respiratory Society Congress held in Milan from September 9th to 13th. The experimental, in vivo or in sillico models presented may be quite helpful for investigating specific pathogenic and therapeutic questions, addressing especially severe forms of sarcoidosis. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 2-4).

Production of IL-12, IL-23 and IL-27p28 by bone marrow-derived conventional dendritic cells rather than macrophages after LPS/TLR4-dependent induction by Salmonella Enteritidis.

Induction of the interleukin-12 (IL-12) cytokine family comprising IL-12, IL-23, IL-27, and IL-12p40 by intracellular pathogens is required for orchestration of cell-mediated immune responses. Macrophages (MPhi) have been shown to be a source of IL-12 following TLR4-dependent activation by Salmonella (S.). In this study another antigen-presenting cell type, the conventional dendritic cell (cDC), was analyzed and its cytokine responses compared with those of MPhi. We generated bone marrow-derived conventional dendritic cells (BMDC) and macrophages (BMMPhi) by incubating murine bone marrow cells with supernatants containing granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), respectively. Stimulation of BMDC and BMMPhi with S. enterica serovar Enteritidis (SE) or LPS resulted in the release of IL-12 and IL-23 by BMDC but not by BMMPhi. Furthermore, BMDC secreted approx. 20-fold more IL-12p40 and IL-27p28 than BMMPhi. However, BMDC and BMMPhi produced similar levels of IL-10. Using BMDC originating from wild-type (wt), TLR2(def) and TLR4(def) mice, we show that in BMDC the induction of IL-12, IL-23, and IL-27p28 by SE is dependent on TLR4, whereas low-level production of p40 is also mediated by pattern recognition receptors (PRR) other than TLR4. Interestingly, LPS- and SE-provoked responses of BMDC were remarkably similar indicating that LPS is the primary danger molecule of SE. Taken together, our results point to cDC rather than MPhi as the major producers of the IL-12 family members during in vitro infection with SE. The mechanisms of recognition of SE, however, appear to be the same for cDC and MPhi.

Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors.IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

Cytotoxic efficacy and influence on cellular phospholipid metabolism of 2-hydroxy- and 2-O-acetyl-octadecylphosphocholines.

The cytotoxic efficacy and influence on phospholipid composition of the new alkylphosphocholines (APC) 2-hydroxy and 2-O-acetyl-octadecylphosphocholines both synthesised in R- and S-configuration (R/S-OH and R/S-O-acetyl) were examined in vitro using HL-60 and MDA-MB-468 cells. IC50- and LC50-values were measured by MTT- and cell count assay. All tested APC showed higher or similar cytotoxic efficacy compared to the well known APC hexadecylphosphocholine (HePC). However, while S-configured APC (IC50) revealed considerably higher cytotoxic activities, only R- (natural)-configured APC caused significant changes in the phospholipid composition of tumour cells. Further investigations revealed an increase in R-O-acyl and loss of PC up to 70% in the membrane of both cell lines. Similar to PC, R-O-acyl bears two long non-polar hydrocarbon chains and stabilises cell membranes structurally, thus, possibly explaining less cytotoxicity and lack of apoptosis induction by R-configured APC. Nevertheless, an enrichment of R-O-acyl up to 70% at the expense of PC in cell membrane is tremendous and may inhibit tumour development by influencing the intracellular lipid signalling. In conclusion, our findings reveal the antitumoral efficacy of all tested new APC and offer new perspectives in drug development targeting phospholipid metabolism.

Lethal effect and apoptotic DNA fragmentation in response of D-GalN-treated mice to bacterial LPS can be suppressed by pre-exposure to minute amount of bacterial LPS: dual role of TNF receptor 1.

AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 microg). Both control and experimental groups simultaneously were challenged with LPS plus D-GalN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GalN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GalN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GalN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.

Role of interferons in LPS hypersensitivity.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria. We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta. Further, IFN-beta can be replaced by IFN-alpha. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta. Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection.