Microglia-derived proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta induce Purkinje neuronal apoptosis via their receptors in hypoxic neonatal rat brain

The developing cerebellum is extremely vulnerable to hypoxia which can damage the Purkinje neurons. We hypothesized that this might be mediated by tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) derived from activated microglia as in other brain areas. One-day-old rats were subjected to hypoxia following, which the expression changes of various proteins in the cerebellum including hypoxia inducible factor-1α, TNF-α, IL-1β, TNF-R₁ and IL-1R₁ were analyzed. Following hypoxic exposure, TNF-α and IL-1β immunoexpression in microglia was enhanced coupled by that of TNF-R₁ and IL-1R₁ in the Purkinje neurons. Along with this, hypoxic microglia in vitro showed enhanced release of TNF-α and IL-1β whose receptor expression was concomitantly increased in the Purkinje neurons. In addition, nitric oxide (NO) level was significantly increased in the cerebellum and cultured microglia subjected to hypoxic exposure. Moreover, cultured Purkinje neurons treated with conditioned medium derived from hypoxic microglia underwent apoptosis but the incidence was significantly reduced when the cells were treated with the same medium that was neutralized with TNF-α/IL-1β antibody. We conclude that hypoxic microglia in the neonatal cerebellum produce increased amounts of NO, TNF-α and IL-1β which when acting via their respective receptors could induce Purkinje neuron death.     

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2⁺ neurons, GFAP⁺ astrocytes, F4/80⁺ microglia, and O1⁺ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection. Received: 25 March 1997 / Accepted: 16 April 1997     

GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia

Background

Microglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer’s disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common β chain (βc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer’s disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear.

Methods

Microglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytechemistry. The levels of IL-1β, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons.

Results

GM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1β, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively.

Conclusions

These results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS.

     

Effect of Cytokines on Growth of Toxoplasma gondii in Murine Astrocytes

Cytokines play a significant role in the regulation of Toxoplasma gondii in the central nervous system. Cytokine-activated microglia are important host defense cells in central nervous system infections. Recent evidence indicates that astrocytes can also be activated by cytokines to inhibit intracellular pathogens. In this study, we examined the effect of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1 on the growth of T. gondii in a primary murine astrocyte culture. Pretreatment of astrocytes with IFN-γ resulted in 65% inhibition of T. gondii growth. Neither TNF-α, IL-1, nor IL-6 alone had any effect on T. gondii growth. IFN-γ in combination with either TNF-α, IL-1, or IL-6 caused a 75 to 80% inhibition of growth. While nitric oxide was produced by astrocytes treated with these cytokines, inhibition of T. gondii growth was not reversed by the addition of the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine. Furthermore, IFN-γ in combination with IL-1, IL-6, or TNF-α also induced inhibition in astrocytes derived from syngeneic mice deficient in the enzyme inducible nitric oxide synthase. This finding suggests that the mechanism of cytokine inhibition is not nitric oxide mediated. Similarly, the addition of tryptophan had no effect on inhibition, indicating that the mechanism was not mediated via induction of the enzyme indoleamine 2,3-dioxygenase. The mechanism of inhibition remains to be elucidated. Results from this study demonstrate that cytokine-activated astrocytes are capable of significantly inhibiting the growth of T. gondii. These data indicate that astrocytes may be important host defense cells in controlling toxoplasmosis in the brain.Toxoplasma gondii is an important pathogen in the central nervous system and causes a severe encephalitis in patients with AIDS. Cytokines play an important role in the regulation of T. gondii replication in the central nervous system (17). Gamma interferon (IFN-γ) has been shown to prevent reactivation of Toxoplasma encephalitis in mice (30). Tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) are up-regulated in the brains of mice with chronic toxoplasmosis (9, 15, 16). Studies indicate that IFN-γ, TNF-α, IL-1, and IL-6 may control the growth of T. gondii in the brain via activation of microglia (4, 5). Studies of mice indicate that cytokines induce anti-Toxoplasma activity in microglia via a nitric oxide (NO)-mediated mechanism (10).Recent evidence indicates that cytokines can also activate astrocytes to inhibit growth of T. gondii (6, 8, 25, 27). For example, IFN-γ has been shown to inhibit growth of T. gondii in the glioblastoma cell line 86HG39 (6). Inhibition was shown to be via induction of indoleamine 2,3-dioxygenase (IDO), resulting in the degradation of intracellular tryptophan (8). Pelloux et al. found that in the astrocytoma cell line GHE, TNF-α inhibited, IL-1 stimulated, and IFN-γ and IL-6 had no effect on growth of T. gondii (25). Finally, in primary human astrocytes, IFN-γ and IL-1 in combination have been shown to inhibit growth of T. gondii via the production of NO (27). Interpretation of these results is difficult due to variability found in astrocyte cell lines and differences between tumor cells and primary astrocytes.For this reason, we have chosen to study the effects of cytokines on growth of T. gondii in a primary astrocyte culture. In this study, we evaluated the effects of IFN-γ, TNF-α, IL-1, and IL-6 on the replication of T. gondii ME49 in a primary murine astrocyte culture. The effects of these cytokines individually, and the effects of IFN-γ in combination with IL-1, TNF-α, and IL-6, on the growth of T. gondii were examined. The ability of each of these cytokines and cytokine combinations to induce nitric oxide production was assessed by using the Griess reagent. A nitric oxide-mediated mechanism of cytokine inhibition of T. gondii growth was addressed by using N^G-monomethyl-l-arginine (NMMA), a nitric oxide inhibitor, and by using astrocytes derived from syngeneic mice deficient in the enzyme inducible nitric oxide synthase (iNOS). The IDO mechanism of inhibition was investigated via the addition of exogenous tryptophan. The aim of this study was to clarify the effect of cytokines on T. gondii replication in astrocytes and increase our understanding of the role of astrocytes in the host defense against T. gondii in the central nervous system.     

Genes, cells and cytokines in resistance against development of toxoplasmic encephalitis

Toxplasma gondii is an intracellular protozoan parasite which invades various organs including the central nervous system. Immunity is crucial for preventing development of toxoplasmic encephalitis (TE) following infection. IFN-gamma-mediated immune response plays a central role in the resistance. The activation of microglia and astrocytes by IFN-gamma or a combination of this cytokine with TNF-alpha appears to be an important effector mechanism in the host immunity. GM-CSF, IL-1 beta and IL-6 may participate in this activation. iNOS is a molecule which mediates the protective activity of the activated murine microglia and human astrocytes; however, another unknown mechanism(s) not mediated by iNOS is involved in the activity of activated murine astrocytes and human microglia. IL-6 plays a protective role, at least in part, by up-regulating IFN-gamma production during the chronic stage of infection in mice. IL-6 also plays a role in regulating the infiltration of T cell subsets into the brain. IL-4 appears to play a protective role, but its role may differ depending on the strains of mice. In contrast to these protective cytokines, IL-10 may play a pathogenic role by down-regulating IFN-gamma production. The resistance against development of TE is under genetic control in both mice and humans. The Ld gene within the D region of the H-2 complex confers resistance in mice. In AIDS patients, HLA-DQ3 appears to be a genetic marker of susceptibility, and HLA-DQ1 appears to be a resistance marker. Strains (genetic variation) of T. gondii is another factor which affects the susceptibility of the host to the development of TE. The genotypes of the parasite may be important for determining the susceptibility. It is possible that the genes of the host and genetic variation of T. gondii affect immune response of the host to the parasite, and thereby contribute to determining the resistance against the development of TE.     

Perioperative cytokine release during coronary artery bypass grafting in patients of different ages

Surgical interventions and cardiopulmonary bypass (CPB) induce a systemic inflammatory response with cytokine release. Ageing is perceived as a process of impaired immune functions: IL-1β, IL-6 and tumour necrosis factor-alpha (TNF-α) secretion are increased while IL-2 release is reduced in advanced age. At present, little information is available about perioperative immune reactions at different stages of ageing. The aim of the present study was to compare IL-6, IL-1β, TNF-α, IL-10 and soluble IL-2 receptor (sIL-2R) in younger and older patients undergoing cardiac surgery. Male patients (n = 14) undergoing elective coronary artery bypass grafting (CABG) surgery employing CPB with moderate hypothermia were divided into two groups according to their age: group 1 included seven patients < 50 years old, group 2 included seven patients > 65 years old. All patients received general anaesthesia using a balanced technique with sufentanil, isoflurane and midazolam. Blood samples were collected pre-operatively (T1); intra-operatively during CPB (T2); post-operatively on the day of surgery (T3); on the first post-operative day (T4). Blood concentrations of IL-6, IL-1β, IL-10, TNF-α and sIL-2R were measured using commercially available ELISA kits and corrected for plasma cell volume. Statistical analysis was performed by non-parametric analysis of variance and Mann–Whitney U-test. Significance level was set to P < 0.05. There were no statistically significant differences in the perioperative release of TNF-α, IL-6, IL-1β, IL-10 and sIL-2R among the two groups. We conclude that the perioperative course of cytokine release in patients undergoing CABG surgery with CPB and comparable perioperative management does not significantly differ in the two age groups.     

Interleukin-12 activates human γδ T cells: synergistic effect of tumor necrosis factor-α

γδ T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium-stimulated monocytes activate cord blood γδ T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for γδ T cell activation in vitro: interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-α and granulocyte/macrophage colony-stimulating factor were examined. Recombinant human IL-12 stimulated γδ T cells, but not αβ T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL-12-primed γδ T cell numbers increased to a greater extent in the culture to which exogenous IL-2 (5 U/ml) was added. Anti-TNF-α monoclonal antibody inhibited IL-12-induced up-regulation of CD25 on γδ T cells, suggesting that endogenous TNF-α may play a role in IL-12-induced activation of γδ T cells. Recombinant TNF-α synergistically augmented IL-12-induced activation of γδ T cells. Furthermore, IL-12 up-regulated TNF receptors on γδ T cells in vitro: TNF-α binding to its receptor induced CD25 expression on the γδ T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL-12 and TNF-α were produced by mycobacterial lysate-stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, γδ T cells can be quickly and antigen-nonspecifically activated by soluble factors including IL-12 and TNF-α, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.     

Psychosis may be associated with toxoplasmosis

Many parasites induce characteristic changes in their host. The effect of Toxoplasma gondii infection on the cerebrum and neuropsychiatric patients has been increasingly emphasized in recent years. T. gondii has a high affinity for brain tissue where tachyzoites may form tissue cysts and persist for a life long time. Some psychiatric symptoms such as schizophrenia and mental retardation may be induced by the infection of T. gondii. Furthermore, experiments demonstrated that some antipsychotics and mood stabilizers used to treat psychosis displayed the function of inhibiting T. gondii replication. Investigations from various regions in China in psychotic patients support the hypotheses that psychosis may be linked to T. gondii infection.     

T cell receptor specificities of Toxoplasma gondii-reactive mouse CD4+ T lymphocytes and Th1 clones

The Toxoplasma gondii-directed CD4⁺ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains Vβ4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4⁺ splenocytes. This repertoire was also detected among T. gondii-specific CD4⁺ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-γ and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4⁺ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates. Received: 3 February 1997     

Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania -infected cells

Intracellular staining for cytokines and parasites, combined with two-color flow cytometric analyses, were used to examine the frequencies of IL-12-, TNF-α- and IL-6-producing macrophages in response to Leishmania major infection and/or activation with IFN-γ/lipopolysaccharide (LPS). Inflammatory macrophages were obtained from nonimmune granulomas, initiated by the injection of polyacrylamide microbeads (Bio-gel P-100) into subcutaneous pouches of different mouse strains. Infection of inflammatory macrophages in vitro using metacyclic promastigotes produced identical effects on cytokine responses regardless of whether cells from genetically resistant or susceptible mouse strains were used: IL-12 was not produced in response to infection itself, virtually every infected cell lost its ability to produce IL-12 in response to IFN-γ/LPS, and the IL-6 response was partially inhibited, while the TNF-α response of infected cells was unimpaired. Low-multiplicity infection of inflammatory macrophages in vivo using either metacyclic promastigotes or tissue amastigotes also resulted in the complete and selective inhibition of IL-12 responses in infected cells. These data establish the physiologic relevance of prior observations regarding the selective impairment of IL-12 induction pathways in infected macrophages, and suggest a mechanisms for the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease.     

Toll-like Receptor Expression and Activation in Astroglia: Differential Regulation by HIV-1 Tat,gp120, and Morphine

In this study, we aimed to determine whether morphine alone or in combination with HIV-1 Tat or gp120 affects the expression of Toll-like receptors (TLRs) by astrocytes and to assess whether TLRs expressed by astrocytes function in the release of inflammatory mediators in vitro. TLR profiling by immunofluorescence microscopy, flow cytometry, in-cell westerns, and RT-PCR showed that subpopulations of astrocytes possessed TLR 2, TLR3, TLR4, and TLR9 antigenicity. Exposure to HIV-1 Tat, gp120, and/or morphine significantly altered the proportion of TLR-immunopositive and/or TLR expression by astroglia in a TLR-specific manner. Subsets of astroglia displayed significant increases in TLR2 with reciprocal decreases in TLR9 expression in response to Tat or gp120 ± morphine treatment. TLR9 expression was also significantly decreased by morphine alone. Exposing astrocytes to the TLR agonists LTA (TLR2), poly I:C (TLR3), LPS (TLR4) and unmethylated CpG ODN (TLR9) resulted in increased secretion of MCP-1/CCL2 and elevations in reactive oxygen species. TLR3 and TLR4 stimulation increased the secretion of TNF-α, IL-6, and RANTES/CCL5, while activation of TLR2 caused a significant increase in nitric oxide levels. The results suggest that HIV-1 proteins and/or opioid abuse disrupt the innate immune response of the central nervous system (CNS) which may lead to increased pathogenicity.     

Bacterial extract (OM-89) specific and non specific immunomodulation in rheumatoid arthritis patients

The Escherichia Coli bacterial extract (OM-89) is used in the treatment of rheumatoid arthritis (RA). We evaluated the immunological changes induced by oral administration of OM-89 in 12 RA patients (polyclonal T cell reactivity to PHA, T cell precursor frequencies specific for OM-89 and Tetanus toxoid (TT), a control antigen and the release of Th1 (IFN-γ, TNF-α), Th2 (IL-4) and T regulatory 1 cell (Tr1) (IL-10) cytokines in the supernatants of PBMC cultures. Stimulation index in response to PHA decreased at month 3 as well as T cell precursor frequencies specific for TT with similar trends for OM-89-specific T cell precursor frequencies. OM-89 induced a strong production of IL-10, a significant decrease in IL-4 production while TNF-α and IFN-γ production tended to decrease during the study.

Our results suggest that OM-89 has immunomodulatory properties by inducing changes in PBMC cytokines release suggestive of an induced Tr1 response to OM-89.     

Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to certain neuronal populations. Since persistent BDV infection of neurons is nonlytic in vitro, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brains remain unclear. Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia. Here, we evaluated the mechanisms whereby astrocytes can contribute to activation of microglia in neuron-glia-microglia mixed cultures. We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES. Activation of astrocytes then produces activation of microglia as evidenced by increased formation of round-shaped, MHCI-, MHCII- and IL-6-positive microglia cells. Our analysis of possible molecular mechanisms of activation of astrocytes and/or microglia in culture indicates that the mediators of activation may be soluble heat-resistant, low molecular weight factors. The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons. The data are consistent with the hypothesis that microglia activation in the absence of neuronal damage may represent initial steps in the gradual neurodegeneration observed in brains of neonatally BDV-infected rats.     

Lipopolysaccharide-induced nigral inflammation leads to increased IL-1β tissue content and expression of astrocytic glial cell line-derived neurotrophic factor

Reactive gliosis and inflammatory change is a key component of nigral dopaminergic cell death in Parkinson's disease (PD). Astrocyte derived glial cell line-derived neurotrophic factor (GDNF) promotes the survival and growth of dopaminergic neurones and it protects against or reverses nigral degeneration induced by 6-OHDA and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rodents and primates. But the effect of increased levels of pro-inflammatory cytokines on the release of GDNF is unknown. This study examined the relationship between release of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and the expression of GDNF in rats following nigral lipopolysaccharide (LPS) administration. Acute nigral administration of LPS led to marked elevation of IL-1β but insignificant TNF-α tissue content and to a prominent expression of GDNF immunoreactivity in astrocytes but not microglia. The results suggest that inflammation is not only involved in neuronal loss but could promote neuronal survival through increased release of GDNF following up-regulation of IL-1β.     

Differences in microglia activation between rats-derived cell and mice-derived cell after stimulating by soluble antigen of IV larva from Angiostrongylus cantonensis in vitro

Angiostrongylus cantonensis is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats. Humans and mice are accidental hosts or named nonpermissive hosts. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningoencephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of the host to parasite during A. cantonensis invasion and development. Microglia is considered to be the key immune cell in the central nervous system like macrophage. To further understand the reasons for why mice and rats attain different outcomes in A. cantonensis infection, we set up the method to isolate and culture newborn rats’ primary microglia and observe the activation of the microglia cells, comparing with mice microglia cell line N9. We treated cells with soluble antigen of the fourth larva of A. cantonensis (L4 larva) and measured mRNA levels of IL-1β, IL-5, IL-6, IL-13, eotaxin, iNOS, and TNF-α by real-time PCR. The results showed that N9 expressed high mRNA level of IL-6, IL-1β, TNF-α, iNOS, IL-5, IL-13, and eotaxin, but primary microglia only had IL-5, IL-13, and eotaxin mRNA level. It implies that microglia from rats and mice had different reaction to soluble antigen of A. cantonensis. Therefore, we supposed that microglia may play an immune modulation role during the brain inflammation induced by A. cantonensis.     

IL-10 up-regulates nitric oxide (NO) synthesis by lipopolysaccharide (LPS)-activated macrophages: improved control of Trypanosoma cruzi infection

We examined the effects of IL-10 on tumour necrosis factor-alpha (TNF-α) and NO production by LPS-activated macrophages and on the ability of these cells to control Trypanosoma cruzi infection. We first observed that the addition of rIL-10 to macrophages of the J774 cell line decreased their synthesis of TNF-α but increased their release of NO in a dose-dependent manner. In parallel, treatment of J774 cells with rIL-10 resulted in a better control of T. cruzi infection involving up-regulation of NO synthesis, as it was not observed in presence of N-nitro- L -arginine methyl ester ( L -NAME), a competitive inhibitor of NO synthase. The enhancing effect of rIL-10 on NO production was not observed on peritoneal macrophages from wild-type C57Bl/6 mice, but well on macrophages from IL-10 knock-out mice. The control of NO production by endogenous IL-10 was confirmed by the demonstration that neutralization of IL-10 secreted by LPS-activated macrophages from wild-type mice inhibited their production of NO and, in parallel, their ability to control T. cruzi infection. Taken together, these data demonstrate that both exogenous and endogenous IL-10 up-regulate the production of NO by LPS-activated macrophages and improve thereby their ability to clear T. cruzi infection.     

Intimate adherence by enteropathogenic Escherichia coli modulates TLR5 localization and proinflammatory host response in intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease by altering enterocyte physiology and producing mucosal inflammation. Many details concerning the host response against EPEC remain unknown. We evaluated the role of EPEC virulence factors on the inflammatory response through an analysis of bacterial recognition, cell signalling, and cytokine production using an in vitro epithelial cell infection model. Interestingly, we found that EPEC infection recruits Toll-like receptor 5 (TLR5) to the cell surface. We confirmed that type 3 secretion system (T3SS) and flagellin (FliC) are necessary for efficient extracellular regulated kinases 1 and 2 (ERK1/2) activation and found that intimin could down-regulate this pathway. Besides flagellin, intimin was required to keep nuclear factor kappa B (NF-κB) activated during infection. EPEC infection activated tumour necrosis factor alpha (TNF-α) production and induced interleukin (IL)-1β and IL-8 release. Virulence factors such as intimin, T3SS, EspA and fliC were required for IL-1β secretion, whereas intimin and T3SS participated in IL-8 release. Flagellin was essential for late secretion of TNF-α and IL-8 and intimin stimulated cytokine secretion. Initial adherence limited TNF-α release, whereas late attachment sustained TNF-α secretion. We conclude that intimin modulates TLR5 activation and intimate adherence alters the proinflammatory response.     

Anti-parasitic effector mechanisms in human brain tumor cells: Role of interferon-γ and tumor necrosis factor-α

Toxoplasma gondii, an obligate intracellular parasite, is able to replicate in human brain cells. We recently showed that interferon (IFN)-γ-activated cells from glioblastoma line 86HG39 were able to restrict Toxoplasma growth. The effector mechanism responsible for this toxoplasmostatic effect was shown by us to be the IFN-γ-mediated activation of indolamine 2,3-dioxygenase (IDO), resulting in the degradation of the essential amino acid tryptophan. In contrast, glioblastoma 87HG31 was unable to restrict Toxoplasma growth after IFN-γ activation, and IFN-γ-mediated IDO activation was weak. We observed that tumor necrosis factor (TNF)-α alone is unable to activate IDO or to induce toxoplasmostasis in any glioblastoma cell line tested. Interestingly, we found that TNF-α and IFN-γ were synergistic in the activation of IDO in glioblastoma cells 87HG31, 86HG39 and U373MG and in native astrocytes. This was shown by the measurement of enzyme activity as well as by the detection of IDO mRNA in TNF-α + IFN-γ activated cells. This IDO activity results in a strong toxoplasmostatic effect mediated by glioblastoma cells activated simultaneously by both cytokines. Antibodies directed against TNF-α or IFN-γ were able to inhibit IDO activity as well as the induction of toxoplasmostasis in glioblastoma cells stimulated with both cytokines. Furthermore, it was found that the addition of L -tryptophan to the culture medium completely blocks the antiparasitic effect. We therefore conclude that both TNF-α and IFN-γ may be involved in the defense against cerebral toxoplasmosis by inducing IDO activity as an antiparasitic effector mechanism in brain cells.