Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.

A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.     

Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.     

Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway

Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.     

Recruitment of CTL activity by tumor-specific antibody-mediated targeting of single-chain class I MHC-peptide complexes

Acute and lethal ileitis can be elicited in certain strains of inbred mice after oral infection with the intracellular protozoan parasite Toxoplasma gondii. The development of this inflammatory process is dependent upon the induction of a robust Th1 response, including overproduction of IFN-gamma, TNF-alpha, and NO, as has been reported in other experimental models of human inflammatory bowel disease. In this study we have investigated the role of CD4(+) T cells from the lamina propria (LP) in the early inflammatory events after T. gondii infection using isolated and primary cultured intestinal cells from infected mice and immortalized mouse mIC(cl2) intestinal epithelial cells. Primed LP CD4(+) T cells isolated from parasite-infected mice produce substantial quantities of both IFN-gamma and TNF-alpha. IFN-gamma- and TNF-alpha-producing LP CD4(+) T cells synergize with infected mIC(cl2) and enhance the production of several inflammatory chemokines including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage-inflammatory protein-1alphabeta, and IFN-gamma-inducible protein-10. Furthermore, primed LP CD4(+) T cells cocultured with infected mIC(cl2) inhibited replication of the parasite in the intestinal epithelial cells. Thus, LP CD4(+) T cells can interact with parasite-infected intestinal epithelial cells and alter the expression of several proinflammatory products that have been associated with the development of intestinal inflammation. The interaction between these two components of the gut mucosal compartment (CD4(+) T cells and enterocytes) may play a role in the immunopathogenesis of this pathogen-driven experimental inflammatory bowel disease model.     

Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling

Toxoplasma gondii is an intracellular parasite that survives and multiplies in professional phagocytes such as macrophages. Therefore, T. gondii has to cope with the panel of antimicrobial host immune mechanisms, among which IFN-gamma plays a crucial role. We report in this study that in vitro infection of murine macrophages with viable, but not with inactivated, parasites results in inhibition of IFN-gamma signaling within the infected cells. Thus, infection of RAW264.7 macrophages with tachyzoites inhibited IFN-gamma-induced STAT-1 tyrosine phosphorylation, mRNA expression of target genes, and secretion of NO. These effects were dependent on direct contact of the host cells with living parasites and were not due to secreted intermediates. In parallel, we report the induction of suppressor of cytokine signaling-1 (SOCS-1), which is a known feedback inhibitor of IFN-gamma receptor signaling. SOCS-1 was induced directly by viable parasites. SOCS overexpression in macrophages did not affect tachyzoite proliferation per se, yet abolished the inhibitory effects of IFN-gamma on parasite replication. The inhibitory effects of T. gondii on IFN-gamma were diminished in macrophages from SOCS-1-/- mice. The results suggest that induction of SOCS proteins within phagocytes due to infection with T. gondii contributes to the parasite's immune evasion strategies.     

Toxoplasma gondii exposes phosphatidylserine inducing a TGF-beta1 autocrine effect orchestrating macrophage evasion

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. Activated macrophages control T. gondii growth by nitric oxide (NO) production. However, T. gondii active invasion inhibits NO production, allowing parasite persistence. Here we show that the mechanism used by T. gondii to inhibit NO production persisting in activated macrophages depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V on parasites or activated macrophages abolished NO production inhibition and parasite persistence. NO production inhibition depended on a transforming growth factor-beta1 (TGF-beta1) autocrine effect confirmed by the expression of Smad 2 and 3 in infected macrophages. TGF-beta1 led to inducible nitric oxide synthase (iNOS) degradation, actin filament (F-actin) depolymerization, and lack of nuclear factor-kappaB (NF-kappaB) in the nucleus. All these features were reverted by TGF-beta1 neutralizing antibody treatment. Thus, T. gondii mimics the evasion mechanism used by Leishmania amazonensis and also the anti-inflammatory response evoked by apoptotic cells.     

Leishmania sp: comparative study with Toxoplasma gondii and Trypanosoma cruzi in their ability to initialize IL-12 and IFN-gamma synthesis

We compared in vitro and in vivo induction of IL-12 (p40) and IFN-gamma by mouse cells stimulated with Toxoplasma gondii, Trypanosoma cruzi, and different species of Leishmania. Spleen cells cultured in vitro with T. cruzi or T. gondii, but not with Leishmania, produced IL-12 (p40) and IFN-gamma. Accordingly, IL-12 (p40) was produced by macrophages stimulated in vitro with live T. cruzi or T. gondii or membrane glycoconjugates obtained from trypomastigotes or tachyzoites. No IL-12 production was detected when macrophages were stimulated with live parasites or glycoconjugates from Leishmania, regardless of priming with IFN-gamma. In vivo, only T. cruzi and T. gondii induced the synthesis of IL-12 and IFN-gamma by mouse spleen cells after intraperitoneal injection of parasites. When injected subcutaneously, live Leishmania sp. induced IL-12 (p40) and IFN-gamma production by draining lymph node cells, albeit the levels were slightly lower than those induced by infection with T. gondii or T. cruzi using the same route. Together our results indicate that under different conditions, the intracellular protozoa T. gondii and T. cruzi are more potent stimulators of IL-12 and IFN-gamma synthesis by host immune cells than parasites of the genus Leishmania.     

Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production

To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774-A.1 macrophage-like cells. The selection, which was based on intracellular growth competition, rapidly yielded isolates that out-competed the wild-type strain during intracellular growth. J774-A.1 cells responded to challenge with S. typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production. Inhibition of NO production with the use of the competitive inhibitor N-monomethyl-L-arginine (NMMA) resulted in a 20-fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth. In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774-A.1 cells, despite an induction of iNOS mRNA and iNOS protein. Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion-competent bacteria. The results indicate that S. typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.     

Impact of foetus and mother on IFN-gamma-induced indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression in murine placenta following Toxoplasma gondii infection

IFN-gamma production is a hallmark of acute infection with the protozoan parasite Toxoplasma gondii. The tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO), as well as inducible nitric oxide synthase (NOS2) are induced by IFN-gamma and can play extremely diverse roles in immune regulation, defence against pathogens and physiological homeostasis. We investigated the regulation of these two central enzymes in the placenta during acute infection of pregnant female mice. Using IFN-gamma receptor knockout (IFNgammaR-/-) mice, we showed that IDO is not constitutively expressed in term placentas. In contrast, NOS2 expression was observed, largely dependent on IFN-gamma signalling. Upon infection with the avirulent PRU strain of T. gondii, IDO mRNA expression was induced in an IFNgammaR-dependent manner. Surprisingly, NOS2 mRNA was severely suppressed. Importantly, we showed in crossing experiments of heterozygote (IFNgammaR+/-) mothers with IFNgammaR-/- males and vice versa that IDO expression largely depends on the presence of IFN-gamma receptors on foetal cells, and to a lesser extent on maternal cells. Immunohistochemical analysis localised foetal IDO production to invasive trophoblasts within the maternal part of the placenta. The placental vascular endothelium only stained positive when the mothers possessed functional IFN-gamma receptors. In contrast, placental NOS2 expression, but also its suppression following infection, seems to be largely dependent on IFN-gamma signalling in maternal cells. Neither factor appears to regulate placental T. gondii growth, as we observed no difference in parasite numbers between (+/-) and (-/-) foetuses. Taken together, our results demonstrate the crucial role of the foetus in placental IDO, but not NOS2, production following T. gondii infection.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.     

The role of protein kinase C in the induction of nitric oxide synthesis by murine macrophages.

The role of protein kinase C (PKC) in the induction of nitric oxide synthesis by interferon-gamma (IFN-gamma) was investigated using two murine macrophage cell lines, J774 and RAW 264.7. Nitric oxide (NO) production was markedly reduced by a PKC inhibitor, Ro31-8220 in a dose-dependent manner. Incubation of cells with IFN-gamma resulted in translocation of PKC to the cell membrane. Prolonged incubation of cells with a high concentration of phorbol ester, which down-regulated PKC activity, also reduced nitric oxide production. These findings provide evidence that PKC is involved in the induction of nitric oxide synthesis by IFN-gamma.     

Molecular Characteristics and Potent Immunomodulatory Activity of Fasciola hepatica Cystatin

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.     

Inhibition of nitric oxide synthase expression by a methanolic extract of Crescentia alata and its derived flavonols.

In order to validate the use of Crescentia alata (Bignoniaceae) in the traditional medicine of Guatemala as an antiinflammatory remedy, the methanolic (MeOH) extract has been evaluated in vivo for antiinflammatory activity on carrageenin paw edema in rats and in vitro on Escherichia coli lipopolysaccharide- (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774.A1 macrophage cell line. This extract exerted in vivo a significant anti-inflammatory activity at the highest dose tested. The same extract showed in vitro an inhibitory activity on inducible nitric oxide synthase expression and on NO formation in LPS-primed J774.A1 cells. Subsequent fractionation and analysis of the extract has led to the isolation and characterization as major constituents of two flavonol glycosides: quercetin 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (rutin) 1, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (kaempferol 3-O-rutinoside) 2, and flavonol aglycone, kaempferol 3. Their structures were elucidated by spectral methods. The bioassay-directed analysis of flavonols 1-3 indicated that kaempferol (3) was the most active compound contained in the MeOH extract because it reduced in vitro both NO production and iNOS expression in LPS-primed J774.A1 cells, whereas rutin (1) and kaempferol 3-O-rutinoside (2) showed no significant activity. The MeOH extract and all of flavonoids tested did not show in vitro significant cytotoxic effect in J774.A1 macrophage cell line.     

Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-kappaB and p38 MAPK signaling pathways.

Mercury is well known to adversely affect the immune system; however, little is known regarding its molecular mechanisms. Macrophages are major producers of nitric oxide (NO) and this signaling molecule is important in the regulation of immune responses. The present study was designed to determine the impact of mercury on NO and cytokine production and to investigate the signaling pathways involved. The murine macrophage cell line J774A.1 was used to study the effects of low-dose inorganic mercury on the production of NO and proinflammatory cytokines. Cells were treated with mercury in the presence or absence of lipopolysaccharide (LPS). Mercury (5-20 microM) dose-dependently decreased the production of NO in LPS-stimulated cells. Concomitant decreases in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein were detected. Treatment of J774A.1 cells with mercury alone did not affect the production of NO nor the expression of iNOS mRNA or protein. Interestingly, mercury alone stimulated the expression of tumor necrosis factor alpha (TNFalpha), and increased LPS-induced TNFalpha and interleukin-6 mRNA expression. Mercury inhibited LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) but had no effect alone. In contrast, mercury activated p38 mitogen-activated protein kinase (p38 MAPK) and additively increased LPS-induced p38 MAPK phosphorylation. These results indicate that mercury suppresses NO synthesis by inhibition of the NF-kappaB pathway and modulates cytokine expression by p38 MAPK activation in J774A.1 macrophage cells.