TNF receptor-associated factor 6-dependent CD40 signaling primes macrophages to acquire antimicrobial activity in response to TNF-alpha

IFN-gamma is considered an essential stimulus that allows macrophages to acquire activity against intracellular pathogens in response to a second signal such as TNF-alpha. However, protection against important pathogens can take place in the absence of IFN-gamma through mechanisms that are still dependent on TNF-alpha. Engagement of CD40 modulates antimicrobial activity in macrophages. However, it is not known whether CD40 can replace IFN-gamma as priming signal for induction of this response. We show that CD40 primes mouse macrophages to acquire antimicrobial activity in response to TNF-alpha. The effect of CD40 was not caused by modulation of IL-10 and TGF-beta production or TNFR expression and did not require IFN-alphabeta signaling. Induction of antimicrobial activity required cooperation between TNFR-associated factor 6-dependent CD40 signaling and TNFR2. These results support a paradigm where TNFR-associated factor 6 signaling downstream of CD40 alters the pattern of response of macrophages to TNF-alpha leading to induction of antimicrobial activity.     

The CD40-autophagy pathway is needed for host protection despite IFN-Γ-dependent immunity and CD40 induces autophagy via control of P21 levels

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40(-/-) mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1(+/-) mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40(-/-) mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ.     

Divergent role for TNF-alpha in IFN-gamma-induced killing of Toxoplasma gondii and Salmonella typhimurium contributes to selective susceptibility of patients with partial IFN-gamma receptor 1 deficiency

Patients with defects in IFN-gamma- or IL-12-mediated immunity are susceptible to infections with Salmonella and non-tuberculous mycobacteria, but rarely suffer from infections with other intracellular pathogens such as Toxoplasma gondii. Here we describe macrophage and T cell function in eight individuals with partial IFN-gamma receptor 1 (IFN-gammaR1) deficiency due to a mutation that results in elevated cell surface expression of a truncated IFN-gammaR1 receptor that lacks the intracellular domain. We show that various effector mechanisms dependent on IFN-gammaR signaling are affected to different extents. Whereas TNF-alpha production was normally up-regulated in response to IFN-gamma, IL-12 production and CD64 up-regulation were strongly reduced, and IFN-gamma-mediated killing of the intracellular pathogens Salmonella typhimurium and T. gondii was completely abrogated in patient's macrophages. Since these patients suffer selectively from infections with non-tuberculous mycobacteria and Salmonella, but not T. gondii, despite sero-immunity in six of eight patients, which indicates previous contact with this pathogen, we next studied the role of TNF-alpha as a possible immune compensatory mechanism. IFN-gamma-induced killing of T. gondii appeared to be partially mediated by TNF-alpha, and addition of TNF-alpha could compensate for the abrogated killing of T. gondii in the patient's macrophages. In contrast, IFN-gamma-mediated killing of S. typhimurium appeared to be independent of TNF-alpha. We propose that the divergent role of TNF-alpha in IFN-gamma-induced killing of T. gondii and S. typhimurium may at least partially explain the highly selective susceptibility of patients.     

CD40-TRAF6 and autophagy-dependent anti-microbial activity in macrophages

A fundamental question in host-pathogen interaction is to determine if the immune system activates fusion with the lysosomes to eradicate pathogens. We recently reported that this task is accomplished by the interaction between CD40 expressed on macrophages and CD154 expressed on activated CD4+ T cells. CD40 stimulation of macrophages induces vacuole-lysosome fusion through autophagy and results in killing of the obligate intracellular pathogen Toxoplasma gondii. This response is independent of IFN-gamma, STAT1 and p47 GTPases. We now report that vacuole-lysosome fusion is dependent on synergy between TRAF6 signaling downstream of CD40 and TNF-alpha. These studies identified a new paradigm by which T cells eradicate an intracellular pathogen within macrophages.     

B cells amplify IFN-gamma production by T cells via a TNF-alpha-mediated mechanism

Aside from being the precursors of the Ab-secreting cells, B cells are engaged in other immune functions such as Ag presentation to T cells or cytokine production. These functions may contribute to the pathogenic role of B cells in a wide range of autoimmune diseases. We demonstrate that B cells acquire the capacity to amplify IFN-gamma production by CD4 and CD8 T cells during the course of the Th1 inflammatory response to Toxoplasma gondii infection. Using the two following different strategies, we observed that B cells from T. gondii-infected mice, but not from naive mice, induce higher IFN-gamma expression by splenic host T cells: 1) reconstitution of B cell-deficient mice with B cells expressing an alloantigen different from the recipients, and 2) adoptive transfer of B and T cells into RAG-/- mice. In vitro assays allowing the physical separation of T and B cells demonstrate that Ag-primed B cells enhance IFN-gamma production by T cells in a contact-dependent fashion. Using an OVA-transgenic strain of T. gondii and OVA-specific CD4 T cells, we observed that the proinflammatory effect of B cells is neither Ag specific nor requires MHCII expression. However, TNF-alpha expressed on the surface of B cells appears to mediate in part the up-regulation of IFN-gamma by the effector T cells.     

Pathogenicity of Toxoplasma gondii through B-2 cell-mediated downregulation of host defense responses

IFN-gamma is the primary mediator of anti-parasite effector mechanisms against Toxoplasma gondii. After intraperitoneal infection with the Fukaya strain of T. gondii, unirradiated IFN-gamma knock-out (GKO) mice transferred with wild type (WT) CD8+ effector T cells from infected mice failed to induce the production of IFN-gamma and died, whereas irradiated (IR) GKO mice transferred with WT CD8+ T cells induced IFN-y production and survived more than 6 months. IR GKO mice transferred with WT CD8+ T cells together with GKO B-2 cells died 8 days after infection, whereas those transferred with WT CD8+ T cells together with B-la or T cells survived. B-2 cells of infected GKO mice activated CD11b+ cells for IL-4 production, and down-regulated NO release, STAT1 phosphorylation, and interferon regulatory factor-1 expression in the peritoneal exudates cells of IR GKO mice transferred with WT CD8+ T cells together with GKO B-2 cells after infection. Thus, B-2 cells in T. gondii-infected mice act as suppressor cells in the host defense of infected mice.     

Toxoplasma gondii partially inhibits nitric oxide production of activated murine macrophages

Activated macrophages produce nitric oxide (NO) and as such are able to control the multiplication of Toxoplasma gondii. Until now, no reports have described a possible modulation of NO production of macrophages after T. gondii infection. To investigate this possibility, murine blood monocyte-derived and peritoneal macrophages were activated in vitro with interferon-gamma and lipopolysaccharide and infected with T. gondii and Trypanosoma cruzi, and NO production was evaluated. NO was produced by monocyte-derived macrophages only if cultured in the presence of macrophage-colony-stimulating factor. Monocyte-derived or peritoneal macrophages infected with T. gondii presented a significant reduction in NO production. NO production inhibition was not detected after T. cruzi infection. Macrophages infected with higher T. gondii/macrophage ratios presented lower NO production. Furthermore, only viable T. gondii could cause partial inhibition of NO production. In macrophages activated 24 h before the interaction, partial inhibition was detected after 3 h of infection and continued for 48 h. In macrophages activated immediately after the interaction, partial inhibition was not detected at 12 h, but was observed at 24 h. T. gondii-infected macrophages present lower inducible nitric oxide synthase expression as assayed by immunofluorescence. T. gondii did not develop in monocyte-derived macrophages producing NO, but were not totally eliminated. These results demonstrate that T. gondii infection partially inhibits NO production by murine macrophages, suggesting that a deactivating macrophage mechanism may be used for better survival into phagocytic cells.     

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.     

Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages

IFN-gamma is an important mediator of cellular resistance against microbial pathogens and tumor cells due in part to its potent capacity to activate macrophages for enhanced cytotoxicity. The present study demonstrates that TNF-alpha regulates the expression of enhanced antimicrobial activity by triggering IFN-gamma primed macrophages to kill or inhibit intracellular Toxoplasma gondii. Resident mouse macrophages stimulated with rIFN-gamma at levels up to 2500 U/ml failed to display enhanced antitoxoplasmal activity when cultured in vitro under low endotoxin conditions (less than 10 pg/ml), but were triggered by addition of small amounts of LPS (0.1 ng/ml). A similar requirement for LPS as a second signal necessary to trigger antitoxoplasmal activity was observed when IFN-gamma was administered to mice in vivo. The essential nature of this triggering step allowed us to explore the role of cytokines that act as endogenous regulators of macrophage activation. rTNF-alpha, although unable to confer antitoxoplasmal activity when used alone to treat macrophages, was capable of triggering IFN-gamma-primed macrophages cultured under low endotoxin conditions. The ability of TNF-alpha to trigger IFN-gamma-primed macrophages was blocked by rabbit anti-TNF-alpha polyclonal antisera but was not affected by polymyxin B indicating that TNF-alpha triggering was not due to contamination with LPS. Collectively, these findings demonstrate that TNF-alpha performs an important regulatory role in the expression of enhanced anti-microbial activity by IFN-gamma-primed macrophages.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

Autophagic elimination of intracellular parasites: convergent induction by IFN-gamma and CD40 ligation?

Autophagy has recently been implicated in the immune elimination of the intracellular protozoan parasite, Toxoplasma gondii. Toxoplasma and other apicomplexan parasites actively invade host cells and form nonfusogenic parasitophorous vacuoles. Nevertheless, following entry into IFN-gamma-activated effector macrophages, vesiculation of the parasite vacuole or PV membrane ensues, in a process dependent upon the activity of p47 GTPases induced by IFN-gamma signaling. Subsequent disruption of the plasma membrane of the stripped parasites precedes autophagolysosomal elimination of T. gondii. In contrast, ligation of the CD40 receptor and autocrine signaling by TNF activate a seemingly distinct, p47 GTPase-independent mechanism leading to autophagic elimination of intracellular T. gondii, without prior disruption of the pathogen vacuole. Thus, two key pathways of the cell-mediated immune response, namely IFN-gamma and CD40/CD40L, trigger a common autophagolysosomal endpoint of parasite elimination, via distinct intermediary mechanisms.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

From cells to signaling cascades: manipulation of innate immunity by Toxoplasma gondii

The intracellular opportunistic protozoan Toxoplasma gondii is a potent stimulus for cell-mediated immunity, and IL-12-dependent IFN-gamma induction is vital in resistance to the parasite. Dendritic cells, neutrophils and macrophages are important sources of IL-12 during infection. T. gondii possesses two mechanisms for triggering IL-12. One is dependent upon the common adaptor protein MyD88, and is likely to involve Toll-like receptors. The other is a more unusual pathway that involves triggering through CCR5 by a parasite cyclophilin molecule. Countering these potent pro-inflammatory activities, T. gondii has several mechanisms to down-regulate immunity. Intracellular infection causes a blockade in the NFkappaB macrophage signaling pathway, correlating with reduced capacity for IL-12 and TNF-alpha production. The parasite also prevents STAT1 activity, resulting in decreased levels of IFN-gamma-stimulated MHC surface antigen expression. Furthermore, infection also induces resistance to apoptosis through inhibition of caspase activity. Extracellular pathways of suppression involve soluble mediators such as IL-10 and lipoxins that have potent IL-12 down-regulatory effects. The balance of pro-inflammatory and anti-inflammatory signaling which T. gondii engages is likely dictated by requirements for a stable host-parasite interaction. First, there is a need for Toxoplasma to induce an immune response robust enough to allow host survival and establish long-term chronic infection. Second, the parasite must avoid immune-elimination and induction of pro-inflammatory pathology that can cause lethality if unchecked. The widespread distribution of T. gondii and the normally innocuous nature of infection indicate the skill with which the parasite achieves the two seemingly contrary goals.     

Ability of T cell subsets and their soluble mediators to modulate the replication of Mycobacterium bovis in bovine macrophages

Peripheral blood mononuclear cells (PBMCs) from cattle vaccinated with Bacillus Calmette-Guerin (BCG) were obtained and expanded in vitro by incubation with purified protein derivative. The ability of these cells to modulate the replication of virulent Mycobacterium bovis in autologous-infected macrophages was compared to cells from non-vaccinated controls. Cells from non-vaccinated animals were shown to confer a significant degree of mycobacteriostatic activity to autologous-infected macrophages. This activity was not inhibited by including a neutralizing antibody versus interferon-gamma (IFN-gamma), and was dependent on direct contact between PBMCs and infected macrophages. Addition of autologous PBMCs from BCG-vaccinated cattle was shown to significantly enhance macrophage resistance to M. bovis, and this increased macrophage resistance was partly abrogated by including a neutralizing antibody to IFN-gamma. Addition of T cells from non-vaccinated animals to infected macrophages was associated with a modest increase in macrophage release of TNF-alpha and nitric oxide, whereas PBMCs from vaccinated animals increased very significantly the release of these factors. Neutralization of nitric oxide (NO), by inclusion of monomethyl-L-arginine, significantly diminished the ability of PBMCs from vaccinated animals to enhance macrophage resistance to M. bovis, but had no impact on the ability of T cells from naive animals to modulate macrophage function. The ability of naive cells to increase macrophage anti-M. bovis activity was largely mediated by CD4+ T cells, whereas both CD4+ T cells and CD8+ T cells conferred macrophage resistance to M. bovis in vaccinated animals. These data highlight the role of IFN-gamma and NO in the immune resistance of cattle to M. bovis.     

Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macrophages to lymphokines and interferon-gamma

To test the hypothesis that tissue macrophages from AIDS patients have no intrinsic defects in either antimicrobial activity or in the capacity to respond to T cell-derived activating stimuli, alveolar macrophages from 11 patients were treated with crude lymphokines produced by healthy donors. After 72 hr of pretreatment with 10% mitogen- or antigen-induced crude lymphokines (which contained 300 U/ml of interferon-gamma [IFN-gamma]), AIDS alveolar macrophages generated twofold to threefold more H2O2 and readily inhibited the replication of the intracellular pathogens Toxoplasma gondii and Chlamydia psittaci. These responses were indistinguishable from those displayed by activated alveolar cells from 12 non-AIDS patients and three healthy volunteers. As judged by the abrogating effects of a neutralizing anti-human IFN-gamma monoclonal antibody, lymphokine-induced alveolar macrophage activation appeared to be largely IFN-gamma-dependent; thus, macrophages were also stimulated with recombinant (r)IFN-gamma alone. Seventy-two hours of treatment with 300 U/ml of rIFN-gamma resulted in both enhanced oxidative and antimicrobial activity comparable to that achieved by crude lymphokines, and the responsiveness of AIDS alveolar macrophages to rIFN-gamma was identical to control cells. These in vitro results suggest that tissue mononuclear phagocytes from AIDS patients a) are free of apparent defects in intracellular antimicrobial activity, b) are fully responsive to activating T cell products, and c) support the use of IFN-gamma as a potential macrophage-activating immunotherapeutic agent in AIDS-related opportunistic infections.     

Roles of Toxoplasma gondii-derived heat shock protein 70 in host defense against T. gondii infection

C57BL/6 mice receiving intraperitoneal injection of Toxoplasma gondii -derived heat shock protein 70 (T.g. HSP70) on day 3 post T. gondii infection succumbed by day 9 post infection, while vector protein-injected control mice survived more than 6 months. The deteriorating effect of T.g. HSP70 on host immune responses was dose-dependent. By T.g. HSP70 injection, T. gondii loads increased in various organs of T. gondii-infected mice. Th2 cytokines such as IL-4 and IL-10 were continuously produced from spleen and peritoneal exudate cells of T. gondii -infected mice by injection of T.g. HSP70. Furthermore, nitric oxide production from peritoneal macrophages in T. gondii-infected mice was reduced by T.g. HSP70.     

Endogenous polyamine levels in macrophages is sufficient to support growth of Toxoplasma gondii

Cytotoxic-activated macrophages control Toxoplasma gondii growth by producing nitric oxide (NO). However, the parasite can partially inhibit NO production. NO is generated from arginine within the polyamine biosynthetic pathway. Two enzymes of this pathway are ornithine, decarboxylase (ODC) and arginine decarboxylase (ADC). The aim of the present work was to investigate whether T. gondii is able to modulate polyamine metabolism in macrophages. Toxoplasma gondii infection did not affect basal ODC or ADC activity. However, lipopolysaccharide induced an increase in ODC activity. Polyamine-treated macrophages exhibited a T. gondii-infection index similar to controls but a higher adhesion index; the parasite did not grow in methyl-ornithine (ODC inhibitor)-treated macrophages. The parasites were able to take up putrescine with a Km of 0.92 microM, indicating the presence of a high-affinity putrescine-transporter system. Putrescine-treated T. gondii actively penetrated macrophages and Vero cells. However, NO production and lysosomal parasitophorous vacuole fusion were not inhibited. Considered together, these results demonstrate that T. gondii requires polyamines for multiplication. However, as opposed to Trypanosoma cruzi and because of a relatively high-affinity putrescine-transporter system in the parasite, constitutive macrophage levels of putrescine seem sufficient to support T. gondii survival and multiplication.