Macrophage activation in murine African trypanosomiasis.

African trypanosomiasis is accompanied by a profound general immunosuppression in which both suppressive T cells and macrophages (M phi) have been implicated. The present studies define changes in the M phi surface, endocytic and secretory properties, during the infection of mice by Trypanosoma brucei. Peritoneal M phi obtained after the control of the first wave of parasitemia displayed characteristics similar to those activated by intracellular pathogens, such as Mycobacterium bovis bacillus Calmette-Guérin, e.g., the enhanced expression of Ia antigen, decreased M phi-specific antigens, receptors mediating the pinocytosis of mannose-terminal glycoproteins, and an increased ability to secrete plasminogen activator, superoxide anion, and H2O2. Some markers of macrophage activation persisted during the subpatent period before the recurrence of parasitemia, whereas others reverted to normal. Mature T cell function appears not to be essential for M phi activation by T. brucei since the infection of athymic nude mice also induced Ia antigens and plasminogen activator. These studies show that M phi activated by different pathways express common features which may contribute to immune dysfunction observed in trypanosomiasis, as well as in other infections.     

In vivo analysis of impaired macrophage bactericidal capacity during experimental African trypanosomiasis

Since innate resistance of mice to Salmonella typhimurium depends on an intact macrophage system, we have used this bacterium to investigate the effect of Trypanosoma brucei subsp. rhodesiense infection on macrophage phagocytic and cytolytic function. CBA/CaJ mice infected with T. brucei subsp. rhodesiense have decreased resistance to S. typhimurium, since doubly infected mice rapidly succumb to sublethal doses of S. typhimurium. Although trypanosomiasis is known to suppress antibody formation, such a suppression of antibody does not seem to play a role in trypanosome-induced sensitivity to S. typhimurium. A trypanosome-induced blockade of the reticuloendothelial system also does not occur, since parasitized and control mice clear S. typhimurium from the blood equally well. Early killing (0 to 48 h) of S. typhimurium in the liver and spleen is mainly macrophage mediated, and mice infected with trypanosomes kill S. typhimurium in the liver and spleen very poorly. Apparently trypanosomiasis inhibits macrophage bactericidal activity, but has no effect on phagocytosis.     

Alternative versus classical macrophage activation during experimental African trypanosomosis

The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e., classically activated macrophages (caMphi, type I) versus alternatively activated macrophages (aaMphi, type II). Recently, we showed that although mice infected with phospholipase C-deficient (PLC-/-) Trypanosoma brucei brucei exhibit a clear shift from type I to the type II cytokine production, wild type (WT)-infected mice remain locked in a type I cytokine response. In the present study, phenotype and accessory cell function of macrophages elicited during WT and PLC-/- T. b. brucei infection were compared. Results indicate that caMphi develop in a type I cytokine environment in the early phase of WT and PLC-/- trypanosome infection, correlating with inhibition of T cell activation triggered by a mitogen, a superantigen, or an antigen. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated aaMphi correlating with impaired antigen- but not mitogen- or superantigen-induced T cell activation.     

Hypocretin and human African trypanosomiasis

OBJECTIVES: To detail clinical and polysomnographic characteristics in patients affected with Trypanosoma brucei gambiense (Tb.g.) human African trypanosomiasis (HAT) at different stages of evolution and to measure and compare cerebrospinal fluid (CSF) levels of hypocretin-1 with narcoleptic patients and neurologic controls. METHODS: Twenty-five untreated patients affected with T.b.g. HAT were included. The patients were evaluated using a standardized clinical evaluation and a specific interview on sleep complaints. Diagnosis of stages I and II and intermediate stage was performed by CSF cell count and/or presence of trypanosomes: 4 patients were classified as stage II, 13 stage I, and 8 "intermediate" stage. Seventeen untreated patients completed continuous 24-hour polysomnography. We measured CSF levels of hypocretin-1 in all patients at different stages and evolutions, and we compared the results with 26 patients with narcolepsy-cataplexy and 53 neurologic controls. RESULTS: CSF hypocretin-1 levels were significantly higher in T.b.g. HAT (423.2 +/- 119.7 pg/mL) than in narcoleptic patients (40.16 +/- 60.18 pg/ mL) but lower than in neurologic controls (517.32 +/- 194.5 pg/mL). One stage I patient had undetectable hypocretin levels and 1 stage II patient showed intermediate levels, both patients (out of three patients) reporting excessive daytime sleepiness but without evidence for an association with narcolepsy. No differences were found in CSF hypocretin levels between patients with HAT stages; however, the presence of major sleep-wake cycle disruptions was significantly associated with lower CSF hypocretin-1 level with a same tendency for the number of sleep-onset rapid eye movement periods. CONCLUSION: The present investigation is not in favor of a unique implication of the hypocretin system in T.b.g. HAT. However, we propose that dysfunction of the hypothalamic hypocretin region may participate in sleep disturbances observed in African trypanosomiasis.     

Suppressor factor of T-cell activation and decreased interleukin 2 activity in experimental African trypanosomiasis.

Spleen cells from Trypanosoma brucei-infected BALB/c mice were unable to respond to a T-cell mitogen, concanavalin A. Moreover, they were unable to produce detectable amounts of the growth factor required for T cell proliferation, interleukin 2. In addition, supernatants from 24-h in vitro cultures of these cells produced a slight but detectable suppressive activity of the interleukin 2-dependent proliferation of a T-cell line. Infected spleen cells also suppressed the response of T. brucei-immunized spleen cells as well as normal spleen cells to concanavalin A. However, a major difference was shown in the mechanism of the suppression in both systems. Suppression of normal spleen cells required cell-to-cell contact. In contrast, suppression of 30-day T. brucei-immune cells could be mediated by a soluble suppressor factor released by in vitro culture of infected spleen cells. This molecule had an apparent molecular weight of 18,000. Finally, similar suppression could be generated in 30-day T. brucei-immune spleen cells but not in normal cells, with living cells but not with extracts of T. brucei.     

African human trypanosomiasis. Therapeutic strategies

Although therapeutic strategies for trypanosomiasis appear to be straightforward, their application in the field raises a number of questions which are successively examined below. After a quick reminder of the available drugs, we discuss: Criteria for eligibility: problems of beginning treatment for immunological suspects and determination of the development phase. Present-day problems: availability of drugs; side effects; therapeutic failures. While efforts should be continued to make existing drugs available, it is important that fundamental research be carried out to enhance their utilisation as well as to develop new active molecules.     

Immunosuppression in experimental African trypanosomiasis. Polyclonal B-cell activation and mitogenicity of trypanosome-derived saturated fatty acids.

Changes in antibody responses in adult mice infected with Trypanosoma congolense and subsequently challenged with unrelated antigens (sheep red blood cells and pneumococcal polysaccharide) were studied. Immune responses were significantly depressed within 1 week of infection, and complete suppression of both IgM and IgG responses to both types of antigen was established 15 days after immunization. Coincidentally with the development of high parasitaemias, background IgM plaque-forming cell responses to sheep red cell antigen significantly increased in non-immunized T. congolense-infected animals. Autolysates of T. congolense and chloroform-soluble extracts of the autolyzed trypanosome were found to be mitogenic in vitro for the spleen cells of normal mice. Fractionation of these extracts by thin-layer chromatography indicated that the mitogenic activity migrated with the free fatty acids. Substitution of the relevant saturated and unsaturated free fatty acids in the autolyzed trypanosome extracts with commercial pure fatty acids in the mouse spleen cultures indicated that the mitogenicity was due to palmitic and stearic acids. It is suggested that the general immunosuppressing effect of trypanosomes may be attributed, at least in part, to the polyclonal activation, and subsequent depletion and/or clonal exhaustion of B-cells as a result of blastogenic stimulus from the parasites. This may operate, at least in part, through the generation of B-cell mitogenic saturated fatty acids.     

T cells and immunopathogenesis of experimental African trypanosomiasis

Summary: African trypanosomes are pathogens for humans and livestock. They are single-cell, extra-cellular parasites that cause persistent infections of the blood and induce profound immunosuppression. Here, we review recent work on experimental African trypanosomiasis, especially infections with Trypanosoma congolense, in mice with regard to mechanisms of immunosuppression and immunopathology. The center of the immunopathology is the T-cell-independent production of antibodies to the variant surface glycoprotein (VSG) of trypanosomes, the anti-VSG antibody-mediated phagocytosis of trypanosomes by macrophages, and the subsequent profound dysregulation of the macrophage system. Depending on the genetics of the host and the parasite load, the malfunction of the macrophage system is enhanced by interferon-γ produced by parasite-specific, major histocompatibility complex class II-restricted, matrix-adherent CD4⁺ T cells or downregulated by interleuin-10 produced by parasite-specific, CD4⁺CD25^high Forkhead box protein 3⁺ regulatory T cells. There is a physiological conflict of the two relevant cytokines interleukin-10 and interferon-γ in regulating the immunopathology versus regulating the induction and effect of protective immune responses. On the basis of very recent work in our laboratory, we propose a hypothetical model suggesting a cross-regulation of natural killer T cells and CD4⁺CD25^high Forkhead box protein 3⁺ regulatory T cells in experimental infections with T. congolense.     

Production of interferons during experimental African trypanosomiasis.

African trypanosomiasis is associated with profound changes in the function of the immune system. In this study we find that alpha/beta and gamma interferon (IFN) are released into the serum of mice infected with Trypanosoma brucei. The parasite-induced rise in serum IFNs is associated with a detectable parasitaemia, but the serum IFN peak precedes the peak parasitaemia in some cases. Unlike other protozoan interferon inducers, no parasite-dependent IFN production was observed in the pre-patent period of infection; while the most virulent clone induced very high IFN levels, no clear difference in stimulation was noted in the first waves of semi-acute and chronic T. brucei clones. However, subsequent IFN augmentation more closely reflected the host parasite load and virulence of infection. The nature of the stimulatory parasite component is as yet unknown, and the parasite surface glycoprotein had no effect on serum IFN. Injection of large quantities of lethally irradiated, but intact organisms did not stimulate IFN production; however this treatment significantly impaired antibody responses to the heterologous antigen SRBC. This suggests that the more severe effects of an actual trypanosome infection are required for induction of IFN synthesis, and that the presence of measurable serum IFN is not a prerequisite for parasite-induced suppression of host antibody responses.     

Human African trypanosomiasis: present and future treatment

During his life General Lapeyssonnie coped with the hazards linked to the therapeutics of the human African trypanosomiasis (HAT), sometimes with passion and disappointment, sometimes with revolt and hope. Because of a lack of political and financial concern during the past decades, a real global policy against the disease and a drug research against HAT didn't emerge. Today, some changes seem to take place. They are the result of the frightening spread of the disease and of the moral obligation that forces pharmaceutical companies to intervene. Drug research needs to be increased. New drugs should present no toxicity and should be able to cross through the blood-brain barrier with efficient cerebrospinal fluid concentrations. Moreover, new drugs should be easy to synthesize, easy to use and at a low cost. Today, megazol is the only one product in preclinical development, which seems to reach each of these goals.     

Experimental African trypanosomiasis: IFN-gamma mediates early mortality

In this study, we demonstrate that Kupffer cells in the livers of highly susceptible BALB/c mice infected with Trypanosoma congolense were loaded with trypanosomal antigen and appeared highly activated. This was associated with an enlarged capillary bed in the livers and decreased blood pressure of these mice towards the terminal stage. Blocking of murine IL-10 receptor (IL-10R)in vivo shortened the survival time of highly susceptible T. congolense-infected BALB/c mice. Anti-IL-10R treatment decreased the survival of relatively resistant T. congolense-infected C57BL/6 mice dramatically. Blocking of the IL-10R also significantly shortened the survival time of mice infected with T. brucei. The acute death of trypanosome-infected mice treated with anti-IL-10R antibodies in vivo was associated with focal liver necrosis, with significantly increased plasma levels of IL-6, IL-10, IL-12p40 and IFN-gamma and enhanced synthesis of IL-6, IL-12p40 and IFN-gamma by spleen cell cultures. Anti-IL-10R-induced death of T. congolense-infected C57BL/6 mice could be prevented by administration of a neutralizing antibody specific forIFN-gamma. We conclude that phagocytosis of a critical number of trypanosomes by Kupffer cells leads to a systemic inflammatory response syndrome and, depending on the degree of Kupffer cell activation, is followed by death that is mediated by IFN-gamma. The role of trypanosome-pulsed macrophages, T cells and genetic influences is discussed in a synopsis.     

Trypanosome-induced suppression of anti-parasite responses during experimental African trypanosomiasis

The effect of Trypanosoma brucei infections on parasite-specific antibody responses was examined by immunization of infected mice with radio-attenuated trypanosomes of a noncross-reacting clone. Antibody liters (total Ig) against variant surface antigen were determined by indirect immunofluorescence. The suppression resulting from acute infection was virtually complete, and is associated with the failure to control the first relapse variant of the acute clone. The suppression resulting from chronic infection was much less severe, and subsequently variant populations of the chronic clone are successfully controlled. Antibody collected 10 days after each of the first 3 peaks of parasitemia during chronic infection was titrated on the homologous parasite preparations isolated from each wave in individual mice. As the infection progressed, both IgG and IgM antibody responses to each successive wave declined. However, the decline of IgG antibody was more rapid than IgM, so that by the third parasite wave the mice were responding with predominantly, if not exclusively, low levels of IgM antibody. We conclude that the severity of trypanosome-induced suppression of the anti-parasite response and IgM response in particular, determines the course of infection by trypanosomes varying in virulence.     

Gene expression profiling in a mouse model for African trypanosomiasis

This study aimed to provide the foundation for an integrative approach to the identification of the mechanisms underlying the response to infection with Trypanosoma congolense, and to identify pathways that have previously been overlooked. We undertook a large-scale gene expression analysis study comparing susceptible A/J and more tolerant C57BL/6 mice. In an initial time course experiment, we monitored the development of parasitaemia and anaemia in every individual. Based on the kinetics of disease progression, we extracted total RNA from liver at days 0, 4, 7, 10 and 17 post infection and performed a microarray analysis. We identified 64 genes that were differentially expressed in the two strains in non-infected animals, of which nine genes remained largely unaffected by the disease. Gene expression profiling at stages of low, peak, clearance and recurrence of parasitaemia suggest that susceptibility is associated with high expression of genes coding for chemokines (e.g. Ccl24, Ccl27 and Cxcl13), complement components (C1q and C3) and interferon receptor alpha (Ifnar1). Additionally, susceptible A/J mice expressed higher levels of some potassium channel genes. In contrast, messenger RNA levels of a few immune response, metabolism and protease genes (e.g. Prss7 and Mmp13) were higher in the tolerant C57BL/6 strain as compared to A/J.     

Echinacea-induced macrophage activation

Public interest in Echinacea is growing rapidly. Unfortunately, there is little scientific evidence to support claims of efficacy of this widely used botanical, and little information about potential mechanism of action. This study examines the ability of Echinacea to upregulate macrophage function and begins to elucidate the mechanism of Echinacea-induced macrophage activation. Murine peritoneal macrophages were cultured with E. purpurea extracts enriched for plant polysaccharide (EP). ELISA was used to measure cytokine production. MAPKs were blocked using specific inhibitors, and Western blotting used to identify phosphorylated proteins involved in signal transduction. To examine in vivo efficacy, EP was administered orally and Listeria monocytogenes given i.v. Mice were sacrificed three days post-infection to determine bacterial load in the spleen. We demonstrate that an endotoxin-free EP extract activates the innate immune response, stimulating production of IL-6, TNF, IL-12, and NO from macrophages in vitro. Along with evidence of enhanced macrophage function, we found that oral EP reduces bacterial burden during infection by Listeria monocytogenes, demonstrating its efficacy in vivo. EP initiates a signaling cascade within macrophages through both TLR4-dependent and -independent mechanisms, involving ERK, p38 and JNK, and ultimately the activation of NF-kappaB.     

Analysis of interferon-gamma-dependent and -independent pathways of macrophage activation

Macrophages are a cellular cornerstone of the innate immune response. The outcome of macrophage activity during development of an immune response to microbes results from macrophage activation by both organism-derived and host-derived factors. In order to more fully understand the spectrum of responses expressed by macrophages when encountering these distinct stimuli, we investigated the similarities and differences between interferon-gamma receptor (IFN-gammaR)-dependent macrophage activation and stimulation of macrophages through the Type A1 scavenger receptor (SR). We observed distinct patterns of macrophage activation depending on the nature of the ligand. IFN-gamma and the SR ligand lipotechoic acid (LTA) induced largely non-overlapping sets of genes. The use of two additional SR ligands, maleylated bovine serum albumin and the polydeoxynucleotide poly dI:dC, revealed differences within SR activation-induced gene expression. We also observed that priming with IFN-gamma resulted in an enhanced response to subsequent SR-mediated activation. These results suggest that full potentiation of macrophage activity during development of an antimicrobial immune response is achieved by activation of these cells through multiple receptors.