Henry Tabel

Experimental African trypanosomiasis: IFN‐γ 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‐γ and enhanced synthesis of IL‐6, IL‐12p40 and IFN‐γ 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‐γ. 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‐γ. The role of trypanosome‐pulsed macrophages, T cells and genetic influences is discussed in a synopsis.

Susceptibility and resistance to Trypanosoma congolense infections.

We have put emphasis on recent findings in experimental Trypanosoma congolense infections in highly susceptible BALB/c and relatively resistant C57Bl/6 mice. Based on various analyses, it has been shown that a major difference in resistance to T. congolense infections is expressed early in infection at the macrophage level. A novel plastic-adherent Thy1.2(+) suppressor lymphocyte, which in absolute synergy with a Thy 1.2(-) cell exerts its suppression via interleukin-10 and interferon-gamma opens up an exciting new field of research.

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+CD25high 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+CD25high Forkhead box protein 3+ regulatory T cells in experimental infections with T. congolense.

Regulatory T Cells Prevent Control of Experimental African Trypanosomiasis

African trypanosomes are single-cell, extra-cellular blood parasites causing profound immunosuppression. Susceptible BALB/c mice infected s.c. into a footpad with 104 Trypanosoma congolense die with fulminating parasitemia within 10 days. We injected BALB/c mice 2 days before such an infection with different doses of a depleting mAb specific for CD25, a surface marker of regulatory T cells (Tregs). Pretreatment with a low, optimal dose of anti-CD25 resulted in a dramatic effect, in that the infected mice did not develop parasitemia, as well as eliminated all parasites and showed no signs of disease. Their spleens showed a 100% reduction of CD4+CD25high T cells and overall a 70% reduction of CD4+CD25+Foxp3+ T cells 7 days postinfection. The protective effect of treatment with an optimal dose of anti-CD25 could be reversed by administration of l-N6-(1-imminoethyl) lysine, a specific inhibitor of inducible NO synthase or administration of anti-CD8 Ab. Analysis of the cytokine patterns and cell surface marker in infected mice pretreated with anti-CD25 Abs pointed to a potential NKT cell response. We then conducted infections in CD1d−/− mice. From our observations, we conclude that CD4+CD25highFoxp3+ Tregs prevent, in normal infected susceptible mice, an early protective response mediated by CD8+ NKT cell-dependent activation of macrophages to kill parasites by production of NO. Our results also indicate that different populations of NKT cells have protective or suppressive effects. Our observations lead us to propose a hypothesis of cross-regulation of NKT cells and Tregs in trypanosome infections.

Intradermal Infections of Mice by Low Numbers of African Trypanosomes Are Controlled by Innate Resistance but Enhance Susceptibility to Reinfection

Antibodies are required to control blood-stage forms of African trypanosomes in humans and animals. Here, we report that intradermal infections by low numbers of African trypanosomes are controlled by innate resistance but prime the adaptive immune response to increase susceptibility to a subsequent challenge. Mice were found 100 times more resistant to intradermal infections by Trypanosoma congolense or Trypanosoma brucei than to intraperitoneal infections. B cell–deficient and RAG2−/− mice are as resistant as wild-type mice to intradermal infections, whereas inducible nitric oxide synthase (iNOS)−/− mice and wild-type mice treated with antibody to tumor necrosis factor (TNF) α are more susceptible. We conclude that primary intradermal infections with low numbers of parasites are controlled by innate defense mediated by induced nitric oxide (NO). CD1d−/− and major histocompatibility complex (MHC) class II−/− mice are more resistant than wild-type mice to primary intradermal infections. Trypanosome-specific spleen cells, as shown by cytokine production, are primed as early as 24 h after intradermal infection. Infecting mice intradermally with low numbers of parasites, or injecting them intradermally with a trypanosomal lysate, makes mice more susceptible to an intradermal challenge. We suggest that intradermal infections with low numbers of trypanosomes or injections with trypanosomal lysates prime the adaptive immune system to suppress protective immunity to an intradermal challenge.

Experimental African trypanosomiasis: differences in cytokine and nitric oxide production by macrophages from resistant and susceptible mice.

Immunosuppression in experimental infections with Trypanosoma congolense is mediated by the synergistic action of macrophages and a novel lymphocyte(s), which involves the activity of IFN-γ as well as IL-10. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant to T. congolense infections. Plasma and/or supernatants of spleen cell cultures of infected susceptible BALB/c mice have more IL-10 but less IL-12 than those of infected relatively resistant C57Bl/6 mice. Cells of a BALB/c macrophage cell line, when pulsed with T. congolense, produce more IL-10 and IL-6, but have less TNF-α mRNA, than equally treated cells of a C57Bl/6 cell line. Peritoneal and/or bone marrow-derived macrophages obtained from BALB/c mice, pulsed with T. congolense in culture, produce less nitric oxide, TNF-α and IL-12, but more IL-6 and IL-10 than equally treated macrophages isolated from C57Bl/6 mice. We suggest that genetic resistance to African trypanosomiasis is expressed at the level of the macrophage.

The alternative pathway of complement in sheep during the course of infection with Trypanosoma congolense and after Berenil treatment

Summary Experimental T. congolense infections in sheep resulted in a striking decrease in parameters of the alternative complement pathway (ACP), ie. factor B, C3 and haemolytic complement activity (HA) initiated via the ACP. The levels of factor B, C3 and HA declined before, during and after the first wave of parasitemia which reached a peak at day 8. Levels of 20 to 25% of normal values (factor B) and 20% (C3, HA) persisted throughout the course of the infection. After Berenil treatment, when no parasites were detected in blood, their serum levels remained low. They returned to normal values about 8 (factor B) to 20 days (C3, HA) after trypanocidal treatment. Serum concentrations of factor B were significantly elevated in some, but not all sheep 6 days after infection with T. congolense. The sheep were tested for their potential state of immuno‐modulation by immunization with Brucella abortus 4 days after trypanocidal treatment. In contrast to other sheep, the sheep which had shown early elevated serum factor B levels were found to express immune enhancement. It is suggested that there might be a positive correlation between the degree of enhanced serum levels of factor B at the early stage of infection and enhanced immune responsiveness.

Immunosuppression: Cause for Failures of Vaccines against African Trypanosomiases

African trypanosomes are extracellular hemoprotozoa that cause disease in humans and livestock. Trypanosoma brucei gambiense and T. b. rhodesiense cause sleeping sickness in humans, also called human African trypanosomiasis (HAT), an emerging disease in East and Central Africa [1], [2]. Infections with T. congolense, T. vivax, or T. b. brucei cause disease in livestock [1]. Various species of tsetse flies (Glossina spp.) can harbor African trypanosomes and act as their intermediate hosts. Humans and animals become infected with trypanosomes by bites of infected tsetse flies. A temporary local inflammation, the so-called chancre, develops in the skin at the site of the bite [1]. The trypanosomes move from the skin into the blood via the lymph system (Figure 1). Figure 1 Mode of natural infections by African trypanosomes. Mice are susceptible to infections by all African trypanosomes pathogenic for humans or livestock. Thus, infection of mice is a relevant model to study the immunobiology of infections by African trypanosomes. Primary intradermal infections by low numbers of parasites in the skin are controlled by innate resistance mediated by induced nitric oxide (iNO) [3]. At this stage, adaptive immune responses are not protective but are immunosuppressive [3] (discussed below). At the blood stage of infection, antibodies are absolutely required for the control of parasitemia [4]–[6]. Antibodies to the VSG control parasitemia by mediating phagocytosis of the trypanosomes by macrophages of the liver and spleen.

Innate resistance to experimental Trypanosoma congolense infection: differences in IL-10 synthesis by macrophage cell lines from resistant and susceptible inbred mice.

BALB/c and C57Bl/6 mice differ in resistance to T. congolense infections. We investigated the production of various cytokines (IL‐10, IL‐6, TNF‐α and TGF‐β) by macrophages from these mice. Macrophage cell lines (BALB.BM cells) of BALB/c mice but not (ANA‐1 cells)of C57BL/6 mice constitutively produced IL‐10. Challenge of these cells with trypanosomes induced the production of 50–100 times more IL‐10 in BALB.BM cells than in ANA‐1 cells. Pre‐incubation of the cell lines with IFN‐γ, prior to the trypanosome challenge, further upregulated this IL‐10 production in BALB.BM but not in ANA‐1 cells. Primary cultures of bone marrow‐derived macrophages (BMDM) from BALB/c mice also produced more IL‐10 following challenge with IFN‐γ and opsonized trypanosomes than did the C57Bl/6 BMDM. Similarly after challenge with trypanosomes, BALB.BM and BALB/c BMDM produced significantly more IL‐6 than did the analogous cells from the C57Bl/6 mice following such challenges. Higher steady state levels of TNF‐α mRNA accumulated in ANA‐1 cells than in BALB.BM cells following challenge with IFN‐γ and opsonized trypanosomes. Findings of this study for the first time indicate a differential regulation of cytokines (IL‐10, IL‐6 and TNF‐α) in macrophages of mice that significantly differ in their susceptibility to infections with T. congolense.

Characterization of major surface protease homologues of Trypanosoma congolense.

Trypanosomes encode a family of proteins known as Major Surface Metalloproteases (MSPs). We have identified six putative MSPs encoded within the partially sequenced T. congolense genome. Phylogenic analysis indicates that T. congolense MSPs belong to five subfamilies that are conserved among African trypanosome species. Molecular modeling, based on the known structure of Leishmania Major GP63, reveals subfamily-specific structural variations around the putative active site despite conservation of overall structure, suggesting that each MSP subfamily has evolved to recognize distinct substrates. We have cloned and purified a protein encoding the amino-terminal domain of the T. congolense homologue TcoMSP-D (most closely related to Leishmania GP63). We detect TcoMSP-D in the serum of T. congolense-infected mice. Mice immunized with the amino-terminal domain of TcoMSP-D generate a persisting IgG1 antibody response. Surprisingly, a low-dose challenge of immunized mice with T. congolense significantly increases susceptibility to infection, indicating that immunity to TcoMSP-D is a factor affecting virulence.