IL-27 Negatively Regulates Tip-DC Development during Infection.

Tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cells (Tip-DCs) have profound impacts on host immune responses during infections. The mechanisms regulating Tip-DC development remain largely unknown. Here, using a mouse model of infection with African trypanosomes, we show that a deficiency in interleukin-27 receptor (IL-27R) signaling results in escalated intrahepatic accumulation of Ly6C-positive (Ly6C+) monocytes and their differentiation into Tip-DCs. Blocking Tip-DC development significantly ameliorates liver injury and increases the survival of infected IL-27R-/- mice. Mechanistically, Ly6C+ monocyte differentiation into pathogenic Tip-DCs in infected IL-27R-/- mice is driven by a CD4+ T cell-interferon gamma (IFN-γ) axis via cell-intrinsic IFN-γ signaling. In parallel, hyperactive IFN-γ signaling induces cell death of Ly6C-negative (Ly6C-) monocytes in a cell-intrinsic manner, which in turn aggravates the development of pathogenic Tip-DCs due to the loss of the negative regulation of Ly6C- monocytes on Ly6C+ monocyte differentiation into Tip-DCs. Thus, IL-27 inhibits the dual-track exacerbation of Tip-DC development induced by a CD4+ T cell-IFN-γ axis. We conclude that IL-27 negatively regulates Tip-DC development by preventing the cell-intrinsic effects of IFN-γ and that the regulation involves CD4+ T cells and Ly6C- monocytes. Targeting IL-27 signaling may manipulate Tip-DC development for therapeutic intervention.IMPORTANCE TNF/iNOS-producing dendritic cells (Tip-DCs) are at the front line as immune effector cells to fight off a broad range of invading microbes. Excessive development of Tip-DCs contributes to tissue destruction. Thus, identifying master regulators of Tip-DC development is fundamental for developing new therapeutic strategies. Here, we identify Tip-DCs as a terminal target of IL-27, which prevents Tip-DC-mediated early mortality during parasitic infections. We demonstrate that IL-27 inhibits Tip-DC development via a dual-track mechanism involving the complex interactions of effector CD4+ T cells, Ly6C- monocytes, and Ly6C+ monocytes. These findings delineate an in-depth view of mechanisms of Tip-DC differentiation that may have significant implications for the ongoing development of IL-27-based immunotherapy.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.

TLR-2 and MyD88-Dependent Activation of MAPK and STAT Proteins Regulates Proinflammatory Cytokine Response and Immunity to Experimental Trypanosoma congolense Infection.

It is known that Trypanosoma congolense infection in mice is associated with increased production of proinflammatory cytokines by macrophages and monocytes. However, the intracellular signaling pathways leading to the production of these cytokines still remain unknown. In this paper, we have investigated the innate receptors and intracellular signaling pathways that are associated with T. congolense-induced proinflammatory cytokine production in macrophages. We show that the production of IL-6, IL-12, and TNF-α by macrophages in vitro and in vivo following interaction with T. congolense is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, JNK, and signal transducer and activation of transcription (STAT) proteins. Specific inhibition of MAPKs and STATs signaling pathways significantly inhibited T. congolense-induced production of proinflammatory cytokines in macrophages. We further show that T. congolense-induced proinflammatory cytokine production in macrophages is mediated via Toll-like receptor 2 (TLR2) and involves the adaptor molecule, MyD88. Deficiency of MyD88 and TLR2 leads to impaired cytokine production by macrophages in vitro and acute death of T. congolense-infected relatively resistant mice. Collectively, our results provide insight into T. congolense-induced activation of the immune system that leads to the production of proinflammatory cytokines and resistance to the infection.

Host Immune Responses and Immune Evasion Strategies in African Trypanosomiasis.

Parasites, including African trypanosomes, utilize several immune evasion strategies to ensure their survival and completion of their life cycles within their hosts. The defense factors activated by the host to resolve inflammation and restore homeostasis during active infection could be exploited and/or manipulated by the parasites in an attempt to ensure their survival and propagation. This often results in the parasites evading the host immune responses as well as the host sustaining some self-inflicted collateral tissue damage. During infection with African trypanosomes, both effector and suppressor cells are activated and the balance between these opposing arms of immunity determines susceptibility or resistance of infected host to the parasites. Immune evasion by the parasites could be directly related to parasite factors, (e.g., antigenic variation), or indirectly through the induction of suppressor cells following infection. Several cell types, including suppressive macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells have been shown to contribute to immunosuppression in African trypanosomiasis. In this review, we discuss the key factors that contribute to immunity and immunosuppression during T. congolense infection, and how these factors could aid immune evasion by African trypanosomes. Understanding the regulatory mechanisms that influence resistance and/or susceptibility during African trypanosomiasis could be beneficial in designing effective vaccination and therapeutic strategies against the disease.

Myeloid-Derived Suppressor Cells Contribute to Susceptibility to Trypanosoma congolense Infection by Suppressing CD4+ T Cell Proliferation and IFN-γ Production.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during Trypanosoma congolense infection has not been studied. Given that immunosuppression is a hallmark of African trypanosomiasis, we investigated the role of MDSCs in immunity to T. congolense infection. We found increased numbers of MDSCs in the spleen and liver of infected mice, which correlated with increased parasitemia. Depletion of MDSCs significantly increased the percentage of proliferating and IFN-γ-producing CD4+ T cells from the spleen of T. congolense-infected mice. Furthermore, MDSCs from T. congolense-infected mice directly suppressed CD4+ T cell proliferation in a coculture setting. This suppressive effect was abolished by the arginase-1 inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), indicating that MDSCs suppress CD4+ T cell proliferation and function in an arginase-1-dependent manner. Indeed, depletion of MDSCs during infection led to control of the first wave of parasitemia and prolonged survival of infected mice. This was also associated with increased CD4+ T cell proliferation and IFN-γ production. Taken together, our findings identify an important role of MDSCs in the pathogenesis of experimental T. congolense infection via suppression of T cell proliferative and effector cytokine responses in an arginase-1-dependent manner.

Ly6C- Monocytes Regulate Parasite-Induced Liver Inflammation by Inducing the Differentiation of Pathogenic Ly6C+ Monocytes into Macrophages.

Monocytes consist of two well-defined subsets, the Ly6C+ and Ly6C- monocytes. Both CD11b+ myeloid cells populations have been proposed to infiltrate tissues during inflammation. While infiltration of Ly6C+ monocytes is an established pathogenic factor during hepatic inflammation, the role of Ly6C- monocytes remains elusive. Mice suffering experimental African trypanosome infection die from systemic inflammatory response syndrome (SIRS) that is initiated by phagocytosis of parasites by liver myeloid cells and culminates in apoptosis/necrosis of liver myeloid and parenchymal cells that reduces host survival. C57BL/6 mice are considered as trypanotolerant to Trypanosoma congolense infection. We have reported that in these animals, IL-10, produced among others by myeloid cells, limits the liver damage caused by pathogenic TNF-producing Ly6C+ monocytes, ensuring prolonged survival. Here, the heterogeneity and dynamics of liver myeloid cells in T. congolense-infected C57/BL6 mice was further dissected. Moreover, the contribution of Ly6C- monocytes to trypanotolerance was investigated. By using FACS analysis and adoptive transfer experiments, we found that the accumulation of Ly6C- monocytes and macrophages in the liver of infected mice coincided with a drop in the pool of Ly6C+ monocytes. Pathogenic TNF mainly originated from Ly6C+ monocytes while Ly6C- monocytes and macrophages were major and equipotent sources of IL-10 within myeloid cells. Moreover, Nr4a1 (Nur77) transcription factor-dependent Ly6C- monocytes exhibited IL-10-dependent and cell contact-dependent regulatory properties contributing to trypanotolerance by suppressing the production of TNF by Ly6C+ monocytes and by promoting the differentiation of the latter cells into macrophages. Thus, Ly6C- monocytes can dampen liver damage caused by an extensive Ly6C+ monocyte-associated inflammatory immune response in T. congolense trypanotolerant animals. In a more general context, Ly6C- or Ly6C+ monocyte targeting may represent a therapeutic approach in liver pathogenicity induced by chronic infection.

Chronic Trypanosoma congolense infections in mice cause a sustained disruption of the B-cell homeostasis in the bone marrow and spleen.

Trypanosoma congolense is one of the main species responsible for Animal African Trypanosomosis (AAT). As preventive vaccination strategies for AAT have been unsuccessful so far, investigating the mechanisms underlying vaccine failure has to be prioritized. In T. brucei and T. vivax infections, recent studies revealed a rapid onset of destruction of the host B-cell compartment, resulting in the loss of memory recall capacity. To assess such effect in experimental T. congolense trypanosomosis, we performed infections with both the cloned Tc13 parasite, which is considered as a standard model system for T. congolense rodent infections and the noncloned TRT55 field isolate. These infections differ in their virulence level in the C57BL/6 mouse model for trypanosomosis. We show that early on, an irreversible depletion of all developmental B cells stages occur. Subsequently, in the spleen, a detrimental decrease in immature B cells is followed by a significant and permanent depletion of Marginal zone B cells and Follicular B cells. The severity of these events later on in infection correlated with the virulence level of the parasite stock. In line with this, it was observed that later-stage infection-induced IgGs were largely nonspecific, in particular in the more virulent TRT55 infection model.

Low-dose intradermal infection with trypanosoma congolense leads to expansion of regulatory T cells and enhanced susceptibility to reinfection.

BALB/c mice are highly susceptible to experimental intraperitoneal Trypanosoma congolense infection. However, a recent report showed that these mice are relatively resistant to primary intradermal low-dose infection. Paradoxically, repeated low-dose intradermal infections predispose mice to enhanced susceptibility to an otherwise noninfectious dose challenge. Here, we explored the mechanisms responsible for this low-dose-induced susceptibility to subsequent low-dose challenge infection. We found that akin to intraperitoneal infection, low-dose intradermal infection led to production of interleukin-10 (IL-10), IL-6, IL-12, tumor necrosis factor alpha (TNF-α), transforming growth factor ß (TGF-ß), and gamma interferon (IFN-γ) by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low-dose intradermal infection led to expansion of CD4+ CD25+ Foxp3+ cells (T regulatory cells [Tregs]) in both the spleens and lymph nodes draining the infection site. Depletion of Tregs by anti-CD25 monoclonal antibody (MAb) treatment during primary infection or before challenge infection following repeated low-dose infection completely abolished the low-dose-induced enhanced susceptibility. In addition, Treg depletion was associated with dramatic reduction in serum levels of TGF-ß and IL-10. Collectively, these findings show that low-dose intradermal infection leads to rapid expansion of Tregs, and these cells mediate enhanced susceptibility to subsequent infection.

Molecular regulation of Trypanosoma congolense-induced nitric oxide production in macrophages.

BALB/c mice are highly susceptible while C57BL/6 mice are relatively resistant to experimental Trypanosoma congolense infection. Several reports show that an early interferon-gamma (IFN-γ) response in infected mice is critically important for resistance via the activation of macrophages and production of nitric oxide (NO). NO is a pivotal effector molecule and possesses both cytostatic and cytolytic properties for the parasite. However, the molecular mechanisms leading to T. congolense (TC)-induced NO release from macrophages are not known. In this study, we investigated the signaling pathways induced by trypanosomes in immortalized macrophage cell lines from the highly susceptible BALB/c (BALB.BM) and relatively resistant C57Bl/6 (ANA-1) mice. We found that T. congolense whole cell extract (TC-WCE) induces significantly higher levels of NO production in IFN-γ-primed ANA-1 than BALB.BM cells, which was further confirmed in primary bone marrow-derived macrophage (BMDM) cultures. NO production was dependent on mitogen-activated protein kinase (MAPK, including p38, Erk1/2, and JNK) phosphorylation and was significantly inhibited by specific MAPK inhibitors in BALB.BM, but not in ANA-1 cells. In addition, T. congolense- and IFN-γ-induced NO production in ANA-1 and BALB.BM cells was dependent on STAT1 phosphorylation and was totally suppressed by the use of fludarabine (a specific STAT1 inhibitor). We further show that T. congolense induces differential iNOS transcriptional promoter activation in IFN-γ-primed cells, which is dependent on the activation of both GAS1 and GAS2 transcription factors in BALB.BM but only on GAS1 in ANA-1 cells. Taken together, our findings show the existence of differential signalling events that lead to NO production in macrophages from the highly susceptible and relatively resistant mice following treatment with IFN-γ and T. congolense. Understanding these pathways may help identify immunomodulatory mechanisms that regulate the outcome of infection during Trypanosome infections.

The CD20 homolog Ms4a8a integrates pro- and anti-inflammatory signals in novel M2-like macrophages and is expressed in parasite infection.

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.

Effect of adjuvants on the humoral immune response to congopain in mice and cattle.

BACKGROUND: We investigated several adjuvants for their effects on the humoral immune response in both mice and cattle using the central domain of congopain (C2), the major cysteine protease of Trypanosoma congolense, as a model for developing a vaccine against animal trypanosomosis. The magnitude and sustainability of the immune response against C2 and the occurrence of a booster effect of infection, an indirect measure of the presence of memory cells, were determined by ELISA, while spectrofluorometry was used to determine and measure the presence of enzyme-inhibiting antibodies. RESULTS: Mice immunized with recombinant C2 in TiterMax™, Adjuphos™, purified saponin Quil A™ or Gerbu™ showed the best response according to the evaluation criteria and the latter three were chosen for the cattle vaccination study. The cattle were challenged with T. congolense four and a half months after the last booster. Cattle immunized with recombinant C2 in purified saponin Quil A™ showed the best antibody response according to the measured parameters. CONCLUSIONS: We identified purified saponin Quil A™ as a good adjuvant for immunizations with C2. The results from this study will be useful in future attempts to develop an effective anti-disease vaccine against African trypanosomosis.

IL-10 limits production of pathogenic TNF by M1 myeloid cells through induction of nuclear NF-κB p50 member in Trypanosoma congolense infection-resistant C57BL/6 mice.

A balance between parasite elimination and control of infection-associated pathogenicity is crucial for resistance to African trypanosomiasis. By producing TNF and NO, CD11b(+) myeloid cells with a classical activation status (M1) contribute to parasitemia control in experimental Trypanosoma congolense infection in resistant C57BL/6 mice. However, in these mice, IL-10 is required to regulate M1-associated inflammation, avoiding tissue/liver damage and ensuring prolonged survival. In an effort to dissect the mechanisms behind the anti-inflammatory activity of IL-10 in T. congolense-infected C57BL/6 mice, we show, using an antibody blocking the IL-10 receptor, that IL-10 impairs the accumulation and M1 activation of TNF/iNOS-producing CD11b(+) Ly6C(+) cells in the liver. Using infected IL-10(flox/flox) LysM-Cre(+/+) mice, we show that myeloid cell-derived IL-10 limits M1 activation of CD11b(+) Ly6C(+) cells specifically at the level of TNF production. Moreover, higher production of TNF in infected IL-10(flox/flox) LysM-Cre(+/+) mice is associated with reduced nuclear accumulation of the NF-κB p50 subunit in CD11b(+) M1 cells. Furthermore, in infected p50(-/-) mice, TNF production by CD11b(+) Ly6C(+) cells and liver injury increases. These data suggest that preferential nuclear accumulation of p50 represents an IL-10-dependent anti-inflammatory mechanism in M1-type CD11b(+) myeloid cells that regulates the production of pathogenic TNF during T. congolense infection in resistant C57BL/6 mice.

Production of congopain, the major cysteine protease of Trypanosoma (Nannomonas) congolense, in Pichia pastoris reveals unexpected dimerisation at physiological pH.

African animal trypanosomosis (nagana) is arguably the most important parasitic disease affecting livestock in sub-Saharan Africa. Since none of the existing control measures are entirely satisfactory, vaccine development is being actively pursued. However, due to antigenic variation, the quest for a conventional vaccine has proven elusive. As a result, we have sought an alternative 'anti-disease vaccine approach', based on congopain, a cysteine protease of Trypanosoma congolense, which was shown to have pathogenic effects in vivo. Congopain was initially expressed as a recombinant protein in bacterial and baculovirus expression systems, but both the folding and yield obtained proved inadequate. Hence alternative expression systems were investigated, amongst which Pichia pastoris proved to be the most suitable. We report here the expression of full length, and C-terminal domain-truncated congopain in the methylotrophic yeast P. pastoris. Differences in yield were observed between full length and truncated proteins, the full length producing 2-4 mg of protein per litre of culture, while the truncated form produced 20-30 mg/l. The protease was produced as a proenzyme, but underwent spontaneous activation when acidified (pH <5). To investigate whether this activation was due to autolysis, we produced an inactive mutant (active site Cys→Ala) by site-directed mutagenesis. The mutant form was produced at a much higher rate, up to 100mg/l culture, as a proenzyme. It did not undergo spontaneous cleavage of the propeptide when subjected to acidic pH suggesting an autocatalytic process of activation for congopain. These recombinant proteins displayed a very unusual feature for cathepsin L-like proteinases, i.e. complete dimerisation at pH >6, and by reversibly monomerising at acidic pH <5. This attribute is of utmost importance in the context of an anti-disease vaccine, given that the epitopes recognised by the sera of trypanosome-infected trypanotolerant cattle appear dimer-specific.

Altered proinflammatory cytokine production and enhanced resistance to Trypanosoma congolense infection in lymphotoxin beta-deficient mice.

BALB/c mice are highly susceptible to Trypanosoma congolense infection, whereas C57BL/6 mice are relatively resistant. Overproduction of interferon-gamma (IFN-gamma) and other proinflammatory cytokines contribute to death in susceptible mice. Here, we show that lymphotoxin beta-deficient (LTbeta(-/-)) mice are more resistant than wild-type (WT) mice to T. congolense infection, as shown by a lower parasitemia level and a longer survival duration. The enhanced resistance of LTbeta(-/-) mice was associated with undetectable or low serum levels of proinflammatory cytokines (i.e., tumor necrosis factor-alpha, interleukin [IL]-6, IL-12, and monocyte chemotactic protein-1). Although infected LTbeta(-/-) mice had high numbers of CD4(+)CD25(+)Foxp3(+) cells and high serum IL-10 levels, these cells were not the major producers of IL-10. Treatment of LTbeta(-/-) mice with anti-IL-10R monoclonal antibody abolished their enhanced resistance, whereas depletion of CD25(+) cells further enhanced resistance among infected WT and LTbeta(-/-) mice. These results suggest that LTbeta plays critical role in regulating the outcome of T. congolense infection in mice.

Modulation of the immunogenicity of the Trypanosoma congolense cysteine protease, congopain, through complexation with alpha(2)-macroglobulin.

The protozoan parasite Trypanosoma congolense is the main causative agent of livestock trypanosomosis. Congopain, the major lysosomal cysteine proteinase of T. congolense, contributes to disease pathogenesis, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. The potential of different adjuvants to facilitate the production of antibodies that would inhibit congopain activity was evaluated in the present study. Rabbits were immunised with the recombinant catalytic domain of congopain (C2), either without adjuvant, with Freund's adjuvant or complexed with bovine or rabbit alpha(2)-macroglobulin (alpha(2)M). The antibodies were assessed for inhibition of congopain activity. Rabbits immunised with C2 alone produced barely detectable anti-C2 antibody levels and these antibodies had no effect on recombinant C2 or native congopain activity. Rabbits immunised with C2 and Freund's adjuvant produced the highest levels of anti-C2 antibodies. These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation. Rabbits receiving C2-alpha(2)M complexes produced moderate levels of anti-C2 antibodies and these antibodies consistently showed the best inhibition of C2 and native congopain activity of all the antibodies, with maximum inhibition of 65%. Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex. This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

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-gamma 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-gamma 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.

Molecular and biochemical characterization of a cathepsin B-like protease family unique to Trypanosoma congolense.

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.

Tumor necrosis factor (TNF) receptor-1 (TNFp55) signal transduction and macrophage-derived soluble TNF are crucial for nitric oxide-mediated Trypanosoma congolense parasite killing.

Control of Trypanosoma congolense infections requires an early cell-mediated immune response. To unravel the role of tumor necrosis factor (TNF) in this process, 6 different T. congolense strains were used in 6 different gene-deficient mouse models that included TNF(-/-), TNF receptor-1 (TNFp55)(-/-), and TNF receptor-2 (TNFp75)(-/-) mice, 2 cell type-specific TNF(-/-) mice, as well as TNF-knock-in mice that expressed only membrane-bound TNF. Our results indicate that soluble TNF produced by macrophages/neutrophils and TNFp55 signaling are essential and sufficient to control parasitemia. The downstream mechanism in the control of T. congolense infection depends on inducible nitric oxide synthase activation in the liver. Such a role for nitric oxide is corroborated ex vivo, because the inhibitor N(G)-monomethyl-l-arginine blocks the trypanolytic activity of the adherent liver cell population, whereas exogenous interferon- gamma that stimulates nitric oxide production enhances parasite killing. In conclusion, the control of T. congolense infection depends on macrophage/neutrophil-derived soluble TNF and intact TNFp55 signaling, which induces trypanolytic nitric oxide.

Cytokine mRNA profiles in bovine macrophages stimulated with Trypanosoma congolense.

It is known that different breeds of cattle display differential susceptibilities to Trypanosome congolense infections, and that N'Dama cattle remain more productive after infection than Boran cattle which are more susceptible to T. congolense. Macrophages from both breeds were cultured in vitro and the expressions of a number of cytokines and iNOS mRNA were analyzed using real time RT-PCR after stimulation with antibody-opsonized trypanosomes. No significant difference was seen between the responses of the two breeds. However, RNA levels of TNF-alpha in the IFN-gamma-primed macrophages were about 100-fold higher than those in the non-primed macrophages. A significant ten-fold decrease was seen for the anti-inflammatory cytokine IL-10. These results indicate that priming of the cells with IFN-gamma cause a serious shift toward an inflammatory response.

Interferon-gamma and nitric oxide in combination with antibodies are key protective host immune factors during trypanosoma congolense Tc13 Infections.

The control of chronic Trypanosoma congolense trypanosomiasis was analyzed using several gene-deficient mouse strains. First, interferon (IFN)-gamma receptor (IFN-gamma-R)-deficient mice were used to show that IFN- gamma -mediated immune activation is crucial for parasitemia control. Second, infections in major histocompatibility complex (MHC) class II-deficient mice indicate that this molecule is needed for initiation of IFN- gamma and subsequent tumor necrosis factor (TNF) production. Downstream of IFN-gamma-R signaling, inducible NO synthase (iNOS)-dependent trypanosome killing occurs, as is shown by the hypersusceptible phenotype of iNOS-deficient mice. Besides proinflammatory responses, B cells and, more specifically, immunoglobulin (Ig) G antibodies are crucial for parasite killing. Hence, parasitemia control is abolished in B cell-deficient mice, whereas IgM-deficient mice control the infection as efficiently as do wild-type mice. In addition, splenectomized mice that have a normal IgM response but an impaired IgG2a/3 response fail to control T. congolense infection. Collectively, these results suggest that host protective immunity against T. congolense is critically dependent on the combined action of the proinflammatory mediators/effectors IFN- gamma , TNF, and NO and antiparasite IgGs.