The initial host response toward the extracellular parasite Trypanosoma brucei is characterized by the early release of inflammatory mediators associated with a type 1 immune response. In this study, we show that this inflammatory response is dependent on activation of the innate immune system mediated by the adaptor molecule MyD88. In the present study, MyD88-deficient macrophages are nonresponsive toward both soluble variant-specific surface glycoprotein (VSG), as well as membrane-bound VSG purified from T. brucei. Infection of MyD88-deficient mice with either clonal or nonclonal stocks of T. brucei resulted in elevated levels of parasitemia. This was accompanied by reduced plasma IFN-γ and TNF levels during the initial stage of infection, followed by moderately lower VSG-specific IgG2a Ab titers during the chronic stages of infection. Analysis of several TLR-deficient mice revealed a partial requirement for TLR9 in the production of IFN-γ and VSG-specific IgG2a Ab levels during T. brucei infections. These results implicate the mammalian TLR family and MyD88 signaling in the innate immune recognition of T. brucei.

It is generally accepted that the triggering of an innate immune response is mediated by recognition of invading microorganisms by pattern recognition receptors (PRRs)3 present on APCs (1, 2). Within the family of PRRs, a family of evolutionarily conserved germline-encoded transmembrane receptors called TLRs initiate intracellular signaling cascades upon exposure to microbial molecules (3, 4). Distinct TLRs stimulate APC function in response to a diversity of ligands, including LPS, bacterial flagellin, lipoteichoic acid, peptidoglycan, and nucleic acid structures present on bacterial, fungal, and viral pathogens (2). In general, the activation of individual TLRs by their corresponding ligands is associated with the recruitment of an intracellular adaptor molecule, namely MyD88 (3). The production of inflammatory cytokines associated with early-phase NF-κB activation is associated with a MyD88-dependent innate immune response (4), although TLR-dependent, MyD88-independent production of IFN-β and expression of IFN-inducible genes has been reported (5, 6). To date, several reports have investigated the role of TLRs in initiating an innate immune response against experimental parasitic infections, including Trypanosoma cruzi, Leishmania major, Plasmodium berghei, and Toxoplasma gondii (7, 8, 9, 10, 11, 12). Collectively, the importance of MyD88-dependent signaling was established both in vitro and in vivo. Using in vitro assays, APCs deficient in MyD88 displayed impaired IL-12 responses upon stimulation with parasite Ags, whereas animals deficient in MyD88 were more susceptible to live infections when compared with wild-type controls. In these models, susceptibility toward a variety of parasitic infections was associated with a decreased Th1 response, the induction of which being associated with the MyD88-dependent activation of the innate immune system.

The necessity to mount an early polarized variant-specific surface glycoprotein (VSG) type 1 immune response following an African trypanosome infection has been well documented (13, 14, 15, 16). During early infection, T. brucei stimulates both IFN-γ and TNF production, which along with a VSG-specific B cell response results in the control of parasitemia and overall host resistance (17, 18, 19, 20). In terms of T. brucei Ags responsible for eliciting an initial macrophage inflammatory response, trypanosomal VSG was found to encompass two distinct macrophage-activating components (21). Although both soluble VSG (sVSG) and membrane-bound VSG (mfVSG) were shown to have similar TNF-inducing capacities, the dimyristoylglycerol moiety of the mfVSG anchor was not required for TNF triggering. This being the case, macrophages require IFN-γ prestimulation before becoming responsive toward sVSG, whereas the TNF-inducing capacity of mfVSG is IFN-γ independent. In an in vivo setting, the absence of either IFN-γ or TNF was associated with a severely diminished capacity to control proliferation of the parasite (19, 20, 22), emphasizing the importance of these proinflammatory cytokines in the initial control of the disease.

The importance of MyD88-dependent signaling in other parasitic infections indicated that the induction of inflammatory cytokines following a T. brucei infection might be mediated by the MyD88-dependent activation of an innate immune response. In the present study, T. brucei-derived sVSG and mfVSG were found to activate macrophages in a MyD88-dependent manner. Mice deficient in MyD88 were unable to control both clonal and nonclonal T. brucei infections. Parasitemia control following clearance of the first peak of parasites was found to be partly mediated by TLR9, and macrophages deficient in this TLR were less responsive toward T. brucei genomic DNA than wild-type controls. Therefore, the data presented here implicates TLR triggering as a critical step in the initiation of innate immunity against T. brucei.

Mouse strains used in this study include animals deficient in TLR1 (23), TLR2 (24), TLR2/4 (25), TLR9 (26), MyD88 (27), CD14 (28), TNF (29), IFN-γR1 (30), IL-1R1 (31), and caspase-1 (32). Mice deficient in TLR1, TLR2, TLR9, MyD88, TNF, IFN-γR1, IL-1R1, and caspase-1 were backcrossed onto a C57BL/6 background five times or more. Mice deficient in TLR2/4 and CD14 were on 129/SvJ × C57BL/6 and CBA/J backgrounds, respectively. Age-matched control littermate mice of the corresponding genetic background were used in all experiments. Mice were obtained from either the University of Cape Town or the Transgenose Institute animal breeding facility. All experiments performed were in accordance with the guidelines of the Animal Research Ethics Board of the University of Cape Town, South Africa, and the Regional Ethics Committee for Animal Experiments of Toulouse, France.

For clonal infections, the Antat1.1E clone of the EATRO 1125 stock of the pleomorphic bloodstream form was originally provided by Dr. N. Van Meirvenne (Institute of Tropical Medicine, Antwerp, Belgium). Frozen stabilate stocks of AnTat1.1 T. brucei used for infections were stored at −80°C at the Free University of Brussels. Mice were infected i.p. with 5 × 103 parasites diluted in PBS (pH 8.0) supplemented with 1.6% glucose. Animals were bled from the tail at intervals of either 1 or 2 days for the duration of the infection, and parasites were counted using a light microscope.

For infections of tsetse flies, male flies from the Glossina morsitans morsitans colony maintained at the Institute of Tropical Medicine were used throughout the study. This colony was maintained on rabbits at 25°C and 65% relative humidity and is characterized by a high intrinsic vectorial capacity. Teneral flies (8–32 h after emergence) were fed their first bloodmeal on immune-suppressed Naval Medical Research Institute mice (80 mg/kg cyclophosphamide (Endoxan)), showing a parasitemia of 108.4 to 108.7 parasites/ml of a pleiomorphic T. brucei brucei AnTAR1 population, containing at least 70% short stumpy forms. After the infective meal, flies were maintained on uninfected rabbits for 28 days, with 3 days/week feeding regime. Twenty-eight days after the infective bloodmeal, flies were starved for 72 h and were forced to salivate on a prewarmed (37°C) glass slide. This drop of saliva was examined for the presence of metacyclic trypanosomes. Metacyclic-infected tsetse flies were retained for the nonclonal infection experiments.

Trypanosomes were harvested from infected blood by DE52 chromatography (33), using sterile PBS (pH 8.0) supplemented with 1.6% glucose for equilibration and elution. After separation, parasites were washed and resuspended in RPMI 1640 medium at a concentration of 109 parasites/ml. sVSG was prepared from DE52-purified parasites by osmotic lysis for 5 min at 37°C in 10 mM sodium phosphate (pH 8.0) containing 0.1 mM Nα-p-tosyl-L-lysin chloromethyl ketone and 0.1 mM PMSF (Boehringer Mannheim). The supernatant was passed through a column of DE52 equilibrated in 10 mM sodium phosphate (pH 8.0). sVSG was further purified on a column of Sephacryl-S200 (Pharmacia Biotech), dialyzed against water overnight at 4°C, and freeze dried. mfVSG was prepared according to the method described previously (34). VSG samples were incubated under gentle shaking for 2 h at room temperature with Prosep-Remtox (Bioprocessing) glass beads to remove possible LPS contamination. Following this, beads were separated by sample filtration over a 22-μm sterile Spin-X centrifuge tube filter (Costar). Protein concentration of VSG was estimated by a detergent-compatible protein assay kit (Bio-Rad) using BSA as a standard. sVSG was resuspended in PBS (pH 8.0), and mfVSG was resuspended in PBS containing 0.02% N-octylglucoside (Sigma-Aldrich). For isolation of T. brucei DNA, DE52-purified parasites were lysed using TRIzol (Invitrogen Life Technologies), and DNA was purified according to the manufacturer’s recommendations. DNA was resuspended in water, and concentrations were determined by spectrophotometric analysis. Endotoxin units < 0.5 pg/mg VSG or DNA were determined using the Limulus amebocyte lysate test (BioWhittaker). VSG preparations contained no T. brucei DNA.

MyD88−/− (27), TLR9−/− (26), and control murine bone marrow cells were isolated from femurs and cultivated (106/ml) for 7 days in DMEM supplemented with 2 mM l-glutamine, 0.2 μM 2-ME, 20% horse serum, and 30% L929 cell-conditioned medium. Following this, cells were resuspended in cold PBS and recultured for 3 days in fresh medium at 37°C and 5% CO2.

MyD88−/− bone marrow-derived macrophages were plated in 96-well microculture plates at a density of 105 cells/well. Culture medium used was DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, 0.2 μM 2-ME, and 2 mM l-glutamine. Cells were stimulated with 1 μg/ml LPS (Escherichia coli, serotype O111:B4; Sigma-Aldrich), 5 or 10 μg/ml sVSG, and 5 or 10 μg/ml mfVSG both in the presence or absence of 30 U/ml IFN-γ. Similarly, TLR9−/− bone marrow-derived macrophages were cultured with 1 μg/ml LPS or 1.0 mg/ml, 2.5 μg/ml, or 12.5 mg/ml T. brucei DNA in the presence of 30 U/ml IFN-γ. All cells were stimulated for 24 in the presence of 50 U/ml polymyxin B sulfate, following which supernatants were harvested and frozen at −80°C.

Cytokine content in supernatants and plasma were assayed using commercially available ELISA reagents for TNF-α, IL-12p40, and IL-6 (Duoset R&D Systems).

Infection-induced anti-VSG serum titers were determined in a VSG solid phase ELISA. ELISA plates (Nalge Nunc International) were coated in PBS (pH 8.0) with purified sVSG (10 μg/ml to 100 μl/well) by overnight incubation at 4°C. Free binding sites were blocked by an additional overcoat of BSA (1 mg/ml to 300 μl/well, 1 h incubation at 37°C). Serial serum dilutions were added to the plates and incubated overnight, following which plates were extensively washed with PBS. Detection of bound serum Abs was done using 1/1000 dilutions of IgM, and IgG isotype-specific HRP-coupled Abs (Southern Biotechnology Associates), followed by tetramethylbenzidine substrate addition. After 20 min, the substrate conversion reaction was stopped by addition of 50 μl of 1 N H2SO4, and ODs were measured at 450 nm.

Nitrite concentrations in cell supernatants were determined using the Griess reaction (3% phosphoric acid, 1% p-aminobenzenesulfonamide, and 1% N-(1-napthyl)ethylenediamide) as described previously (25).

The statistical significance of differences in data means was analyzed using an unpaired Student’s t test. Values of p < 0.05 were considered significant.

It has been shown previously that the glycosyl-inositol-phosphate moiety of sVSG is the main trypanosome-derived, TNF-inducing component (21). However, the TNF-inducing capacity of sVSG required IFN-γ priming, suggesting that in an in vivo setting, macrophage responsiveness toward sVSG occurs after an IFN-γ prestimulation event. In the present study, all sVSG macrophage stimulation experiments were performed using a concentration of protein no higher than 10 μg/ml, a dose corresponding to a trypanosome load of ∼2 × 107 parasites during infection. To determine whether macrophage TNF responses toward sVSG might be mediated by TLRs, bone marrow-derived macrophages deficient in the adaptor molecule MyD88 were stimulated with sVSG either in the presence or absence of IFN-γ (Fig. 1,a). Neither wild-type nor MyD88-deficient macrophages responded to sVSG in the absence of IFN-γ. Upon prestimulation with IFN-γ, wild-type macrophages produced TNF in a dose-dependent manner, whereas macrophages deficient in MyD88 produced none. TNF production in response to LPS was reduced in MyD88-deficient macrophages (Fig. 1,a) but not absent. sVSG fractions were free of endotoxin, indicating that TNF production was solely due to sVSG stimulation. The production of nitrite by wild-type macrophages in response to sVSG was dependent on IFN-γ priming but was reduced in MyD88-deficient macrophages (Fig. 1,b). In contrast to TNF and nitrite, no IL-6 or IL-12p40 was detected in culture supernatants (Fig. 1, c and d), and prestimulation with IFN-γ did not induce macrophages to secrete IL-6 or IL-12p40 in response to sVSG. In general, macrophages were far less responsive toward sVSG than they were to LPS (Fig. 1, a–d). In the present study, cytokine production induced by 1 μg/ml LPS far exceeded that produced by 10 μg/ml sVSG.

FIGURE 1.

Impaired responses to T. brucei Ags in MyD88−/− macrophages. a–d, Bone marrow-derived macrophages from wild-type (▪) or MyD88−/− (□) mice were stimulated with LPS (1 μg/ml) or AnTat1.1 T. brucei sVSG (5 or 10 μg/ml) in the presence or absence of 30 U/ml IFN-γ for 24 h. TNF, nitrite, IL-6, and IL-12p40 in culture supernatants were determined by ELISA. e–h, Bone marrow-derived macrophages from wild-type and MyD88−/− mice were stimulated with LPS (1 μg/ml) or AnTat1.1 T. brucei membrane fraction VSG (5 or 10 μg/ml) in the presence or absence of 30 U/ml IFN-γ for 24 h. Concentrations of TNF, nitrite, IL-6, and IL-12p40 in culture supernatants were determined by ELISA. Each bar represents the average of four wells, and all experiments were repeated twice. n.d., not detected.

FIGURE 1.

Impaired responses to T. brucei Ags in MyD88−/− macrophages. a–d, Bone marrow-derived macrophages from wild-type (▪) or MyD88−/− (□) mice were stimulated with LPS (1 μg/ml) or AnTat1.1 T. brucei sVSG (5 or 10 μg/ml) in the presence or absence of 30 U/ml IFN-γ for 24 h. TNF, nitrite, IL-6, and IL-12p40 in culture supernatants were determined by ELISA. e–h, Bone marrow-derived macrophages from wild-type and MyD88−/− mice were stimulated with LPS (1 μg/ml) or AnTat1.1 T. brucei membrane fraction VSG (5 or 10 μg/ml) in the presence or absence of 30 U/ml IFN-γ for 24 h. Concentrations of TNF, nitrite, IL-6, and IL-12p40 in culture supernatants were determined by ELISA. Each bar represents the average of four wells, and all experiments were repeated twice. n.d., not detected.

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In response to environmental stress, trypanosomes release their VSG via a VSG lipase (35). In doing so, the dimyristoylglycerol VSG anchor remains in the trypanosomal membrane while the glycosyl-inositol-phosphate-VSG is released (36). However, during a trypanosomal infection, it is also likely that macrophages are exposed to the VSG-GPI anchor due to lyses of trypanosomes. Within this context, it has already been shown that mfVSG can elicit a macrophage response and that this response is independent of IFN-γ prestimulation (21). To determine whether mfVSG-induced macrophage responsiveness was dependent on MyD88 signaling, macrophages deficient in MyD88 were stimulated with mfVSG either in the absence or presence of IFN-γ (Fig. 1, e–h). First, macrophages required MyD88 to produce TNF in response to mfVSG stimulation (Fig. 1,e), but in contrast to sVSG, this response was not dependent on prestimulation with IFN-γ. Second, macrophages did require MyD88 and IFN-γ prestimulation to produce nitrite upon mfVSG stimulation (Fig. 1,f). Third, unlike macrophages stimulated with sVSG (Fig. 1, c and d), macrophages stimulated with mfVSG secreted both IL-6 and IL-12p40 (Fig. 1, g and h), the production of which was not dependent on IFN-γ prestimulation. However, while MyD88 was required by macrophages to produce IL-6 in response to mfVSG stimulation (Fig. 1,g), the production of IL-12p40 was MyD88 independent (Fig. 1 h). Therefore, macrophages in general were far more responsive toward mfVSG than sVSG, and the response required MyD88 for the production of TNF, IL-6, and nitrite but not IL-12p40.

To evaluate the role of MyD88 in host resistance to T. brucei, mice deficient in MyD88 were infected with pleiomorphic AnTat1.1 T. brucei. The development of parasitemia and survival of these animals were compared with wild-type controls, as well as mice deficient in either IFN-γR1 or TNF (Fig. 2). In terms of development of parasitemia, mice deficient in MyD88 could not efficiently control the height of first peak parasitemia, a phenotype similar to mice deficient in either IFN-γR1 or TNF (Fig. 2, a and c). Following clearance of the first peak of parasitemia, wild-type animals developed a reduced second peak of parasitemia at days 13–15 postinfection and succumbed to the final lethal peak of parasitemia at ∼40–50 days postinfection (Fig. 2,a). Animals deficient in either MyD88 or IFN-γR1 had significantly elevated parasitemia levels after first peak (Fig. 2,a), which remained elevated for the duration of the infection with both gene-deficient animals succumbing to infection before wild-type controls (p < 0.01 and p < 0.001, respectively; Fig. 2,b). Mice deficient in TNF controlled parasitemia development after first peak more effectively than either MyD88- or IFN-γR1-deficient animals (Fig. 2,c) and displayed similar survival kinetics when compared with wild-type controls (p > 0.10; Fig. 2,d). Thus, MyD88-deficient animals succumbed to the infection before TNF-deficient animals (p < 0.01). In addition, IL-12p40 did not contribute significantly to first-peak parasitemia control as animals deficient in this cytokine controlled the first peak of parasitemia as well as wild-type controls (Table I). However, parasitemia levels did increase slightly in the absence of IL-12p40 following clearance of the second peak of infection (data not shown; F. Brombacher, unpublished observation). To determine whether the impaired resistance of MyD88-deficient mice toward a T. brucei infection could be attributed to a reduced type 1 immune response, IFN-γ and TNF were measured in the plasma at various time points postinfection (Fig. 3, a and b). At 4 days postinfection, MyD88-deficient mice had reduced plasma IFN-γ levels when compared with wild-type controls (Fig. 3,a), which then increased marginally at day 6 postinfection. A similar trend was observed for plasma TNF levels in MyD88-deficient mice (Fig. 3,b). For comparative purposes, plasma IFN-γ levels were analyzed in TNF-deficient animals. In the present study, plasma IFN-γ levels were also reduced in mice lacking TNF at days 4 and 6 postinfection (Fig. 3,a). Furthermore, parasite clearance is also mediated by a VSG-specific Ab response. In the present study, VSG-specific IgM and IgG2a titers at days 4, 6, and 20 postinfection were moderately reduced in MyD88-deficient animals (Fig. 3, d–f). Ab titers for IgG1, IgG2b, and IgG3 in MyD88- and TNF-deficient mice were comparable to wild-type controls for the duration of infection (data not shown). Thus, the development of a type 1 cytokine and VSG-specific IgG2a response during a clonal T. brucei infection is mediated in part by the MyD88-dependent activation of the innate immune system.

FIGURE 2.

MyD88 is required to control a pleomorphic T. brucei AnTat1.1 infection. Parasitemia de-velopment and survival in (a and b) wild-type (▪), MyD88−/− (□), and IFN-γR1−/− (○) and (c and d) wild-type (▪), MyD88−/− (□), and TNF−/− (○) mice. Five mice per group were infected at day 0 i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Parasitemia data indicate mean ± of five mice per group from one of three independent experiments, while survival data are representative of two to three pooled experiments.

FIGURE 2.

MyD88 is required to control a pleomorphic T. brucei AnTat1.1 infection. Parasitemia de-velopment and survival in (a and b) wild-type (▪), MyD88−/− (□), and IFN-γR1−/− (○) and (c and d) wild-type (▪), MyD88−/− (□), and TNF−/− (○) mice. Five mice per group were infected at day 0 i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Parasitemia data indicate mean ± of five mice per group from one of three independent experiments, while survival data are representative of two to three pooled experiments.

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Table I.

Systemic responses of PRR- and cytokine-deficient mice following a clonal T. brucei infectiona

Mouse StrainFirst Peak Parasitemia (×2.106 par./ml)Second Peak Parasitemia (×2.106 par./ml)
C57BL/6b 56 ± 7 9 ± 5 
 TLR1−/− 58 ± 6 10 ± 7 
 TLR2−/− 37 ± 4 12 ± 9 
 TLR9−/− 51 ± 14 25 ± 4 
 MyD88−/− 167 ± 47 38 ± 14 
 TNF−/− 197 ± 41 15 ± 13 
 IFN-γR1−/− 141 ± 38 111 ± 83 
 IL-1R1−/− 43 ± 10 14 ± 8 
 Caspase-1−/− 55 ± 20 7 ± 2 
 IL-12p40−/− 53 ± 9 16 ± 4 
TLR2/4+/+c 135 ± 48 10 ± 5 
 TLR2/4−/− 121 ± 23 5 ± 4 
CBA/Jd 66 ± 31 15 ± 10 
 CD14−/− 58 ± 18 8 ± 5 
Mouse StrainFirst Peak Parasitemia (×2.106 par./ml)Second Peak Parasitemia (×2.106 par./ml)
C57BL/6b 56 ± 7 9 ± 5 
 TLR1−/− 58 ± 6 10 ± 7 
 TLR2−/− 37 ± 4 12 ± 9 
 TLR9−/− 51 ± 14 25 ± 4 
 MyD88−/− 167 ± 47 38 ± 14 
 TNF−/− 197 ± 41 15 ± 13 
 IFN-γR1−/− 141 ± 38 111 ± 83 
 IL-1R1−/− 43 ± 10 14 ± 8 
 Caspase-1−/− 55 ± 20 7 ± 2 
 IL-12p40−/− 53 ± 9 16 ± 4 
TLR2/4+/+c 135 ± 48 10 ± 5 
 TLR2/4−/− 121 ± 23 5 ± 4 
CBA/Jd 66 ± 31 15 ± 10 
 CD14−/− 58 ± 18 8 ± 5 
a

Five wild-type and PPR/cytokine-deficient mice were infected i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Results are representative of two to three similar experiments. par., parasitemia; bold, wild-type parasitemia counts; bold, gene deficient mice with elevated parasitemia counts. The corresponding numbers have been underlined accordingly.

b

Gene-deficient mice backcrossed onto a C57BL/6 background five times or more.

c

Gene-deficient mice on a 129/SvJ × C57BI/6 background.

d

Gene-deficient mice on a CBA/J background.

FIGURE 3.

MyD88 is required to mount an early inflammatory response following a T. brucei infection. a and b, Wild-type (▪), MyD88−/− (□), and TNF−/− (▤) were infected i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Sera was collected at days 4 and 6 postinfection and analysed for IFN-γ and TNF by ELISA. Results from one of two experiments performed are the mean of sera samples from three mice. n.d., not detected. c–e, Sera was collected from wild-type (▪), MyD88−/− (□), and TNF−/− (○) mice at days 4, 6, and 20 postinfection and analyzed for VSG-specific IgM and IgG2a Abs. Results are means of sera samples from three mice from one of two similar experiments performed.

FIGURE 3.

MyD88 is required to mount an early inflammatory response following a T. brucei infection. a and b, Wild-type (▪), MyD88−/− (□), and TNF−/− (▤) were infected i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Sera was collected at days 4 and 6 postinfection and analysed for IFN-γ and TNF by ELISA. Results from one of two experiments performed are the mean of sera samples from three mice. n.d., not detected. c–e, Sera was collected from wild-type (▪), MyD88−/− (□), and TNF−/− (○) mice at days 4, 6, and 20 postinfection and analyzed for VSG-specific IgM and IgG2a Abs. Results are means of sera samples from three mice from one of two similar experiments performed.

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MyD88 is an integral component of the TLR-signaling pathway (37), but this adaptor molecule is also an important component of the IL-1 and IL-18-signaling cascade (38, 39, 40). In the present study, animals deficient in MyD88 have been reported to have reduced responsiveness toward either IL-1 and/or IL-18 (41). To determine whether the increased susceptibility of MyD88-deficient animals during a clonal T. brucei infection was associated with defective IL-1 and/or IL-18 signaling, mice deficient in IL-1R1 and caspase-1 were infected with clonal T. brucei. In contrast to MyD88-deficient mice, these mice controlled the first and second peaks of parasitemia as well as wild-type controls (Table I), indicating that the increased susceptibility toward T. brucei observed for MyD88-deficient animals was due to defective TLR signaling. Screening of several TLR-deficient animals revealed that neither TLR1, TLR2, the combination of both TLR2 and TLR4, or TLR9 were required by the host to control the first peak of parasitemia following a clonal T. brucei infection (Table I). Interestingly, animals deficient in TLR9 developed elevated levels of parasitemia following clearance of the first peak of parasites (Table I), indicating that TLR9 could be involved in parasitemia control after first peak.

Following the first wave of parasitemia, lysis of the parasite results in the release of several parasite components into the bloodstream. Apart from sVSG and mfVSG that promote a macrophage response, it has been reported that DNA from T. brucei is not only mitogenic for B lymphocytes (42) but induces macrophages to produce TNF, NO, and IL-12p40 in the presence of IFN-γ. In the present study, the extent of B cell proliferation and macrophage activation was associated with CG dinucleotide content, a feature associated with bacterial DNA and TLR9 responsiveness (26). Therefore, it was postulated that induction of an inflammatory response following a T. brucei infection might be mediated, in part, by TLR9-dependent macrophage activation. Indeed, when stimulated with T. brucei DNA, macrophages deficient in TLR9 produced less TNF (Fig. 4,a), IL-6 (Fig. 4,b), IL-12p40 (Fig. 4,c), and nitrite (Fig. 4,d) than their wild-type counterparts. Macrophages deficient in TLR9 were unresponsive toward CpG DNA (Fig. 4) but responded normally to stimulation with LPS. In general, macrophages were far less responsive toward T. brucei genomic DNA than they were to CpG DNA (Fig. 4), possibly because the frequency of CG dinucleotides in T. brucei genomic DNA is only 3.9% (42). These results suggest that macrophage responsiveness toward T. brucei DNA is mediated in part by TLR9.

FIGURE 4.

Impaired responses to AnTat1.1 T. brucei genomic DNA in TLR9−/− macrophages. a–d, Bone marrow-derived macrophages from wild-type or TLR9−/− mice were stimulated with CpG ODN (1.0 μM), LPS (1.0 μg/ml), or the indicated concentrations of T. brucei genomic DNA in the presence of 30 U/ml IFN-γ for 24 h. Concentrations TNF, IL-6, IL-12p40, and nitrite in culture supernatants were determined by ELISA. Each bar represents the average of four wells, and all experiments were repeated twice. n.d., not detected.

FIGURE 4.

Impaired responses to AnTat1.1 T. brucei genomic DNA in TLR9−/− macrophages. a–d, Bone marrow-derived macrophages from wild-type or TLR9−/− mice were stimulated with CpG ODN (1.0 μM), LPS (1.0 μg/ml), or the indicated concentrations of T. brucei genomic DNA in the presence of 30 U/ml IFN-γ for 24 h. Concentrations TNF, IL-6, IL-12p40, and nitrite in culture supernatants were determined by ELISA. Each bar represents the average of four wells, and all experiments were repeated twice. n.d., not detected.

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Not only did macrophages deficient in TLR9 respond poorly to T. brucei genomic DNA, but animals deficient in TLR9 infected with clonal T. brucei had increased numbers of parasites following clearance of the first peak of parasitemia (Fig. 5,a), but no significant difference was observed in the mean survival time of both wild-type and TLR9-deficient animals (p > 0.10; Fig. 5,b). IFN-γ is a critical factor in contributing to parasitemia control following first peak clearance (Table I; Fig. 2,a). Analysis of plasma IFN-γ levels in TLR9-deficient mice showed reduced IFN-γ levels during the second parasitemia peak (Fig. 5,c). Reduced plasma IFN-γ levels coincided with clearance of the first peak of parasitemia at days 10–13 at a point where 108 parasites would release genomic DNA into circulation. This was accompanied by lower VSG-specific IgG2a Ab titers at day 13 postinfection (Fig. 5,d), which were restored to wild-type levels by day 16 postinfection (Fig. 5 e).

FIGURE 5.

Reduced resistance of TLR9−/− mice following an T. brucei infection. a and b, Parasitemia development and survival in wild-type (▪) and TLR9−/− (□) mice infected i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Results from one of three similar experiments are shown. c–e, Sera was collected at days 13 and 16 postinfection and analyzed for IFN-γ and VSG-specific IgG2a Abs by ELISA. Results are means of sera samples from three mice from one of two similar experiments performed. n.d., not detected.

FIGURE 5.

Reduced resistance of TLR9−/− mice following an T. brucei infection. a and b, Parasitemia development and survival in wild-type (▪) and TLR9−/− (□) mice infected i.p. with 5 × 103 AnTat1.1 T. brucei parasites. Results from one of three similar experiments are shown. c–e, Sera was collected at days 13 and 16 postinfection and analyzed for IFN-γ and VSG-specific IgG2a Abs by ELISA. Results are means of sera samples from three mice from one of two similar experiments performed. n.d., not detected.

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In the field, T. brucei parasites that infect livestock initially express metacyclic VSG and not the clonal form of VSG first seen by the immune response as present on AnTat1.1 T. brucei parasites (16, 43, 44). Seeing that the murine host required both TLR9 and MyD88 to control a clonal T. brucei infection, we sought to determine whether this was also true for a nonclonal metacyclic T. brucei infection. Using a tsetse fly as a vector, mice deficient in either TLR9 or MyD88 were infected with metacyclic AnTar1.1 T. brucei parasites (Fig. 6,a). When compared with the clonal AnTat1.1 T. brucei infection (Figs. 2,a and 5,a), the nonclonal AnTar1.1 T. brucei infection resulted in similar parasitemia profiles (Fig. 6,a). Mice deficient in MyD88 were unable to control both first and second parasitemia peaks. In the present study, nonclonal parasitemia levels in MyD88-deficient animals reached 4 × 108 parasites/ml blood at day 20 postinfection (Fig. 6,a), apposed to 2 × 108 parasites/ml blood for the clonal infection (Fig. 2,a). Additionally, all MyD88-deficient animals succumbed to nonclonal infection by day 20 postinfection (Fig. 6,b), while these animals survived for 40 days following a clonal infection (Fig. 2,b). Mice deficient in TLR9 controlled the first peak of parasitemia, as well as wild-type controls, but were unable to effectively clear parasites following first peak parasitemia (Fig. 6,a). As was seen for MyD88-deficient mice, TLR9-deficient mice succumbed to the infection before their wild-type controls (p < 0.002 and p < 0.005, respectively; Fig. 6 b). These results suggest that in a nonclonal T. brucei infection both TLR9 and MyD88 are important components of an innate immune response and are required by the host to control parasite replication.

FIGURE 6.

Differential requirement for MyD88 and TLR9 in controlling a nonclonal T. brucei infection. a and b, Parasitemia development and survival of wild-type (▪), MyD88−/− (□), and TLR9−/− (○) mice. Five mice per group were infected with metacyclic AnTar1.1 T. brucei brucei parasites using Glossina species. as a vector. Results are expressed as means ± SDs.

FIGURE 6.

Differential requirement for MyD88 and TLR9 in controlling a nonclonal T. brucei infection. a and b, Parasitemia development and survival of wild-type (▪), MyD88−/− (□), and TLR9−/− (○) mice. Five mice per group were infected with metacyclic AnTar1.1 T. brucei brucei parasites using Glossina species. as a vector. Results are expressed as means ± SDs.

Close modal

There is a growing body of evidence that indicates that recognition of several parasitic infections leads to the MyD88-dependent activation of an innate immune response (7, 8, 9, 10, 11, 12). In models analyzing experimental T. gondii, T. cruzi, P. berghei, L. major, or B. burgdorferi infections, a deficiency in MyD88 was correlated with reduced plasma IL-12p40 and/or IFN-γ levels during the infection. This was accompanied by decreased production of IL-12p40 and IFN-γ following splenocyte restimulations with parasitic Ags (7, 9, 10, 12). Thus, in these models, the activation of the MyD88-signaling pathway results in the early production of IFN-γ and IL-12, both of which are essential components in host resistance against parasitic infections.

The cytokines TNF and IFN-γ have been shown to be critical factors in determining the relative level of host resistance against the extracellular parasite T. brucei (19, 20, 22). Deficiencies in either of these cytokines have been associated with elevated levels of parasitemia and, in terms of IFN-γ, shorter survival times. Results presented here link the initial production of TNF and IFN-γ following a T. brucei infection to the MyD88-dependent activation of an innate immune response. The production of TNF by macrophages in response to either sVSG or mfVSG requires signaling via MyD88. Thus, the release of parasite Ags in the time period preceding the first peak of parasitemia stimulates macrophages to produce TNF, which in turn contributes to controlling the height of first-peak parasitemia. Lower systemic TNF levels present in mice lacking MyD88 is indicative of a failure by the host to respond to parasite Ags. However, the activation state of macrophages during infection may influence the ability of the host to respond to a Trypanosome infection (45). Indeed, macrophage responsiveness toward T. brucei sVSG required IFN-γ prestimulation, which suggests that direct sensitization of host T cells by T. brucei to produce IFN-γ (46) occurs before macrophages are fully responsive to sVSG. However, IL-12p40-mediated activation of the host T cell compartment does not appear to play a crucial role during a T. brucei infection as mice deficient in this cytokine control first-peak parasitemia as well as wild-type controls (Table I; F. Brombacher, unpublished observation). In addition, stimulation of macrophages with mfVSG did induce the production of IL-12p40, but this response was independent of MyD88 signaling. These results suggest that the induction of an IFN-γ environment following a T. brucei infection is independent of the action of IL-12, as previously shown in experimental infection models using T. cruzi (47), Listeria monocytogenes (48), and Mycobacterium tuberculosis (49), but that the induction of IFN-γ requires signaling via MyD88. This initial IFN-γ environment not only controls first peak parasitemia but also modulates VSG-specific IgM to IgG2a switching (15), thereby contributing to parasitemia control post first-peak.

Within this context, MyD88 is required for responsiveness toward IL-1α, IL-1β, as well as IL-18 (38, 39, 40, 41). Deficiencies in these cytokines have been associated with a diminished capacity to clear intracellular pathogens and fungi (50, 51, 52), with reports attributing a reduced early protective response to decreased levels of IFN-γ. Mice deficient in IL-1R1 and caspase-1 were used to control for possible decreased responsiveness toward IL-1α, IL-1β, and IL-18, respectively. The protease caspase-1 is required for processing of IL-1β and IL-18 into their biologically active forms (53). Infection of mice deficient in either IL-1R1 or caspase-1 showed little or no differences in terms of parasitemia control when compared with wild-type animals (Table I), and although a role for MyD88 in IL-1 and IL-18 signaling during infection cannot be excluded, the necessity for these cytokines appear to be minimal when compared with TNF and IFN-γ. Therefore, we attribute the reduced early protective response observed in MyD88-deficient animals to defective Toll signaling. However, deficiencies in several individual TLRs failed to reproduce the elevated parasitemia levels observed during first-peak parasitemia in MyD88-deficient animals (Table I). This would indicate that the control of parasitemia is not dependent on activation of an innate immune response via TLRs 1, 2, or 4. Although it is possible that these receptors function cooperatively with one another in response toward parasite components such as sVSG and mfVSG, this study shows no individual role in vivo either before or after the first peak of parasitemia. However, a partial role for TLR9 was found to contribute to parasitemia control following first peak clearance. During this stage of infection, lyses of 108 parasites would release more nuclear material into circulation when compared with lyses of parasites before the first peak of infection. In the present study, IL-12p40 produced by macrophages in response to T. brucei DNA could, in turn, induce IFN-γ production by PBMCs (42) and could account for the reduced systemic IFN-γ levels found in TLR9-deficient mice following clearance of the first peak of parasitemia. In addition to activating macrophages via TLR9, parasite DNA may also be involved in triggering B cells to proliferate and differentiate (42, 54), thereby contributing to the differentiation of Th1 cells and isotype switching. Therefore, both sVSG and parasitic DNA released systemically during infection contribute to macrophage activation and the resulting inflammatory response. The extent to which they contribute to macrophage activation and the inflammatory response could be more substantially addressed once a receptor for sVSG has been identified. This being said, attempts to identify individual TLRs involved in the activation of a protective innate immune response against other parasitic infections have been inconclusive. In some cases, purified parasitic components such as T. cruzi-derived GPI anchor were shown to activate TLR2 from both mouse and human origin in vitro (55), although within an in vivo setting, mice deficient in this TLR do not display a severely reduced capacity to control a T. cruzi infection compared with mice deficient in MyD88 (7). Furthermore, in murine infection models using P. berghei, T. gondii, and L. major, investigators have been unable to formally demonstrate that individual, or combinations of TLRs, are involved in innate recognition of the aforementioned parasites.

It is likely that the induction of an innate immune response following a T. brucei infection is mediated by interaction between several TLRs that signal via MyD88. Indeed, the requirement for the MyD88-dependent activation of an innate immune response is far more pronounced in nonclonal T. brucei infections. In the field, this would indicate that the VSG repertoire presented by the parasite is far greater, emphasizing the requirement for the TLR protein family in initiating an immune response. However, it is also plausible that recognition of T. brucei is not solely mediated by the TLR family but could include PRRs involved in recognizing sugar moieties within the variant surface glycoprotein. An example of this type of complex formation between the TLR protein family and proteins unrelated to the TLR family exists for recognition of endotoxin by the CD14-TLR4-MD2 complex (56, 57). In the case of T. gondii, parasite-induced IL-12 production by dendritic cells was found to be mediated by the dual interaction between MyD88 and the CCR5 (12, 58), indicating that TLRs function in concert with additional cell surface receptors to effect the appropriate immune response. Analysis of the VSG coat covering T. brucei revealed that the galactose side chain composition appears to be important in terms of the TNF-inducing capacity of sVSG (21). Thus, while the generation of a protective type 1 immune response following a T. brucei infection is MyD88 dependent, it is likely that the initial recognition event is mediated by member(s) of the TLR family functioning cooperatively with a surface protein(s) involved in galactose recognition.

We thank the animal facility staff at the Health Science Faculty and University of Cape Town and Centre National de la Recherché Scientifique for technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by a Flanders-South-African Research Cooperation Program (to S.M.), the Wellcome Trust and National Research Foundation (to F.B.), the Centre National de la Recherché Scientific (to B.R.), and an Interuniversity Attraction Pole Program. S.M. is a research fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.

3

Abbreviations used in this paper: PRR, pattern recognition receptor; VSG, variant-specific surface glycoprotein; sVSG, soluble VSG; mfVSG, membrane form VSG.

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