Effects of exogenous transforming growth factor beta on Trypanosoma congolense infection in mice

Abstract

The socioeconomic implications of trypanosomosis in sub-Saharan Africa and the limitations of its current control regimes have stimulated research into alternative control methods. Considering the pro- and anti-inflammatory properties of transforming growth factor beta1 (TGF-beta1) and its potential to enhance immunity against protozoan parasites, we examined the effects of intraperitoneally delivered TGF-beta1 in C57BL/6 mice infected with Trypanosoma congolense, the hemoprotozoan parasite causing nagana in cattle. A triple dose of 10 ng TGF-beta1 significantly reduced the first parasitemic peak and delayed mortality of infected mice. Furthermore, exogenous TGF-beta1 significantly decreased the development of trypanosome-induced anemia and splenomegaly. The apparent TGF-beta1-induced antitrypanosome protection, occurring mainly during the early stage of infection, correlated with an enhanced parasite antigen-specific Th1 cell response characterized by a skewed type I cytokine response and a concomitant stronger antitrypanosome immunoglobulin G2a antibody response. Infected TGF-beta1-pretreated mice exhibited a significant reduction in the trypanosome-induced hyperexpansion of B cells. Furthermore, evidence is provided herein that exogenous TGF-beta1 activates macrophages that may contribute to parasite control. Collectively, these data indicate that exogenous TGF-beta1 is immunostimulative, inducing partial protection against T. congolense infection, possibly through mechanisms involving innate immune responses.

FIG. 1.

Effects of exogenous TGF-β1 on parasitemia, development of pathology, and survival rate of T. congolense-infected mice. Female C57BL/6 mice were pretreated i.p. with a single dose of 5 to 100 ng TGF-β1 on day −5 p.i. (A) or with triple doses of 10 ng TGF-β1 on days −5, −1, and +3 p.i. (B and C), while control animals received PBS. On day 0, mice were challenged with T. congolense via the i.p. route. (D) To test whether TGF-β1 had any direct effects against parasites, about 10⁵/ml DE52-purified bloodstream forms of trypanosomes were cocultured in 500 ng/ml TGF-β1 or 10% NHS diluted in EMEM containing 2 mg/ml BSA. Levels of parasites in blood (A and B) or culture medium (D), the survival rate (C), and the development of anemia (E) and splenomegaly (F) were monitored at different times p.i. Statistical analysis was performed by two-tailed Student's t test, with P values of <0.05 considered statistically significant. Dashed horizontal lines represent average PCV levels and spleen weights in noninfected mice or viable parasite count in untreated medium. *, statistically significant difference compared to noninfected mice; #, statistically significant difference between infected TGF-β1- and PBS-pretreated mice; $, statistically significant difference compared to PBS cocultures.

FIG. 2.

Effects of exogenous TGF-β1 on NK cell activity in T. congolense-infected mice. NK cell cytotoxic activity was measured in TGF-β1-pretreated mice by using alamarBlue dye to assess cell viability during preinfection (A), early (week 2 p.i.) (B), and late (week 4 p.i.) (C) stages of infection. Pooled data from five independent experiments are expressed as the mean cytotoxicities of the nonadherent spleen cells from untreated (SPC-N), PBS-pretreated (SPC-PBS), and TGF-β1-pretreated (SPC-β) uninfected mice or from infected PBS-pretreated (SPC-I) or infected TGF-β1-pretreated (SPC-βI) mice against YAC-1 target cells at the indicated E:T ratios (±SE). Statistical analysis was performed by two-tailed Student's t test, with P values of <0.05 considered statistically significant. *, statistically significant difference compared to noninfected mice.

FIG. 3.

Effects of exogenous TGF-β1 on cytokine levels in the spleen compartment and peritoneal cavity. The mRNA expression levels of GAPDH and type I (IFN-γ, TNF-α, and IL-12p40) and type II (IL-4, IL-10, and TGF-β1) cytokines were determined in adherent PEC (A) or SPC (B) from untreated (lane 1), PBS-pretreated (lane 2), or TGF-β1-pretreated (lane 3) noninfected mice or from noninfected (lane 1), infected PBS-pretreated (SPC-I) (lane 2), or infected TGF-β1-pretreated (SPC-βI) (lane 3) mice on day 9 p.i. (C) by RT-PCR. The product size of each gene is shown in parentheses. Secreted bioactive TGF-β1 (D), IL-10 (E), and IFN-γ (F) levels in culture supernatants of SPC or IFN-γ/IL-10 ratios (G), in the presence or absence of TPL, were quantified by ELISA. Statistical analysis was performed by two-tailed Student's t test, with P values of <0.05 considered statistically significant. Pooled data from five independent experiments are expressed as means ± SE. Dashed horizontal lines represent average cytokine levels in noninfected mice. #, statistically significant difference between infected TGF-β1- and PBS-pretreated mice.

FIG. 4.

Effects of exogenous TGF-β1 on serum cytokine responses in T. congolense-infected mice. At week 2 and week 4 p.i., TGF-β1, IL-10, and IFN-γ levels as well as IFN-γ/IL-10 ratios in Serum-N, Serum-I, and Serum-βI were quantified by ELISA. Statistical analysis was performed by two-tailed Student's t test, with P values of <0.05 considered statistically significant. Pooled data from five independent experiments are expressed as means ± SE. Dashed horizontal lines represent average cytokine levels in noninfected mice. #, statistically significant difference between infected TGF-β1- and PBS-pretreated mice.

FIG. 5.

Effects of exogenous TGF-β1 on anti-T. congolense serum antibody production in infected mice. At week 2 p.i., anti-TPL-specific antibody isotypes (IgM, IgG1, and IgG2a) or IgG2a/IgG1 ratios were determined by ELISA, using Serum-N, Serum-I, and Serum-βI. Statistical analysis was performed by two-tailed Student's t test, with P values of <0.05 considered statistically significant. Pooled data from five independent experiments are expressed as means ± SE. Serum-N had no detectable antibody titers. #, statistically significant difference between infected TGF-β1- and PBS-pretreated mice.