Control of experimental Trypanosoma brucei infections occurs independently of lymphotoxin-alpha induction

Abstract

Trypanosome infections are marked by severe pathological features, including anemia, splenomegaly, and suppression of T-cell proliferation. We have used lymphotoxin-alpha-deficient (LT-alpha(-/-)) mice, as well as LT-alpha-tumor necrosis factor-double-deficient (LT-alpha(-/-) TNF(-/-)) mice, to analyze the contributions of these related cytokines in both induction of trypanosomosis-associated immunopathology and infection control. Moreover, as the cytokine-deficient mice used have no detectable lymph nodes and lack germinal-center formation upon immune stimulation, we have analyzed the functional importance of both the lymph nodes and spleen during experimental Trypanosoma brucei infections. First, we show that the absence of LT-alpha does not significantly alter early trypanosomosis development or pathology but does result in better control of late-stage parasitemia levels and slightly prolonged survival. This increased survival of infected LT-alpha(-/-) mice coincides with the appearance of increased chronic-stage anti-trypanosome immunoglobulin M (IgM)-IgG2a serum titers that are generated in the absence of functional peripheral lymphoid tissue and do not require germinal-center formation. Second, we show that splenectomized mice control their parasitemia to the same extent as fully immune-competent littermates. Finally, using LT-alpha(-/-) TNF(-/-) double-deficient mice, we show that in these mice T. brucei infections are very well controlled during the chronic infection stage and that infection-induced pathology is minimized. Together, these findings indicate that while increased IgM-IgG2a anti-trypanosome antibody titers (generated in the absence of LT-alpha, peripheral lymph nodes, and germinal-center formation) coincide with improved parasitemia control, it is TNF that has a major impact on trypanosomosis-associated immunopathology.

FIG. 1.

(A and B) Parasitemia development in LT-α-deficient mice compared to parasitemia development in control WT mice (A), as well as survival of infected individual mice in both groups (B). p.i., postinfection. (C and D) For reference, parasitemia development in parental C56BL/6J and 129SvImJ (129) mice is also shown (C), as well as the individual survival of these mice (D). (E to H) Parasitemia development (E) and survival (F) of T. brucei-infected TNF-deficient and control (WT) mice (data from reference 20), as well as parasitemia development (G) and survival (H) of infected LT-α-TNF-double-deficient and WT mice, are shown. All results were obtained with experimental groups of 10 mice, and individual experiments were repeated at least two times with similar results. Parasitemia data are presented as mean parasite counts (# Par.) per milliliter of blood ± standard deviations. In the experiment represented in panel H, one LT-α-TNF-double-deficient mouse (∗) died early on day 15 for unknown reasons. This mouse was not taken into account for the calculation of the mean survival time of the whole group.

FIG. 2.

Development of trypanosomosis-associated anemia in LT-α-deficient mice and control WT mice (A) and in LT-α-TNF-double-deficient as well as TNF-deficient mice (B). The values presented are mean RBC counts ± standard deviations, obtained from the same mice as those used for the data presented in Fig. 1 and expressed as percentages compared to mice before infection. p.i., postinfection. Trypanosomosis-induced serum TNF levels (C) in both LT-α-deficient mice and control WT mice were measured. Sera were collected on days 7, 14, and 28 and just prior to the lethal end of the infection. (∗), no WT mice survived the infection as long as 55 days.

FIG. 3.

Infection-induced anti-VSG (A and B) and anti-trypanosome (C and D) responses. Specific IgM (A) and IgG2a and -2b (B) isotype serum endpoint titers were determined in a VSG solid-phase ELISA system (0.2 μg/well), using sera collected on days 7, 14, and 28 and just prior to the lethal end of the infection. The same sera were used to determine specific IgM (C) and IgG2a and -2b (D) isotype serum endpoint titers in an ELISA system that used total soluble trypanosome extracts as a solid-phase coating (0.5 μg/well).

FIG. 4.

(A) Parasitemia development (left axis) and occurrence of trypanosomosis-associated anemia (RBC; right axis) in splenectomized B6129SF2 mice. The parasitemia values presented are mean parasite counts (# Par.)/ml of blood ± standard deviations. (B) Survival was followed for five individual mice, and five other mice were sacrificed for serum collection on days 7, 14, 28, 36, and 40, respectively. (C and D) Infection-induced specific anti-VSG IgM (C) and IgG2a and -2b (D) isotype serum endpoint titers were determined in a VSG solid-phase ELISA system (0.2 μg/well), using sera collected on days 7, 14, and 28 and just prior to the lethal end of the infection. (E and F) The same sera were used to determine specific IgM (E) and IgG2a and -2b (F) isotype serum endpoint titers in an ELISA system that used total soluble trypanosome extracts as a solid-phase coating (0.5 μg/well).