Resistant mice lacking interleukin-12 become susceptible to Trypanosoma cruzi infection but fail to mount a T helper type 2 response

Abstract

Interleukin-12 (IL-12) is essential to resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma) that activates macrophages to a parasiticidal effect. Investigation of mice deprived of IL-12 genes (IL-12 knockout mice) has confirmed the important role of IL-12 and IFN-gamma in controlling parasitism in T. cruzi infection. However, it has not yet been addressed whether a shift towards a T helper type 2 (Th2) pattern of cytokine response occurred in these mice that might have contributed to the aggravation of the infection caused by IL-12 deprivation. We examined the course of T. cruzi (Y strain) infection and the regulation of cytokine responses and nitric oxide production in C57BL/6 IL-12 p40-knockout mice. The mutant mice were extremely susceptible to the infection as evidenced by increased parasitaemia, tissue parasitism and mortality in comparison with the control C57BL/6 mouse strain (wild-type) that is resistant to T. cruzi. A severe depletion of parasite-antigen-specific IFN-gamma response, without an increase in IL-4 or IL-10 production, accompanied by reduced levels of nitric oxide production was observed in IL-12 knockout mice. We found no evidence of a shift towards a Th2-type cytokine response. In IL-12 knockout mice, the residual IFN-gamma production is down-regulated by IL-10 but not by IL-4 and nitric oxide production is stimulated by tumour necrosis factor-alpha. Parasite-specific immunoglobulin G1 antibody levels were similar in IL-12 knockout and wild-type mice, whereas IL-12 knockout mice had much higher levels of immunoglobulin G2b.

Figure 1

Parasite counts in the blood from C57BL/6 IL-12 knockout mice and in wild-type C57BL/6 controls infected intraperitoneally with 5000 blood forms of Trypanosoma cruzi (Y strain). Arithmetic means from eight mice per group ± SD; *P < 0·05.

Figure 2

Survival rates of C57BL/6 IL-12 knockout and wild-type C57BL/6 mice infected intraperitoneally with 5000 blood forms of T. cruzi (Y strain). Mean per cent survival rates of eight mice per group.

Figure 3

Production of the cytokines IFN-γ, IL-10 and IL-4 and of nitrite by spleen cells from C57BL/6 IL-12 knockout and wild-type C57BL/6 mice infected with T. cruzi. Cultures were performed on days 7 and 14 after infection and were kept only in culture medium or stimulated with parasite-antigen, T-Ag, or with plate-bound anti-CD3. Cytokines and nitrite were measured in the supernatants. Results are expressed as the arithmetic means from duplicate cultures ± SD. The results are representative of three experiments.

Figure 4

Regulation by cytokines of splenic cells IFN-γ production in C57BL/6 IL-12 knockout and wild-type C57BL/6 mice infected with T. cruzi. Cultures were performed on days 7 and 14 after infection and stimulated with parasite-antigen, T-Ag. At the beginning of the cultures the neutralizing anti-cytokine mAbs, anti-IL-10, anti-IL-4 and anti-TNF-α, were added; GL.113 was used as isotype control mAb. The supernatants were harvested after 72 hr and IFN-γ was measured by enzyme-linked immunosorbent assay. Results are expressed as the arithmetic means from duplicate cultures ± SD. *P < 0·05. The results are representative of three experiments.

Figure 5

Regulation by cytokines of splenic cell nitrite production in C57BL/6 IL-12 knockout and wild-type C57BL/6 mice infected with T. cruzi. Cultures were performed on days 7 and 14 after infection and stimulated with parasite-antigen, T-Ag. At the beginning of the cultures the neutralizing anti-cytokine mAbs, anti-IL-10, anti-IL-4 and anti-TNF-α, were added; GL.113 was used as isotype control mAb. The supernatants were harvested after 72 hr and nitrite was measured by the Griess reaction. Results are expressed as the arithmetic means from duplicates ± SD. *P < 0·05. The results are representative of three experiments.

Figure 6

Parasite-specific antibodies determination in the sera from day 10 infected C57BL/6 IL-12 knockout and wild-type C57BL/6 mice. Day 0 refers to uninfected mice. IgG1, IgG2a and IgG2b antibody levels are expressed by the mean OD accompanied by the SD, obtained in the sera from three mice at the reciprocal of the tested serum dilutions. *P < 0·05.