Parasite-induced lipoxin A4 is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection

Abstract

The production of interleukin (IL)-12 is critical for the development of interferon (IFN)-gamma-dependent resistance to Toxoplasma gondii. Nevertheless, when this response is dysregulated, such as occurs in the absence of IL-10, the uncontrolled inflammation that results can have lethal consequences for the host. Recently, we demonstrated that lipoxin (LX)A(4), an eicosanoid mediator that depends on 5-lipoxygenase (LO) for its biosynthesis, exerts a regulatory role on dendritic cell IL-12 production triggered artificially by a T. gondii extract. We now formally establish the physiological relevance of this pathway in the systemic control of IL-12 production induced by live T. gondii infection and demonstrate its function to be distinct from that of IL-10. Thus, T. gondii-exposed wild-type, but not 5-LO-deficient animals, produced high levels of serum LXA(4) beginning at the onset of chronic infection. Moreover, 5-LO(-/-), in contrast to wild-type mice, succumbed during the same period displaying a marked encephalitis. The increased mortality of the 5-LO(-/-) animals was also associated with significant elevations of IL-12 and IFN-gamma and was completely prevented by the administration of a stable LXA(4) analogue. Together, these findings demonstrate a new pathway involving the induction of host LXs for the in vivo regulation of proinflammatory responses during microbial infection.

Figure 1.

LXA4 and IL-10 are induced with different kinetics during T. gondii infection. Wild-type and 5-LO–deficient mice were infected intraperitoneally with 20 cysts of T. gondii (ME49 strain) and at the indicated times the animals were bled and LXA₄ and IL-10 levels were assessed by ELISA in the serum. The values represent mean ± SD of triplicate samples of at least three animals per time point per group. The experiment shown is representative of two performed.

Figure 2.

5-LO^−/− mice infected with T. gondii display profound weight loss and accelerated mortality despite the presence of lower parasite burdens and elevated proinflammatory cytokine production. Wild-type (B6, 129J F2) and 5-LO–deficient (B6, 129J Alox-5) animals (males and females) were infected as described in Fig. 1 and (A) body weight and (B) survival rates were monitored during the times indicated (n = 15 animals per group, per experiment). (C) To evaluate parasite load, the animals were killed and cysts were counted in brain homogenates. For systemic cytokine production, mice were bled at the indicated time points and the levels of (D) IL-12p40, (E) IFN-γ, and (F) TNF were measured in serum by ELISA. The values presented are the mean ± SD of at least three animals per time point per group. The experiments shown are representative of at least two performed that gave similar results. *, statistically significant (P < 0.05) differences between the means of the values obtained with 5-LO–deficient versus wild-type control mice.

Figure 3.

Uncontrolled encephalitis and leukocyte infiltration in the brains of 5-LO^−/− mice chronically infected with T. gondii. (A) Frozen sections of brains of wild-type controls (a, b, and c) and 5-LO^−/− (d, e, and f) mice infected for 25 d with T. gondii stained with anti-CD4 (a and d), anti-CD8 (b and e), or anti-CCR5 (c and f). Representative micrographs (×200) from at least three animals per group are presented. Arrows indicate reactive cells in the section. The mean ± SD of the total counts of (B) CD3⁺ CD4⁺, CD3⁺ CD8⁺ cells or of the (C) CD11c⁺ CD11b^lo, CD11c⁺ CD11b^hi cells and the intensity of the (D) latter cells obtained from brain homogenates (n = 3) of the same experimental groups is also shown. *, statistically significant (P < 0.05) differences between group means.

Figure 3.

Uncontrolled encephalitis and leukocyte infiltration in the brains of 5-LO^−/− mice chronically infected with T. gondii. (A) Frozen sections of brains of wild-type controls (a, b, and c) and 5-LO^−/− (d, e, and f) mice infected for 25 d with T. gondii stained with anti-CD4 (a and d), anti-CD8 (b and e), or anti-CCR5 (c and f). Representative micrographs (×200) from at least three animals per group are presented. Arrows indicate reactive cells in the section. The mean ± SD of the total counts of (B) CD3⁺ CD4⁺, CD3⁺ CD8⁺ cells or of the (C) CD11c⁺ CD11b^lo, CD11c⁺ CD11b^hi cells and the intensity of the (D) latter cells obtained from brain homogenates (n = 3) of the same experimental groups is also shown. *, statistically significant (P < 0.05) differences between group means.

Figure 4.

Enhanced IL-12p40 production by CD11c⁺ and CD11c⁻ cells in the brains of 5-LO^−/− mice chronically infected with T. gondii. (a) The same frozen sections of brain from wild-type and (b) 5-LO^−/− mice analyzed in Fig. 3 were double stained with anti-CD11c (green) or anti–IL-12p40 (red) and counterstained with DAPI (blue). Representative micrographs (×200) from at least three animals per group are presented.

Figure 5.

In vivo administration of a stable analogue of LXA₄ rescues 5-LO^−/− animals from mortality induced by T. gondii infection. Wild-type controls or 5-LO^−/− mice were infected with T. gondii as described above and treated every other day from days 10 to 20 with vehicle (0.2 ml/animal/time), LXA₄ analogue (1 μg/animal/time), or IL-10 (0.1 μg/animal/time). (A) Survival rates, (B) cysts counts, (C) serum IL-12, and (D) serum IFN-γ levels were assessed as previously described. Values presented are the mean ± SD of triplicate samples of five animals per group. *, statistically significant (P < 0.05) differences between group means.

Figure 6.

Redundant and nonredundant roles of IL-10 and LXA₄ in the regulation of proinflammatory function. Thioglycollate-elicited macrophages were incubated with medium alone, 100 ng/ml IL-10, or 100 ng/ml LXA₄. After 1 h, cells were infected with tachyzoites of the RH strain of T. gondii (5:1 parasite/macrophage ratio) with or without the addition of 100 ng/ml IFN-γ. (A) After an additional 48-h incubation, cells were fixed, Giemsa stained, and the number of intracellular parasites was determined by microscopic examination. In a parallel set of macro-phage cultures, supernatants were harvested at 24 h and (B) NO₂, (C) TNF, and (D) MIP-1α production was determined as described in Materials and Methods. Bars represent mean ± SD of triplicate measurements performed on cells pooled from five animals. The results shown are representative of two independent experiments performed. *, statistically significant (P < 0.05) differences between group means.